Chapter One opens with an overview of the role of pyridines in the pharmaceutical industry. This is followed by an in-depth survey of literature methods that have been reported for pyridine synthesis, including the Bohlmann-Rahtz reaction. The chapter concludes with the aims of this project.

Chapter Two delineates the development and optimization of a new ZnBr2-catalyzed method for the synthesis of 2,3,6-trisubstituted and 2,3,4,6-tetrasubstituted 3-nitropyridines using the Bohlmann-Rahtz reaction. This culminates in the synthesis and characterization of two libraries of 3-nitropyridines. The scope of this new Bohlmann-Rahtz reaction is then expanded to facilitate the synthesis of 2,3-disubstituted and 2,3,4-trisubstituted 3-nitropyridines.

Chapter Three describes a DPPE-mediated reaction for the cyclization of 3-nitropyridines into substituted ẟ-carbolines. The rest of the chapter then describes the development of a PPh3-mediated method for the cyclization of 3-nitropyridines into substituted β-carbolines. It concludes with the synthesis of the β-carboline natural product harmine.

EMBARGOED - expected end date 14.09.2023

We have previously shown that the thermolabile, cavity-creating p53 cancer mutant Y220C can be reactivated by small-molecule stabilizers. In our ongoing efforts to unearth druggable variants of the p53 mutome, we have now analyzed the effects of other cancer-associated mutations at codon 220 on the structure, stability and dynamics of the p53 DNA-binding domain (DBD). We found that the oncogenic Y220H, Y220N and Y220S mutations are also highly destabilizing, suggesting that they are largely unfolded under physiological conditions.
A high-resolution crystal structure of the Y220S mutant DBD revealed a mutation-induced surface crevice similar to that of Y220C, whereas the corresponding pocket’s accessibility to small molecules was blocked in the structure of the Y220H mutant. Accordingly, a series of carbazole-based small molecules, designed for stabilizing the Y220C mutant, also bound to and stabilized the folded state of the Y220S mutant, albeit with varying affinities due to structural differences in the binding pocket of the two mutants. Some of the compounds also bound to and stabilized the Y220N mutant, but not the Y220H mutant. Our data validate the Y220S and Y220N mutant as druggable targets and provide a framework for the design of Y220S or Y220N-specific compounds as well as compounds with dual Y220C/Y220S specificity for use in personalized cancer therapy

We present a short review of some of our recent work mainly targeting cancer-related oncoproteins through the development of primarily novel air- and water- stable iron-based organometallic agents. This work was presented at the recent ISBOMC19 conference at York as an invited lecture.

In an effort to characterise the human NUDIX family SGC Oxford has expressed recombinant human NUDT7 as part of the SGC chemical probe programme and solved the first crystal structure of this enzyme. This enabled a crystallographic fragment screen which in conjunction with a separate covalent fragment approach yielded a first-in-class small molecule inhibitor of NUDT7 with activity in the single-digit micromolar range in a catalytic assay. This compound paves the way for chemical probe development and further functional exploration of NUDT7 in physiological and disease contexts.

Tyrosyl DNA phosphodiesterase 2 (TDP2) facilitates the repair of topoisomerase II (TOP2)-linked DNA double-strand breaks and, as a consequence, is required for cellular resistance to TOP2 “poisons”. Recently, a deazaflavin series of compounds were identified as potent inhibitors of TDP2, in vitro. Here, however, we show that while some deazaflavins can induce cellular sensitivity to the TOP2 poison etoposide, they do so independently of TDP2 status. Consistent with this, both the cellular level of etoposide-induced TOP2 cleavage complexes and the intracellular concentration of etoposide was increased by incubation with deazaflavin, suggesting an impact of these compounds on etoposide uptake/efflux. In addition, deazaflavin failed to increase the level of TOP2 cleavage complexes or sensitivity induced by m-AMSA, which is a different class of TOP2 poison to which TDP2-defective cells are also sensitive. In conclusion, while deazaflavins are potent inhibitors of TDP2 in vitro, their limited cell permeability and likely interference with etoposide influx/efflux limits their utility in cells.

Bacillus thuringiensis produces a range of toxins that include both the insecticidal Cry toxins, non-insecticidal, non-haemolytic, and Parasporins. The latter exhibits cytocidal activity to some cancer cell lines. Parasporins 3 or Cry41Aa is cytocidal to human hepatic HepG2 cell lines. It contains the five conserved sequence blocks found in many insecticidal 3-domain Cry toxins and is also believed to possess the same 3-domain structure. In addition, it has an extra loop in its domain II as well as an additional ricin domain at its C-terminus. studies on insecticidal Cry toxins have implicated domain II loops in the specificity of a toxin to target a particular cell. In this study the specificity of Cry41Aa towards HepG2 cell lines was investigated. Bioinformatic tools were used to predict domain II loops of Cry41Aa. A number of mutants were created to investigate its specificity. Loop exchange mutants between loop 3 Cry41Aa and Cry loop 3 of insecticidal were created but did not result in a proteolytically stable protein. Domain II hybrids of Cry41Aa and insecticidal Cry toxins were created but these did not result in a proteolytic stable protein. Finally, residue substitutions with alanine in loop 1,3, and the extra loop resulted in stable activated toxins. loop 1 mutants retained toxicity. The extra loop mutant lost toxicity towards HepG2 cell lines. A number of Loop 3 mutants were made. Recombinant, Y514A and W511F retained toxicity towards HepG2 cells. Recombinant W511A and several recombinants at position 509 including F509A did not exert toxicity as confirmed by cell viability assays. Despite the lack of toxicity, membrane damage assays and western blots on HepG2 incubated with F509A revealed the likely presence of pores and phosphorylation of p38. Cell electrophysiology tools were applied to investigate the effect that nontoxic recombinant F509A on artificial and cell membrane. Cry41Aa induced the formation of stable pores, cell membrane damage and subsequent cell death. F509A induced the formation of unstable pores and did not compromise the integrity of cell membrane. The study Findings indicated that Cry41Aa is likely to have a similar mode of action as insecticidal Cry toxins.

Eukaryotic initiation factor 4E (eIF4E) is a key focus in cancer research due to its central role in controlling the translation of tumour-associated proteins that drive an aggressive migratory phenotype. eIF4E activity, modulated via its availability and phosphorylation are regulated by the PI3K/AKT/mTOR and mitogen-activated protein kinase interacting protein kinases (MNK1/2). The latter phosphorylates eIF4E on Ser209 whereas mTORC1 phosphorylates and de-activates the eIF4E inhibitor, 4E-BP1, to release translational repression. The work presented here describes the synthesis and characterisation of 4-((4- fluoro-2-isopropoxyphenyl)amino)-5-methyl thieno[2,3-d] pyrimidine-6- carboylic acid, known as compound 1, a MNK1/2 inhibitor.
Further analysis of compound 1 in combination with mTORC1/2 inhibitors show that inhibiting these pathways simultaneously effectively slows the rate of cell migration in MDA-MB-231 triple negative breast cancer (TNBC) cells. As an alternative approach, novel, cleavable dual MNK1/2 and PI3K/mTOR inhibiting hybrids were synthesised and characterised in MDA-MB-231 cells. These were found to be less effective at slowing cell migration than the combination of individual inhibitors. Molecular modelling of compound 1 revealed a large hydrophobic pocket which was exploited with a bulkier ferrocene group. Two novel ferrocene-containing compounds based upon compound 1 were synthesised and screened for MNK1/2 inhibition.
To target migration more specifically, work was also carried out with an alternative translational protein, DDX3X. Both genetic knockdown and pharmacological inhibition alone and in combination with compound 1 reveal its potential as an anti-cancer target.

The Epstein-Barr virus (EBV) is one of the oldest and most successful human viruses, infecting over 95% of the human population. Exposure usually occurs during early childhood and leads to mild, or no, acute symptoms. After primary infection, the virus enters naive B cells, causing differentiation and proliferation, finally leading to lifelong persistence of the virus in these cells (latency). Infectious virions are produced during the lytic phase of the EBV life cycle, leading to spread of the virus via oral secretions. There is a strong link between EBV infection and over a dozen malignant diseases. A large amount of our EBV knowledge has been obtained in B cells. However, this virus also infects epithelial cells and is associated with a range of epithelial malignancies. This thesis aims to elucidate some of the ambiguity surrounding EBV lytic cycle in epithelial cells and to extend the knowledge surrounding the switch from latency to lytic cycle. For this purpose EBV was induced into lytic cycle, in the epithelial cell line HONE1-EBV, with the histone deacetylase inhibitor SAHA, which is used in the treatment of non-Hodgkin lymphoma and is in Phase II clinical trials for the treatment of nasopharyngeal carcinoma. The binding of the early lytic cycle protein, Zta, to the epithelial host cell genome was investigated using Chromatin Immunoprecipitation followed by high-throughput sequencing. This led to the identification of several host genes regulated by the lytic virus. In addition, results previously obtained in B cells and other public ChIP sequencing data were included in the data analysis and revealed a potentially significant role of the host transcription co-factor and protooncogene candidate BCL3. Further experiments led to the proposal of a model for the regulation of Zta gene activation involving Zta, BCL3 and TORC.

Two novel ferrocene-containing compounds based upon a known MNK1/2 kinase (MAPK-interacting kinase) inhibitor have been synthesized. The compounds were designed to use the unique shape of ferrocene to exploit a large hydrophobic pocket in MNK1/2 that is only partially occupied by the original compound. Screening of the ferrocene analogues showed that both exhibited potent anticancer effects in several breast cancer and AML (acute myeloid leukemia) cell lines, despite a loss of MNK potency. The most potent ferrocene-based compound 5 was further analysed in vitro in MDA-MB-231 (triple negative breast cancer cells). Dose–response curves of compound 5 for 2D assay and 3D assay generated IC50 values (half maximal inhibitory concentration) of 0.55 µM and 1.25 µM, respectively.

Chaperones play a pivotal role in protein homeostasis, but with age their ability to clear aggregated and damaged protein from cells declines. Tau pathology is a driver of a variety of neurodegenerative disease and in Alzheimer's disease (AD) it appears to be precipitated by the formation of amyloid-β (Aβ) aggregates. Aβ-peptide appears to trigger Tau hyperphosphorylation, formation of neurofibrillary tangles and neurotoxicity. Recently, dihydropyridine derivatives were shown to upregulate the heat shock response (HSR) and provide a neuroprotective effect in an APPxPS1 AD mouse model. The HSR response was only seen in diseased cells and consequently these compounds were defined as co-inducers since they upregulate chaperones and co-chaperones only when a pathological state is present. We show for compounds tested herein, that they target predominantly the C-terminal domain of Hsp90, but show some requirement for its middle-domain, and that binding stimulates the chaperones ATPase activity. We identify the site for LA1011 binding and confirm its identification by mutagenesis. We conclude, that binding compromises Hsp90's ability to chaperone, by modulating its ATPase activity, which consequently induces the HSR in diseased cells. Collectively, this represents the mechanism by which the normalization of neurofibrillary tangles, preservation of neurons, reduced tau pathology, reduced amyloid plaque, and increased dendritic spine density in the APPxPS1 Alzheimer's mouse model is initiated. Such dihydropyridine derivatives therefore represent potential pharmaceutical candidates for the therapy of neurodegenerative disease, such as AD.

A series of novel oxazolo[5,4-d]pyrimidines was designed via a scaffold hopping strategy and synthesized through a newly developed approach. All these compounds were evaluated for their biological activity toward CB1/CB2 cannabinoid receptors, their metabolic stability in mice liver microsomes and their cytotoxicity against several cell lines. Eight compounds have been identified as CB2 ligands with Ki values less than 1 μM. It is noteworthy that 2-(2-chlorophenyl)-5-methyl-7-(4-methylpiperazin-1-yl) oxazolo[5,4-d]pyrimidine 47 and 2-(2-chlorophenyl)-7-(4-ethylpiperazin-1-yl)- 5-methyloxazolo[5,4-d]pyrimidine 48 showed CB2 binding affinity in the nanomolar range and significant selectivity over CB1 receptors. Interestingly, functionality studies imply that they behave as competitive neutral antagonists. Moreover, all tested compounds are devoid of cytotoxicity toward several cell lines, including Chinese hamster ovary cells (CHO) and human colorectal adenocarcinoma cells HT29.

While the molecular details by which Hsp90 interacts with Sgt1 and Rar1 were previously described the exact stoichiometric complex that is formed remains elusive. Several possibilities remain that include two asymmetric complexes, Sgt12-Hsp902-Rar12 (two molecules of Sgt1 and Rar1 and one Hsp90 dimer) or Sgt12-Hsp902-Rar11 (with a single Rar1 molecule) and an asymmetric complex (Sgt11-Hsp902-Rar11). The Hsp90-mediated activation of NLR receptors (Nucleotide-binding domain and Leucine-rich Repeat) in the innate immunity of both plants and animals is dependent on the co-chaperone Sgt1 and in plants on Rar1, a cysteine- and histidine-rich domain (CHORD)-containing protein. The exact stoichiometry of such a complex may have a direct impact on NLR protein oligomerization and thus ultimately on the mechanism by which NLRs are activated. CD spectroscopy was successfully used to determine the stoichiometry of a ternary protein complex among Hsp90, Sgt1, and Rar1 in the presence of excess ADP. The results indicated that a symmetric Sgt12-Hsp902-Rar11 complex was formed that could allow two NLR molecules to simultaneously bind. The stoichiometry of this complex has implications on, and might promote, the dimerization of NLR proteins following their activation.

Ferrocene analogues of known fatty acid amide hydrolase inhibitors and CB2 ligands have been synthesized and characterized spectroscopically and crystallographically. The resulting bioorganometallic isoxazoles were assayed for their effects on CB1 and CB2 receptors as well as on FAAH. None had any FAAH activity but compound 3, 5-(2-(pentyloxy)phenyl)-N-ferrocenylisoxazole- 3-carboxamide, was found to be a potent CB2 ligand (Ki = 32.5 nM).

Abstract Alzheimer's disease is characterised by the self-assembly of tau and amyloid β proteins into oligomers and fibrils. Tau protein assembles into paired helical filaments (PHFs) that constitute the neurofibrillary tangles observed in neuronal cell bodies in individuals with Alzheimer's disease. The mechanism of initiation of tau assembly into {PHFs} is not well understood. Here we report that a truncated 95-amino acid tau fragment (corresponding to residues 297-391 of full-length tau) assembles into PHF-like fibrils in vitro without the need for other additives to initiate or template the process. Using electron microscopy, circular dichroism and X-ray fibre diffraction, we have characterised the structure of the fibrils formed from truncated tau for the first time. To explore the contribution of disulphide formation to fibril formation, we have compared the assembly of tau(297-391) under reduced and non-reducing conditions and for truncated tau carrying a {C322A} substitution. We show that disulphide bond formation inhibits assembly and that the {C322A} variant rapidly forms long and highly ordered PHFs.

The R2TP complex, comprising the Rvb1p-Rvb2p AAA-ATPases, Tah1p, and Pih1p in yeast, is a special- ized Hsp90 co-chaperone required for the assembly and maturation of multi-subunit complexes. These include the small nucleolar ribonucleoproteins, RNA polymerase II, and complexes containing phosphati- dylinositol-3-kinase-like kinases. The structure and stoichiometry of yeast R2TP and how it couples to Hsp90 are currently unknown. Here, we determine the 3D organization of yeast R2TP using sedimenta- tion velocity analysis and cryo-electron microscopy. The 359-kDa complex comprises one Rvb1p/Rvb2p hetero-hexamer with domains II (DIIs) forming an open basket that accommodates a single copy of Tah1p-Pih1p. Tah1p-Pih1p binding to multiple DII do- mains regulates Rvb1p/Rvb2p ATPase activity. Using domain dissection and cross-linking mass spectro- metry, we identified a unique region of Pih1p that is essential for interaction with Rvb1p/Rvb2p. These data provide a structural basis for understanding how R2TP couples an Hsp90 dimer to a diverse set of client proteins and complexes.

Telomeres protect the ends of the linear chromosomes by assembling a nucleoprotein complex called shelterin. This complex regulates the action of telomerase, a reverse transcriptase enzyme that adds telomeric DNA to the G-rich 3’ end of the chromosomes. CST (CTC1/Cdc13-Stn1-Ten1) complex, also associated with telomeres, counteracts telomerase mediated telomere overhang elongation by recruiting lagging strand DNA polymerases for C-Strand synthesis. SUMOylation, a post-translational modification, is known to contribute towards negative regulation of telomerase in both budding yeast and fission yeast. It has previously been shown in budding yeast that SUMOylation of Cdc13 enhances its interaction with Stn1 to restrain telomerase function. In humans, CST interacts with overhang-binding shelterin proteins TPP1/POT1, but there is no evidence whether this interaction is regulated. The work in this thesis provides a mechanistic insight into the role of SUMOylation in telomere length regulation in fission yeast.

It was established in this study that fission yeast telomeric protein Tpz1, an ortholog of human TPP1, is SUMOylated at lysine 242 by SUMO E3 ligase Pli1. The mutation of this lysine residue leads to telomere elongation in a telomerase-dependent manner. Chromatin immunoprecipitation (ChIP) analysis indicated that the association of telomerase with telomeres is increased in a tpz1-K242R mutant, whereas that of Stn1 and Ten1 is greatly reduced. In addition, a SUMO-Tpz1 fusion protein showed increased affinity for Stn1, in the yeast two-hybrid assays. The assay was also used to define minimal region in Tpz1 required for its interaction with Stn1. This data indicated that SUMOylation of Tpz1 facilitates the recruitment and function of Stn1-Ten1 at telomeres, which in turn down-regulates telomerase. Collectively, this study highlight the evolutionary conservation of the regulation of (C)ST function by SUMOylation and provides further insight into the role of Tpz1 in telomerase regulation.

In order to obtain further insight into the dynamics of telomerase action and overhang fill-in by C-strand synthesis I also developed and validated a PCR-based assay for precise measurement of telomere overhang i budding and fission yeast.

Epstein-Barr virus (EBV) immortalises resting B-lymphocytes to lymphoblastoid cell lines (LCLs) and is associated with many cancers. The cell-cycle regulator response gene to complement 32 (RGC-32) is upregulated in EBV-infected cells, binds the mitotic kinases CDK1 and PLK1 and disrupted cell cycle checkpoints. RGC-32 may therefore play a role in EBV-mediated cell-cycle deregulation. RGC-32 has no homology with any other known proteins, so affinity-tagged forms of RGC-32 were expressed in E.coli for structure-function studies. Replacing a polyhistidine tag with a glutathione S-Transferase (GST) tag and optimising expression conditions improved RGC-32 solubility. Purified soluble RGC-32 was produced for structural studies, but no crystals were obtained. Using the GST-RGC-32 fusion protein I showed that RGC-32 interacts with CDK1, Plk1 and the kinetochore component Spc24 from B-cell lysates. Interestingly, RGC-32 did not interact with cyclin B1, suggesting that it may activate CDK1 in a cyclin-independent manner. Mapping the regions of interaction between RGC-32 and CDK1 and Plk1 revealed these kinases bind to different but adjacent regions of RGC-32. To investigate the role of RGC-32 in cell cycle disruption by EBV, I made a series of cell-lines stably expressing inducible RGC-32 constructs and RGC-32 disrupted G2/M checkpoint in an additional B-cell line. Investigating the mechanism of RGC-32 transcription control in EBV-infected cells demonstrated that although EBV transcription factors bind to intronic regions of the RGC-32 gene, no regulation was detected in reporter assay. These data reveal a novel aspect of CDK1 activation by RGC-32, identify the sites of protein-protein interactions and provide new cell-lines for further investigation of RGC-32 function.

Meiotic recombination is a complex process that requires tight regulation to ensure accurate chromosomal segregation and to prevent DNA double-strand breaks (DSBs), introduced to initiate meiotic recombination, from becoming damaging. Spo11 introduces DSBs via a topoisomerase-like reaction during meiosis. In this thesis I present work investigating the mechanisms that regulate the formation and repair of the protein-linked DSBs created by Spo11 and topoisomerase II (Top2). Initiation of Spo11-DSB resection is conducted by the Mre11-Rad50-Xrs2 (MRX) complex and Sae2 protein, which nucleolytically removes Spo11 covalently bound to oligonucleotides via a phosphotyrosine bond. Sae2 activity is controlled by post-translational modifications and regulation of its oligomeric state. Here I present data characterising the phenotype of Sae2 proteins mutated at putative Mec1/Tel1 phosphorylation sites (Chapter 3). The human TDP2 protein, hydrolytically removes proteolysed topoisomerase II (Top2) from the 5ʹ end of Top2-DSBs. Here I show that TDP2 is also active upon the phosphotyrosine bond between Spo11 and DNA in vitro (Chapter 4). Removal of Spo11 from the 5ʹ′end of doublestranded DNA by TDP2 permits resection by lambda exonuclease but no resection is observed by the primary meiotic 5ʹ to 3ʹ exonuclease, Exo1, in vitro. This suggests an evolutionary benefit of Spo11-DSB processing by the MRX complex and Sae2 at generating a substrate that permits Exo1 resection instead of the hydrolytic Spo11 removal mechanism by TDP2 (Chapter 4). Utilising TDP2 activity, I have developed a novel method to map Spo11-DSBs genome-wide with single nucleotide resolution (Chapter 5). The spatial patterning of meiotic DSBs is controlled in yeast by the ATM/ATR homologs Tel1/Mec1. Results from this new genome-wide DSB mapping method suggest that the kinase activity of Tel1 regulates hyper-local repression of coincident Spo11-DSBs (Chapter 6). v Utilising TDP2 and the nucleotide resolution mapping procedure for Spo11, Saccharomyces cerevisiae topoisomerase II (Top2) was also mapped genome-wide, which indicated that there are preferential sites for Top2 cleavage in vivo (Chapter 7). In the future this procedure can be adapted to map other protein-DNA complexes in vivo in a wide range of organisms. Collectively the work presented in this thesis further elucidates the mechanisms underpinning the spatial patterning of Spo11-DSBs in meiosis, the subsequent repair of meiotic DSBs, and also contributes to our understanding of the location of Top2 cleavage sites in vivo.

Epstein-Barr virus (EBV) is a widespread human tropic B cell virus that is linked to several malignancies. EBV modulates the transcriptome of B lymphocytes to drive immortalisation and viral persistence. EBV nuclear antigens (EBNA) 2,3A, 3B and 3C are transcriptional regulators of both viral and cellular genes and are the primary drivers of the immortalisation and the continued proliferation of infected B-cells. EBNA 2 activates all EBV gene promoters and cellular growth control genes while EBNA3A, 3B and 3C activates or represses transcription. EBNA2 and 3 proteins do not bind directly to DNA. They bind through cellular DNA-binding proteins like RBP-Jκ and PU.1. The focus of this research was to investigate how EBNA 2 promotes immortalisation through the epigenetic reprogramming of cellular genes and how EBNA 3A, 3B and 3C antagonise or cooperate with EBNA 2 in gene regulation. Previous ChIP-seq results in our lab identified significant binding sites for EBNA 2 and EBNA 3s. I targeted three important novel shared EBNA 2 and EBNA 3s binding sites; the integrin ITGAL, cell cycle kinase WEE1 and transcription repressor CTBP2 genes. I investigated if these shared sites are functional as EBNA 2 response elements in reporter assay by transiently transfecting the endogenous promoter and any associated long range enhancer region of genes and performing luciferase assays. EBNA 2 activates the ITGAL promoter and EBNA 3s inhibits the activation while WEE1 and CTBP2 does not respond in reporter assay. I also performed site-directed mutagenesis to determine which cellular transcription factor was important for the activation of EBNA 2 at the ITGAL promoter. RBP-Jk site mutation disrupted the EBNA 2 activation. Another research focus was EBNA 2 association with gene activation and repression. BCR components CD79A and CD79B are involved in signal transduction and the regulation of B-cell growth and survival and transcription factor EBF1 plays an important role in B cell differentiation. I investigated the association of EBNA 2 with these repressed gene targets and if EBF1 plays a role in the mechanism of repression using reporter assay. CD79A and CD79B activates EBNA 2 and EBF1 does not significantly repress the activation in luciferase reporter assay. EBNA 2 have been mapped binding to enhancers at a new target gene interferon response factor 4 IRF4 and microarray data implicates EBNA 2 in its activation. When IRF4 expression is reduced in EBV transformed cells, cell proliferation rate is decreased and apoptosis enhanced so this activation may be important for B-cell transformation by EBV. I carried out reporter assays to determine if the site is EBNA 2 responsive and whether it interacts with the IRF4 promoter and enhancers. EBNA 2 slightly activates the promoter and enhancers.

Linear DNA is packaged into higher-order structures termed chromatin, in which the majority of DNA sequences are structurally inaccessible and functionally inactive. Hence, chromatin must exist in a dynamic state to govern the accessibility of the DNA to various regulatory factors that control nuclear processes, such as transcription, DNA replication, and DNA repair. Accessibility in turn is mediated by post-translational modifications, histone variants, histone chaperones, and ATP-dependent chromatin remodelling complexes. In particular, these complexes, such as the multi-subunit INO80 complex, are characterized by their ability to utilise ATP hydrolysis to alter histone-DNA contact by the sliding, eviction, or exchange of histones or nucleosomes. Since its identification in 1999, many INO80 subunits have been purified and structurally characterized. Here, we structurally and biochemistry characterized the recently identified human INO80 YY1 subunit and demonstrated a role for YY1 together with RUVBL2, another INO80 subunit, in promoting DNA repair by homologous recombination (HR). Consequential to its chromatin-remodelling activity, INO80 plays multiple roles in cellular metabolism including DNA repair and chromosomal stability. Although evidence from yeast and mammals has indicated the involvement of INO80 in HR repair, the exact mechanism affected by INO80 therein remained unclear. Through a combination of live cell imaging and in vivo techniques, we revealed that in human cells the histone variant H2AZ is rapidly removed from damaged chromatin by INO80. Furthermore, we found that INO80 together with the histone chaperone, ANP32E, promotes HR by removing H2AZ from damaged chromatin. Finally, we verified that INO80 is required for the maintenance of chromosomal stability and that loss of INO80 in CIN+ tumour cells induced cell death. Therefore, INO80 may serve as a therapeutic target for the selective elimination of CIN+ tumour cells.

TDP2, a DNA phosphodiesterase that removes trapped topoisomerase 2 (TOP2) from 5’-DNA termini, is required for efficient repair of TOP2-induced DNA double-strand breaks (DSBs). Cellular depletion of TDP2 was shown to result in a substantially increased sensitivity to TOP2-induced DSBs and TOP2 poisons, such as etoposide, in various types of human cancer cell lines. In addition, over-expression of TDP2 has been shown to increase resistance to etoposide. Recent data suggest that expression levels of TDP2 vary greatly in different cancer cell lines. However, there are no reported studies addressing the possible role of TDP2 as a clinical predictor of anti-cancer therapy outcome, or wider studies correlating TDP2 over-expression with resistance to TOP2 poisons. TDP2 has the potential to be a good target for pharmacological inhibition potentially increasing tumour sensitivity to TOP2 poisons, particularly those that develop resistance during the course of treatment. There is already a lot of interest in the development of TDP2 inhibitors and the in vitro results are very promising with many possible small molecule inhibitors showing selectiveness in their target. In my thesis I aim to further establish the range of TDP2 and TOP2 mRNA and protein levels in a panel of lung and breast cancer cell lines. In addition, I will explore the possibility of a correlation between TDP2 protein levels or TOP2/TDP2 protein ratios and sensitivity to the TOP2 poison etoposide. This likely complex relationship will be further defined by possible mutation effects based on available literature and studies for the cancer cell lines accessible for this project. Furthermore, as etoposide has been tested in clinical trials not only alone but also in combination with other treatments, I aim to include other accessible drugs, either currently in use for cancer treatment or at a promising clinical trial stage, such as estradiol and PARP1 inhibitors.

There is a recent model, which suggests that induction of transcriptional programs by stimulating breast cancer cells with estrogens, or prostate cancer cells with androgens, can involve the formation of TOP2B mediated DSBs and the recruitment of DSB repair proteins. TOP2B is believed to be recruited with the estrogen/androgen receptor to regulatory sites on target genes. It is hypothesized that the formation of these DSBs by TOP2B could also be exploited therapeutically. In my thesis I aim to utilise a combination treatment that will first induce transcription with estradiol in breast cancer cells, which will cause transient TOP2B-mediated DSBs, and then transform those breaks into abortive breaks with etoposide. Cells will then be overwhelmed with DSBs and apoptosis will be promoted. This will also be explored in TDP2-depleted MCF7 breast cancer cells. Such a strategy could possibly find particular use in hormone dependent cancers, where other types of treatment have failed.

In eukaryotes, DNA is packaged into a highly condensed structure, known as chromatin. Several complexes facilitate the remodelling of chromatin, for example, INO80, NURD and SWI/SNF, which attach to tightly bound chromatin, allowing its relaxation by nucleosome sliding, unwrapping, histone eviction and exchange of histone variants. The activities carried out by these chromatin remodelling complexes are thought to be integral in the prevention of cancer cell formation. Recently, whole exome sequencing has identified frequent mutations in subunits of the SWI/SNF chromatin remodelling complex, at a frequencythat rivals p53. Strikingly, the BAF180 (PBRM1) subunit of the PBAF variant of SWI/SNF remodelers is mutated in over 40% of clear cell renal cell carcinoma (ccRCC), a cancer with typically poor prognosis and limited treatment options to date.This work embodiesfour main results chapters that aim to identify novel synthetic lethal gene candidates with BAF180, with a view to targeting these gene candidates with chemotherapeutic drugs. In the first chapter we work through a short list of hypothesis driven potential synthetic lethal candidates and identify the genes KAT2A, RNF4, EZH2 and BAP1 as potential synthetic lethal partners for BAF180. Chapter twodescribes the development of both stableshRNAand CRISPR/Cas9-derived BAF180-deficient cell lines that were used both inthis studyas well as for other ongoing projects. The third chapter outlines the set-up of a high-throughput synthetic lethal siRNA (HTS) screen and determinespotential synthetic lethal interactions identified here. The final chapter examinesvarious PARP genes, identified ashits in HTS screening, to further explore the interaction between PARP and BAF180. We find that PARP1 and PARP3are synthetic lethal with BAF180 and treatment with various siRNA’s and PARP inhibitors in BAF180 deficient mammalian cells results in specific cell death. A phenotype that could be clinically exploited for treatment of ccRCC.

The Structural Maintenance of Chromosome (SMC) family of proteins are required to regulate almost all aspects of chromosome biology and are critical for genomic stability. The SMC5/6 complex, a member of this family, is composed of two SMC heterodimers and six additional Non-SMC Elements 1-6. The components of SMC5/6 possess activities including ATPases, ubiquitin and SUMO ligases. SMC5/6 is required in homologous recombination and for
accurate chromosome segregation. Loss of SMC5/6 is lethal in yeasts, embryonic lethal in mice and mutations in NSMCE2 leads to primordial dwarfism and insulin resistance.

This thesis focuses on a mutation in NSMCE3, found in American and Dutch families, that results in a novel chromosomal breakage syndrome characterized by fatal pulmonary disease. Another focus is the development, execution and validation of a microscopy based synthetic sick/lethal screen using cells with knockdown of NSMCE4a. Studies of SMC5/6 in yeasts predict that compromising SMC5/6 function would lead to a dependence on other DNA
repair pathways. The results combined with patient data confirm that SMC5/6 is important in the absence of repair by non-homologous end joining and is particularly important under conditions of replication stress.

Heat shock protein 90 (HSP90) is an ATP-dependent molecular chaperone that
plays critical roles in regulating the folding, stabilization, post-translational
modification, activation and maturation of its various client proteins, of which many are
oncoproteins. Impairing the function of HSP90 by the inhibition of its ATPase cycle
with inhibitors such as AUY922 promotes the ubiquitylation and proteasomal
degradation of its client proteins. However, we currently do not fully understand the
mechanism for ATPase-inhibited triggered degradation of client proteins, and which E3
ligase systems are involved.
Although previous studies revealed a number of E3 ligases including CHIP and
CUL5 as potentially E3 ligases involved in the degradation of HSP90-dependent client
protein, these have often used cancer cells that may have dysregulated systems.
Additionally, other components of such E3 ligase systems have not been well
characterised.
Using a Reverse Transfection Format (RTF) siRNA screen system we identified
two E3 ligases that are involved in two independent pathways for mediating
proteasomal degradation of the HSP90-dependent protein kinase CRAF in HEK293
cells. The elongin BC-CUL5-SOCS-box protein (ECS) complex operates one pathway
for the degradation of CRAF, while a novel but poorly described HECTD3 from the
HECT-family was identified as the main E3 ligase for degrading CRAF following the
pharmaceutical inhibition of HSP90. We revealed a potential complexes consisting of
CRAF, HSP90 and HECTD3, which may contribute towards identifying the pathway
for the degrading of such HSP90-dependent client protein kinases. We were also able to
show that depriving access of CRAF to CDC37 and therefore HSP90 resulted in an
HECTD3 and CUL5 independent degradation pathway. These studies form the basis of
establishing the complex network of pathways that help to regulate CRAF protein levels.

Accurate chromosome segregation during mitosis prevents aneuploidy and cancer. The Wee1-Cdc25-Cdk1 feedback loop ensures cells enter mitosis in a timely and orderly manner. This signalling system has been proposed to work as a bistable switch that is maintained by the counterbalancing action of kinases and phosphatases. However, the phosphatases critical for this transition in mammalian cells are yet to be identified. Cdc14 is the major phosphatase antagonising CDK in yeast. But its eukaryotic homologue does not appear to play an essential role in mitotic control. Studies in Drosophila and Xenopus have shown that PP2A/B55 inhibition is crucial for MPF activation, and that this is brought about by Greatwall kinase and its substrates, the closely related 17kDa proteins Ensa and Arpp19. However, it remains unclear, if PP2A/B55 inhibition by phosphorylated Ensa/Arpp19 is a critical event in regulating mitotic entry in somatic cells.
In this thesis I have performed a cell biological, genetic and biochemical characterisation of Ensa/Arpp19. I have generated and optimised tools to study the function of these two proteins and characterised their localisation, their depletion phenotypes, the nature of their interaction with the PP2A/B55 phosphatase and their protein interactome in metaphase and anaphase in mammalian cells. In parallel I have been optimising a novel protein transfection based method to study the effects of constitutively phosphorylated Ensa/Arpp19 in cells.
The experiments described in this thesis show a surprising divergence of the functions of Ensa/Arpp19 and their upstream kinase Greatwall, suggesting a more complex signaling cascade involving these proteins. Our biochemical characterization of the Ensa/B55 interaction also suggests a novel mechanism of action for these proteins. Overall this work sheds light on how the balanced activity of the Cdk1 activation switch and its counteracting phosphatases contribute to the regulation of mitotic entry.

Fluorescence microscopy is a popular biological technique because it allows the study of cells in great detail. However, the resolution achievable is limited by the diffraction properties of light, meaning that fine detail cannot be resolved. Various super-resolution microscopy methods have been developed to break this resolution limit. This thesis focuses on the single molecule localisation microscopy techniques. My host laboratory focuses on DNA replication and repair pathways using the model organism Schizosaccharomyces pombe (fission yeast). The aim of this thesis is thus to apply the technique of photo-activatable localisation microscopy (PALM) to specific biological questions in order to establish its benefits and limitations.
In theory, in PALM every molecule will be imaged once and, as such, could be counted. So far this has been largely limited to membrane proteins. Using a combination of artificially created fluorescent oligomers, endogenous ribonucleotide reductase proteins tagged with mEos and computer simulations I studied the feasibility of counting highly expressed cytoplasmic proteins and assigning them to complexes of known or unknown stoichiometry. I established that density of expression is a significant limiting factor when using PALM to resolve complex stoichiometry.
I thus went on to develop a variation of fluorescence correlation spectrometry to study the same protein complexes to see if we could determine their stoichiometry by diffusion speed. I established that the technique could differentiate between quite small changes in size. However the endogenous complex did not respond well to the fluorophore used so I was not able to establish its size.
Using the PALM system I also studied a biological molecule, Rrp2, which was expressed at such low levels it was not possible to observe with conventional fluorescence microscopy. I established that we were able to observe this protein at endogenous levels and characterised its behaviour in response to stress.

The SMC protein family (Structural Maintenance of Chromosomes) consists of a group of highly conserved protein complexes, central to chromosome dynamics and key cell cycle events. Condensin is a member of the SMC protein family, best known for its role in chromosome condensation and segregation in mitosis. The condensin complex becomes enriched at specific chromosome loci in a cell-cycle specific manner. However, the details of how it becomes associated with chromatin remain unclear. A particular area of interest regarding condensin association and activity is at the centromeres and pericentromeres, where condensin has been consistently shown to be enriched specifically during mitosis.

This work is comprised of four results chapters, investigating factors affecting condensin association with mitotic centromeres in Saccharomyces cerevisiae, using chromatin immunoprecipitation (ChIP). We started by establishing a robust ChIP assay suitable for probing condensin enrichment at the centromeric regions. We conducted genetic control experiments to ensure the functionality of the experimental technique. In the next chapter we explored the importance of the kinetochore with regards to condensin enrichment, and found that perturbing the budding yeast kinetochore results in a loss of centromeric condensin association during mitosis. We then used condensin phosphorylation site and mitotic kinase mutants to examine the role of condensin subunit phosphorylation in its association with chromatin. Our results showed that Ipl1 (Aurora B kinase) and condensin phosphorylation is important for its enrichment at the centromere, but rather surprisingly that Cdc5 (polo-like kinase) a known activator of condensin does not appear to be. The final chapter investigates the function of condensin’s intrinsic ATPase activity, and we found that ATP-binding activity but not ATP-hydrolysis is important for condensin association with chromatin.

Bacillus thuringiensis (Bt) crystal (Cry) proteins, used for decades as insecticidal toxins worldwide, are well known to be toxic to certain insects, but not to mammals. The three domain Cry toxins represent the biggest group with pore formation as a widely accepted model for insect killing. A novel group of Cry proteins has been identified known as parasporins. They do not show insecticidal or hemolytic activity, but exert a strong cytocidal effect against some human cancer cells. The preferential activity of parasporins has potential for anticancer drug design but at the same time the knowledge that some Bt toxins are able to kill mammalian cells may raise concerns about the use of Bt-based pesticides in the future. Out of 19 parasporins Parasporin-3 (PS-3) most closely resembles the commercially used insecticidal toxins and is toxic to a narrow range of human cancer cell lines. In this study the effect of recombinant PS-3 on the human hepatic cancer cell line HepG2 was investigated to elucidate its mode of action. Results are consistent with PS-3 being a pore forming toxin. The toxin induced: pore formation in artificial and biological membranes, irreparable membrane damage, cell swelling, rapid decrease in ATP levels and drop in metabolic activity. The toxin did not induce activation of caspases or oxidative stress. In response to the toxin, cells activated p38 MAPK, a conserved signalling pathway induced in host cells by pore forming toxins. PS-3 induced very stable lesions and p38 MAPK did not facilitate cell recovery. Identification of a proteinous receptor was unsuccessful, but the toxin interaction with the membrane was prevented by EGTA facilitated chelation of membrane associated cations, suggesting the existence of a cation dependent receptor.

Ruthenium complexes are considered as potential replacements for platinum compounds in oncotherapy. Their clinical development is handicapped by a lack of consensus on their mode of action. In this study, we identify three histones (H3.1, H2A, H2B) as possible targets for an anticancer redox organoruthenium compound (RDC11). Using purified histones, we confirmed an interaction between the ruthenium complex and histones that impacted on histone complex formation. A comparative study of the ruthenium complex versus cisplatin showed differential epigenetic modifications on histone H3 that correlated with differential expression of histone deacetylase (HDAC) genes. We then characterized the impact of these epigenetic modifications on signaling pathways employing a transcriptomic approach. Clustering analyses showed gene expression signatures specific for cisplatin (42%) and for the ruthenium complex (30%). Signaling pathway analyses pointed to specificities distinguishing the ruthenium complex from cisplatin. For instance, cisplatin triggered preferentially p53 and folate biosynthesis while the ruthenium complex induced endoplasmic reticulum stress and trans-sulfuration pathways. To further understand the role of HDACs in these regulations, we used suberanilohydroxamic acid (SAHA) and showed that it synergized with cisplatin cytotoxicity while antagonizing the ruthenium complex activity. This study provides critical information for the characterization of signaling pathways differentiating both compounds, in particular, by the identification of a non-DNA direct target for an organoruthenium complex.

The alternative oxidase (AOX) is a respiratory protein found in the mitochondrial electron transfer pathway throughout plants, sporadically in fungi and protozoa and within a small percentage of animals and proteobacteria. The AOX branches from the classical respiratory chain at the ubiquinone pool by coupling the oxidation of two molecules of ubiquinol to the complete 4-electron reduction of oxygen to water. The AOX can be found within several pathogenic organisms such as Trypanosoma bruceii and Cryptosporidium parvum and due to its absence in the mammalian host the AOX proves to be a potential therapeutic target in these systems. Recent crystallisation of the trypanosomal alternative oxidase (TAO) has enabled a more detailed examination of the structure of all AOXs, through homology modelling. Comparisons of sequence alignments of multiple AOXs and AOX homology models has revealed high conservation of residues predicted to be involved in ubiquinol binding and a proton coupled electron transport (PCET) network proposed to be involved in oxygen reduction.

Recombinant wild type TAO and Sauromatum guttatum AOX (SgAOX) have been expressed in a haem deficient strain of E. coli, in addition to a number of mutants that contain mutations occuring within the ubiquinol binding pocket and the PCET. Over-expression and purification of recombinant wild type AOX and mutants has enabled the characterisation of residues within the ubiquinol binding site and PCET network, contained within the secondary ligation sphere of the AOX. The results of which confirmed previously proposed roles for these residues, drawn from the crystal structure of TAO and models of the redox cycle mechanisms.

A novel AOX was identified within the fungus that causes Ash Dieback, Hymenoscyphus fraxineus. Protocols for the over expression and purification were established for the recombinant H. fraxineus AOX. This protein revealed significantly lower ubiquinol oxidase activities but a similar Km for oxygen and ubiquinol when compared to that previously seen with TAO and SgAOX. Typical potent AOX inhibitors, such as ascofuranone, were shown to be significantly less effective at inhibiting HfAOX.

This led to testing inhibitors against multiple AOXs from different kingdoms. It was found that not only did the efficiency of inhibitors vary between AOXs but the ubiquinol oxidase rates varied as well. The results presented in this thesis suggest that the differences in inhibitor efficacy and ubiquinol oxidase activity are caused by the functional structure of the residues lining the inhibitor/substrate channel.

Fused-in-sarcoma (FUS) is an RNA binding protein, thought to be involved in a wide variety of cellular processes, and mutations in FUS are known to be causative for amyotrophic lateral sclerosis (ALS). The mechanism of pathogenesis for ALS has not been established but it has been proposed that dysfunction in cellular functions involving RNA could be responsible. Investigations into a FUS-ALS patient cell line showed sensitivity to the transcriptional inhibitor camptothecin (CPT) and demonstrated constitutively fragmented nucleoli, a phenotype that has been associated with rRNA dysfunction, as well as a possible defect in ribosomal RNA (rRNA) maturation. In addition a reversible relocalisation of FUS to the nucleolus in response to inhibition of RNA polymerase II was observed in all cell lines examined. This relocalisation appeared to be dependent on the activity of phosphodiesterase 8 (PDE8) and on the presence of rRNA, as pre-inhibition of RNAP I (which produces rRNA) prevented relocalisation of FUS. However treatment of both RNAP I and RNAP II at the same time resulted in FUS relocalisation and the protein remaining in the nucleolus for hours if inhibition was maintained - long after RNA would be depleted at the site were RNAP I inhibited in isolation. These findings suggest that FUS may have a role in protecting pre-rRNA transcripts from degradation during transcriptional stress.

Parkinson’s disease (PD) is characterized by intracellular, insoluble Lewy bodies composed of highly stable α-synuclein (α-syn) amyloid fibrils. α-synuclein is an intrinsically disordered protein that has the capacity to assemble to form β-sheet rich fibrils. Oxidiative stress and metal rich environments have been implicated in triggering assembly. Here, we have explored the composition of Lewy bodies in post-mortem tissue using electron microscopy and immunogold labeling and revealed dityrosine crosslinks in Lewy bodies in brain tissue from PD patients. In vitro, we show that dityrosine cross-links in α-syn are formed by covalent ortho-ortho coupling of two tyrosine residues under conditions of oxidative stress by fluorescence and confirmed using mass-spectrometry. A covalently cross-linked dimer isolated by SDS-PAGE and mass analysis showed that dityrosine dimer was formed via the coupling of Y39-Y39 to give a homo dimer peptide that may play a key role in formation of oligomeric and seeds for fibril formation. Atomic force microscopy analysis reveals that the covalent dityrosine contributes to the stabilization of α-syn assemblies. Thus, the presence of oxidative stress induced dityrosine could play an important role in assembly and toxicity of α-syn in PD.

Fish can be exposed to a complex mixture of chemical contaminants, including pharmaceuticals, present in discharges of wastewater treatment works (WwTWs) effluents. There is little information on the effects of effluent exposure on fish metabolism, especially the small molecule signaling compounds which are the biological target of many pharmaceuticals. We applied a newly developed sensitive nanoflow-nanospray mass spectrometry nontargeted profiling technique to identify changes in the exposome and metabolome of roach (Rutilus rutilus) exposed to a final WwTWs effluent for 15 days. Effluent exposure resulted in widespread reduction (between 50% and 90%) in prostaglandin (PG) profiles in fish tissues and plasma with disruptions also in tryptophan/serotonin, bile acid and lipid metabolism. Metabolite disruptions were not explained by altered expression of genes associated with the PG or tryptophan metabolism. Of the 31 pharmaceutical metabolites that were detected in the effluent exposome of fish, 6 were nonsteroidal anti-inflammatory drugs but with plasma concentrations too low to disrupt PG biosynthesis. PGs, bile acids, and tryptophan metabolites are important mediators regulating a diverse array of physiological systems in fish and the identity of wastewater contaminants disrupting their metabolism warrants further investigation on their exposure effects on fish health.

Improved understanding of normal human ageing will provide important insights into major risk factors for many age-related diseases. Using the progeroid disease Werner syndrome (WS) as a model for accelerated ageing, this project aimed to investigate how modulation of protein function in the p38α stress-signalling kinase cascade could affect ageing on the cellular level.
New rapid routes towards p38α and MK2 inhibitors have been developed to evaluate their use as chemical probes to investigate the mechanisms of cellular ageing. The p38α inhibitor RO3201195 has been synthesized and used as a probe to corroborate previously published evidence that inhibition of the p38α protein kinase reverses the aged morphology of WS fibroblasts.
A new route for the synthesis of the MK2 inhibitor PF-3644022 was developed, exploring new methods for building the heterocyclic scaffold through a number of retrosynthetic strategies.
Microwave-assisted organic synthesis was used extensively as a tool towards halogenated benzothiophenes and fused quinoline systems, and in rapid Suzuki-Miyaura coupling and Buchwald-Hartwig N-arylation chemistry. This MK2 inhibitor can now be used as a readily accessible probe to further investigate the role of kinases downstream of p38α and the role of MK2 in the premature ageing of WS cells.
The final part of the project focussed on the potential of a known p38α inhibitor, BIRB 796, in the development of new chemical probes. BIRB 796 binds allosterically outside of the ATPbinding pocket of p38α MAPK, which drives a conformational change of the kinase into an inactive form. Using rapid synthetic methods towards pyrazole and urea formation, the selectivity elements of the inhibitor were reviewed and a new library of analogues was synthesized. Upon testing against a small panel of kinases, it was found that through small changes the inhibitor potency for p38α could be dramatically altered, and binding affinity for alternative kinases could be selectively enhanced.

In mitotic cells, the cyclin-dependent kinase (CDK) subunit protein CKS1 regulates S phase entry by mediating degradation of the CDK inhibitor p27. Although mature neurons lack mitotic CDKs, we found that CKS1 was actively expressed in post-mitotic neurons of the adult hippocampus. Interestingly, Cks1 knockout (Cks1−/−) mice exhibited poor long-term memory, and diminished maintenance of long-term potentiation in the hippocampal circuits. Furthermore, there was neuronal accumulation of cofilin-actin rods or cofilin aggregates, which are associated with defective dendritic spine maturation and synaptic loss. We further demonstrated that it was the increased p27 level that activated cofilin by suppressing the RhoA kinase-mediated inhibitory phosphorylation of cofilin, resulting in the formation of cofilin aggregates in the Cks1−/− neuronal cells. Consistent with reports that the peptidyl-prolyl-isomerase PIN1 competes with CKS1 for p27 binding, we found that inhibition of PIN1 diminished the formation of cofilin aggregates through decreasing p27 levels, thereby activating RhoA and increasing cofilin phosphorylation. Our results revealed that CKS1 is involved in normal glutamatergic synapse development and dendritic spine maturation in adult hippocampus through modulating p27 stability.

Protein-protein interactions (PPIs) present a challenging target for the development of
inhibitors, due to the large size and lipophilicity of protein binding sites. Because of this,
work that is able to increase our understanding of how PPI inhibitors can be better
developed is of great value.
In unusual cases, approved drug compounds are able to exhibit excellent pharmacological
(PK) profiles despite having unfavourable physical properties. New methodology was
developed whereby the solubilising side chains found in one such compound, daclatasvir,
could be incorporated onto aryl-bromides using palladium-catalysed C-H activation. The
scope of the reaction was tested, and in 18 different examples the C-H activation was
found to be successful 11 times.1 This methodology has application in the potential
synthesis of cell-permeable PPI inhibitors.
Inhibiting the PALB2/BRCA2 PPI has the potential to produce synthetic lethality in
cancerous cells with damaged DNA repair mechanisms. Computational techniques were
used to design small-molecule and miniprotein mimetics of BRCA2, which could
potentially competitively inhibit PALB2/BRCA2. This resulted in the purchase of 10
miniprotein sequences ready for testing, one of which has been proven to have significant
helical secondary structure. The synthesis of small molecule α-helical mimetics of
BRCA2 has also been carried out, with one compound ready for biological testing and
the synthesis of two others close to completion.

Publications:

1 T. O. Moore, M. Paradowski and S. E. Ward, Org. Biomol. Chem., 2016, 14,
3307–3313.

A small library of ferrocene-containing amides has been synthesized using standard amide coupling chemistry with ferrocenylamine. Ferrocene analogues of known bioactive adamantylamides were shown to be effective cannabinoid receptor (CB1 and CB2) agonists, displaying, in many cases, single-digit nanomolar potency. Three final ferrocene-containing derivatives have been characterized in the solid state by X-ray crystallography and display intramolecular hydrogen bonding of the type NH---C═O. N-Methylation of the amide, confirmed by X-ray crystallography, leads to both loss of hydrogen bonding and biological activity.

PARP3 is a member of the ADP-ribosyl transferase superfamily that we show accelerates the repair of chromosomal DNA single-strand breaks in avian DT40 cells. Two-dimensional nuclear magnetic resonance experiments reveal that PARP3 employs a conserved DNA-binding interface to detect and stably bind DNA breaks and to accumulate at sites of chromosome damage. PARP3 preferentially binds to and is activated by mononucleosomes containing nicked DNA and which target PARP3 trans-ribosylation activity to a single-histone substrate. Although nicks in naked DNA stimulate PARP3 autoribosylation, nicks in mononucleosomes promote the trans-ribosylation of histone H2B specifically at Glu2. These data identify PARP3 as a molecular sensor of nicked nucleosomes and demonstrate, for the first time, the ribosylation of chromatin at a site-specific DNA single-strand break.

Background and Purpose

Flecainide is a use-dependent blocker of cardiac Na+ channels. Mechanistic analysis of this block showed that the cationic form of flecainide enters the cytosolic vestibule of the open Na+ channel. Flecainide is also effective in the treatment of catecholaminergic polymorphic ventricular tachycardia but, in this condition, its mechanism of action is contentious. We investigated how flecainide derivatives influence Ca2+-release from the sarcoplasmic reticulum through the ryanodine receptor channel (RyR2) and whether this correlates with their effectiveness as blockers of Na+ and/or RyR2 channels.
Experimental Approach

We compared the ability of fully charged (QX-FL) and neutral (NU-FL) derivatives of flecainide to block individual recombinant human RyR2 channels incorporated into planar phospholipid bilayers, and their effects on the properties of Ca2+ sparks in intact adult rat cardiac myocytes.
Key Results

Both QX-FL and NU-FL were partial blockers of the non-physiological cytosolic to luminal flux of cations through RyR2 channels but were significantly less effective than flecainide. None of the compounds influenced the physiologically relevant luminal to cytosol cation flux through RyR2 channels. Intracellular flecainide or QX-FL, but not NU-FL, reduced Ca2+ spark frequency.
Conclusions and Implications

Given its inability to block physiologically relevant cation flux through RyR2 channels, and its lack of efficacy in blocking the cytosolic-to-luminal current, the effect of QX-FL on Ca2+ sparks is likely, by analogy with flecainide, to result from Na+ channel block. Our data reveal important differences in the interaction of flecainide with sites in the cytosolic vestibules of Na+ and RyR2 channels.

We examined the effects of the histone deacetylase inhibitor (HDACi) suberoylanilide
hydroxamic acid (SAHA) combined with the vascular endothelial growth factor receptor-1/2 inhibitor
(3Z)-5-hydroxy-3-(1H-pyrrol-2-ylmethylidene)-2,3-dihydro-1H-indol-2-one on MDA-MB-231 breast
cancer cells (triple-negative) in the form of both a cocktail of the separate compounds and a chemically
synthesized hybrid (N-hydroxy-N'-[(3Z)-2-oxo-3-(1H-pyrrol-2-ylmethylidene)-2,3-dihydro-1H-indol-
5-yl]octanediamide). Comparative flow cytometric and Western blot analyses were performed on
cocktail- and hybrid-treated cells to evaluate cell cycle distribution, autophagy/apoptosis modulation,
and mitochondrial metabolic state in order to understand the cellular basis of the cytotoxic effect.
Cell cycle analysis showed a perturbation of the rate of progression through the cycle, with
aspects of redistribution of cells over different cycle phases for the two treatments. In addition,
the results suggest that the two distinct classes of compounds under investigation could induce
cell death by different preferential pathways, i.e., autophagy inhibition (the cocktail) or apoptosis
promotion (the hybrid), thus confirming the enhanced potential of the hybrid approach vs. the
combination approach in finely tuning the biological activities of target cells and also showing the
hybrid compound as an additional promising drug-like molecule for the prevention or therapy of
“aggressive” breast carcinoma.

Fallopia japonica, or Japanese knotweed is a rhizomatous perennial herb native to East Asia; most notably Japan, China and Korea. Upon discovery of this species and subsequent import to Europe in the 1840’s, it was considered an esteemed ornamental plant – winning the medal for the ‘most interesting new plant of the year’ in 1847.

F. japonica soon became known as a menace rather than a champion, when it began to spread throughout its new environment, spreading to gardens and nurseries and regenerating from discarded plant fragments. The species ability to cause environmental damage has earnt it a place in the ‘top 100 world’s worst most invasive alien species’ list.

Commercially available herbicides have proven have little effect on F. japonica, and to be successful require many repeat applications. The plant can grow up to 10 cm per day during the early budding and shoot stage and can easily dominate an environment when left unchecked. A key objective of this research was to determine the biochemical pathways of energy generation particularly during the rapid phase of growth with the longer term goal of identifying potential inhibitors of this process which may have commercial opportunities.

Very little research is available regarding the biochemistry of growth of F. japonica, thus detailed protocols were required to be established and optimised prior to biochemical investigations.

Mitochondrial isolations and following respiratory activity measurements were performed on F. japonica prepared from naturalised plants. Such mitochondrial samples were found to have a very low respiratory rates when compared to mitochondria isolated from other species such as Arum maculatum. This was confirmed following an analysis of the respiratory complexes via electrophoresis, which revealed that all complexes were of low abundance in comparison with other plant species. Transmission electron microscopy also revealed that the numbers and volumes of mitochondria in budding tissue were considerably fewer and larger than those observed in other rapidly expanding plant tissues - providing further confirmation of the respiratory measurements.

In an attempt to overcome the small yield associated with mitochondrial isolations, research is also presented on the generation, optimisation and characterisation of suspension cultures from F. japonica explants. Suspension cultures were shown to have almost identical characteristics in terms of mitochondrial protein complement and respiratory capacity as observed in bud and shoot isolations. Preliminary mass spectroscopy data indicated a large proportion of ATP synthase subunits were present in the isolated mitochondrial fractions from leaf, bud, shoot and suspension cultures. Glycolytic analysis of fractions isolated from suspension cultures were also undertaken the outcome of which are discussed in terms of the energy generation pathways within F. japonica and the implications of how such pathways may be controlled.

The INO80 complex is a large ATPase chromatin remodeller which contains 15 accessory subunits in S.cerevisiae. Its subunits include the highly conserved ATPases Ruvb1 and Ruvb2, the actin-related proteins Arp5, Arp8, Act1 and Arp4, Actin, and a number of IES (I̱noE̱ighty S̱pecific) subunits Ies1, Ies2, Ies3, Ies4, Ies5 and Ies6, in addition to subunits Nhp10 and Taf14. All 15 of the accessory subunits are assembled around a catalytic core component known as Ino80.
The INO80 complex has roles in transcription, DNA repair, replication, and chromosome segregation. These roles are in addition to its traditional nucleosome remodelling activities and the dispacement of H2A.Z from chromatin. Recent studies in S. cerevisiae have identified the subunit Ies6 as a critical component of the INO80 complex. Deletion of IES6, which encodes the small accessory subunit, clearly mimics the deletion f the gene encoding the catalytic subunit, INO80. Surprisingly, only one domain within Ies6 has been formally identified based on sequence analysis. This domain belongs to the L1_C class of domains. Such domains are commonly associated with DNA binding activity and transcription factors.
This stud has further characterised the Ies6 subunit both genetically and biochemically. Genetically, it has demonstrated that single point mutations at regions of proposed subunit-subunit interaction between the Arp5 or Rvb2 subunits, or within the YL1_C are not sufficient to disrupt Ies6 function. However, expression of a double point mutation, ies6(K114E/Y125A), in combination with rad50 deletion, caused a sensitivity to replication inhibition, but not chromosome segregation inhibition, indicating a potential separation of function in this utant due to the loss due of only one of the biological functions of Ies6.
Biochemically, we have confirmed that DBA binding capacity of Ies6 resides within the YL_C domain. In addition, although it has been demonstrated that the removal of H2A.Z acetylation exacerbates the increase in cellular ploidy observed in ies6 null cells, we found that overall levels of H2A.Z acetylation were not influenced by the loss of Ies6. This indicates that the role of H2A.Z acetylation in chromosome segregation may only affect ploidy status upon the loss of Ies6.
In addition, work on the R2TP complex (which contains the INO80 APases Ruvb1/Ruvb2, and subunits Tah1 and Phi1) has revealed the recruitment mechanism for the molecular chaperone, Hsp90, and the telomere length regulation protein, Tel2. Together, the R2TP complex, Hsp90 and Tel2 promote the stabilisation and maturation of multi-protein complexes. These include Phosphatidylinositol 3-kinase-related kinases (PIKKs, a family of kinases involved i Serine and Threonine phosphorylation), subunits of the INO80 complex and subunits of the SWR1 chromatin remodelling complex (a partner comlex to INO80 that incorporates H2A.Z into chromatin).

Many oncogenic mutants of the tumor suppressor p53 are conformationally unstable, including the frequently occurring Y220C mutant. We have previously developed several small-molecule stabilizers of this mutant. One of these molecules, PhiKan083, 1-(9-ethyl-9H-carbazole-3-yl)-N-methylmethanamine, binds to a mutationinduced surface crevice with a KD = 150 μM, thereby increasing the melting temperature of the protein and slowing its rate of aggregation. Incorporation of fluorine atoms into small molecule ligands can substantially improve binding affinity to their protein targets. We have, therefore, harnessed fluorine−protein interactions to improve the affinity of this ligand. Step-wise introduction of fluorines at the carbazole ethyl anchor, which is deeply buried within the binding site in the Y220C−PhiKan083 complex, led to a 5-fold increase in affinity for a 2,2,2-trifluoroethyl anchor (ligand efficiency of 0.3 kcal mol−1 atom−1).
High-resolution crystal structures of the Y220C−ligand complexes combined with quantum chemical calculations revealed favorable interactions of the fluorines with protein backbone carbonyl groups (Leu145 and Trp146) and the sulfur of Cys220 at the mutation site. Affinity gains were, however, only achieved upon trifluorination, despite favorable interactions of the mono- and difluorinated anchors with the binding pocket, indicating a trade-off between energetically favorable protein−fluorine interactions and increased desolvation penalties. Taken together, the optimized carbazole scaffold provides a promising starting point for the development of high-affinity ligands to reactivate the tumor suppressor function of the p53 mutant Y220C in cancer cells.

Recent studies into the stringent response and the discovery of a number of RNA polymerase binding proteins suggests that the model for bacterial transcription initiation in Actinobacteria may differ from that in Escherichia coli. In E. coli, the alarmone ppGpp, together with DksA, binds to RNA polymerase to elicit the stringent response. However, the ppGpp binding site on RNA polymerase is not conserved in S. coelicolor, although the organism possesses a DksA homologue. Deletion of DksA did not affect the growth and development of S. coelicolor, although its overexpression stimulated antibiotic production. Evidence is presented that suggests that this occurs through binding to the RNA polymerase secondary channel. The biological role of this protein remains unknown. CarD and RbpA are two RNA polymerase­‐binding proteins present in all Actinobacteria, including S. coelicolor and M. tuberculosis. Both proteins are critical for growth and have been identified as transcriptional activators from σHrdB­‐dependent promoters in vitro. Here it was demonstrated that CarD and RbpA activate transcription from rRNA promoters with a poorly conserved ­‐35 element. Surprisingly it was also found that both proteins can inhibit transcription from synthetic promoters with highly conserved ­‐35 elements. Chromatin immunoprecipitation followed by high throughput sequencing (ChIP­‐seq) experiments revealed that CarD and RbpA are found exclusively at promoter regions. RbpA is localised only at promoters recognised by σHrdB, whereas CarD also co‐localises with the alternative sigma factor σR during oxidative stress indicating that it lacks RNA polymerase holoenzyme specificity. The sigma specificity of RbpA was tested by the generation of sigma mutants that were defective in binding. In vivo, in vitro and ChIP­‐seq data presented in this study suggest that CarD and RbpA have an overlapping role in transcription initiation at σHrdB­‐dependent promoters in S. coelicolor.

Meiosis is characterized by one round of DNA replication followed by two successive rounds of cell division, resulting in a halving of the genome. During meiotic prophase I, many events occur to allow faithful chromosome segregation. At the chromosomal level, homologs align and become closely juxtaposed along their entire lengths via a proteinaceous structure called the synaptonemal complex (SC). DNA double-strand breaks are induced during prophase I, resulting in the formation of crossover between homologs, which leads to the correct segregation during meiosis I. A well-characterized protein, termed Zip1, is the major component of the SC. Zip1 is known to involve in several different processes of meiosis. Firstly, Zip1 is involved during non- homologous centromere coupling. Secondly, Zip1 synapse homologs together. Thirdly, Zip1 promotes crossing over during prophase I as well as required for interference. The work described in this thesis has characterized the functions and regulation of several Zip1-phospho mutants during meiosis. In particular, Zip1-T114 was shown to involve partially during non-homologous centromere coupling. Zip1-S144 is a putative consensus site for Cdc5 phosphorylation and was found to have a role in SC disassembly.

Zip1 has also been known to involve in non-exchange chromosome segregation (NECS). The work described here used time lapse imaging to further study the characteristics of NECS. This study has generated another homeologous chromosome in SK1 strain background. This homeologous chromosome diploid contains one chromosome III from Saccharomyces cerevisiae and one chromosome III from Saccharomyces paradoxus. Both species share 85% homology. Using live cell imaging has revealed that the NECS is very dynamic. This dynamic movement distinguishes from exchange chromosome segregation where stable centromere pairing between homologs was observed. Therefore a model has proposed for NECS, whereby non-exchange centromeres constant been associate and dissociate from prophase until anaphase segregation
during meiosis I.

Although manganese is an essential trace metal, little is known about its transport and homeostatic regulation. Here we have identified a cohort of patients with a novel autosomal recessive manganese transporter defect caused by mutations in SLC39A14. Excessive accumulation of manganese in these patients results in rapidly progressive childhood-onset parkinsonism–dystonia with distinctive brain magnetic resonance imaging appearances and neurodegenerative features on post-mortem examination. We show that mutations in SLC39A14 impair manganese transport in vitro and lead to manganese dyshomeostasis and altered locomotor activity in zebrafish with CRISPR-induced slc39a14 null mutations. Chelation with disodium calcium edetate lowers blood manganese levels in patients and can lead to striking clinical improvement. Our results demonstrate that SLC39A14 functions as a pivotal manganese transporter in vertebrates.

Comprising 16 chapters and almost 470 pages, this is the fiftieth title of the RSC Drug Discovery Series. The overarching theme of this volume is the synthesis, applications and medicinal chemistry of these so-called special scaffolds recurrent in numerous low molecular weight bioactive molecules.

Project 1: Small molecule inhibitors of TDP2

DNA Topoisomerase II (TOP2) has important roles in many cellular processes such as DNA replication and transcription, as well as in chromosome segregation. The main enzymatic function of TOP2 is to alter DNA topology and release torsional stress, by transiently introducing a double strand break (DSB) into a DNA duplex, passing a second intact duplex through the break, and then re-sealing the break. This enzymatic process involves the formation of TOP2-DNA covalent complexes, where the catalytic tyrosine (Y821) is linked to the 5’ phosphate group of a substrate DNA. TOP2 ‘poisons’ such as etoposide, doxorubicin and mitozantrone, which have found utility as anti-cancer agents, lead to an accumulation of these covalent complexes, leading eventually to cell death in rapidly replicating and dividing cells.

As many tumours treated with TOP2 poisons go on to develop chemo-resistance, it is postulated that dual-combination therapy with inhibitors of a second enzyme, 5'-tyrosyl DNA phosphodiesterase-2 (TDP2) may prevent this from occurring; TDP2 acts to remove TOP2-DNA adducts, liberating DNA ends for repair. Inhibitors of TDP2 may also prove useful as a mono-therapy in defined tumour types.

As part of an ongoing collaboration with the Sussex Drug Discovery Centre (SDDC), the aim of the project was to determine high-resolution X-ray crystal structures of TDP2 in complex with a series of deazaflavin inhibitors. The information acquired will guide ongoing structure-based drug design, with the aim of developing and nominating a hit-to lead compound in the near future.

Project 2: The XRCC1 phosphate-binding pocket binds poly(ADP-ribose)

In living organisms, genomic DNA is constantly exposed to both endogenous and exogenous sources of DNA damaging agents, which if not repaired, can result in the accumulation of mutations and chromosomal aberrations. Cells have evolved a series of DNA-damage repair enzymes and pathways, to cope with this perpetual threat. Poly(ADP-ribose) polymerase 1 (PARP1) is the founding member of the large ADP ribosyl transferase superfamily. Among its broad range of functions, PARP1 can detect the presence of both single- and double-strand breaks (SSBs and DSBs) in DNA, upon which it becomes catalytically activated. As a result, PARP1 then synthesises poly(ADP-ribose) polymer using NAD+ as a co-factor, thereby modifying both itself (auto-ribosylation ) and other proteins (trans-ribosylation) in the vicinity of the DNA break.

During the initial phases of the single-strand break repair (SSBR), the scaffold protein XRCC1 is recruited by PARP1, via an interaction between poly(AD-ribose) (PAR) and the central BRCT1 domain in XRCC1. However, further investigation is required to elucidate the mechanism by which the BRCT1 domain interacts with PAR. This project aims to address this question.

Meiotic recombination is initiated by self-inflicted DNA breaks and primarily involves homologous chromosomes, whereas mitotic recombination involves sister chromatids. Whilst the mitotic recombinase Rad51 exists during meiosis, its activity is suppressed in favour of the meiosis-specific recombinase, Dmc1, thus establishing a meiosis-specific mode of homologous recombination (HR). A key contributor to the suppression of Rad51 activity is the synaptonemal complex (SC), a meiosis-specific chromosomal structure that adheres homologous chromosomes along their entire lengths. Here, in budding yeast, we show that two major cell cycle kinases, Dbf4-dependent Cdc7 kinase (DDK) and Polo-kinase (Cdc5), collaborate to link the mode change of HR to the meiotic cell cycle by. This regulation of HR is through the SC. During prophase I, DDK is shown to maintain SC integrity and thus inhibition of Rad51. Cdc5, which is produced during the prophase I/metaphase I transition, interacts with DDK to cooperatively destroy the SC and remove Rad51 inhibition. By enhancing the interaction between DDK and Cdc5 or depleting DDK at late prophase I, meiotic DNA breaks are repaired even in the absence of Dmc1 by utilising Rad51. We propose that the interplay between DDK and Polo-kinase reactivates mitotic HR mechanisms to ensure complete repair of DNA breaks before meiotic chromosomem segregation.

Epstein - Barr virus (EBV) is a member of the γ-herpesvirus subfamily of Herpesviridae. EBV is a double stranded DNA virus infecting humans causing a variety of disease from asymptomatic infection to association with certain tumours including Burkitts lymphoma, Hodgkin’s disease and nasopharyngeal carcinoma. EBV encodes an immediate-early protein called Zta (BZLF1, EB1, ZEBRA), which is an important transcription factor and replication factor direct in disrupting latency. EBV encodes viral proteins that assemble as a replisome at the viral lytic origin recognition site (Ori-Lyt). Zta binds Ori-Lyt and it is unclear how Zta interacts and recruits the complex to the site of DNA replication, while coordinating and recruiting host factors. After a mutation to three alanines (ZtaAAA) data implicates that the extreme C-terminus of Zta is essential for replication.

The question posed is how does Zta assemble the replisome? Identification of the lytic changes that contribute to lytic replication, including cellular components that may contribute to EBV replication is attempted.

Transfected control, Zta and ZtaAAA in HEK293-BZLF1-KO cells was compared. Size exclusion chromatography identified a higher molecular weight complex containing Zta during viral replication. SILAC (Stable isotope labelling by amino acids in cell culture) coupled to proteomics analysis identified the elution fraction composition. An interpretation of these cellular components in the context of lytic replication is explored. Identification of interactions of Zta with cellular proteins was attempted by SILAC histidine tagged Zta with pull down assay. Quantitative data was returned and a confirmation of interactions was attempted. A global proteomics approach was also performed. An enrichment method to isolate SILAC labeled Burkitts Lymphoma cells undergoing EBV lytic replication was coupled to mass spectrometry analysis to identify changes in host and viral proteins.

Overall, cellular targets that may interact with Zta are to be confirmed. The global proteomics study recognized for the first time by proteomic analysis the identification of three EBV lytic replication cycle protein

Chelating agents offer a versatile and effective means of removing toxic metal ions from the human body. Mounting evidence suggests that exposure to nickel and cobalt causes a number of adverse nasal/pulmonary health effects, skin hazards and allergies. In this context, the development of chelators that are able to bind nickel and cobalt with high stability constants is clearly crucial. Herein, the interaction of cobalt(II) and nickel(II) with a 8-hydroxyquinoline-cyclodextrin conjugate was investigated in aqueous solution at neutral pH. This family of chelator under study is potentially relevant in the treatment of diseases related to metal dyshomeostasis. Stability constants for the cobalt(II) and nickel(II) complexes were determined by competition experiments. The data indicate the formation of stable complexes, which were characterized by spectroscopic techniques. The collected information may provide new insights and suggest further strategies for the development of hydroxyquinoline derivatives for the control of cobalt and nickel toxicity.

The ability to produce multi-milligram quantities of a recombinant ‘target’ protein in a proteolytically stable, soluble, and functional form is often necessary for subsequent biochemical, biophysical and structure-based analyses. Sub-constructs expressing only part of a large target protein can often be useful. Combinatorial Domain Hunting (CDH) is a methodology that allows the rapid production of sub- constructs via a random DNA fragmentation technique. One particular issue with CDH is that it can be used to identify globular regions or domains of a target protein, but does not take account of the functional properties of such domains; therefore some ‘hits’ are not useful, because they exclude these functional regions. Here, we have attempted to enhance the CDH methodology by including an additional screening step that could specifically identify those constructs expressing functional protein domains. However, whilst rigorous testing of this functionality screen proved it to be successful under selective conditions, it was not considered suitable for inclusion in the CDH method.

CDH was also used to identify highly expressed, proteolytically stable regions of a previously largely uncharacterized protein, and to investigate their functionality. Human Claspin is a large, highly charged, S=phase specific ‘molecular scaffold’ protein, with no identifiable sub-domains or enzymatic function(s). However, Claspin is known to make multiple different protein-protein interactions at replication forks during the intertwined processes of DNA replication and DNA replication-coupled repair. CDH successfully identified a number of N-terminal expression constructs that could be expressed and purified to a high degree of homogeneity. Structural and functional analyses of these protein fragments indicated that the N-terminus of human Claspin is intrinsically disordered, and elongated in nature. However, these regions may become ordered upon binding to their respective protein or macromolecular partner(s). Furthermore, several N-terminal fragments were found to be able to bind to both single- or double-stranded DNA when longer than 16 nucleotides/base-pairs in length. Additionally, the phospho-specific protein-protein interaction made by human Claspin, with the checkpoint kinase Chk1 was further investigated.

The destabilizing p53 cancer mutation Y220C creates an extended crevice on the surface of the protein that can be targeted by small-molecule stabilizers. Here, we identify different classes of small molecules that bind to this crevice and determine their binding modes by X-ray crystallography. These structures reveal two major conformational states of the pocket and a cryptic, transiently open hydrophobic subpocket that is modulated by Cys220. In one instance, specifically targeting this transient protein state by a pyrrole moiety resulted in a 40-fold increase in binding affinity. Molecular dynamics simulations showed that both open and closed states of this subsite were populated at comparable frequencies along the trajectories. Our data extend the framework for the design of high-affinity Y220C mutant binders for use in personalized anticancer therapy and, more generally, highlight the importance of implementing protein dynamics and hydration patterns in the drug-discovery process.

Proteomics techniques for analysing the redox status of individual proteins in complex mixtures tend to identify the same proteins due to their high abundance. We describe here an array-based technique to identify proteins undergoing glutathionylation and apply it to the secretome and the proteome of human monocytic cells. The method is based on incorporation of biotinylated glutathione (GSH) into proteins, which can then be identified following binding to a 1000-protein antibody array. We thus identify 38 secreted and 55 intracellular glutathionylated proteins, most of which are novel candidates for glutathionylation. Two of the proteins identified in these experiments, IL-1 sRII and Lyn, were then confirmed to be susceptible to glutathionylation. Comparison of the redox array with conventional proteomic methods confirmed that the redox array is much more sensitive, and can be performed using more than 100-fold less protein than is required for methods based on mass spectrometry. The identification of novel targets of glutathionylation, particularly in the secretome where the protein concentration is much lower, shows that redox arrays can overcome some of the limitations of established redox proteomics techniques.

The type three-secretion system (T3SS) is a large and complex protein nano-machine
that many gram-negative pathogens employ to infect host cells. A key structure of this
machine is a proteinaceous pore that inserts into the target membrane and forms a
channel for bacterial toxins to flow from bacteria into the host cell. The pore is mainly
formed from two large membrane proteins called “translocators”. Importantly,
effective secretion and thus pore formation of the translocators depends on their
binding to and being transported by small specialized chaperones after synthesis in the
bacterial cytosol. Recent crystal structures have shown these chaperones are formed
from modular tetratricopeptide repeats (TPRs). However, each crystal structure
produced different homodimeric structures, suggesting flexibility in their topology that
may be of importance to function.

Given the crucial role of the translocator chaperones, we investigated the
conformational stability of the chaperone LcrH (Yersinia pestis). Mutational analysis
coupled with analytical ultra-centrifugation and equilibrium chemical denaturations
showed that LcrH is a weak and thermodynamically unstable dimer (KD ≈ 15 μM, ΔGH2O
= 7.4 kcalmol-1). The modular TPR structure of the dimer allows it to readily unfold in a
non-cooperative manner to a one-third unfolded dimeric intermediate (ΔGH2O = 1.7
kcalmol-1), before cooperatively unfolding to a monomeric denatured state (ΔGH2O =
5.7 kcalmol-1). Thus under physiological conditions the chaperone is able to populate
C-terminally unravelled partially folded states, whilst being held together by its dimeric
interface. Such ability suggests a “fly-casting” mechanism as a route to binding their far
larger translocator cargo.

The SelectBio 2015: Academic Drug Discovery Conference was held in Cambridge, UK, on 19–20 May 2015. Building on the success of academic drug discovery events in the USA, this conference aimed to showcase the exciting new research emerging from academic drug discovery and to help bridge the gap between basic research and commercial application. At the event the authors heard from a number of speakers on a broad array of topics, from partnering models for academia and industry to novel drug discovery approaches across various therapeutic areas, with a few talks, such as those by Susanne Muller-Knapp (Structure Genomics Consortium, Oxford University, Oxford, UK) and Julian Blagg (Institute of Cancer Research, UK), covering both remits, by highlighting a number of such partnerships and then delving into some case studies. The conference concluded with a heated debate on whether phenotypic discovery should be favored over targeted discovery in academia and pharma, in a panel discussion chaired by Roland Wolkowicz (San Diego State University, USA)

The histone deacetylase inhibitor N1-(ferrocenyl)-N8-hydroxyoctanediamide (JAHA) down-regulates extracellular-signal-regulated kinase (ERK) and its activated form in triple-negative MDA-MB231 breast cancer cells after 18 h and up to 30 h of treatment, and to a lesser extent AKT and phospho-AKT after 30 h and up to 48 h of treatment. Also, DNA methyltransferase 1 (DNMT1), 3b and, to a lesser extent, 3a, downstream ERK targets, were down-regulated already at 18 h with an increase up to 48 h of exposure. Methylation-sensitive restriction arbitrarily-primed (MeSAP) polymerase chain reaction (PCR) analysis confirmed the ability of JAHA to induce genome-wide DNA hypomethylation at 48 h of exposure. Collective data suggest that JAHA, by down-regulating phospho-ERK, impairs DNMT1 and 3b expression and ultimately DNA methylation extent, which may be related to its cytotoxic effect on this cancer cytotype.

Rats are shown to acquire a preference for protein- predictive olfactory cues which depends on a state of mild deficit in protein intake - i. e. a learned protein-specific appetite.

At doses which do not reduce food intake elicited by prior food deprivation, puromycin (12 mg/kg) and cycloheximide (0.5 mg/kg) reduce the food intake elicited from the rat by subcutaneous injection of solutions of bovine crystalline insulin (15–20 units/kg). Counteraction of hypoglycaemia by puromycin is not the cause of its blockade of insulin-induced feeding. The water intake evoked by insulin injection is, in contrast, not more sensitive to these drugs than is drinking after deprivation. Food intakes after insulin injection or after food deprivation are not differentially disrupted by actinomycin D (0.03–0.5 mg/kg) or 6-dimethylaminopurine (12 mg/kg). These results are consistent with the hypothesis that some metabolic adaptation to insulin-induced hypoglycaemia mediates the elicitation of feeding. They suggest that this effect of insulin depends on protein synthesis but is not mediated by ribonucleic acid synthesis dependent on deoxyribonucleic acid.

The greater the dose of insulin given in a single injection the deeper the induced hypoglycaemia, whereas over the same dose range the amount of eating elicited comes to a maximum and then declines. The maximum rate of insulin-induced drinking occurs during rapid fall in blood glucose concentration but the induced eating coincides with a period of relatively constant blood glucose concentration. Injection of concentrated glucose with the insulin delays the induction of feeding, but co-injection of approximately isotonic glucose gives shorter feeding latencies than co-injection of more dilute solutions. Glucose ingestion at the time of insulin injection, but not before or after, eliminates induced feeding. Gastric intubation of glucose can block both eating and drinking responses to insulin. Unmetabolizable 3-methylglucose is not so effective at blocking the eating. Glycerol diminishes and intragastric palmitate augments the induced feeding. It is suggested that elicitation of eating by injected insulin is mediated by metabolic signals generated by adaptation to changed body glucose distribution.

Protein-free diet flavored with saccharin or salt was presented to rats following a single intragastric administration of a histidine-devoid, but otherwise balanced, amino acid mixture. A control load was paired with the alternate flavor on another day. When they were subsequently given a choice of flavors, the rats preferred the control flavor to the one paired with the deficient load.

Studies using metabolic inhibitors have led to the widespread belief that protein synthesis during learning is essential for the establishment of long-term memory. However such studies have been criticized on the grounds that actions of the drugs other than on protein synthesis or on memory formation ma~ account for the observed effects. We have reasoned that non-specific effects of inhCbltors would be minimized by using a localized, intracranial administration of the drugs. Rats whose spontaneous paw-preference had previously been determined were trained to reach with the non-preferred paw in a single session of 200 reaches or 2hr duration, whichever occurred sooner. Using a chronically implanted cannula which will be described, a solution of cycloheximide (CHX) at a dose of 0.2 mg/kg was applied to the pla mater overlying the forelimb sensorimotor cortex. With saline or CHX on the pla mater, rats showed a paw-preference switch when tested within 2hr of the end of training. The salinetreated rats continued to show the new preference for weeks, whereas those animals treated with CHX failed to retain it for more than 3 days. Administering CHX again, 1.5 or 3hr before retest at 7 days, did not restore the new preference. Estimates of the degree and extent of inhibition of protein synthesis were made by assessing the incorporation rate of lhC-lysine at different times after the application of CHX. Brains were dissected into 8 regions. Inhibition of lhC-lysine incorporation was maximal in that region containing the crucial forelimb sensorimotor cortex. The results suggest that at least near-normal rates of protein synthesis are necessary for the establishment and retention of a newly acquired motor skill.

Intravenous infusion of palmitic acid in the day or in the night was found neither to inhibit nor to facilitate feeding in the rat. The results showed only a night-phase inhibition when infusion approached a calorically substantial rate and this effect was produced both by the palmitic acid-albumin complex and by the albumin vehicle alone. The well-known increase in rate of feeding at light offset was closely controlled by lighting, rather than by duration of the daytime fat mobilizing phase. Pent-4-enoic acid, an inhibitor of fat oxidation, did not increase food intake during the day. Insulin increased food intake not only by day when it would prevent fat mobilization, but also at night, when no mobilization normally occurs. Propranolol, a blocker of beta adrenergic fat mobilization, had only inhibitory effects on feeding. Under isolation conditions, propranolol injection shortened the latency of meal by day, but so did saline injection. It is concluded that, although fat no doubt ultimately enters into the caloric control of feeding, there is as yet no evidence that circulating fatty acids have large short-term effects on food intake.

Relation of fatty acids to feeding behaviour: Effects of palmitic acid infusions, lighting variation and Pent4-enoate, insulin or propranolol injection1 - ResearchGate.

In adult rats, the first meal on restoring access to food following the complete absorption of an intragastric carbohydrate load is smaller than the meal following a nonnutritive load. The weanling rat does not show this postabsorptively induced satiation. The effect does not appear until above a body weight of about 200 g. Subcutaneous injection of a moderate dose of insulin (0.2 units/kg) at the time of glucose intubation results in postabsorptive satiety appearing in the immature rat. The inhibitor of insulin secretion, D-mannoheptulose, injected shortly before glucose intubation, considerably reduces the satiety effect in the 300 g rat. It is suggested that an abundant secretion of insulin during absorption is necessary to establish the parenteral satiety signal operative under these conditions.

Latencies to feed and meal sizes were observed in recently satiated rats injected intraperitoneally with an isotonic solution of 2-deoxyglucose, using 3-O-methyl glucose for the control injection. The feeding response latency shortened monotonically with dose from about 0.3 mmole/kg (50 mg/kg) to 0.5–0.6 mmole/kg, with an extended plateau up to debilitating doses. At modest doses, the latency to feed was shorter in adrenal demedullated rats than in intact rats, suggesting that the hyperglycemic response to 2DG partially inhibits the feeding response. Food-deprived rats took larger meals following 2DG injection, if there had been recent rapid intestinal absorption of glucose. It is suggested that a major action of 2DG is to block the satiating consequences of the process of absorption, possibly at hepatic receptors.

Daily protein intakes in the rat compensate rather precisely for current over- or under-supply of amino acids to provide energy and protein, when feeding is not dominated by energy need, extremes of palatability, or inappropriate selection habits. Interactions between protein-induced satiety and acquired protein appetites may possibly be sufficient to explain the observed compensatory behavior. The results confirm that control of food intake can play the primary role in regulation of nitrogen exchange as well as in the regulation of energy exchange in the rat.

Rats become averse to the odor of a protein-free diet presented after gastric administration of loads devoid of histidine and isoleucine but otherwise balanced in amino acid composition. The specific sensory aversion may in these cases explain the intake decrement seen shortly after loading. The suppression of intake shortly after a threonine-devoid load was, in contrast, not allied with an acquired aversion under the experimental conditions used. There were signs of aversion or anorexia following loads devoid of methionine, valine, phenylalanine of lysine. A complete balanced load, and to a lesser extent tryptophan- and perhaps glycine-devoid loads, induced a preference for the associated odor over an odor paired with saline intubation. Omission of leucine or arginine from the balanced mixture produced niether preference nor aversion. The results support a suggestion that reduced synthesis of a brain protein is aversion-inducing.

1. A protein-free diet, to which an odour had been added, was offered to rats immediately after giving a gastric load of an histidine-free, but otherwise balanced, amino acid mixture. The same diet with a different odour was offered to the rats on another day, after administration of a control load of saline or a balanced amino acid mixture. After access to stock diet for 6 h of 1 d, the rats were offered two samples of protein-free diet, each with one of the two odours.

2. The rate of consumption of odorized protein-free diet was depressed 2–4 h after administration of the histidine-free load. In the later preference test, the dietary sample with odour which had been offered after the deficient load was rejected in favour of the sample with the odour which had been offered after the control load.

3. Rejection of the deficiency-paired odour in the final preference test did not occur when the histidine-free load had been given 2 h before the rats were first offered odorized diet. Also, there was in these instances no depression of rate of food consumption 2–4 h after loading. This indicated that aversive reactions to the odour were established by association of the odour with some effect of the histidine-free load which had occurred within 2 h of its administration, and that the early depression of intake and the much later rejection during choice were both expressions of these acquired reactions.

4. This rapid conditioning of selective rejection did not depend on previous prolonged protein deprivation or on the use of immature rats but did depend upon an intermittent supply of amino acids during 3 d before conditioning. Subcutaneous injection of deficient amino acid mixture did not establish rejection, suggesting that conditioning depended on rapid delivery of the deficient mixture into the circulation.

5. It is concluded that the critical biochemical events which lead to the rejection of diets that are imbalanced or deficient in essential amino acids occur soon after ingestion of the diet, and may have been effective in producing a conditioned aversion before any suppression of food intake appears. It is suggested that the depression of food consumption, which is the normal response to an imbalanced diet, is in this instance the result of conditioned response to sensory qualities of the diet rather than to the direct effect of biochemical stimuli.

A cellophane sac containing a mixture of amino acids was implanted under the skin of the freely fed and watered rat. After its dissolution in body fluid, the load diffused out of the sac according to an exponential function. Water intake increased maximally during the period of most rapid release of amino acids into the body. Food intake was depressed most effectively somewhat later. The anorexigenic effect of a mixture consisting solely of essential amino acids was greater than that of a mixture containing a proportion of dispensable amino acids. Effects of amino acids which facilitate the urea cycle indicated that ammonia toxicity contributed to the suppression of intake by the highest dose of essential amino acids (90 mmoles/kg). The moderate suppression of feeding by a mixture containing both essential and dispensable amino acids was potentiated by inclusion of glucose. This suggested that metabolic effects of the amino acids, other than ammonia production, contributed to the observed inhibition of feeding.

Rat pups come to accept a starch-containing diet and to prefer its flavor or other sensory properties, even when they also have access to the laboratory chow on which their mother is maintained. Protein induces acceptance somewhat more weakly, triglyceride not at all under the conditions used. Nutrient-free material becomes aversive. It is concluded that at least part of the acceptability of a food is an augmentation of its power to elicit ingestatory reactions which has been established by the contingency of physiological effects of ingested carbohydrate or protein on experiences of that foodstuff’s distinctive sensory qualities.

Protein-deprived rats were given, on one day, a balanced mixture of amino acids followed by access to protein-free food having a distinctive odour. On another day, an imbalanced (histidine-free) amino acid mixture was given just before food having another odour. The rats afterwards preferred the balance-paired odour to the imbalance-paired odour. The preference was acquired whether the duration of odour presentation, or the amount of odourised food taken, was kept constant on the two conditioning days. Retention of the preference seemed unattenuated after 4 weeks. An attraction to the balance- paired odour (relative to odours paired with a water load) contributed to the acquired preference. There was also a relative aversion to unfamiliar odours when they had been paired with imbalance. Such acquired chemosensory control of preferences, together with an anorexigenic effect of imbalanced amino acid mixtures, can account for characteristics of feeding behaviour under conditions in which the diet is deficient in an essential amino acid.

Human subjects took a protein-rich or a protein-poor lunch and 2–3 hr later a supplementary meal of average protein content. Total caloric intake for the two meals was lower when the main meal contained a high proportion of protein. Therefore, calorie for calorie, amino acids contribute more than carbohydrates and fats to the suppression of hunger in the post-absorptive period.

A cellophane dialysis bag containing small molecular weight solids is implanted under the skin of the rat. Sodium chloride, glucose and hydrolyzed casein give differential effects on food and water intakes over the day following implantation. An uncompensated decrease in food intake is seen after administering glucose by this technique, as after rapid intraperitoneal or subcutaneous injection and unlike after either continuous intravenous infusion or rapid gastric intubation.

Global metabolomic analysis of urine offers great potential for detection of early warning markers of disease. Current methods focus on rapid sample preparation and high throughput analyses at the expense of the detection of low abundance metabolites. The aim of this study was to develop sensitive analytical methods for metabolomic profiling. Methods were developed to use nanoflow ultra high performance liquid chromatography-nanospray ionization-mass spectrometry (nUHPLC-nESI-TOFMS), normally used for proteomics, for metabolomic analyses of urine samples. Compared with a conventional UHPLC-ESI-TOFMS, the use of a nanoflow-nanospray platform increased the sensitivity to a standard mixture of metabolites by 2-2000 fold. Highly repeatable results for retention time and metabolome peak area were achieved, where the coefficients of variation were <0.2% and <30% respectively for the majority of peaks present in the urine metabolome. To further increase sensitivity and enable small injection volumes, a sample preparation method was developed using polymeric anion and cation exchange mixed mode solid phase extraction with pre-concentration. Combined with the nano platform, this enabled the detection of low abundance signalling molecules (estrogens, eicosanoids and unconjugated androgens) not usually detected with conventional methods. A pre-analysis normalisation technique based on osmolality concentrations was used to reduce sample variability due to differing urine concentrations. These methods were used to investigate the metabolomic consequences of HIV infection and patient response to combined antiretroviral therapy (cART). No significant differences in metabolomic profiles between HIV positive and negative patients were observed. However, disruption of bile acid profiles and decreased concentrations of selected carnitines, steroid conjugates, polypeptides and nucleosides were detected in patients on cART therapy indicating disrupted lipid and protein metabolism but improved immunological function associated with antiretroviral medication. These finding highlight the importance of these newly developed SPE sample preparation and nUHPLC-nESI-TOFMS analysis methods for global profiling of the urinary metabolome.

The gastric emptying of normally sized chow meals taken after minimal food deprivation appeared to be almost constant in rate for most of the period of emptying, although a short initial acceleration was not excluded and a slowing was generally observed late in emptying. Intestinal contents of dry matter and carbohydrate remained about constant and so under these conditions absorption rate equalled the recent gastric emptying rate. When the rat's meals are frequent, in the dark period of the 24 hour, the stomach emptied much more rapidly. When measured post mortem in groups of rats, blood concentrations of glucose did not vary markedly after a meal in the dark period, nor did blood concentrations of alanine, glycerol and other gluconeogenic precursors. An increase in rates of cerebral glucose uptake and conversion to glutamate and lactate was occasionally observed after meals, by comparisons of specific activities 5 min after subcutaneous injection of U-14C-glucose, but the increase was not regionally localised. Hepatic glycogen concentration did not vary up to the time the next meal would have been taken in the mid dark period. However, gluconeogenic capacity was reduced by 20–30% for about 90 min following the meal, as measured by conversion of a loading dose of 14C-alanine to 14C-glucose in blood. Gluconeogenesis or regulation of hepatic glucose output may protect the brain and other tissues, including even the liver, from the minor and brief variations in absorption between meals ad lib. Normal satiety and hunger may be anticipatory responses, established by the metabolic and/or hormonal consequences of occasional bursts or drops in absorption rate.

The theory that current supply of readily used energy is the primary control in feeding is embodied in a computer model of energy flows from the gut and to or from storage as fat. Values for all parameters are derived from physiological data. Meal patterns and cumulative food intakes are realistically predicted for normal and VMH- lesioned rats.

Faithful genome duplication and inheritance require the complete resolution of all intertwines within the parental DNA duplex. This is achieved by topoisomerase action ahead of the replication fork or by fork rotation and subsequent resolution of the DNA precatenation formed. Although fork rotation predominates at replication termination, in vitro studies have suggested that it also occurs frequently during elongation. However, the factors that influence fork rotation and how rotation and precatenation may influence other replication-associated processes are unknown. Here we analyze the causes and consequences of fork rotation in budding yeast. We find that fork rotation and precatenation preferentially occur in contexts that inhibit topoisomerase action ahead of the fork, including stable protein–DNA fragile sites and termination. However, generally, fork rotation and precatenation are actively inhibited by Timeless/Tof1 and Tipin/Csm3. In the absence of Tof1/Timeless, excessive fork rotation and precatenation cause extensive DNA damage following DNA replication. With Tof1, damage related to precatenation is focused on the fragile protein–DNA sites where fork rotation is induced. We conclude that although fork rotation and precatenation facilitate unwinding in hard-to-replicate contexts, they intrinsically disrupt normal chromosome duplication and are therefore restricted by Timeless/Tipin.

Dysregulation of translation has a direct effect on growth control in mammalian cells. The initiation step of translation is an important point of control of gene expression and a rate-limiting step for protein synthesis. Initiation requires initiation factors (eIFs) that are important for the activation of both mRNA and the recruitment of ribosomal subunits. Previously, post-translational modifications (PTMs), such as phosphorylation, have been shown to be an important part in fine-tuning translation. SUMOylation is another PTM that affects a number of fundamental cellular processes, including the response to DNA damage, metabolic regulation and protein trafficking. Many eIFs have been identified in proteomic screens as SUMOylated targets, but to date most of these modifications have not been confirmed. Preliminary work from the Watts lab indicated that some eIFs co-purified with the S. pombe SUMO protease Ulp2. The aim of my project was to determine whether components of the eIF4F complex are SUMOylated and to initiate studies to investigate the role of this modification. The first results chapter investigates eIF4G, eIF3h and Sla1 (the La protein homologue) and demonstrates that eIF4G and Sla1, but not eIF3h, are SUMOylated in S. pombe. These experiments were then extended to mammalian cells. The effects of stress conditions on protein synthesis and SUMOylation in a range of cell lines were first analysed. SUMO localisation was altered in response to sodium arsenite (AR) and ionising radiation (IR). In most cell types tested, for example, after IR treatment, SUMO1 went to nuclear foci in HeLa cells, but was more abundant in the cytoplasm following exposure to AR. Next, in vivo and in vitro SUMOylation assays were used to demonstrate that mammalian eIF4G and eIF4A are both SUMOylated. Mass spectrometric analysis identified the SUMOylation sites in eIF4G, as K1386 and K1588. Those of eIF4AI and eIF4AII are K225 and K226, respectively. Mutated eIF4AII was introduced into cells to investigate the role of SUMOylation of this factor. Colocalisation of eIF4A/eIF4G and SUMO1 shows that, in AR-treated cells, SUMO1 colocalises with eIF4A and eIF4G in the cytoplasmic stress granules, especially at their edges. In contrast, in IR-treated cells, the colocalisation of eIF4G/eIF4A with SUMO1 is much more in the nucleus, compared to that in untreated cells, suggesting that eIF4G/eIF4A and SUMO1 may have a cellular role in some aspects in response to AR and IR.

DNA non-homologous end-joining (NHEJ) is the major DNA double strand break (DSB) repair pathway inmammalian cells. Defects in NHEJ proteins confer marked radiosensitivity in cell lines and mice models,since radiation potently induces DSBs. The process of V(D)J recombination functions during the devel-opment of the immune response, and involves the introduction and rejoining of programmed DSBs togenerate an array of diverse T and B cells. NHEJ rejoins these programmed DSBs. Consequently, NHEJdeficiency confers (severe) combined immunodeficiency – (S)CID – due to a failure to carry out V(D)Jrecombination efficiently. NHEJ also functions in class switch recombination, another step enhancing Tand B cell diversity. Prompted by these findings, a search for radiosensitivity amongst (S)CID patientsrevealed a radiosensitive sub-class, defined as RS-SCID. Mutations in NHEJ genes, defining human syn-dromes deficient in DNA ligase IV (LIG4 Syndrome), XLF-Cernunnos, Artemis or DNA-PKcs, have beenidentified in such patients. Mutations in XRCC4 or Ku70,80 in patients have not been identified. RS-SCIDpatients frequently display additional characteristics including microcephaly, dysmorphic facial featuresand growth delay. Here, we overview the clinical spectrum of RS-SCID patients and discuss our currentunderstanding of the underlying biology.

Following in utero exposure to low dose radiation
(10 – 200 mGy), we recently observed a linear induction of DNA
double-strand breaks (DSB) and activation of apoptosis in the
embryonic neuronal stem/progenitor cell compartment. No
signifi cant induction of DSB or apoptosis was observed following
exposure to magnetic fi elds (MF). In the present study, we
exploited this in vivo system to examine whether exposure to MF
before and after exposure to 100 mGy X-rays impacts upon DSB
repair rates.
Materials and methods : 53BP1 foci were quantifi ed following
combined exposure to radiation and MF in the embryonic neuronal
stem/progenitor cell compartment. Embryos were exposed
in utero to 50 Hz MF at 300 m T for 3 h before and up to 9 h after
exposure to 100 mGy X-rays. Controls included embryos exposed
to MF or X-rays alone plus sham exposures.
Results : Exposure to MF before and after 100 mGy X-rays did not
impact upon the rate of DSB repair in the embryonic neuronal
stem cell compartment compared to repair rates following radiation
exposure alone.
Conclusions : We conclude that in this sensitive system MF do not
exert any signifi cant level of DNA damage and do not impede
the repair of X-ray induced damage.

The seminal discovery by James Cleaver of defective DNA repair in xeroderma pigmentosum (XP) opened up an everexpanding field of DNA repair-related disorders. In addition, it put XP on the map and has led to improved diagnosis, care and management of affected patients. In the United Kingdom, we recently established a multidisciplinary specialist clinic for XP patients. All XP patients in the United Kingdom are able to visit the clinic where they are examined and advised by a team of specialists with detailed knowledge of the different aspects of XP.

The MAGE (Melanoma-associated antigen) protein family members are structurally related to each other by a MAGEhomology domain comprised of 2 winged helix motifs WH/A and WH/B. This family specifically evolved in placental mammals although single homologs designated NSE3 (non-SMC element) exist in most eukaryotes. NSE3, together with its partner proteins NSE1 and NSE4 form a tight subcomplex of the structural maintenance of chromosomes SMC5–6 complex. Previously, we showed that interactions of the WH/B motif of the MAGE proteins with their NSE4/EID partners are evolutionarily conserved (including the MAGEA1-NSE4 interaction). In contrast, the interaction of the WH/A motif of NSE3 with NSE1 diverged in the MAGE paralogs. We hypothesized that the MAGE paralogs acquired new RING-finger containing partners through their evolution and form MAGE complexes reminiscent of NSE1-NSE3-NSE4 trimers. In this work, we employed the yeast 2-hybrid system to screen a human RING-finger protein library against several MAGE baits. We identified a number of potential MAGE-RING interactions and confirmed several of them (MDM4, PCGF6, RNF166, TRAF6, TRIM8, TRIM31, TRIM41) in co-immunoprecipitation experiments. Among these MAGE-RING pairs, we chose to examine MAGEA1-TRIM31 in detail and showed that both WH/A and WH/B motifs of MAGEA1 bind to the coiled-coil domain of TRIM31 and that MAGEA1 interaction stimulates TRIM31 ubiquitin-ligase activity. In addition, TRIM31 directly binds to NSE4, suggesting the existence of a TRIM31-MAGEA1-NSE4 complex reminiscent of the NSE1-NSE3-NSE4 trimer. These results suggest that MAGEA1 functions as a co-factor of TRIM31 ubiquitin-ligase and that the TRIM31-MAGEA1-NSE4 complex may have evolved from an ancestral NSE1-NSE3-NSE4 complex.

Although the application of the concept of a threshold to risk assessment is widespread, there remains little experimental evidence for the existence of thresholds for genotoxic compounds, other than aneugens. The clastogenicity of topoisomerase inhibitors is believed to result from the transient stabilization of the topoisomerase enzyme with DNA during the catalytic cycle. This leads to the formation of a stabilized cleavage complex, which, in turn, may result in the formation of a DNA strand break. This indirect mechanism of clastogenicity is the basis for the concept of threshold for this class of drug. Using micronucleus induction in L5178Y mouse lymphoma cells as a genotoxic end-point, a three pronged approach was used to examine whether the concept of a threshold for clastogenicity could be demonstrated for topoisomerase type II inhibitors in vitro. This involved (i) the study of mechanism (TARDIS assay), (ii) hypothesis testing versus estimation (i.e. scoring up to 10,000 cells/treatment at concentrations immediately above and below the NOEL for micronucleus induction) and (iii) statistical modelling of the concentration-response curves for micronucleus induction. Several topoisomerase type II inhibitors were investigated with varying clastogenic potencies (etoposide = doxorubicin < genistein < ciprofloxacin). Pragmatic thresholds for clastogenicity in L5178Y cells were defined at 0.00236 microg/ml for etoposide, 0.00151 microg/ml for doxorubicin, 1 microg/ml for genistein and 50 microg/ml for ciprofloxacin. In addition, it was demonstrated that etoposide-induced clastogenicity was concentration and time dependent. These results, along with mechanistic data showing that all of the compounds induced concentration-dependent increases in the formation of topoisomerase II stabilized cleavage complexes, provide a weight of evidence to support a threshold concept for clastogenicity with topoisomerase II poisons.

Amyotrophic lateral sclerosis (ALS) and spinal muscular atrophy with lower extremity predominance (SMA-LED) are two forms of motor neuron diseases at the opposite ends of the age spectrum, with ALS being mainly an adult-onset progressive and fatal neurodegenerative disease, and SMA-LED being a childhood-onset neuromuscular disease, manifested by muscle weakness, joint contracture, abnormal gait, and in some cases combined with intellectual disability. This thesis represents my research on the role of mutant forms of two proteins, namely Tar-DNA binding protein 43 (TDP-43) and cytoplasmic dynein heavy chain 1 (DYNC1H1) in the pathogenesis of ALS and SMA-LED, respectively.
TDP-43 – an RNA/DNA binding protein – has been implicated in ALS. The function of TDP-43 in the nucleus is to regulate RNA processing, including RNA splicing and editing. Abnormal expression of the peripherin splice variant per61 has been found in transgenic mouse models of ALS. In addition, aberrant expression of EAAT2 (excitatory amino acid glutamate transporter 2) protein has been reported in some ALS cases. Thus, I investigated splicing of peripherin and GLT-1 (the murine homologue of EAAT2) RNAs, as potential targets of TDP-43 and examined whether mutations in TDP-43 alter the expression levels of the genes encoding the two proteins. My data show that per61 is expressed in wild type mice at both RNA and protein levels. This suggests a role for this isoform in the assembly of peripherin filaments. Moreover, overexpression of TDP-43A315T increases the expression level of per45 (an alternative translated isoform of peripherin) and leads to the instability of the filament network. Analysis of GLT-1B, the neuronal splice variant of GLT-1, reveals significant down-regulation in TDP-43A315T transgenic mice, indicating impaired RNA processing of GLT-1B. Collectively, these data show that the expression of peripherin and EAAT2 is regulated by TDP-43, and that aberrant expressions of these two genes caused by TDP-43 mutations could have a role in the pathology of ALS.
A Phe580Tyr mutation in the mouse gene Dync1h1 impairs growth factor-induced endocytic trafficking in Dync1h1+/F580Y mouse motor neurons, resulting in aberrant activation of extracellular-signal-related kinases 1 and 2 (ERK1/2) and phosphorylation of the immediate early gene c-Fos. My data show that the induction of c-Fos upon
serum starvation and/or growth factor stimulation is ERK1/2 dependent and that the mitogen-activated protein kinase p38 is also likely to be involved in c-Fos activation during starvation. Moreover, the activation of autophagy is reduced in Dync1h1+/F580Y motor neurons, suggesting a role for cytoplasmic dynein in autophagy induction/formation.
In addition, the Dync1h1F580Y/F580Y mouse embryonic fibroblasts (MEFs) exhibit a defect in cell migration, as manifested by a delayed wound closure and reduced levels of paxillin phosphorylation at Tyr118 (p-paxillin). They also show abnormal and increased number of focal adhesions in spreading assays. Interestingly, human SMA-LED DYNC1H1R399G/R399G fibroblasts show defective lamellipodia formation, as well as reduced levels of p-paxillin. Moreover, Dync1h1+/F580Y mouse motor neurons show a defect in exploratory microtubules in the peripheral domain of their growth cones. As the molecular mechanism of growth cone motility is analogous to that found in fibroblasts, the molecular pathogenesis of SMA-LED caused by mutations in cytoplasmic dynein heavy chain 1, is likely to involve impaired growth cone development and axonal pathfinding, which could be exacerbated by the aberrant endocytic trafficking and signalling in mutant motor neurons.

Epstein-Barr virus (EBV) is a widespread human B cell virus that is linked to many malignancies. EBV modulates the transcriptome of B lymphocytes to drive immortalisation and viral persistence. This is primarily coordinated by the EBV nuclear antigens (EBNA) 2 and the EBNA 3 family (3A, 3B and 3C), which regulate overlapping sets of cellular genes. Using Chromatin immunoprecipitation (ChIP) coupled to next generation sequencing we found >21000 EBNA 2 and >7000 EBNA 3 binding sites in the human genome, providing the first evidence of EBNA 3 association with the human genome in vivo. Binding sites were predominantly distal to transcription start sites (TSS) indicating a key role in long-range gene control. This was especially pronounced for EBNA 3 proteins (84% of sites over 4kb from any TSS). 56% of genes previously reported to be regulated by these EBNA proteins in micro array experiments were bound by an EBNA. Using ChIP-QPCR we confirmed EBNA 3C bound to and promoted epigenetic silencing of a subset of integrin receptor signalling genes (ITGA4, ITGB1, ADAM28, ADAMDEC1). Indirect silencing of CXCL10 and CXCL11 chemokines by EBNA 3C was also demonstrated. 75% of sites bound by EBNA 3 were also bound by EBNA 2 implicating extensive interplay between EBNA proteins in gene regulation. By examining novel (WEE1, CTBP2) and known (BCL2L11, ITGAL) targets of EBNA 3 proteins bound at promoter-proximal or distal binding sites, we found both cell-type and locus-specific binding and transcriptional regulation. Importantly, genes differentially regulated by a subset EBNA 3 proteins were bound by the same subset, providing a mechanism for selective regulation of host genes by EBNA 3 proteins. In summary, this research demonstrates that EBNA proteins primarily act through long-range enhancer elements and regulate gene expression in a locus and gene-specific manner through differential binding.

PrimPol is a recently identified member of the archaeo-eukaryotic primase (AEP) family
of proteins. It possesses both primase and polymerase activities and is involved in the
replication of both nuclear and mitochondrial DNA. PrimPol is predicted to possess an
AEP polymerase and a UL52-like zinc finger domain. This thesis establishes the roles
of these domains in the context of PrimPol’s catalytic activities. Although apparently
dispensable for polymerase activity, the zinc finger is essential for maintaining primase
activity and also appears to play an important role in regulating the processivity and
fidelity of PrimPol’s extension activities. A recently study identified a PrimPol mutation
(Y89D) that is potentially associated with the development of high myopia in humans.
Here, the biochemical defects associated with this mutant are analysed and described.
This protein variant has a significant reduction in polymerase activity. Mutational
analysis suggests that the hydrophobic ring of tyrosine is important for retaining wildtype
DNA extension activity. Biophysical analysis of the secondary structure and
stability of this PrimPol variant suggests that this PrimPol variant has reduced α-helical
content and is less stable than the wild-type protein.

Finally, the interaction of PrimPol with single-stranded DNA binding protein replication
protein A (RPA) is investigated. Previous studies have identified an interaction of
PrimPol with RPA. Here, it is demonstrated that PrimPol has two separate RPA
interaction motifs and a crystal structure is presented of one such motif in PrimPol
bound to RPA that reveals the molecular basis for this interaction.
Together, these studies provide molecular insights into the catalytic mechanism of
PrimPol as well as some of the key intramolecular and intermolecular mechanisms of
that regulate the activities of PrimPol.

Abstract: BC-11 is an easily synthesized simple thiouronium-substituted phenylboronic acid, which has been shown to be cytotoxic on triple negative MDA-MB231 breast cancer cells by inducing a perturbation of cell cycle when administered at a concentration equal to its ED50 at 72 h (117 μM). Exposure of cells to BC-11, either pre-absorbed with a soluble preparation of the N-terminal fragment of urokinase-plasminogen activator (uPa), or in co-treatment with two different EGFR inhibitors, indicated that: (i) BC-11 acts via binding to the N-terminus of the enzyme where uPa- and EGF receptor-recognizing sites are present, thereby abrogating the growth-sustaining effect resulting from receptor binding; and (ii) the co-presence of the EGFR inhibitor PD153035 potentiates BC-11’s cytotoxicity. Exposure of cells to a higher concentration of BC-11 corresponding to its ED75 at 72 h (250 μM) caused additional impairment of mitochondrial activity, the production of reactive oxygen species and promotion of apoptosis. Therefore, BC-11 treatment appears to show potential for the development of this class of compounds in the prevention and/or therapy of “aggressive” breast carcinoma.

For most plants, submergence in water is a rare occurrence, but for plants that grow on salt marshes flooding with seawater may be a twice-daily event. This is the case for plants of the halophyte Suaeda maritima, growing at low elevations on salt marshes. These plants are, however, smaller than those growing at higher elevations, where flooding is less frequent and the soil is better drained. We investigated whether the reduced growth brought about by flooding with saline water was a consequence of toxicity of manganese or iron. Seedlings of S. maritima were grown both in a sold medium (a mixture of salt-marsh mud and sand) that was either submerged twice a day or continuously flooded with half-strength seawater and in a hydroponic solution where the oxygen concentration was adjusted by bubbling with nitrogen or air. Hypoxia, reduced the growth of plants in both solid and liquid media and resuted in increases in manganese and iron in the shoots and roots. Experiments in culture solution showed that elevated levels of manganese were unlikely to be toxic, but that iron did reach toxic concentrations in flooded plants.

The medium chain triglyceride (MCT) ketogenic diet is a major treatment of drug-resistant epilepsy but is problematic, particularly in adults, because of poor tolerability. Branched derivatives of octanoic acid (OA), a medium chain fat provided in the diet have been suggested as potential new treatments for drug-resistant epilepsy, but the structural basis of this functionality has not been determined. Here we investigate structural variants of branched medium chain fatty acids as new seizure-control treatments. We initially employ a series of methyl-branched OA derivatives, and using the GABAA receptor antagonist pentylenetetrazol to induce seizure-like activity in rat hippocampal slices, we show a strong, branch-point–specific activity that improves upon the related epilepsy treatment valproic acid. Using low magnesium conditions to induce glutamate excitotoxicity in rat primary hippocampal neuronal cultures for the assessment of neuroprotection, we also show a structural dependence identical to that for seizure control, suggesting a related mechanism of action for these compounds in both seizure control and neuroprotection. In contrast, the effect of these compounds on histone deacetylase (HDAC) inhibition, associated with teratogenicity, shows no correlation with therapeutic efficacy. Furthermore, small structural modifications of the starting compounds provide active compounds without HDAC inhibitory effects. Finally, using multiple in vivo seizure models, we identify potent lead candidates for the treatment of epilepsy. This study therefore identifies a novel family of fatty acids, related to the MCT ketogenic diet, that show promise as new treatments for epilepsy control and possibly other MCT ketogenic diet-responding conditions, such as Alzheimer disease.

Lytic replication of the human gamma herpes virus Epstein-Barr virus (EBV) is an essential prerequisite for the spread of the virus. Differential regulation of a limited number of cellular genes has been reported in B-cells during the viral lytic replication cycle. We asked whether a viral bZIP transcription factor, Zta (BZLF1, ZEBRA, EB1), drives some of these changes. Using genome-wide chromatin immunoprecipitation coupled to next-generation DNA sequencing (ChIP-seq) we established a map of Zta interactions across the human genome. Using sensitive transcriptome analyses we identified 2263 cellular genes whose expression is significantly changed during the EBV lytic replication cycle. Zta binds 278 of the regulated genes and the distribution of binding sites shows that Zta binds mostly to sites that are distal to transcription start sites. This differs from the prevailing view that Zta activates viral genes by binding exclusively at promoter elements. We show that a synthetic Zta binding element confers Zta regulation at a distance and that distal Zta binding sites from cellular genes can confer Zta-mediated regulation on a heterologous promoter. This leads us to propose that Zta directly reprograms the expression of cellular genes through distal elements.

Dis3 is a highly conserved exoribonuclease which degrades RNAs in the 3'-5' direction. Mutations in Dis3 are associated with a number of human cancers including multiple myeloma and acute myeloid leukaemia. In this work, we have assessed the effect of a Dis3 knockdown on Drosophila imaginal disc development and on expression of mature microRNAs. We find that Dis3 knockdown severely disrupts the development of wing imaginal discs in that the flies have a "no wing" phenotype. Use of RNA-seq to quantify the effect of Dis3 knockdown on microRNA expression shows that Dis3 normally regulates a small subset of microRNAs, with only 11 (10.1%) increasing in level > 2-fold and 6 (5.5%) decreasing in level >2-fold. Of these microRNAs, miR-252-5p is increased 2.1-fold in Dis3-depleted cells compared to controls while the level of the miR-252 precursor is unchanged, suggesting that Dis3 can act in the cytoplasm to specifically degrade this mature miRNA. Furthermore, our experiments suggest that Dis3 normally interacts with the exosomal subunit Rrp40 in the cytoplasm to target miR-252-5p for degradation during normal wing development. Another microRNA, miR-982-5p, is expressed at lower levels in Dis3 knockdown cells, while the miR-982 precursor remains unchanged, indicating that Dis3 is involved in its processing. Our study therefore reveals an unexpected specificity for this ribonuclease towards microRNA regulation, which is likely to be conserved in other eukaryotes and may be relevant to understanding its role in human disease.

The cellular activity of several regulatory and signal transduction proteins, which depend on the Hsp90 molecular chaperone for folding, is markedly decreased by geldanamycin and by radicicol (monorden). We now show that these unrelated compounds both bind to the N-terminal ATP/ADP-binding domain of Hsp90, with radicicol displaying nanomolar affinity, and both inhibit the inherent ATPase activity of Hsp90 which is essential for its function in vivo. Crystal structure determinations of Hsp90 N-terminal domain complexes with geldanamycin and radicicol identify key aspects of their nucleotide mimicry and suggest a rational basis for the design of novel antichaperone drugs.

The HSP90 molecular chaperone plays a key role in the maturation, stability and activation of its clients, including many oncogenic proteins. Kinases are a substantial and important subset of clients requiring the key cochaperone CDC37. We sought an improved understanding of protein kinase chaperoning by CDC37 in cancer cells. CDC37 overexpression in human colon cancer cells increased CDK4 protein levels, which was negated upon CDC37 knockdown. Overexpressing CDC37 increased CDK4 protein half-life and enhanced binding of HSP90 to CDK4, consistent with CDC37 promoting kinase loading onto chaperone complexes. Against expectation, expression of C-terminus-truncated CDC37 (ΔC-CDC37) that lacks HSP90 binding capacity did not affect kinase client expression or activity; moreover, as with wild-type CDC37 overexpression, it augmented CDK4-HSP90 complex formation. However, although truncation blocked binding to HSP90 in cells, ΔC-CDC37 also showed diminished client protein binding and was relatively unstable. CDC37 mutants with single and double point mutations at residues M164 and L205 showed greatly reduced binding to HSP90, but retained association with client kinases. Surprisingly, these mutants phenocopied wild-type CDC37 overexpression by increasing CDK4-HSP90 association and CDK4 protein levels in cells. Furthermore, expression of the mutants was sufficient to protect kinase clients CDK4, CDK6, CRAF and ERBB2 from depletion induced by silencing endogenous CDC37, indicating that CDC37's client stabilising function cannot be inactivated by substantially reducing its direct interaction with HSP90. However, CDC37 could not compensate for loss of HSP90 function, showing that CDC37 and HSP90 have their own distinct and non-redundant roles in maintaining kinase clients. Our data substantiate the important function of CDC37 in chaperoning protein kinases. Furthermore, we demonstrate that CDC37 can stabilise kinase clients by a mechanism that is not dependent on a substantial direct interaction between CDC37 and HSP90, but nevertheless requires HSP90 activity. These results have significant implications for therapeutic targeting of CDC37.

The inhibition of feeding that follows intragastric administration of glucose had previously been shown to result in an ultimate net decrement in food intake which was equivalent in available energy to the amount of glucose loaded. Stomach loads of acetate, alanine, citrate, ethanol, glucose, glutamate, glycerol, lactate, oleate or valine (10 mmoles of each) were given in the present experiments. Food was withheld for 1 or 2 hr after gavage and then continuous access was restored. Relative to sodium chloride or urea loads, the metabolizable loads all inhibited feeding at some time in the subsequent few hr—in some cases at a time which followed complete absorption of the load. The net food intake decrement over 24 or 48 hr following gavage reliably correlated with the expected energy yield of the load. It is suggested that the primary metabolic control of food intake is an adjustment of the meal pattern which brings the current energy balance towards the null point.

ERK5 is a mitogen-activated protein (MAP) kinase regulated in human cells by diverse mitogens and stresses but also suspected of mediating the effects of a number of oncogenes. Its expression in the slt2Delta Saccharomyces cerevisiae mutant rescued several of the phenotypes caused by the lack of Slt2p (Mpk1p) cell integrity MAP kinase. ERK5 is able to provide this cell integrity MAP kinase function in yeast, as it is activated by the cell integrity signaling cascade that normally activates Slt2p and, in its active form, able to stimulate at least one key Slt2p target (Rlm1p, the major transcriptional regulator of cell wall genes). In vitro ERK5 kinase activity was abolished by Hsp90 inhibition. ERK5 activity in vivo was also lost in a strain that expresses a mutant Hsp90 chaperone. Therefore, human ERK5 expressed in yeast is an Hsp90 client, despite the widely held belief that the protein kinases of the MAP kinase class are non-Hsp90-dependent activities. Two-hybrid and protein binding studies revealed that strong association of Hsp90 with ERK5 requires the dual phosphorylation of the TEY motif in the MAP kinase activation loop. These phosphorylations, at positions adjacent to the Hsp90-binding surface recently identified for a number of protein kinases, may cause a localized rearrangement of this MAP kinase region that leads to creation of the Hsp90-binding surface. Complementation of the slt2Delta yeast defect by ERK5 expression establishes a new tool with which to screen for novel agonists and antagonists of ERK5 signaling as well as for isolating mutant forms of ERK5.

ATP hydrolysis by the Hsp90 molecular chaperone requires a connected set of conformational switches triggered by ATP binding to the N-terminal domain in the Hsp90 dimer. Central to this is a segment of the structure, which closes like a "lid" over bound ATP, promoting N-terminal dimerization and assembly of a competent active site. Hsp90 mutants that influence these conformational switches have strong effects on ATPase activity. ATPase activity is specifically regulated by Hsp90 co-chaperones, which directly influence the conformational switches. Here we have analyzed the effect of Hsp90 mutations on binding (using isothermal titration calorimetry and difference circular dichroism) and ATPase regulation by the co-chaperones Aha1, Sti1 (Hop), and Sba1 (p23). The ability of Sti1 to bind Hsp90 and arrest its ATPase activity was not affected by any of the mutants screened. Sba1 bound in the presence of AMPPNP to wild-type and ATPase hyperactive mutants with similar affinity but only very weakly to hypoactive mutants despite their wild-type ATP affinity. Unexpectedly, in all cases Sba1 bound to Hsp90 with a 1:2 molar stoichiometry. Aha1 binding to mutants was similar to wild-type, but the -fold activation of their ATPase varied substantially between mutants. Analysis of complex formation with co-chaperone mixtures showed Aha1 and p50cdc37 able to bind Hsp90 simultaneously but without direct interaction. Sba1 and p50cdc37 bound independently to Hsp90-AMPPNP but not together. These data indicated that Sba1 and Aha1 regulate Hsp90 by influencing the conformational state of the "ATP lid" and consequent N-terminal dimerization, whereas Sti1 does not.

The molecular chaperone heat shock protein 90 (HSP90) has emerged as an exciting molecular target. Derivatives of the natural product geldanamycin, such as 17-allylamino-17-demethoxy-geldanamycin (17-AAG), were the first HSP90 ATPase inhibitors to enter clinical trial. Synthetic small-molecule HSP90 inhibitors have potential advantages. Here, we describe the biological properties of the lead compound of a new class of 3,4-diaryl pyrazole resorcinol HSP90 inhibitor (CCT018159), which we identified by high-throughput screening. CCT018159 inhibited human HSP90beta with comparable potency to 17-AAG and with similar ATP-competitive kinetics. X-ray crystallographic structures of the NH(2)-terminal domain of yeast Hsp90 complexed with CCT018159 or its analogues showed binding properties similar to radicicol. The mean cellular GI(50) value of CCT018159 across a panel of human cancer cell lines, including melanoma, was 5.3 mumol/L. Unlike 17-AAG, the in vitro antitumor activity of the pyrazole resorcinol analogues is independent of NQO1/DT-diaphorase and P-glycoprotein expression. The molecular signature of HSP90 inhibition, comprising increased expression of HSP72 protein and depletion of ERBB2, CDK4, C-RAF, and mutant B-RAF, was shown by Western blotting and quantified by time-resolved fluorescent-Cellisa in human cancer cell lines treated with CCT018159. CCT018159 caused cell cytostasis associated with a G(1) arrest and induced apoptosis. CCT018159 also inhibited key endothelial and tumor cell functions implicated in invasion and angiogenesis. Overall, we have shown that diaryl pyrazole resorcinols exhibited similar cellular properties to 17-AAG with potential advantages (e.g., aqueous solubility, independence from NQO1 and P-glycoprotein). These compounds form the basis for further structure-based optimization to identify more potent inhibitors suitable for clinical development.

In this second of a two-part review encompassing random, combinatorial methods for soluble protein 'domain hunting', we focus upon the expression screening from DNA fragment libraries. Given a library of domain length-encoding DNA fragments assembled in expression vectors, it is necessary to devise reliable means to screen the sample DNA fragment population to find those that express stable, soluble target protein fragments, suitable for the required downstream aims. This review summarizes a variety of alternative strategies that have been employed to identify such stable truncates of full-length proteins. In addition, we review measures that can determine the quality of the expressed protein, the likely reliability of these measures, and the apparent extent of their application within the featured studies.

The molecular chaperone heat-shock protein 90 (HSP90) plays a key role in the cell by stabilizing a number of client proteins, many of which are oncogenic. The intrinsic ATPase activity of HSP90 is essential to this activity. HSP90 is a new cancer drug target as inhibition results in simultaneous disruption of several key signaling pathways, leading to a combinatorial approach to the treatment of malignancy. Inhibitors of HSP90 ATPase activity including the benzoquinone ansamycins, geldanamycin and 17-allylamino-17-demethoxygeldanamycin, and radicicol have been described. A high-throughput screen has been developed to identify small-molecule inhibitors that could be developed as therapeutic agents with improved pharmacological properties. A colorimetric assay for inorganic phosphate, based on the formation of a phosphomolybdate complex and subsequent reaction with malachite green, was used to measure the ATPase activity of yeast HSP90. The Km for ATP determined in the assay was 510+/-70 microM. The known HSP90 inhibitors geldanamycin and radicicol gave IC(50) values of 4.8 and 0.9 microM respectively, which compare with values found using the conventional coupled-enzyme assay. The assay was robust and reproducible (2-8% CV) and used to screen a compound collection of approximately 56,000 compounds in 384-well format with Z' factors between 0.6 and 0.8.

Recruitment of protein kinase clients to the Hsp90 chaperone involves the cochaperone p50(cdc37) acting as a scaffold, binding protein kinases via its N-terminal domain and Hsp90 via its C-terminal region. p50(cdc37) also has a regulatory activity, arresting Hsp90's ATPase cycle during client-protein loading. We have localized the binding site for p50(cdc37) to the N-terminal nucleotide binding domain of Hsp90 and determined the crystal structure of the Hsp90-p50(cdc37) core complex. Dimeric p50(cdc37) binds to surfaces of the Hsp90 N-domain implicated in ATP-dependent N-terminal dimerization and association with the middle segment of the chaperone. This interaction fixes the lid segment in an open conformation, inserts an arginine side chain into the ATP binding pocket to disable catalysis, and prevents trans-activating interaction of the N domains.

Exploitation of potential new targets for drug and vaccine development has an absolute requirement for multimilligram quantities of soluble protein. While recombinant expression of full-length proteins is frequently problematic, high-yield soluble expression of functional subconstructs is an effective alternative, so long as appropriate termini can be identified. Bioinformatics localizes domains, but doesn't predict boundaries with sufficient accuracy, so that subconstructs are typically found by trial and error. Combinatorial Domain Hunting (CDH) is a technology for discovering soluble, highly expressed constructs of target proteins. CDH combines unbiased, finely sampled gene-fragment libraries, with a screening protocol that provides "holistic" readout of solubility and yield for thousands of protein fragments. CDH is free of the "passenger solubilization" and out-of-frame translational start artifacts of fusion-protein systems, and hits are ready for scale-up expression. As a proof of principle, we applied CDH to p85alpha, successfully identifying soluble and highly expressed constructs encapsulating all the known globular domains, and immediately suitable for downstream applications.

Heat shock protein 90 (Hsp90) is a molecular chaperone essential for activating many signaling proteins in the eukaryotic cell. Biochemical and structural analysis of Hsp90 has revealed a complex mechanism of ATPase-coupled conformational changes and interactions with cochaperone proteins, which facilitate activation of Hsp90's diverse "clientele." Despite recent progress, key aspects of the ATPase-coupled mechanism of Hsp90 remain controversial, and the nature of the changes, engendered by Hsp90 in client proteins, is largely unknown. Here, we discuss present knowledge of Hsp90 structure and function gleaned from crystallographic studies of individual domains and recent progress in obtaining a structure for the ATP-bound conformation of the intact dimeric chaperone. Additionally, we describe the roles of the plethora of cochaperones with which Hsp90 cooperates and growing insights into their biochemical mechanisms, which come from crystal structures of Hsp90 cochaperone complexes.

Until recently, Hsp90 was one of the least well understood of the molecular chaperones, but considerable progress is now being made in unravelling its biochemistry. Hsp90 has now been shown to possess an inherent ATPase that is essential for the activation of authentic 'client' proteins in vivo and in vitro. The molecular detail of Hsp90's interactions with co-chaperones is also becoming clearer and the identification of key roles in assembling regulatory and signalling pathways has made it a target for anticancer drug development. Despite this, a clear understanding of how Hsp90 contributes to the folding and/or activation of its client proteins remains some way off.

During infections with a number of important eukaryotic pathogens the Hsp90 molecular chaperone of the pathogen is recognized as an immunodominant antigen by the host immune system. Yeast molecular genetics should allow study of the extent of sequence variation within conserved immunodominant epitopes on pathogen Hsp90s that is compatible with essential Hsp90 functions, as well as the processes that generate antigenic subfragments of these Hsp90s. The Hsp90 of the fungal pathogen Candida albicans was shown in this study to provide both essential and nonessential (pheromone signalling and mammalian steroid receptor activation) Hsp90 functions in Saccharomyces cerevisiae cells. Much of the C. albicans Hsp90 expressed in respiratory S. cerevisiae cells was shown to undergo a partial degradation in vivo, a degradation that closely resembles that of the native Hsp82 (one isoform of the homologous Hsp90) in S. cerevisiae. Allowing for the differences in the length of the charged linker region between the N- and C-terminal domains of C. albicans Hsp90 and S. cerevisiae Hsp82, these two proteins expressed in S. cerevisiae appear to give the same major degradation products. These Hsp90 fragments are similar to the products of incomplete Hsp90 degradation found in C. albicans cultures.

Client protein activation by Hsp90 involves a plethora of cochaperones whose roles are poorly defined. A ubiquitous family of stress-regulated proteins have been identified (Aha1, activator of Hsp90 ATPase) that bind directly to Hsp90 and are required for the in vivo Hsp90-dependent activation of clients such as v-Src, implicating them as cochaperones of the Hsp90 system. In vitro, Aha1 and its shorter homolog, Hch1, stimulate the inherent ATPase activity of yeast and human Hsp90. The identification of these Hsp90 cochaperone activators adds to the complex roles of cochaperones in regulating the ATPase-coupled conformational changes of the Hsp90 chaperone cycle.

Hsp90 is an abundant molecular chaperone essential to the establishment of many cellular regulation and signal transduction systems, but remains one of the least well described chaperones. The biochemical mechanism of protein folding by Hsp90 is poorly understood, and the direct involvement of ATP has been particularly contentious. Here we demonstrate in vitro an inherent ATPase activity in both yeast Hsp90 and the Escherichia coli homologue HtpG, which is sensitive to inhibition by the Hsp90-specific antibiotic geldanamycin. Mutations of residues implicated in ATP binding and hydrolysis by structural studies abolish this ATPase activity in vitro and disrupt Hsp90 function in vivo. These results show that Hsp90 is directly ATP dependent in vivo, and suggest an ATP-coupled chaperone cycle for Hsp90-mediated protein folding.

The Hsp90 chaperone cycle involves sequential assembly of different Hsp90-containing multiprotein complexes, the accessory proteins ("cochaperones") that are associated with these complexes being exchanged as the cycle proceeds from its early to its late stages. To gain insight as to whether the 2-hybrid system could be used to probe the interactions of this Hsp90 system, yeast transformants were constructed that express the Gal4p deoxyribonucleic acid-binding domain (BD) fused to the 2 Hsp90 isoforms and the various Hsp90 system cochaperones of yeast. These "bait" fusions were then introduced by mating into other transformants expressing nearly all the 6000 proteins of yeast expressed as fusions to the Gal4p activation domain (AD). High throughput 2-hybrid screening revealed the ability of Hsp90 and Hsp90 system cochaperones to engage in stable interactions in vivo, both with each other and with the various other proteins of the yeast proteome. Consistent with the transience of most chaperone associations, interactions to Hsp90 itself were invariably weak and generally influenced by stress. Mutations within a Hsp90-BD bait fusion and an AD-Cdc37 "prey" fusion were used to provide in vivo confirmation of the in vitro data that shows that Cdc37p is interacting with the "relaxed" conformation of Hsp90 and also to provide indications that Cdc37p needs to be phosphorylated at its N-terminus for any appreciable interaction with Hsp90. A number of potentially novel cochaperone interactions were also identified, providing a framework for these to be analyzed further using other techniques.

The Hsp90 chaperone cycle catalyzes the final activation step of several important eukaryotic proteins (Hsp90 "clients"). Although largely a functional form of Hsp90, an Hsp90-Gal4p DNA binding domain fusion (Hsp90-BD) displays no strong interactions in the yeast two-hybrid system, consistent with a general transience of most Hsp90-client associations. Strong in vivo interactions are though detected when the E33A mutation is introduced into this bait, a mutation that should arrest Hsp90-client complexes at a stage where the client is stabilized, yet prevented from attaining its active form. This E33A mutation stabilized the two-hybrid interactions of the Hsp90-BD fusion with approximately 3% of the Saccharomyces cerevisiae proteome in a screen of the 6,000 yeast proteins expressed as fusions to the Gal4p activation domain (AD). Among the detected interactors were the two stress-activated mitogen-activated protein (MAP) kinases of yeast, Hog1p and Slt2p (Mpk1p). Column retention experiments using wild-type and mutant forms of Hsp90 and Slt2p MAP kinase, as well as quantitative measurements of the effects of stress on the two-hybrid interaction of mutant Hsp90-BD and AD-Slt2p fusions, revealed that Hsp90 binds exclusively to the dually Thr/Tyr-phosphorylated, stress-activated form of Slt2p [(Y-P,T-P)Slt2p] and also to the MAP kinase domain within this (Y-P,T-P)Slt2p. Phenotypic analysis of a yeast mutant that expresses a mutant Hsp90 (T22Ihsp82) revealed that Hsp90 function is essential for this (Y-P,T-P)Slt2p to activate one of its downstream targets, the Rlm1p transcription factor. The interaction between Hsp90 and (Y-P,T-P)Slt2p, characterized in this study, is probably essential in this Hsp90 facilitation of the Rlm1p activation by Slt2p.

Hsp90 is a molecular chaperone essential for the activation and assembly of many key eukaryotic signalling and regulatory proteins. Hsp90 is assisted and regulated by co-chaperones that participate in an ordered series of dynamic multiprotein complexes, linked to Hsp90 conformationally coupled ATPase cycle. The co-chaperones Aha1 and Hch1 bind to Hsp90 and stimulate its ATPase activity. Biochemical analysis shows that this activity is dependent on the N-terminal domain of Aha1, which interacts with the central segment of Hsp90. The structural basis for this interaction is revealed by the crystal structure of the N-terminal domain (1-153) of Aha1 (equivalent to the whole of Hch1) in complex with the middle segment of Hsp90 (273-530). Structural analysis and mutagenesis show that binding of N-Aha1 promotes a conformational switch in the middle-segment catalytic loop (370-390) of Hsp90 that releases the catalytic Arg 380 and enables its interaction with ATP in the N-terminal nucleotide-binding domain of the chaperone.

Activation of client proteins by the Hsp90 molecular chaperone is dependent on binding and hydrolysis of ATP, which drives a molecular clamp via transient dimerization of the N-terminal domains. The crystal structure of the middle segment of yeast Hsp90 reveals considerable evolutionary divergence from the equivalent regions of other GHKL protein family members such as MutL and GyrB, including an additional domain of new fold. Using the known structure of the N-terminal nucleotide binding domain, a model for the Hsp90 dimer has been constructed. From this structure, residues implicated in the ATPase-coupled conformational cycle and in interactions with client proteins and the activating cochaperone Aha1 have been identified, and their roles functionally characterized in vitro and in vivo.

We demonstrate a role for Qri2 in the essential DNA repair function of the Smc5/6 complex in Saccharomyces cerevisiae. We generated temperature-sensitive (ts) mutants in QRI2 and characterized their properties. The mutants arrest after S phase and prior to mitosis. Furthermore, the arrest is dependant on the Rad24 checkpoint, and is also accompanied by phosphorylation of the Rad53 checkpoint effector kinase. The mutants also display genome instability and are sensitive to agents that damage DNA. Two-hybrid screens reveal a physical interaction between Qri2 and proteins that are non-Smc elements of the Smc5/6 DNA repair complex, which is why we propose the name NSE4 for the open reading frame previously known as QRI2. A key role for Nse4 in Smc5/6 function is likely, as overexpressing known subunits of the Smc5/6 complex suppresses nse4(ts) cell cycle arrest. The nse4(ts) growth arrest is non-lethal and unlike the catastrophic nuclear fragmentation phenotype of smc6(ts) mutants, the nucleus remains intact; replicative intermediates and sheared DNA are not detected. This could imply a role for Nse4 in maintenance of higher order chromosome structure.

The molecular chaperone HSP90 is currently under investigation as a promising target for anticancer drug discovery. It constitutes 1–2% of total cellular protein and is present in the cell as a dimer in association with a number of other proteins (1). HSP90 is involved in ensuring adequate protein folding and preventing non-specific aggregation of proteins following chemical mutation or stress (2). Under physiological conditions, together with its endoplasmic reticulum homolog GRP94, HSP90 plays a housekeeping role in the cell, maintaining the conformational stability and maturation of several key client proteins, including oncogenic kinases (e.g., ERBB2, RAF-1, CDK4, and LCK), steroid receptors, and mutant TP53 (3). A number of HSP90 inhibitors have already been identified. These include the benzoquinone ansamycin natural product geldanamycin and its analog, 17-allylamino-17-demethoxy-geldanamycin (17AAG), together with the chemically dissimilar natural product radicicol. The predominant mechanism of action of these agents involves binding to HSP90 at the ATP binding site in the N-terminal domain of the protein, leading to inhibition of the intrinsic ATPase activity of HSP90 (4–6). Inhibition of HSP90 ATPase activity prevents recruitment of co-chaperones and encourages the formation of a type of HSP90 heterocomplex from which these client proteins are targeted for degradation via the ubiquitin proteosome pathway (3,7).

The in vivo function of the heat shock protein 90 (Hsp90) molecular chaperone is dependent on the binding and hydrolysis of ATP, and on interactions with a variety of co-chaperones containing tetratricopeptide repeat (TPR) domains. We have now analysed the interaction of the yeast TPR-domain co-chaperones Sti1 and Cpr6 with yeast Hsp90 by isothermal titration calorimetry, circular dichroism spectroscopy and analytical ultracentrifugation, and determined the effect of their binding on the inherent ATPase activity of Hsp90. Sti1 and Cpr6 both bind with sub-micromolar affinity, with Sti1 binding accompanied by a large conformational change. Two co-chaperone molecules bind per Hsp90 dimer, and Sti1 itself is found to be a dimer in free solution. The inherent ATPase activity of Hsp90 is completely inhibited by binding of Sti1, but is not affected by Cpr6, although Cpr6 can reactivate the ATPase activity by displacing Sti1 from Hsp90. Bound Sti1 makes direct contact with, and blocks access to the ATP-binding site in the N-terminal domain of Hsp90. These results reveal an important role for TPR-domain co-chaperones as regulators of the ATPase activity of Hsp90, showing that the ATP-dependent step in Hsp90-mediated protein folding occurs after the binding of the folding client protein, and suggesting that ATP hydrolysis triggers client-protein release.

In addressing a new drug discovery target, the generation of tractable protein substrates for functional and structural analyses can represent a significant hurdle. Traditional approaches rely on protein expression trials of multiple variants in various systems, frequently with limited success. The increasing knowledge base derived from genomics and structural proteomics initiatives assists the bioinformatics-led design of these experiments. Nevertheless, for many eukaryotic polypeptides, particularly those with relatively few homologues, the generation of useful protein products can still be a major challenge. This review describes the basis of efforts to forge an alternative 'domain-hunting' paradigm, based upon combinatorial sampling of expression construct libraries derived by fragmentation of the encoding DNA template, namely the methods and considerations in generating fragment length DNA from target genes. An accompanying review focuses upon the expression screening of such combinatorial DNA libraries for the sampling of the corresponding set of protein fragments.

Hsp90 is a highly specific chaperone for many signal transduction proteins, including steroid hormone receptors and a broad range of protein kinases. The crystal structure of the N-terminal domain of the yeast Hsp90 reveals a dimeric structure based on a highly twisted sixteen stranded beta-sheet, whose topology suggests a possible 30-domain-swapped structure for the intact Hsp90 dimer. The opposing faces of the beta-sheets in the dimer define a potential peptide-binding cleft, suggesting that the N-domain may serve as a molecular 'clamp' in the binding of ligand proteins to Hsp90.

Understanding the mode of action of Hsp90 requires that molecular detail of its interactions with client proteins and co-chaperones are known. The structure determination of the N-terminal domain of Hsp90/Hsp90beta, proof that it is an ATPase, that this activity is regulated and the identification of co-chaperones that facilitate Hsp90 function were landmarks towards understanding conformational changes in Hsp90 brought about by ATP, co-chaperones and client proteins. Sti1 and Cdc37/p50, which associate with early Hsp90 complexes, were shown to be inhibitors of Hsp90 ATPase activity and therefore promote its 'open' state, whereas Sba1/p23, which associates with mature complexes, inhibits ATPase activity and stabilises the 'closed' state. The isolation and characterisation of Aha1, the only known strong activator of Hsp90 ATPase activity, which promotes the 'closed' state of Hsp90, will also be of major importance in understanding Hsp90 function. The structure determination of the middle region of Hsp90 has shed further light on the complex ATP-cycle of Hsp90, identifying a catalytic loop, with key residues that are essential for ATP hydrolysis. These studies, together with biochemical ones, suggest that ATP hydrolysis, is dependent on a complex rate-limiting step, involving N-terminal dimerization and association of the middle region, and therefore the catalytic loop, of Hsp90 with the N-terminal domains. The structure of the middle region of Hsp90 will also accelerate our understanding of client protein interactions since this region is implicated in their recognition and in particular their active-site openings.

How the ATPase activity of Heat shock protein 90 (Hsp90) is coupled to client protein activation remains obscure. Using truncation and missense mutants of Hsp90, we analysed the structural implications of its ATPase cycle. C-terminal truncation mutants lacking inherent dimerization displayed reduced ATPase activity, but dimerized in the presence of 5'-adenylamido-diphosphate (AMP-PNP), and AMP-PNP- promoted association of N-termini in intact Hsp90 dimers was demonstrated. Recruitment of p23/Sba1 to C-terminal truncation mutants also required AMP-PNP-dependent dimerization. The temperature- sensitive (ts) mutant T101I had normal ATP affinity but reduced ATPase activity and AMP-PNP-dependent N-terminal association, whereas the ts mutant T22I displayed enhanced ATPase activity and AMP-PNP-dependent N-terminal dimerization, indicating a close correlation between these properties. The locations of these residues suggest that the conformation of the 'lid' segment (residues 100-121) couples ATP binding to N-terminal association. Consistent with this, a mutation designed to favour 'lid' closure (A107N) substantially enhanced ATPase activity and N-terminal dimerization. These data show that Hsp90 has a molecular 'clamp' mechanism, similar to DNA gyrase and MutL, whose opening and closing by transient N-terminal dimerization are directly coupled to the ATPase cycle.

The aconitase of Escherichia coli was purified to homogeneity, albeit in low yield (0.6%). It was shown to be a monomeric protein of Mr 95,000 or 97,500 by gel filtration and SDS-PAGE analysis, respectively. The N-terminal amino acid sequence resembled that of the Bacillus subtilis enzyme (citB product), but the similarity at the DNA level was insufficient to allow detection of the E. coli acn gene using a 456 bp citB probe. Phages containing the acn gene were isolated from a lambda-E. coli gene bank by immunoscreening with an antiserum raised against purified bacterial enzyme. The acn gene was located at 28 min (1350 kb) in the physical map of the E. coli chromosome by probing Southern blots with a fragment of the gene. Attempts to locate the gene using the same procedure with oligonucleotide probes encoding segments of the N-terminal amino acid sequence were complicated by the lack of probe specificity and an inaccuracy in the physical map of Kohara et al. (Cell 50, 495-508, 1987). Aconitase specific activity was amplified some 20-200-fold in cultures transformed with pGS447, a derivative of pUC119 containing the acn gene, and an apparent four-fold activation-deactivation of the phagemid-encoded enzyme was observed in late exponential phase. The aconitase antiserum cross-reacted with both the porcine and Salmonella typhimurium (Mr 120,000) enzymes.

The nucleotide sequence of the aconitase gene (acn) of Escherichia coli was determined and used to deduce the primary structure of the enzyme. The coding region comprises 2670 bp (890 codons excluding the start and stop codons) which define a product having a relative molecular mass of 97,513 and an N-terminal amino acid sequence consistent with those determined previously for the purified enzyme. The acn gene is flanked by the cysB gene and a putative riboflavin biosynthesis gene resembling the ribA gene of Bacillus subtilis. The 1004-bp cysB--acn intergenic region contains several potential promoter and regulatory sequences. The amino acid sequence of the E. coli aconitase is similar to the mitochondrial aconitases (27-29% identity) and the isopropylmalate isomerases (20-21% identity) but it is most similar to the human iron-responsive-element-binding protein (53% identity). The three cysteine residues involved in ligand binding to the [4Fe-4S] centre are conserved in all of these proteins. Of the remaining 17 active-site residues assigned for porcine aconitase, 16 are conserved in both the bacterial aconitase and the iron-responsive-element-binding protein and 14 in the isopropylmalate isomerases. It is concluded that the bacterial and mitochondrial aconitases, the isopropylmalate isomerases and the iron-responsive-element-binding protein form a family of structurally related proteins, which does not include the Fe-S-containing fumarases. These relationships raise the possibility that the iron-responsive-element-binding protein may be a cytoplasmic aconitase and that the E. coli aconitase may have an iron-responsive regulatory function.

Although the heat shock protein 90 (HSP90) inhibitor 17-allylamino-17-demethoxygeldanamycin (17-AAG) shows clinical promise, potential limitations encourage development of alternative chemotypes. We discovered the 3,4-diarylpyrazole resorcinol CCT018159 by high-throughput screening and used structure-based design to generate more potent pyrazole amide analogues, exemplified by VER-49009. Here, we describe the detailed biological properties of VER-49009 and the corresponding isoxazole VER-50589. X-ray crystallography showed a virtually identical HSP90 binding mode. However, the dissociation constant (K(d)) of VER-50589 was 4.5 +/- 2.2 nmol/L compared with 78.0 +/- 10.4 nmol/L for VER-49009, attributable to higher enthalpy for VER-50589 binding. A competitive binding assay gave a lower IC(50) of 21 +/- 4 nmol/L for VER-50589 compared with 47 +/- 9 nmol/L for VER-49009. Cellular uptake of VER-50589 was 4-fold greater than for VER-49009. Mean cellular antiproliferative GI(50) values for VER-50589 and VER-49009 for a human cancer cell line panel were 78 +/- 15 and 685 +/- 119 nmol/L, respectively, showing a 9-fold potency gain for the isoxazole. Unlike 17-AAG, but as with CCT018159, cellular potency of these analogues was independent of NAD(P)H:quinone oxidoreductase 1/DT-diaphorase and P-glycoprotein expression. Consistent with HSP90 inhibition, VER-50589 and VER-49009 caused induction of HSP72 and HSP27 alongside depletion of client proteins, including C-RAF, B-RAF, and survivin, and the protein arginine methyltransferase PRMT5. Both caused cell cycle arrest and apoptosis. Extent and duration of pharmacodynamic changes in an orthotopic human ovarian carcinoma model confirmed the superiority of VER-50589 over VER-49009. VER-50589 accumulated in HCT116 human colon cancer xenografts at levels above the cellular GI(50) for 24 h, resulting in 30% growth inhibition. The results indicate the therapeutic potential of the resorcinylic pyrazole/isoxazole amide analogues as HSP90 inhibitors.

In vivo activation of client proteins by Hsp90 depends on its ATPase-coupled conformational cycle and on interaction with a variety of co-chaperone proteins. For some client proteins the co-chaperone Sti1/Hop/p60 acts as a "scaffold," recruiting Hsp70 and the bound client to Hsp90 early in the cycle and suppressing ATP turnover by Hsp90 during the loading phase. Recruitment of protein kinase clients to the Hsp90 complex appears to involve a specialized co-chaperone, Cdc37p/p50(cdc37), whose binding to Hsp90 is mutually exclusive of Sti1/Hop/p60. We now show that Cdc37p/p50(cdc37), like Sti1/Hop/p60, also suppresses ATP turnover by Hsp90 supporting the idea that client protein loading to Hsp90 requires a "relaxed" ADP-bound conformation. Like Sti1/Hop/p60, Cdc37p/p50(cdc37) binds to Hsp90 as a dimer, and the suppressed ATPase activity of Hsp90 is restored when Cdc37p/p50(cdc37) is displaced by the immunophilin co-chaperone Cpr6/Cyp40. However, unlike Sti1/Hop/p60, which can displace geldanamycin upon binding to Hsp90, Cdc37p/p50(cdc37) forms a stable complex with geldanamycin-bound Hsp90 and may be sequestered in geldanamycin-inhibited Hsp90 complexes in vivo.

Yeast is rendered temperature sensitive with loss of the C-terminal (CT) domain of heat shock transcription factor (Hsf1). This domain loss was found to abrogate heat stimulation of Slt2 (Mpk1), the mitogen-activated protein kinase that directs the reinforced cell integrity gene expression needed for high-temperature growth. In Hsf1 CT domain-deficient cells, Slt2 still undergoes Mkk1/2-directed dual-Thr/Tyr phosphorylation in response to the heat stimulation of cell integrity pathway signaling, but the low Hsp90 expression level suppresses any corresponding increase in Slt2 kinase activity due to Slt2 being a "client" of the Hsp90 chaperone. A non-Hsf1-directed Hsp90 overexpression restored the heat induction of Slt2 activity in these cells, as well as both Slt2-dependent (Rlm1, Swi4) and Slt2-independent (MBF) transcriptional activities. Their high-temperature growth was also rescued, not just by this Hsp90 overexpression but by osmotic stabilization, by the expression of a Slt2-independent form of the Rlm1 transcriptional regulator of cell integrity genes, and by a multicopy SLT2 gene vector. In providing the elevated Hsp90 needed for an efficient activation of Slt2, heat activation of Hsf1 indirectly facilitates (Slt2-directed) heat activation of yet another transcription factor (Rlm1). This provides an explanation as to why, in earlier transcript analysis compared to chromatin immunoprecipitation studies, many more genes of yeast displayed an Hsf1-dependent transcriptional activation by heat than bound Hsf1 directly. The levels of Hsp90 expression affecting transcription factor regulation by Hsp90 client protein kinases also provides a mechanistic model for how heat shock factor can influence the expression of several non-hsp genes in higher organisms.

CHIP is a dimeric U box E3 ubiquitin ligase that binds Hsp90 and/or Hsp70 via its TPR-domain, facilitating ubiquitylation of chaperone bound client proteins. We have determined the crystal structure of CHIP bound to an Hsp90 C-terminal decapeptide. The structure explains how CHIP associates with either chaperone type and reveals an unusual asymmetric homodimer in which the protomers adopt radically different conformations. Additionally, we identified CHIP as a functional partner of Ubc13-Uev1a in formation of Lys63-linked polyubiquitin chains, extending CHIP's roles into ubiquitin regulation as well as targeted destruction. The structure of Ubc13-Uev1a bound to the CHIP U box domain defines the basis for selective cooperation of CHIP with specific ubiquitin-conjugating enzymes. Remarkably, the asymmetric arrangement of the TPR domains in the CHIP dimer occludes one Ubc binding site, so that CHIP operates with half-of-sites activity, providing an elegant means for coupling a dimeric chaperone to a single ubiquitylation system.

The mitochondrial (MT) genome is a potential means of gene delivery to human cells for therapeutic expression. As a first step towards this, we have synthesized a gene coding for mature human ornithine transcarbamylase (OTC) by recursive PCR using 18 oligodeoxyribonucleotides, each 70-80 nucleotides in length, using codons which should allow translation in accordance with both mammalian mt and universal codon usage. Flanking mt DNA sequences were incorporated which are designed to facilitate site-specific cloning into the mt genome. Expression of this human gene in Escherichia coli leads to an immunoreactive OTC product of the correct size and N-terminal amino-acid sequence, but which forms inclusion bodies and lacks enzymatic activity.

1. Ingestion of phenothiazine drugs did not detectably disturb the urinary excretion of purine bases by normal subjects. No significant differences in purine excretion were found between groups of normal subjects matched with schizophrenic patients untreated with phenothiazine drugs. Drug treatment had thus not obscured differences between normal and schizophrenic populations in a previous study, and conclusions drawn then were supported.

2. Excretion of 3-methylxanthine was more frequently detected among schizophrenic patients than among normal subjects, and this fact was probably attributable to increased ingestion of cocoa products by some patients.

3. Variations and correlations among the daily urinary excretions of endogenous and dietary purine bases are collated.

Small ubiquitin like modifier (SUMO) is post-translationally attached to target proteins, forming a covalent bond between its C-terminal glycine and one or more lysine residues on the target protein. SUMO modification of target proteins can affect protein-protein interactions, protein activity, localistation and stability. This study set out to develop an efficient in vitro SUMOylation system to enable the identification of target lysine residues in S. pombe proteins by mass spectrometry. This involved incorporating a trypsin cleavage site adjacent to the SUMO di-glycine motif to improve peptide coverage during mass spectrometry. Several SUMOylated target proteins were identified here, including the E2 SUMO conjugating enzyme Hus5, the E3 SUMO ligase Nse2 and PCNA.
The second part of this study focused on the characterisation of unSUMOylatable E3 SUMO ligase nse2 mutants. Integration of lysine to arginine mutations into the genome did not result in any mutant phenotypes and a function for auto-SUMOylation of Nse2 was not identified. During this study, human patients with mutations in the nse2 gene were reported and the equivalent mutations were integrated into the S. pombe nse2 gene to investigate the effect of the mutations.
The final part of this work involved the analysis of the SUMOylation of S. pombe PCNA. Using the in vitro system, four target lysine residues for SUMO were identified. SUMOylation of PCNA was also observed in vivo following pull-down studies and 2D gel analysis of wild type and unSUMOylatable mutants. Extensive epistasis analysis was undertaken using these mutants to investigate the role of SUMOylation of S. pombe PCNA.

Endophytic fungi live asymptomatically within plants. They are usually regarded as non-pathogenic or even mutualistic, but whether plants respond antagonistically to their presence remains unclear, particularly in the little-studied associations between endophytes and nong-raminoid herbaceous plants.

We investigated the effects of the endophyte Chaetomium cochlioides on leaf chemistry in Cirsium arvense. Plants were sprayed with spores; leaf material from both subsequent new growth and the sprayed leaves was analysed 2 wk later. Infection frequency was 91% and63% for sprayed and new growth, respectively, indicating that C. cochlioides rapidly infects new foliage.

Metabolomic analyses revealed marked changes in leaf chemistry with infection, especially in new growth. Changes in several novel oxylipin metabolites were detected, including arabi-dopsides reported here for the first time in a plant species other than Arabidopsis thaliana,and a jasmonate-containing galactolipid.

The production of these metabolites in response to endophyte presence, particularly in newly infected foliage, suggests that endophytes elicit similar chemical responses in plants to those usually produced following wounding, herbivory and pathogen invasion. Whether en-dophytes benefit their hosts may depend on a complex series of chemically mediated interactions between the plant, the endophyte, other microbial colonists and natural enemies.

Regulation of protein synthesis is of fundamental
importance to cells. It has a critical role in the control of gene expression, and consequently cell growth and proliferation. The importance of this control is supported by the fact that protein synthesis is frequently upregulated in tumor cells. The major point at which regulation occurs is the initiation stage.
Initiation of translation involves the interaction of several proteins to form the eIF4F complex, the recognition of the mRNA by this complex, and the subsequent recruitment of the 40S ribosomal subunit to the mRNA. This results in the formation of the 48S complex that then scans the mRNA for the start codon, engages the methionyl-tRNA and eventually forms the mature 80S ribosome which is elongationcompetent.
Formation of the 48S complex is regulated by the
availability of individual initiation factors and through specific protein-protein interactions. Both of these events can be regulated by post-translational modification by ubiquitin or Ubls (ubiquitin-like modifiers) such as SUMO or ISG15. We provide here a summary of translation initiation factors that are modified by ubiquitin or Ubls and, where they have been
studied in detail, describe the role of these modifications and their effects on regulating protein synthesis.

Through their association with a kleisin subunit (Scc1), cohesin’s Smc1 and Smc3 subunits
are thought to form tripartite rings that mediate sister chromatid cohesion. Unlike the
structure of Smc1/Smc3 and Smc1/Scc1 interfaces, that of Smc3/Scc1 is not known.
Disconnection of this interface is thought to release cohesin from chromosomes in a
process regulated by acetylation. We show here that the N-terminal domain of yeast Scc1
contains two a helices, forming a four-helix bundle with the coiled coil emerging from
Smc3’s adenosine triphosphatase head. Mutations affecting this interaction compromise
cohesin’s association with chromosomes. The interface is far from Smc3 residues,
whose acetylation prevents cohesin’s dissociation from chromosomes. Cohesin complexes
holding chromatids together in vivo do indeed have the configuration of hetero-trimeric
rings, and sister DNAs are entrapped within these.

Transgenic crop pyramids producing two or more Bacillus thuringiensis (Bt) toxins that kill the same insect pest have been widely used to delay evolution of pest resistance. To assess the potential of pyramids to achieve this goal, we analyze data from 38 studies that report effects of ten Bt toxins used in transgenic crops against 15 insect pests. We find that compared with optimal low levels of insect survival, survival on currently used pyramids is often higher for both susceptible insects and insects resistant to one of the toxins in the pyramid. Furthermore, we find that cross-resistance and antagonism between toxins used in pyramids are common, and that these problems are associated with the similarity of the amino acid sequences of domains II and III of the toxins, respectively. This analysis should assist in future pyramid design and the development of sustainable resistance management strategies.

Myogenic differentiation in the C2C12 myoblast model system reflects a concerted and controlled activation of transcription and translation following the exit of cells from the cell cycle. Previously we have shown that the mTORC1 signaling inhibitor, RAD001, decreased protein synthesis rates, delayed C2C12 myoblast differentiation, decreased p70S6K activity but did not affect the hypermodification of 4E-BP1. Here we have further investigated the modification of 4E-BP1 during the early phase of differentiation as cells exit the cell cycle, using inhibitors to target mTOR kinase and siRNAs to ablate the expression of raptor and rictor. As predicted, inhibition of mTOR kinase activity prevented p70S6K, 4E-BP1 phosphorylation and was associated with an inhibition of myogenic differentiation. Surprisingly, extensive depletion of raptor did not affect p70S6K or 4E-BP1 phosphorylation, but promoted an increase in mTORC2 activity (as evidenced by increased Akt Ser473 phosphorylation). These data suggest that an mTOR kinase-dependent, but raptor-independent regulation of downstream signaling is important for myogenic differentiation.

Fanconi anemia (FA) is an autosomal recessive genetic disorder caused by defects in any of 15 FA genes responsible for processing DNA interstrand cross-links (ICLs). The ultimate outcome of the FA pathway is resolution of cross-links, which requires structure-selective nucleases. FA-associated nuclease 1 (FAN1) is believed to be recruited to lesions by a monoubiquitinated FANCI–FANCD2 (ID) complex and participates in ICL repair. Here, we determined the crystal structure of Pseudomonas aeruginosa FAN1 (PaFAN1) lacking the UBZ (ubiquitin-binding zinc) domain in complex with 5′ flap DNA. All four domains of the right-hand-shaped PaFAN1 are involved in DNA recognition, with each domain playing a specific role in bending DNA at the nick. The six-helix bundle that binds the junction connects to the catalytic viral replication and repair (VRR) nuclease (VRR nuc) domain, enabling FAN1 to incise the scissile phosphate a few bases distant from the junction. The six-helix bundle also inhibits the cleavage of intact Holliday junctions. PaFAN1 shares several conserved features with other flap structure-selective nucleases despite structural differences. A clamping motion of the domains around the wedge helix, which acts as a pivot, facilitates nucleolytic cleavage. The PaFAN1 structure provides insights into how archaeal Holliday junction resolvases evolved to incise 5′ flap substrates and how FAN1 integrates with the FA complex to participate in ICL repair.

Structural biology studies typically require large quantities of pure, soluble protein. Currently the most widely-used method for obtaining such protein involves the use of bioinformatics and experimental methods to design constructs of the target, which are cloned and expressed. Recently an alternative approach has emerged, which involves random fragmentation of the gene of interest and screening for well-expressing fragments. Here we describe the application of one such fragmentation method, combinatorial domain hunting (CDH), to a target which historically was difficult to express, human MEK-1. We show how CDH was used to identify a fragment which covers the kinase domain of MEK-1 and which expresses and crystallizes significantly better than designed expression constructs, and we report the crystal structure of this fragment which explains some of its superior properties. Gene fragmentation methods, such as CDH, thus hold great promise for tackling difficult-to-express target proteins.

The multi-subunit DNA-dependent protein kinase (DNA-PK), a crucial player in DNA repair by non-homologous end-joining in higher eukaryotes, consists of a catalytic subunit (DNA-PKcs) and the Ku heterodimer. Ku recruits DNA-PKcs to double-strand breaks, where DNA-PK assembles prior to DNA repair. The interaction of DNA-PK with DNA is regulated via autophosphorylation. Recent SAXS data addressed the conformational changes occurring in the purified catalytic subunit upon autophosphorylation. Here, we present the first structural analysis of the effects of autophosphorylation on the trimeric DNA-PK enzyme, performed by electron microscopy and single particle analysis. We observe a considerable degree of heterogeneity in the autophosphorylated material, which we resolved into subpopulations of intact complex, and separate DNA-PKcs and Ku, by using multivariate statistical analysis and multi-reference alignment on a partitioned particle image data set. The proportion of dimeric oligomers was reduced compared to non-phosphorylated complex, and those dimers remaining showed a substantial variation in mutual monomer orientation. Together, our data indicate a substantial remodelling of DNA-PK holo-enzyme upon autophosphorylation, which is crucial to the release of protein factors from a repaired DNA double-strand break.

Cyclin/cyclin-dependent kinase (CDK) complexes are critical regulators of cellular proliferation. A complex network of regulatory mechanisms has evolved to control their activity, including activating and inactivating phosphorylation of the catalytic CDK subunit and inhibition through specific regulatory proteins. Primate herpesviruses, including the oncogenic Kaposi sarcoma herpesvirus, encode cyclin D homologues. Viral cyclins have diverged from their cellular progenitor in that they elicit holoenzyme activity independent of activating phosphorylation by the CDK-activating kinase and resistant to inhibition by CDK inhibitors. Using sequence comparison and site-directed mutagenesis, we performed molecular analysis of the cellular cyclin D and the Kaposi sarcoma herpesvirus-cyclin to delineate the molecular mechanisms behind their different behavior. This provides evidence that a surface recognized for its involvement in the docking of CIP/KIP inhibitors is required and sufficient to modulate cyclin-CDK response to a range of regulatory cues, including INK4 sensitivity and CDK-activating kinase dependence. Importantly, amino acids in this region are critically linked to substrate selection, suggesting that a mutational drift in this surface simultaneously affects function and regulation. Together our work provides novel insight into the molecular mechanisms governing cyclin-CDK function and regulation and defines the biological forces that may have driven evolution of viral cyclins.

Heat shock protein 90 (Hsp90) is an essential molecular chaperone whose activity is regulated not only by cochaperones but also by distinct posttranslational modifications. We report here that casein kinase 2 phosphorylates a conserved threonine residue (T22) in α helix-1 of the yeast Hsp90 N-domain both in vitro and in vivo. This α helix participates in a hydrophobic interaction with the catalytic loop in Hsp90's middle domain, helping to stabilize the chaperone's ATPase-competent state. Phosphomimetic mutation of this residue alters Hsp90 ATPase activity and chaperone function and impacts interaction with the cochaperones Aha1 and Cdc37. Overexpression of Aha1 stimulates the ATPase activity, restores cochaperone interactions, and compensates for the functional defects of these Hsp90 mutants.

The innate immune system is critical in the response to infection by pathogens and it is activated by pattern recognition receptors (PRRs) binding to pathogen associated molecular patterns (PAMPs). During viral infection, the direct recognition of the viral nucleic acids, such as the genomes of DNA viruses, is very important for activation of innate immunity. Recently, DNA-dependent protein kinase (DNA-PK), a heterotrimeric complex consisting of the Ku70/Ku80 heterodimer and the catalytic subunit DNA-PKcs was identified as a cytoplasmic PRR for DNA that is important for the innate immune response to intracellular DNA and DNA virus infection. Here we show that vaccinia virus (VACV) has evolved to inhibit this function of DNA-PK by expression of a highly conserved protein called C16, which was known to contribute to virulence but by an unknown mechanism. Data presented show that C16 binds directly to the Ku heterodimer and thereby inhibits the innate immune response to DNA in fibroblasts, characterised by the decreased production of cytokines and chemokines. Mechanistically, C16 acts by blocking DNA-PK binding to DNA, which correlates with reduced DNA-PK-dependent DNA sensing. The C-terminal region of C16 is sufficient for binding Ku and this activity is conserved in the variola virus (VARV) orthologue of C16. In contrast, deletion of 5 amino acids in this domain is enough to knockout this function from the attenuated vaccine strain modified vaccinia virus Ankara (MVA). In vivo a VACV mutant lacking C16 induced higher levels of cytokines and chemokines early after infection compared to control viruses, confirming the role of this virulence factor in attenuating the innate immune response. Overall this study describes the inhibition of DNA-PK-dependent DNA sensing by a poxvirus protein, adding to the evidence that DNA-PK is a critical component of innate immunity to DNA viruses.

Amyloid fibrils are associated with a large number of diseases in which proteins and
peptides abnormally assemble to form insoluble amyloid that deposit in the tissues.
However, oxidative stress has been implicated in the pathogenesis of a number of
neurodegenerative diseases and is believed to play an important role in the amyloid
deposition through protein cross-linkings. Under oxidative stress conditions, tyrosyl
radicals can be formed and coupled to form dityrosine cross-linkage. The formation of
dityrosine cross-linked oligomers is one of the oxidative modifications that may
mediate the toxicity of amyloid β (Aβ) and α-synuclein (α-syn) in Alzheimer’s disease
(AD) and Parkinson’s disease (PD) respectively. In this thesis, I explored the oxidative
modification of two short peptides, HYFNIF and VIYKI, using a Cu2+/H2O2 oxidation
system, and studied the morphological and conformational changes of these amyloid
fibrils during the oxidation process. These peptides were selected as simple amyloid
model systems that have been previously structurally characterised, to better understand
the dityrosine formation at a structural level and to optimise the oxidation conditions.

Oxidative stress has been implicated in AD. Here, I have explored the formation of
dityrosine cross-linked Aβ42 in vitro. We have shown that dityrosine is generated in
internalised Aβ in cell cultures. Results also revealed the prevalence of dityrosine
crosslinks in amyloid plaques in brain tissue and cerebrospinal fluid from AD patients,
indicating that dityrosine could be used as a biomarker of oxidative stress in AD.

The ability of the Cu2+ ion to promote the formation of in vitro dityrosine cross-linked
α-syn was also explored and the effect on α-syn fibrillogenesis and conformation
induced by Cu2+ was investigated. The results revealed the possibility of involvement
the dityrosine cross-linked α-syn dimer as a nucleus to initiate the polymerisation
process of α-syn to form amyloid fibrils.

Dityrosine cross-linkages can be generated in vitro using oxidation system of
Cu2+/H2O2, and might play an important role in the solubility and assembly of
amyloidogenic peptides and proteins that are associated in the pathogenesis of many
neurodegenerative disease including AD and PD. Dityrosine cross-linkages can lend a
further stability to the already stable amyloid fibrils, and this may explain their protease
resistance. Dityrosine cross-links formation represents one of the possible pathways by
which oligomers can be formed. Dityrosine cross-linked oligomers represent a good
bio-index of oxidatively coupled tyrosine-contained proteins due to their high stability.

Self-assembly of proteins and peptides into amyloid structures has been the subject of intense and focused research due to their association with neurodegenerative, age-related human diseases and transmissible prion diseases in humans and mammals. Of the disease associated amyloid assemblies, a diverse array of species, ranging from small oligomeric assembly intermediates to fibrillar structures, have been shown to have toxic potential. Equally, a range of species formed by the same disease associated amyloid sequences have been found to be relatively benign under comparable monomer equivalent concentrations and conditions. In recent years, an increasing number of functional amyloid systems have also been found. These developments show that not all amyloid structures are generically toxic to cells. Given these observations, it is important to understand why amyloid structures may encode such varied toxic potential despite sharing a common core molecular architecture. Here, we discuss possible links between different aspects of amyloidogenic structures and assembly mechanisms with their varied functional effects. We propose testable hypotheses for the relationship between amyloid structure and its toxic potential in the context of recent reports on amyloid sequence, structure, and toxicity relationships.

Development of single-molecule localization microscopy techniques has allowed nanometre scale localization accuracy inside cells, permitting the resolution of ultra-fine cell structure and the elucidation of crucial molecular mechanisms. Application of these methodologies to understanding processes underlying DNA replication and repair has been limited to defined in vitro biochemical analysis and prokaryotic cells. In order to expand these techniques to eukaryotic systems, we have further developed a photo-activated localization microscopy-based method to directly visualize DNA-associated proteins in unfixed eukaryotic cells. We demonstrate that motion blurring of fluorescence due to protein diffusivity can be used to selectively image the DNA-bound population of proteins. We designed and tested a simple methodology and show that it can be used to detect changes in DNA binding of a replicative helicase subunit, Mcm4, and the replication sliding clamp, PCNA, between different stages of the cell cycle and between distinct genetic backgrounds.

We employ stable‐isotope labeling and quantitative mass spectrometry to track histone methylation stability. We show that H3 trimethyl K9 and K27 are slow to be established on new histones and slow to disappear from old histones, with half‐lives of multiple cell divisions. By contrast, the transcription‐associated marks K4me3 and K36me3 turn over far more rapidly, with half‐lives of 6.8 h and 57 h, respectively. Inhibition of demethylases increases K9 and K36 methylation, with K9 showing the largest and most robust increase. We interpret different turnover rates in light of genome‐wide localization data and transcription‐dependent nucleosome rearrangements proximal to the transcription start site.

SUMO is a small post-translational modifier, that is attached to lysine residues in target proteins. It acts by altering proteinprotein
interactions, protein localisation and protein activity. SUMO chains can also act as substrates for ubiquitination,
resulting in proteasome-mediated degradation of the target protein. SUMO is removed from target proteins by one of a
number of specific proteases. The processes of sumoylation and desumoylation have well documented roles in DNA
metabolism and in the maintenance of chromatin structure. To further analyse the role of this modification, we have
purified protein complexes containing the S. pombe SUMO protease, Ulp2. These complexes contain proteins required for
ribosome biogenesis, RNA stability and protein synthesis. Here we have focussed on two translation initiation factors that
we identified as co-purifying with Ulp2, eIF4G and eIF3h. We demonstrate that eIF4G, but not eIF3h, is sumoylated. This
modification is increased under conditions that produce cytoplasmic stress granules. Consistent with this we observe partial
co-localisation of eIF4G and SUMO in stressed cells. Using HeLa cells, we demonstrate that human eIF4GI is also sumoylated;
in vitro studies indicate that human eIF4GI is modified on K1368 and K1588, that are located in the C-terminal eIF4A- and
Mnk-binding sites respectively.

A large number of proteins are modified posttranslationally
by the ubiquitin-like protein (Ubl) SUMO.
This process, known as sumoylation, regulates the function,
localisation and activity of target proteins as part of normal
cellular metabolism, e.g., during development, and through
the cell cycle, as well as in response to a range of stresses. In
order to be effective, the sumoylation pathway itself must also
be regulated. This review describes how the SUMOylation
process is regulated. In particular, regulation of the SUMO
conjugation and deconjugation machinery at the level of transcription
and by post-translational modifications is discussed.

Xeroderma pigmentosum (XP) is a rare, autosomal recessive disorder of DNA repair. Affected
individuals are unable to repair ultraviolet radiation (UVR)-induced DNA damage, leading to a variety
of clinical manifestations: a dramatic increase in mucocutaneous malignancies, increased lentigines,
extreme photosensitivity (in approximately 50% of cases), and neurodegeneration (in approximately
30% of affected individuals). Incidence in Western Europe is recorded as 2.3 per million live births. There
are eight different complementation groups, XP-A to XP-G, and XP-variant (XP-V) corresponding to the
eight affected genes. Classically, XP patients were identified by clinicians for their tendency to develop
severe and exaggerated sunburn on minimal sun exposure, however recently it has been shown that
XP-C, XP-E and XP-V patients have normal sunburn reactions for skin type compared to the other
groups, who suffer not only with severe, exaggerated sunburn, but also have an increased incidence
of neurodegeneration.
A diagnosis of XP should be considered in a child with either severe sunburn, increasing lentigines at
exposed sites, or development of multiple skin cancers at an early age. Skin biopsy and subsequent
testing in cell cultures for defective DNA repair, confirms or excludes the diagnosis. Mean life expectancy
is reduced; the two main causes of mortality are skin cancer and neurodegeneration. These clinical
features distinguish XP from other disorders of DNA repair, namely Trichothiodystrophy and Cockayne
syndrome, although overlapping syndromes do occur. Instigation of meticulous photoprotection for
all XP patients has been shown to reduce both the lentigines and number of skin cancers dramatically
and would be presumed to increase life expectancy. Compliance with photoprotection is a recognised
problem amongst XP patients, particularly in those without easy sunburn. This is further accentuated by
lack of social acceptance for people who wear UVR-protective visors. Increased awareness of XP,
both within the medical and media spheres will benefit current and future XP patients; this will
aid earlier diagnosis and timely photoprotection, with better compliance, and therefore, result in an
improved prognosis.

Elongation of the telomeric overhang by telomerase is counteracted by synthesis of the complementary strand by the CST complex, CTC1(Cdc13)/Stn1/Ten1. Interaction of budding yeast Stn1 with overhang-binding Cdc13 is increased by Cdc13
SUMOylation. Human and fission yeast CST instead interact with overhang-binding TPP1/POT1. We show that the fission yeast TPP1 ortholog, Tpz1, is SUMOylated. Tpz1 SUMOylation restricts telomere elongation and promotes Stn1/Ten1 telomere association,and a SUMO-Tpz1 fusion protein has increased affinity for Stn1. Our data suggest that SUMO inhibits telomerase through stimulation of Stn1/Ten1 action by Tpz1, highlighting the evolutionary conservation of the regulation of CST function by SUMOylation.

2-Oxoglutarate (2OG)-dependent oxygenases have important roles in the regulation of gene expression via demethylation of N-methylated chromatin components1,2 and in the hydroxylation of transcription factors3 and splicing factor proteins4. Recently, 2OG-dependent oxygenases that catalyse hydroxylation of transfer RNA5,6,7 and ribosomal proteins8 have been shown to be important in translation relating to cellular growth, TH17-cell differentiation and translational accuracy9,10,11,12. The finding that ribosomal oxygenases (ROXs) occur in organisms ranging from prokaryotes to humans8 raises questions as to their structural and evolutionary relationships. In Escherichia coli, YcfD catalyses arginine hydroxylation in the ribosomal protein L16; in humans, MYC-induced nuclear antigen (MINA53; also known as MINA) and nucleolar protein 66 (NO66) catalyse histidine hydroxylation in the ribosomal proteins RPL27A and RPL8, respectively. The functional assignments of ROXs open therapeutic possibilities via either ROX inhibition or targeting of differentially modified ribosomes. Despite differences in the residue and protein selectivities of prokaryotic and eukaryotic ROXs, comparison of the crystal structures of E. coli YcfD and Rhodothermus marinus YcfD with those of human MINA53 and NO66 reveals highly conserved folds and novel dimerization modes defining a new structural subfamily of 2OG-dependent oxygenases. ROX structures with and without their substrates support their functional assignments as hydroxylases but not demethylases, and reveal how the subfamily has evolved to catalyse the hydroxylation of different residue side chains of ribosomal proteins. Comparison of ROX crystal structures with those of other JmjC-domain-containing hydroxylases, including the hypoxia-inducible factor asparaginyl hydroxylase FIH and histone Nε-methyl lysine demethylases, identifies branch points in 2OG-dependent oxygenase evolution and distinguishes between JmjC-containing hydroxylases and demethylases catalysing modifications of translational and transcriptional machinery. The structures reveal that new protein hydroxylation activities can evolve by changing the coordination position from which the iron-bound substrate-oxidizing species reacts. This coordination flexibility has probably contributed to the evolution of the wide range of reactions catalysed by oxygenases.

ATR activation is dependent on temporal and spatial interactions with partner proteins. In the budding yeast model, three proteins – Dpb11TopBP1, Ddc1Rad9 and Dna2 - all interact with and activate Mec1ATR. Each contains an ATR activation domain (ADD) that interacts directly with the Mec1ATR:Ddc2ATRIP complex. Any of the Dpb11TopBP1, Ddc1Rad9 or Dna2 ADDs is sufficient to activate Mec1ATR in vitro. All three can also independently activate Mec1ATR in vivo: the checkpoint is lost only when all three AADs are absent. In metazoans, only TopBP1 has been identified as a direct ATR activator. Depletion-replacement approaches suggest the TopBP1-AAD is both sufficient and necessary for ATR activation. The physiological function of the TopBP1 AAD is, however, unknown. We created a knock-in point mutation (W1147R) that ablates mouse TopBP1-AAD function. TopBP1-W1147R is early embryonic lethal. To analyse TopBP1-W1147R cellular function in vivo, we silenced the wild type TopBP1 allele in heterozygous MEFs. AAD inactivation impaired cell proliferation, promoted premature senescence and compromised Chk1 signalling following UV irradiation. We also show enforced TopBP1 dimerization promotes ATR-dependent Chk1 phosphorylation. Our data suggest that, unlike the yeast models, the TopBP1-AAD is the major activator of ATR, sustaining cell proliferation and embryonic development.

Epstein-Barr virus (EBV) is a ubiquitous pathogen that infects most of the adult population and persists for life after the initial contact. The extreme success of the virus is attributed to its bipartite life cycle, which consists of a dormant-like state of latency, with periodical reactivation to the virus producing, lytic phase. Zta (BZLF1, Z, Zebra or EB1) is a multifunctional viral protein that belongs to the bZIP family of transcription factors and is known as the master lytic regulator of EBV. Together with transcriptional activation, Zta has been shown to be involved in DNA binding-dependent transcriptional repression, particularly of the host class II major histocompatibility complex transactivator, CIITA. Distinct protein domains, as well as various post translational modifications, like phosphorylation of Serine 209 by the viral protein kinase (VPK), have been linked to different functional roles of Zta.
In the present study, it was shown that VPK can partially inhibit SUMOylation of Zta on Lysine 12, in a manner which was not dependent on Serine 209 phosphorylation. However, no direct interaction of VPK and Zta could be observed and no significant effect of either proteins on histone H2AX phosphorylation was seen.
Interestingly, in vitro reporter assays revealed that fusion of a SUMO moiety to the amine-terminus of Zta inhibited repression of the CIITA promoter, but not the activation of the viral BHLF1 promoter, pointing at divergent mechanisms of action of transcriptional repression and activation by Zta. Moreover, truncation of the carboxy-terminal dimerisation domain of Zta (crucial for protein-DNA interaction) abrogated BHLF1 transactivation but not CIITA down-regulation, revealing underlying diffe rences in DNA binding requirements for the two processes.
Further in silico sequence analysis, coupled with a mutation approach of the CIITA promoter, confirmed that an alternate route to the Zta DNA binding-dependent repression exists. Finally, no single promoter element could be linked to down-regulation of CIITA, suggesting sequestration of a possible, yet unknown cofactor, by Zta.

The alternative oxidase (AOX) is the terminal protein in the alternative oxidation pathway found in plants, fungi and some protozoa. One of the more prominent protozoa that contain AOX within the bloodstream form is Trypanosoma brucei, the causative agent of human African trypanosomiasis (HAT), in which the parasite has demonstrated to be totally dependent upon the protein for continued respiration. Given the lack of AOX in mammalian cells, the protein represents an attractive chemotherapeutic target for trypanosidal activity.

Ascofuranone is a known inhibitor of AOX, but its complex synthesis precludes it from industrial production. To this end colletochlorin B, an analogue of ascofuranone, was synthesised and its inhibitory efficacy against AOX examined. IC50 values obtained demonstrate that removal of the problematic furanone ring does not reduce inhibitor efficacy to a large degree. Derivatives of colletochlorin B were synthesised to assess the importance of structural moieties present. The compounds were also tested against the cytochrome bc1 complex, an important respiratory chain complex, and compared with known fungicides. Using these compounds assays against fungal species has yielded promising results for the use of colletochlorin B as a lead fungicide.

Recombinant wild type trypanosomal alternative oxidase (TAO) and Sauromatum guttatum alternative oxidase (SgAOX) have been expressed in E.coli. in addition to a number of mutants. Respiratory activities of these mutants were measured in order to assess the importance of highly conserved amino acids, with all mutants showing a decline in specific activity. Three of the mutants generated were also shown to affect the apparent affinity for oxygen, the implications of which are discussed.

Recent crystallisation of TAO has enabled a more detailed examination of the structure of all AOXs. Work is presented relating structure to the overall function of the protein, taking into account conservation throughout the entire AOX family. Comparisons to other di-iron proteins revealed a conserved His-Asp-Tryp motif that could facilitate proton coupled electron transport. A full catalytic cycle based on these findings has been postulated.

Solar generated hydrogen as an energy source is green, sustainable, with a high
energy density. One day the majority of current fossil fuel based technology could
be replaced with hydrogen technology reducing CO2 emission drastically. The goal
in this research is to explore hybrid metal oxide photocatalysts in the pursuit of
achieving highly efficient photoanodes for use in photoelectrochemical cells (PEC).
Achieving high efficiencies of hydrogen production in photoelectrochemical cells is
the key challenge for the commercialisation of PEC technology as a viable, sustainable,
hydrogen source; limited only by the lifetime of the sun and the resources of
the metal oxide materials.
In this research TiO2, Fe-Ti-O, ZnO, and Zn2TiO4 are the photocatalysts explored.
Alloys of Ti-Fe-O showed improvement over TiO2, whilst a hybrid heterostructure
of ZnO/Zn2TiO4/TiO2 enhanced photocurrent densities significantly. A
barrier layer in the photoanode achieved localised exciton separation and reduction
of recombination rates by inhibiting back flow of electrons after injection into the
TiO2 layer.
Nanotubes are created by the simple electrochemical process of anodisation. The
nanotube composition depends on the anode material. To control the composition ofthe anode, iron and titanium are co-deposited onto a substrate using electron beam
evaporation. The introduction of iron into titania nanotubes engineered the band
gap, lowering the band gap energy to that of iron oxide whilst the positions of the
conduction and valence bands with respect to the oxidation and reduction potentials
of water remained favourable. Fe-Ti-O nanotubes showed remarkable photocurrent
density improvement compared to TiO2 nanotubes.
ZnO nanostructures deposited by vapour transport mechanisms showed variability
in the morphology of the structures, as governed by the growth dynamics.
Herein, it is shown that an electronically favourable situation arises by the formation
of a ZnO-Zn2TiO4-TiO2 heterostructure and a high photocatalytic activity is
reported. The structure is composed of a large surface area ZnO nanorod photoabsorber
formed on a Ti foil which forms a Zn2TiO4 barrier layer between ZnO and
TiO2. The Zn2TiO4 layer inhibits electron transport toward the surface of the photoanode
whilst encouraging charge transport to the hydrogenation electrode. The
heterostructure interfacial surface area is extended through the utilisation of TiO2
nanotubes, which demonstrated a 20.22 % photoelectrochemical efficiency under UV
illumination.
Surface modification of ZnO nanorods with aerosol assisted chemical vapour
deposited TiO2 nanoparticles enhanced photocurrent densities of the ZnO rods,
improving charge separation of excitons created within the TiO2 nanoparticles.
ZnO nanotubes formed via a novel route using chemical bath deposition of ZnO
is investigated, an annulus ZnO seed layer facilitated the site specific growth of ZnO
nanotubes whilst a uniform seed layer formed ZnO nanorods.

Telomeres protect the ends of chromosomes from the activity of DNA repair machinery and provide a solution to the end-replication problem. In humans, the core protein complex located at telomeres is known as shelterin and consists of six protein subunits. Although variation is seen in the telomeric complex between species, in fission yeast the complex has notable similarities to that of humans. Separately to shelterin, the CST complex (Cdc13/Stn1/Ten1) is conserved in budding yeast, plants and mammals and is thought to negatively regulate telomerase, in addition to being required for telomere protection. However, unlike Stn1 and Ten1, Cdc13 has not yet been identified in fission yeast.

Poz1 is a bridging molecule equivalent to TIN2 in human shelterin, which links the Taz1-Rap1 and the Pot1-Tpz1-Ccq1 sub-complexes, respectively bound to double- and single-stranded DNA at telomeres. Poz1 is required for the regulation of telomerase activity, and it has been hypothesised that it might do so by playing a structural role in the switching of telomeres from an open to a closed state. In this study, a reverse-2-hybrid approach was used to generate Poz1 alleles unable to interact with Rap1 or Tpz1 specifically. These alleles were subjected to phenotypic and biochemical analysis which indicated that neither individual interaction is sufficient to maintain telomere homeostasis. With telomere lengths similar to a Poz1 deletion, it is proposed that negative regulation cannot occur without the ability to form a closed complex.

Given that Cdc13 is currently the only missing component in fission yeast, a second study was initiated aiming to identify a homologue by yeast-2-hybrid screening of a cDNA library, using Stn1 and Ten1 as baits. However, this approach did not yield any positive candidates. In an alternative approach, Stn1 temperature-sensitive (ts) alleles were generated and characterised. These were used to screen a genomic library for suppressors of the Stn1 ts phenotype. Several candidates were identified that require further examination while the ts allele analysis indicated that telomeres are lost in their entirety at non-permissive temperatures and that survivors of this process did so by chromosome circularisation, similar to Pot1 mutants.

With 9 million people in the UK alone suffering from deafness or hearing impairment, there is a concerted effort to develop effective treatments. To address this, one strategy is to recruit mechanisms naturally occurring during inner ear development, but this first requires a clear picture of the normal molecular mechanisms underlying this developmental process.

Fibroblast growth factors (FGFs) are short-range extracellular signalling molecules, with Fgf3 and Fgf10 already shown to be critical for the earliest event of inner ear induction. Interestingly these ligands are also expressed in the inner ear itself, and mutations in both Fgf3 and Fgf10 have independently been linked to sensorineural deafness in humans. This project is focussed on unravelling the molecular mechanisms controlling their expression in the inner ear.

Bioinformatic analysis of the Fgf3 and Fgf10 enhancer regions revealed the presences of putative binding sites for retinoic acid (RA). RA is a key signalling molecule in inner ear development with both excess and deficit leading to inner ear abnormalities. First, a novel, non-invasive method of RA administration via sugar pellet was tested and proved to be efficient and reliable alternative to gavage. Using an Fgf3-lacZ reporter mouse, the effects of RA excess on Fgf3 expression were investigated in detail. In addition, preliminary studies of the effects of RA on Fgf10 were also carried out. Both Fgf3 and Fgf10 were downregulated with high doses of RA, confirming previous in vitro studies. In addition, detailed analysis of Fgf3-lacZ embryos exposed to RA revealed that downregulation critically depends on the dose and time of administration.

To further explore other direct regulators of Fgf10 expression, additional reporter constructs were also generated for functional analysis in mouse and in chick. To date, analysis of electroporated chick embryos shows that ear specific Fgf10 regulation may not be conserved between two species.

Defects in centrosome, centrosomal-associated and spindle-associated proteins are the most frequent cause of primary microcephaly (PM) and microcephalic primordial dwarfism (MPD) syndromes in humans. Mitotic progression and segregation defects, microtubule spindle abnormalities and impaired DNA damage-induced G2-M cell cycle checkpoint proficiency have been documented in cell lines from these patients. This suggests that impaired mitotic entry, progression and exit strongly contribute to PM and MPD. Considering the vast protein networks involved in coordinating this cell cycle stage, the list of potential target genes that could underlie novel developmental disorders is large. One such complex network, with a direct microtubule-mediated physical connection to the centrosome, is the kinetochore. This centromeric-associated structure nucleates microtubule attachments onto mitotic chromosomes. Here, we described novel compound heterozygous variants in CENPE in two siblings who exhibit a profound MPD associated with developmental delay, simplified gyri and other isolated abnormalities. CENPE encodes centromere-associated protein E (CENP-E), a core kinetochore component functioning to mediate chromosome congression initially of misaligned chromosomes and in subsequent spindle microtubule capture during mitosis. Firstly, we present a comprehensive clinical description of these patients. Then, using patient cells we document abnormalities in spindle microtubule organization, mitotic progression and segregation, before modeling the cellular pathogenicity of these variants in an independent cell system. Our cellular analysis shows that a pathogenic defect in CENP-E, a kinetochore-core protein, largely phenocopies PCNT-mutated microcephalic osteodysplastic primordial dwarfism-type II patient cells. PCNT encodes a centrosome-associated protein. These results highlight a common underlying pathomechanism. Our findings provide the first evidence for a kinetochore-based route to MPD in humans.

The underlying etiologies of genetic congenital microcephaly are complex and multifactorial. Recently, with the exponential growth in the identification and characterization of novel genetic causes of congenital microcephaly, there has been a consolidation and emergence of certain themes concerning underlying pathomechanisms. These include abnormal mitotic microtubule spindle structure, numerical and structural abnormalities of the centrosome, altered cilia function, impaired DNA repair, DNA Damage Response signaling and DNA replication, along with attenuated cell cycle checkpoint proficiency. Many of these processes are highly interconnected. Interestingly, a defect in a gene whose encoded protein has a canonical function in one of these processes can often have multiple impacts at the cellular level involving several of these pathways. Here, we overview the key pathomechanistic themes underlying profound congenital microcephaly, and emphasize their interconnected nature.

Wolf-Hirschhorn syndrome (WHS) represents an archetypical example of a contiguous gene deletion disorder – a condition comprising a complex set of developmental phenotypes with a multigenic origin. Epileptic seizures, intellectual disability, growth restriction, motor delay and hypotonia are major co-morbidities in WHS. Haploinsufficiency of LETM1, which encodes a mitochondrial inner-membrane protein functioning in ion transport, has been proposed as an underlying pathomechanism, principally for seizures but also for other core features of WHS, including growth and motor delay. Growing evidence derived from several model organisms suggests that reduced LETM1 expression is associated with some element of mitochondrial dysfunction. Surprisingly, LETM1-dependent mitochondrial functional deficits have not previously been described in cells from individuals with WHS. Here, using a unique panel of WHS-patient-derived cell lines with deletions of differing sizes,incorporating LETM1 or not, we show, for the first time, that LETM1 expression is reduced in mitochondria isolated from WHS-patient cells. Furthermore, we show that this is associated with distinct mitochondrial phenotypes, including altered intracellular [Ca2+] levels, dysfunctional mitochondrial transition-pore opening, hyperpolarizationand superoxide leakage from resting mitochondria. Interestingly, we find that these phenotypes segregate with seizures in our WHScohort. Our findings identify novel cellular phenotypes in WHSattributable to a 50% reduction in LETM1 expression level; thesephenotypes could underlie and/or contribute to some of the core clinical features of this condition.

Topoisomerase II (TOP2) removes torsional stress from DNA and facilitates gene transcription by introducing transient DNA double-strand breaks (DSBs). Such DSBs are normally rejoined by TOP2 but on occasion can become abortive and remain unsealed. Here we identify homozygous mutations in the TDP2 gene encoding tyrosyl DNA phosphodiesterase-2, an enzyme that repairs 'abortive' TOP2-induced DSBs, in individuals with intellectual disability, seizures and ataxia. We show that cells from affected individuals are hypersensitive to TOP2-induced DSBs and that loss of TDP2 inhibits TOP2-dependent gene transcription in cultured human cells and in mouse post-mitotic neurons following abortive TOP2 activity. Notably, TDP2 is also required for normal levels of many gene transcripts in developing mouse brain, including numerous gene transcripts associated with neurological function and/or disease, and for normal interneuron density in mouse cerebellum. Collectively, these data implicate chromosome breakage by TOP2 as an endogenous threat to gene transcription and to normal neuronal development and maintenance.

Background
Alzheimer’s disease (AD) is characterized by the deposition of insoluble amyloid plaques in the neuropil composed of highly stable, self-assembled Amyloid-beta (Aβ) fibrils. Copper has been implicated to play a role in Alzheimer’s disease. Dimers of Aβ have been isolated from AD brain and have been shown to be neurotoxic.

Results
We have investigated the formation of dityrosine cross-links in Aβ42 formed by covalent ortho-ortho coupling of two tyrosine residues under conditions of oxidative stress with elevated copper and shown that dityrosine can be formed in vitro in Aβ oligomers and fibrils and that these links further stabilize the fibrils. Dityrosine crosslinking was present in internalized Aβ in cell cultures treated with oligomeric Aβ42 using a specific antibody for dityrosine by immunogold labeling transmission electron microscopy. Results also revealed the prevalence of dityrosine crosslinks in amyloid plaques in brain tissue and in cerebrospinal fluid from AD patients.

Conclusions
Aβ dimers may be stabilized by dityrosine crosslinking. These results indicate that dityrosine cross-links may play an important role in the pathogenesis of Alzheimer’s disease and can be generated by reactive oxygen species catalyzed by Cu2+ ions. The observation of increased Aβ and dityrosine in CSF from AD patients suggests that this could be used as a potential biomarker of oxidative stress in AD.

The essential Smc5/6 complex is structurally related to cohesin and condensin. It is required for homologous recombination (HR), rDNA stability and telomere maintenance. In Schizosaccharomyces pombe, two hypomorphic smc6 mutants, smc6-X and smc6-74, haven been shown to be deficient in HR-dependent processing of collapsed replication forks. Collapsed replication forks can generate single-ended DNA double strand breaks (se-DSB) which require HR to restore replication. In this study the requirement for Smc5/6 at a site-specific se-DSB at the mating-type locus and in the mating-type switch process were analysed.

In S.pombe mating-type switching occurs over two S phases; in the first S phase replication fork stalling at mat1 leads to an imprint, which is converted to an se-DSB during the next S phase. This initiates the copying of the donor cassette using HR. In the absences of donors the sister chromatid is used for repair. Mating-type switching analysis showed that snc6-74 had a defect in switching dependent on the genotype of the smc6-74 parent.

Both smc6 mutants had reduced viability in the absence of donors, consistent with a defect in HR repair of an se-DSB. analysis in an inducible system (Holmes et al., 2005) showed that in response to a se-DSB Rad52 foci appeared with wild type kinetics but the smc6 mutants delayed entry into mitosis for approximately 2hrs, dependent on the DNA damage checkpoint kinase Chk1. In order to test whether this delay facilitated rescue by a converging replication fork a novel inducible converged fork (cf) DSB system was developed. The cf-DSB required HR and the RecQ helicase Rqh1 for repair but did not require Mus81. The converging fork rescued smc6-74 but not smc6-X showing Smc5/6 to be required for repair of both types of replication-associated DSBs.

In yeasts, small intrinsically disordered proteins (IDPs) modulate ribonucleotide reductase (RNR) activity to ensure an optimal supply of dNTPs for DNA synthesis. The S. pombe Spd1 protein can directly inhibit the large RNR subunit (R1), import the small subunit (R2) into the nucleus and induce an architectural change in the R1-R2 holocomplex. Here, we report the characterization of Spd2, a protein with homology to Spd1. We show that Spd2 is a CRL4Cdt2 controlled IDP that functions together with Spd1 in the DNA damage response and in modulation of RNR architecture. However, Spd2 does not regulate dNTP pools and R2 nuclear import. Furthermore, deletion of spd2 only weakly suppresses the Rad3ATR checkpoint dependency of CRL4Cdt2 mutants. However, when we raised intracellular dNTP pools by inactivation of RNR feedback inhibition, deletion of spd2 could suppress the checkpoint dependency of CRL4Cdt2 mutant cells to the same extent as spd1. Collectively, these observations suggest that Spd1 on its own regulates dNTP pools, while it together with Spd2 modulates RNR architecture and sensitizes cells to DNA damage.

DNA damage can lead to the accumulation of mutations and diseases such as cancer. It
is therefore integral for cells to identify this damaged DNA and promote its repair. To
carry out this function eukaryotic cells have evolved signal transduction pathways
known as the DNA structure checkpoints. Much of the molecular mechanism
underlying these pathways is still far from understood. The work in this thesis uses the
model organism Schizosaccharomyces pombe to investigate these mechanisms, with a
particular focus on the TopBP1 homolog Rad4.
TopBP1 plays an essential scaffolding role in the initiation of DNA replication, but is
also a key protein in the DNA structure checkpoints. It has previously been shown in
metazoans and budding yeast to stimulate the kinase activity of ATR, via its ATR
Activation Domain (AAD), an early event in checkpoint activation. The work presented
in here, along with initial work carried by previous members of the Carr Laboratory;
Su-Jiun Lin and Valerie Garcia, shows that the Rad4TopBP1 AAD acts in a chromatin
dependent pathway to amplify the checkpoint signal in G1/S-phase, where DNA
resection is limited. A second AAD is also identified in the checkpoint clamp protein
Rad9, which acts redundantly with the Rad4 AAD.
As well as its AAD function, Rad4 also plays a scaffolding role in the DNA structure
checkpoint pathways. The work in this thesis, in collaboration with the Laurence Pearl
and Li Lin Du laboratories, identifies the molecular mechanism of the interaction
between Rad4 and the mediator protein Crb253BP1. It is shown that sequential
phosphorylation of Crb2 by Cdc2CDK is required for the interaction with BRCT domains
1 and 2 of Rad4 and checkpoint activation. It is also shown that Rad4 most likely does
not interact with Mrc1 or Slx4 in the S. pombe checkpoint pathways.

PrimPol is a primase–polymerase involved in nuclear and mitochondrial DNA replication in eukaryotic cells. Although PrimPol is predicted to possess an archaeo-eukaryotic primase and a UL52-like zinc finger domain, the role of these domains has not been established. Here, we report that the proposed zinc finger domain of human PrimPol binds zinc ions and is essential for maintaining primase activity. Although apparently dispensable for its polymerase activity, the zinc finger also regulates the processivity and fidelity of PrimPol's extension activities. When the zinc finger is disrupted, PrimPol becomes more promutagenic, has an altered translesion synthesis spectrum and is capable of faithfully bypassing cyclobutane pyrimidine dimer photolesions. PrimPol's polymerase domain binds to both single- and double-stranded DNA, whilst the zinc finger domain binds only to single-stranded DNA. We additionally report that although PrimPol's primase activity is required to restore wild-type replication fork rates in irradiated PrimPol−/− cells, polymerase activity is sufficient to maintain regular replisome progression in unperturbed cells. Together, these findings provide the first analysis of the molecular architecture of PrimPol, describing the activities associated with, and interplay between, its functional domains and defining the requirement for its primase and polymerase activities during nuclear DNA replication.

The ability to study protein function in vivo often relies on systems that regulate the presence and absence of the protein of interest. Two limitations for previously described transcriptional control systems that are used to regulate protein expression in fission yeast are: the time taken for inducing conditions to initiate transcription and the ability to achieve very low basal transcription in the "OFF-state". In previous work, we described a Cre recombination-mediated system that allows the rapid and efficient regulation of any gene of interest by the urg1 promoter, which has a dynamic range of approximately 75-fold and which is induced within 30-60 minutes of uracil addition. In this report we describe easy-to-use and versatile modules that can be exploited to significantly tune down P urg1 "OFF-levels" while maintaining an equivalent dynamic range. We also provide plasmids and tools for combining P urg1 transcriptional control with the auxin degron tag to help maintain a null-like phenotype. We demonstrate the utility of this system by improved regulation of HO-dependent site-specific DSB formation, by the regulation Rtf1-dependent replication fork arrest and by controlling Rhp18(Rad18)-dependent post replication repair.

Aberrant chromosome structures can promote tumors in the early stages of carcinogenesis and lead to tumor cells becoming resistant to chemotherapy, for example by changing in drug metabolism. Dicentric (containing two centromeres) and acentric (containing no centromeres) chromosomes are two abnormal chromosome structures that consider as precursors of a variety of gross chromosomal rearrangements (GCRs) generated by subsequent recombination events [1-7]. However, the mechanism of the dicentric and acentric palindromic chromosome formation and their subsequent metabolism is difficult to directly visualise. The previous results from our lab shows that replication forks stalled at a specific replication termination sequence (RTS1) can result in the formation of the dicentric and acentric palindromic chromosomes in the fission yeast Schizosaccharomyces pombe [48-52]. However, the formation of acentric and dicentric chromosomes results in a significant visability loss, due to instability and miss-segregation of the chromosomes in the yeast cells. Thus, their fate is difficult or impossible to follow. To resolve this problem, a non-essential mini-chromosome (Ch16) was developed as a novel model system in this project. The behaviour of rearranged chromosome in vivo and their subsequent fate have been visualised by integrating the lac operator (lacO) and tetracycline operator (tetO) arrays with auxotropic makers, adjacent to the RTSI locus on Ch16. The results reveal imbalanced segregation of a dicentric chromosome and subsequently undergoes a breakage event. An acentric chromosome appears to be decoupled or lost rapidly from the nucleus.

Glioblastoma multiforme (GBM) is the most common and most aggressive type of primary brain tumour. It is currently treated by a mixture of ionising radiation and Temozolomide (TMZ) chemotherapy, however virtually all patients experience disease recurrence and 75% die within two years of diagnosis.

Tumours which express elevated levels of the DNA repair protein O6-methylguanine DNA methyltransferase (MGMT) have a particularly poor prognosis, suggesting that levels of MGMT and the inefficacy of treatment are linked.

MGMT is a “suicide” repair protein that binds irreversibly to a DNA adduct (such as those caused by TMZ) and is destroyed by the proteasome once repair has taken place. Therefore the ability of the cell to repair DNA damage relies on the rate at which it can resynthesise MGMT. Previous research has shown that reducing MGMT levels via promoter silencing increases the effectiveness of treatment, however this causes toxicity in bone marrow stem cells and is therefore unable to be used as a possible treatment option.

My preliminary data suggests that inhibition of mTOR signalling reduces the steady state levels of MGMT without affecting mRNA levels, potentially making them more sensitive to TMZ treatment. I am therefore using inhibitors of the mTOR pathway and associated proteins, which have been selected via a novel bioinformatic technique, to ascertain how these affect MGMT protein levels and to determine whether a mixture of these inhibitors with DNA damaging agents could be used to increase the efficacy of TMZ treatment.

Epstein Barr Virus (EBV) is a γ-herpesvirus infecting around 95% of the human
population. EBV infection is life-long and asymptomatic in the majority of individuals,
however EBV is associated with Nasopharyngeal Carcinoma, Burkitt's
lymphoma and Hodgkin’s lymphoma. The transcription factor Zta is an immediate early
gene of EBV able to reactivate the virus from latency, cause cell cycle arrest and bind to
sequence specific DNA elements. Cercopithecine herpesvirus-15 is a closely related virus,
infecting rhesus monkeys, with a homologue to Zta; ZtaRh. A comparison of features of
these proteins may be informative about critical residues in each protein. Binding to
almost all response elements is conserved between the two proteins. A Zta response
element (ZRE3Rh) in the CeHV-15 Rta gene promoter that is not functional for either
protein was identified. ZRE3 from EBV is methylation dependent for Zta binding.
Analysis of ZRE3 using competition EMSA assays has shown the importance of
methylation of individual CpG motifs. ZtaRh is compromised in reactivating EBV from
latency and this appears to be mediated by changes at the extreme C-terminus. ZtaRh is
unable to cause G1 cell cycle arrest, however this function maps to the transactivation
domain. Known binding partners of Zta were cloned to enable investigation of binding by
ZtaRh or other mutants. Co-transfection of p53 and Zta resulted in rapid degradation of
both proteins. Co-transfection of C/EBPα and Zta produced a larger additional Zta species.
Neither effect was seen with a Zta CT mutant. RNA from HEK293-ZKO cells transfected
with Zta or a C-terminal Zta mutant was analysed using a QPCR array probing 595 human
genes associated with cancer, revealing possible host cell proteins influenced by Zta.
Further information on the precise mechanisms of Zta could contribute to the development
of future therapies for the prevention or treatment of EBV related diseases.

The alternative oxidase (AOX) is an integral monotopic membrane protein which
branches from respiratory chain at the point of the Q-pool in the mitochondria of all
flowers, some fungi, and some protists such as the human parasite Trypanosoma brucei.
The aim of this project is threefold: to establish an over-expression and purification
protocol for recombinant Sauromatum guttatum alternative oxidase (SgrAOX); to use
expressed SgrAOX for structural analysis such as crystallography; and finally to use in
silico methods to model the alternative oxidase protein. Of these three, only the first and
last have been attempted previously, with varying success. The second, namely
structural analysis, has never been attempted with SgrAOX.
In order to achieve the aims of this project, primarily laboratory-based protein
production were used, in conjunction with downstream analysis using structural biology
techniques. The in silico modelling was carried out using a wide range of algorithms
freely available on the World Wide Web.
Results of this project are: the determination of an over-expression system and
purification protocols in two E.coli strains, producing enough protein to use for the
second objective detailed above. While no crystal structure has been obtained,
significant steps toward identifying a protocol for rAOX crystallisation have been made.
Results from structural analysis support modelling predictions and give novel insights
into the thermostability of the protein. New and detailed homology models have been
created and critically evaluated, with a very recent crystal structure from our
collaborators providing a unique set of data for model evaluation.
The outcome of this project has contributed towards the determination of conditions
under which SgrAOX protein may form crystals, and therefore bringing the acquisition
of a SgrAOX protein structure closer.

The cytotoxic activities of proteins from the normally insecticidal Bacillus thuringiensis against human cancer cell lines were investigated. Cry41Aa toxin derived from the Bacillus thuringiensis strain A1462, which shows activity against human cancer cell lines is structurally related to the toxins synthesized by commercially produced transgenic insect-resistant plants, with the exception of an additional C-terminal beta-trefoil ricin domain. To test whether this putative carbohydrate binding domain is responsible for the cytotocidal activity of Cry41Aa against cancer cell lines, we developed an efficient expression system for the toxin and created a deletion mutant lacking the ricin domain. Both Cry41Aa and its deletion mutant were stably expressed and found to have almost identical activities against the HepG2 cancer cell line in vitro. Our results suggest that the acquisition of the ricin domain was not responsible for Cry41Aa having toxicity to human cancer cells and more subtle changes have resulted in the evolution of mammalian cancer cell toxicity. We further attempted to identify those differences that are responsible for the divergence in activity between the cancer-killing toxins and their insecticidal counterparts. We also studied the cell-killing activity of other uncharacterized Bacillus thuringiensis crystal toxins including Cry51 and Cry65 whose targets are not known so far.

Nonhomologous end-joining (NHEJ) pathways repair DNA double-strand breaks (DSBs) in eukaryotes and many prokaryotes, although it is not reported to operate in the third domain of life, archaea. Here, we describe a complete NHEJ complex, consisting of DNA ligase (Lig), polymerase (Pol), phosphoesterase (PE), and Ku from a mesophillic archaeon, Methanocella paludicola (Mpa). Mpa Lig has limited DNA nick-sealing activity but is efficient in ligating nicks containing a 3′ ribonucleotide. Mpa Pol preferentially incorporates nucleoside triphosphates onto a DNA primer strand, filling DNA gaps in annealed breaks. Mpa PE sequentially removes 3′ phosphates and ribonucleotides from primer strands, leaving a ligatable terminal 3′ monoribonucleotide. These proteins, together with the DNA end-binding protein Ku, form a functional NHEJ break-repair apparatus that is highly homologous to the bacterial complex. Although the major roles of Pol and Lig in break repair have been reported, PE’s function in NHEJ has remained obscure. We establish that PE is required for ribonucleolytic resection of RNA intermediates at annealed DSBs. Polymerase-catalyzed strand-displacement synthesis on DNA gaps can result in the formation of nonligatable NHEJ intermediates. The function of PE in NHEJ repair is to detect and remove inappropriately incorporated ribonucleotides or phosphates from 3′ ends of annealed DSBs to configure the termini for ligation. Thus, PE prevents the accumulation of abortive genotoxic DNA intermediates arising from strand displacement synthesis that otherwise would be refractory to repair.

Sumoylation is an essential posttranslational modification involved in many cellular
processes such as DNA replication, chromosomal stability, cytokinesis, DNA damage
responses and many others. The process of sumoylation is conserved in all eukaryotic
organisms. This study involves the analysis of various aspects of sumoylation in the
unicellular model organism Schizosaccharomyces pombe.

The first part of this study is concerned with elucidating the functional and structural
importance of a SUMO-like domain (SLD) and a putative SUMO-binding domain (SBM)
present in the essential protein Rad60. Biochemical and genetical analysis reveals that
SLD2 is required for the DNA damage response function of Rad60 but that the putative
SBM3 is a key structural feature of the hydrophobic core of SLD2 and therefore unlikely
to function as a SUMO-interacting motif. As Rad60 interacts with the SUMO E3 ligase
Pli1, which facilitates overall sumoylation and SUMO chain formation, further analysis
was undertaken to identify the function of SUMO chain formation and the function of
Pli1 in maintaining chromosomal stability. A SUMO chain mutant, Pmt3-K14R; K30R,
was characterized and shown to be sensitive to the DNA replication inhibitor
hydroxyurea. Analysis of the pli1 null mutant reveals that Pli1 dependent sumoylation
has multiple functions at the centromeric repetitive sequences as the mutant displays
increased gene conversion at centromeric regions and increased loss of an artificial
minichromosome rates compared to a wild type strain.

The second part of this study is concerned with identifying specific modified lysine
residues in the sumoylation pathway components Fub2, Hus5 and Nse2 and the target
proteins Rtf2 and PCNA. After identification of in vitro sumoylation sites, an analysis of
the sumoylation of the SUMO conjugating enzyme Hus5 and the sumoylation of the Rtf2
protein is carried out. In vivo and genetical analysis of the hus5-K50R mutant suggests
that the sumoylation of the conjugating enzyme is required for maintaining the
homeostasis of the pathway and is essential for cell viability when the homologous
recombination machinery is impaired. Sumoylation of Rtf2 protein is required for the
response to the DNA alkylating agent MMS and, like the sumoylation of Hus5, is
essential for cell viability in homologous recombination mutant backgrounds.

Maintenance of the genome is essential for life to prosper. Regular insults to the genome are sustained by all cellular life and can foster genetic instability if left unrepaired. The most lethal genetic damage is a double strand break (DSB), the cleavage of the phosphate backbone on both strands of the DNA double helix. Two main pathways exist which provide mechanisms for coping with DSBs; precise repair utilising the identical sister chromatid as a template to recreate the broken segment (homologous recombination; HR), and direct fusion of the broken ends in the absence of an intact template (nonhomologous end joining; NHEJ). NHEJ was first characterised in eukaryotes, and an analogous system has been found to exist in bacteria during the past decade. The bacterial NHEJ pathway is composed of four key proteins; the DNA end binding Ku homodimer, a DNA Ligase, a DNA polymerase and a phosphoesterase (PE). The first results chapter of this thesis details the identification of an orthologous set of proteins in the archaeon Methanocella paludicola, and their subsequent isolation and characterisation. The second results chapter expands on the individual activities of the proteins by combining them, and asserting the ability of archaeal NHEJ to join discontinuous ends in vitro. The role of the PE has been unclear in the bacterial system, but in vitro assays described here suggest that the enzyme plays a role in processing NHEJ intermediates formed by the NHEJ polymerase. The PE is found to optimise repair intermediates for ligation, and to reverse potentially genotoxic DNA strand displacements. The final results chapter investigates the structural aspects of the archaeal NHEJ enzymes. Together these studies establish a functional NHEJ system in an archaeon for the first time, and expand our knowledge of the bacterial system by proposing a standard model of archaeo--‐prokaryotic NHEJ.

A significantly improved DNA polymerase fidelity assay, based on a gapped plasmid containing the lacZα reporter gene in a single-stranded region, is described. Nicking at two sites flanking lacZα, and removing the excised strand by thermocycling in the presence of complementary competitor DNA, is used to generate the gap. Simple methods are presented for preparing the single-stranded competitor. The gapped plasmid can be purified, in high amounts and in a very pure state, using benzoylated-naphthoylated DEAE-cellulose, resulting in a low background mutation frequency (~1 × 10(-4)). Two key parameters, the number of detectable sites and the expression frequency, necessary for measuring polymerase error rates have been determined. DNA polymerase fidelity is measured by gap filling in vitro, followed by transformation into Escherichia coli and scoring of blue/white colonies and converting the ratio to error rate. Several DNA polymerases have been used to fully validate this straightforward and highly sensitive system.

The protein kinase mammalian target of rapamycin (mTOR) regulates the phosphorylation and activity of several proteins that have the potential to control translation, including p70S6 kinase and the eIF4E binding proteins 4E-BP1 and 4E-BP2. In spite of this, in exponentially growing cells overall protein synthesis is often resistant to mTOR inhibitors. We report here that sensitivity of wild-type mouse embryonic fibroblasts (MEFs) to mTOR inhibitors can be greatly increased when the cells are subjected to the physiological stress imposed by hypertonic conditions. In contrast, protein synthesis in MEFs with a double knockout of 4E-BP1 and 4E-BP2 remains resistant to mTOR inhibitors under these conditions. Phosphorylation of p70S6 kinase and protein kinase B (Akt) is blocked by the mTOR inhibitor Ku0063794 equally well in both wild-type and 4E-BP knockout cells, under both normal and hypertonic conditions. The response of protein synthesis to hypertonic stress itself does not require the 4E-BPs. These data suggest that under certain stress conditions: (i) translation has a greater requirement for mTOR activity and (ii) there is an absolute requirement for the 4E-BPs for regulation by mTOR. Importantly, dephosphorylation of p70S6 kinase and Akt is not sufficient to affect protein synthesis acutely.

During cell spreading, mammalian cells migrate using lamellipodia formed from a large dense branched actin network which produces the protrusive force required for leading edge advancement. The formation of lamellipodia is a dynamic process and is dependent on a variety of protein cofactors that mediate their local regulation, structural characteristics and dynamics. In the present study, we show that mRNAs encoding some structural and regulatory components of the WAVE [WASP (Wiskott-Aldrich syndrome protein) verprolin homologous] complex are localized to the leading edge of the cell and associated with sites of active translation. Furthermore, we demonstrate that steady-state levels of ArpC2 and Rac1 proteins increase at the leading edge during cell spreading, suggesting that localized protein synthesis has a pivotal role in controlling cell spreading and migration.

Translation mechanisms at different stages of the cell cycle have been studied for many years, resulting in the dogma that translation rates are slowed during mitosis, with cap-independent translation mechanisms favored to give expression of key regulatory proteins. However, such cell culture studies involve synchronization using harsh methods, which may in themselves stress cells and affect protein synthesis rates. One such commonly used chemical is the microtubule de-polymerization agent, nocodazole, which arrests cells in mitosis and has been used to demonstrate that translation rates are strongly reduced (down to 30% of that of asynchronous cells). Using synchronized HeLa cells released from a double thymidine block (G 1/S boundary) or the Cdk1 inhibitor, RO3306 (G 2/M boundary), we have systematically re-addressed this dogma. Using FACS analysis and pulse labeling of proteins with labeled methionine, we now show that translation rates do not slow as cells enter mitosis. This study is complemented by studies employing confocal microscopy, which show enrichment of translation initiation factors at the microtubule organizing centers, mitotic spindle, and midbody structure during the final steps of cytokinesis, suggesting that translation is maintained during mitosis. Furthermore, we show that inhibition of translation in response to extended times of exposure to nocodazole reflects increased eIF2α phosphorylation, disaggregation of polysomes, and hyperphosphorylation of selected initiation factors, including novel Cdk1-dependent N-terminal phosphorylation of eIF4GII. Our work suggests that effects on translation in nocodazole-arrested cells might be related to those of the treatment used to synchronize cells rather than cell cycle status.

Genome stability is of upmost importance to life. DNA polymerases are essential for the
duplication and maintenance of the genome but they cannot themselves begin synthesis
of a DNA chain, and require the activity of specialised RNA polymerases called primases.
In eukaryotic cells distinct enzymes catalyse these two essential processes. This thesis
contains the characterisation of coiled-coil domain containing protein (CCDC)111, a
previously uncharacterised protein conserved in a broad range of unicellular and
multicellular eukaryotes including humans. CCDC111 is a member of the archaeaoeukaroytic
primase (AEP) superfamily and uniquely for a eukaryotic enzyme possesses
both primase and polymerase activities, and was thus renamed PrimPol. The work in this
thesis implicates PrimPol in the process of DNA damage tolerance, a universal
mechanism by which cells complete genome duplication in spite of potentially lethal DNA
damage. The first results chapters detail the essential role of a PrimPol homologue
(TbPrimPol2) in the important protozoan pathogen Trypanosoma brucei. A combination
of molecular, cell biology, and biochemical analyses indicate a role for TbPrimPol2 in the
post-replication tolerance of endogenously occurring DNA damage using its trans-lesion
DNA synthesis activity. The remaining results chapters characterise PrimPol in human
cultured cells, and demonstrate that this enzyme is present in both the nucleus and
mitochondria. In the nucleus PrimPol functions in the cellular tolerance of ultraviolet (UV)-
induced DNA damage, and is required to protect xeroderma pigmentosum variant (XP-V)
cells, deficient in the UV lesion bypass polymerase Pol !, from the cytotoxic affects of
UV radiation. Together, this thesis establishes the involvement of PrimPol in DNA
damage tolerance from one of the earliest diverging eukaryotic organisms to man.

Genome duplication is an essential task our cells have to achieve prior to cell division, and requires a highly specialized replication machinery to ensure it is performed in an accurate and complete manner. DNA primase and polymerases are essential components of the replisome. Primases initiate DNA replication by synthesising short RNA primers that are then elongated by faithful and processive replicative DNA polymerases. However, both exogenous and endogenous agents can damage DNA and hinder progression of the replicative machinery. Translesion synthesis DNA polymerases assist in bypassing these DNA lesions in a process called DNA damage tolerance that enables chromosomal replication to proceed in in spite of damaged templates. This thesis details the characterisation of a novel eukaryotic DNA primase, coiled-coil domain containing protein (CCDC111), a member of the Archaeo Eukaryotic Primase (AEP) superfamily. Preliminary in vitro characterisation of CCDC111 demonstrated that the recombinant protein is capable of both DNA-dependant priming and polymerase activities, which is unprecedented for a eukaryotic polymerase, and it was therefore renamed Primase-polymerase (PrimPol). The aim of this thesis was to provide one of the first cellular characterisations of PrimPol by generating a knockout of the gene in avian DT40 cells and also depleting the protein in human cells using RNAi. In vivo evidence supports the involvement of this novel polymerase in replication fork progression following replicative stress, such as exposure to UV light, but also during unperturbed DNA replication. Work in this thesis also indicates a role for PrimPol in mitochondrial DNA maintenance. Together, the data presented here establish a role for PrimPol in DNA damage tolerance in avian and human cells.

During meiosis, formation and repair of programmed DNA double-strand breaks (DSBs) create genetic exchange between homologous chromosomes-a process that is critical for reductional meiotic chromosome segregation and the production of genetically diverse sexually reproducing populations. Meiotic DSB formation is a complex process, requiring numerous proteins, of which Spo11 is the evolutionarily conserved catalytic subunit. Precisely how Spo11 and its accessory proteins function or are regulated is unclear. Here, we use Saccharomyces cerevisiae to reveal that meiotic DSB formation is modulated by the Mec1(ATR) branch of the DNA damage signalling cascade, promoting DSB formation when Spo11-mediated catalysis is compromised. Activation of the positive feedback pathway correlates with the formation of single-stranded DNA (ssDNA) recombination intermediates and activation of the downstream kinase, Mek1. We show that the requirement for checkpoint activation can be rescued by prolonging meiotic prophase by deleting the NDT80 transcription factor, and that even transient prophase arrest caused by Ndt80 depletion is sufficient to restore meiotic spore viability in checkpoint mutants. Our observations are unexpected given recent reports that the complementary kinase pathway Tel1(ATM) acts to inhibit DSB formation. We propose that such antagonistic regulation of DSB formation by Mec1 and Tel1 creates a regulatory mechanism, where the absolute frequency of DSBs is maintained at a level optimal for genetic exchange and efficient chromosome segregation.

Chapter 1: Chapter One provides an overview on the Bohlmann-Rahtz pyridine synthesis. New procedures, implementing metal based Lewis acids, Brønsted acids and metal-free Lewis acid catalysts have been used in this process. Also, new one-pot two- and three-component methodologies have been developed for the synthesis of various natural products containing the pyridine motif and these have been compared and contrasted.

This chapter also discusses signalling pathways in Werner syndrome cells. The inhibitor SB203580 has been shown to prevent the phosphorylation of the p38α kinase in a ATP competitive manner and this implicates this mechanism in accelerated ageing and gives potential to the prospect of targeting this pathway in a drug discovery programme, if better mechanistic understanding can be garnered.

Chapter 2: Chapter Two discusses the Bohlman–Rahtz synthesis of various substituted pyridines. The process has been modified to be simple, involves mild conditions and provides the heterocyclic targets in high yield. We have shown that substituted pyridines could be synthesised efficiently under microwave conditions using a relatively short reaction time. The process was also successful for the production of a range of fused heterocycles containing the pyridine moiety in high yield, including pyrido[2,3-d]pyrimidin-4(3H)-ones and pyrido[2,3-d]pyrimidine-2,4(1H,3H)-diones.

Chapter 3: Chapter Three describes the efficient synthesis of the p38 MAPK inhibitor UR-13756 using a Hantzsch-type three component cyclocondensation. Microwave irradiation of a mixture of 3-amino-1-methylpyrazole hydrochloride, 1-(4-fluorophenyl)-2-(pyridine-4-yl)ethanone and 4-fluorobenzaldhyde for 4 hours in ethanol under acidic catalytic conditions provided UR-13756 in high yield (71%) after purification by column chromatography.

Chapter 4: Chapter Four shows the synthesis of 4-(3-amino-1-(4-methoxyphenyl)-1H-pyrazol-4-yl)benzamide in three steps by the use of rigorous experimental procedures under microwave conditions. This technique led to faster heating rates and allowed the rapid optimization of yields. These advantages were observed in all steps and allow formation of products in high yields. Biological study of the inhibitor 4-(3-amino-1-(4-methoxyphenyl)-1H-pyrazol-4-yl)benzamide showed, by ELISA analysis, that p38 signalling was inhibited in control dermal cells. Some progress was made towards the synthesis of 3-amino-4-[1-(3-1H-pyrazol-4-yl)]benzamide.

Chapter 5: Chapter Five investigates the synthesis of the chemotherapeutic agent RO3201195, a highly selective inhibitor of p38α, in seven steps under microwave conditions. The procedure provides a relatively high overall yield of the desired product and all other intermediates involved in individual steps compared with conventional heating methods.

Chapter 6: Chapter Six provides the experimental procedures and various spectroscopic data for the synthesized compounds.

Budding yeast centromeres are sequence-defined point centromeres and are, unlike in many other organisms, not
embedded in heterochromatin. Here we show that Fun30, a poorly understood SWI/SNF-like chromatin remodeling factor
conserved in humans, promotes point centromere function through the formation of correct chromatin architecture at
centromeres. Our determination of the genome-wide binding and nucleosome positioning properties of Fun30 shows that
this enzyme is consistently enriched over centromeres and that a majority of CENs show Fun30-dependent changes in
flanking nucleosome position and/or CEN core micrococcal nuclease accessibility. Fun30 deletion leads to defects in histone
variant Htz1 occupancy genome-wide, including at and around most centromeres. FUN30 genetically interacts with CSE4,
coding for the centromere-specific variant of histone H3, and counteracts the detrimental effect of transcription through centromeres on chromosome segregation and suppresses transcriptional noise over centromere CEN3. Previous work has shown a requirement for fission yeast and mammalian homologs of Fun30 in heterochromatin assembly. As centromeres in budding yeast are not embedded in heterochromatin, our findings indicate a direct role of Fun30 in centromere chromatin by promoting correct chromatin architecture.

Meiotic recombination is initiated by DNA double-strand breaks (DSBs) created by the topoisomerase-like protein Spo11. During DSB formation, Spo11 becomes covalently attached to the 5’ DSB ends. Removal of Spo11 is essential to repair the DSB by homologous recombination. Spo11 is removed endonucleolytically creating short-lived Spo11-oligonucleotide products. Here I demonstrate that:

1. Spo11-oligonucleotide products are not detected in recombination mutants believed to be defective in meiotic DSB formation.
2. When DSB repair is delayed, Spo11-oligonucleotides persist for longer.
3. Processing of Spo11-DSB ends to create Spo11-oligonucleotides is largely dependent on Mec1 and Tel1 activity.

In the process of investigating Spo11-oligonucleotide degradation, it was observed that a mutant defective in both the meiotic recombination checkpoint and in DSB repair failed to accumulate the expected level of DSBs. Work described here leads to the proposal of a DSB feedback mechanism that functions though the Mec1 (ATR) pathway to increase the efficiency of DSB formation. By contrast, Tel1 (ATM) functions to inhibit DSB formation, agreeing with recently published data. However, the data presented also suggests that Tel1 acts alongside the Mec1 pathway to promote DSB formation. It is therefore proposed that such positive and negative regulation creates a homeostatic mechanism to ensure that an optimum frequency of DSBs is formed.

In wildtype cells, single –stranded DNA resection relies only on the Exo1 nuclease. In checkpoint defective cells resection length is increased. Results described here demonstrate that in a checkpoint defective strain, resection functions through Exo1, Sgs1/Dna2 and a third currently untested resection mechanism, likely to be Mre11 dependent.

Controlling the order and spatial distribution of self-assembly in multicomponent supramolecular systems could underpin exciting new functional materials, but it is extremely challenging. When a solution of different components self-assembles, the molecules can either coassemble, or self-sort, where a preference for like-like intermolecular interactions results in coexisting, homomolecular assemblies. A challenge is to produce generic and controlled ‘one-pot’ fabrication methods to form separate ordered assemblies from ‘cocktails’ of two or more self-assembling species, which might have relatively similar molecular structures and chemistry. Self-sorting in supramolecular gel phases is hence rare. Here we report the first example of the pH-controlled self-sorting of gelators to form self-assembled networks in water. Uniquely, the order of assembly can be predefined. The assembly of each component is preprogrammed by the pKa of the gelator. This pH-programming method will enable higher level, complex structures to be formed that cannot be accessed by simple thermal gelation

Amyloid fibril formation is associated with misfolding diseases, as well as fulfilling a functional role. The cross-ß molecular architecture has been reported in increasing numbers of amyloid-like fibrillar systems. The Waltz algorithm is able to predict ordered self-assembly of amyloidogenic peptides by taking into account residue type and position. This algorithm has expanded the amyloid sequence space and here we characterise the structures of amyloid-like fibrils formed by three peptides identified by Waltz that form fibrils but not crystals. The structural challenge is met by combining electron microscopy, linear and circular dichroism and X-ray fibre diffraction. We propose structures that reveal a cross-ß conformation with 'steric-zipper' features, giving insights into the role for side chains in peptide packing and stability within fibrils. The amenity of these peptides to structural characterisation makes them compelling model systems to use for understanding the relationship between sequence, self-assembly, stability and structure for amyloid fibrils

Sufficient and balanced pools of deoxyribonucleotide triphophates (dNTPs) is
crucial for high-fidelity DNA replication as well as correct DNA repair. The enzyme
RiboNucleotide Reductase (RNR) catalyses NDP to dNDP and is therefore an
essential enzyme by providing the “building blocks” to the cells. dNTPs production
needs to be tightly regulated in order to minimize mutation frequencies and prevent
genome instability.

RNR in S. pombe is composed of two proteins, Cdc22R1 and Suc22R2, and has
been described as a heterotetramer with a dimer of each subunit: the big subunit
Cdc22R1 and the small subunit Suc22R2. S. pombe also posseses an RNR inhibitor:
Spd1, as well as a second RNR regulator Spd2 which has been newly discovered.
Spd1 has been demonstrated to inhibit RNR and to regulate its activity throughout the
cell cycle. The detailed mechanism of the RNR regulation during the cell cycle or after
DNA damage is not entirely clear, as are the means of inhibition by Spd1. In order to
shed some light on the RNR complex and its regulation, we used various microscopybased
methods to study RNR in vivo as well as in vitro.

The data of this thesis suggest there are different forms of active RNR
heterocomplexes, found throughout the cell cycle in the cytoplasm as well as in the
nucleus. We propose that the precise stoichiometry of subunits in the complexes may
vary, or that the complex conformation may be modified in an Spd1-dependent
manner. In addition, treatment of the cells with a UV mimetic agent, 4NQO, seems to
promote RNR regulation in an Spd1-dependent manner. On the contrary, inhibition of
RNR by HydroxyUrea (HU) affects the RNR in a possible structure-related manner,
independently of Spd1 or Spd2. The in vivo observations correlate with structural
and/or oligomerization modifications of the RNR, representing a novel RNR regulation
in S. pombe.

Repair of damage to the DNA is essential for the maintenance of genomic stability, both during embryonic development and normal growth. The cell has therefore evolved a complex array of interconnected pathways to ensure the appropriate response to DNA damage is initiated, such as cell cycle checkpoint arrest, activation of DNA repair pathways or induction of apoptotic processes. These co-ordinated signal transduction pathways have been termed the DNA damage response (DDR). A previous study showed that ATR-dependent damage responses were frequently defective in cell lines from patients with Microcephalic Primordial Dwarfism (MPD) disorders. In this thesis I have further characterised ATR–dependent damage response signalling in several cell lines from patients with various MPD disorders. I have shown that novel mutations in PCNT, which encodes a structural centrosomal protein, result in an MPD disorder and have characterised the associated ATRdependent DNA damage responses. I also contributed to the identification of mutations in ORC1, encoding a component of the DNA replication Origin Recognition Complex, in further MPD patients and examined origin licensing and Sphase progression in the patient derived cell lines. As a novel finding, I observed defects in the ATR-dependent G2/M checkpoint response in these cells. Additionally, I have characterised novel mutations in ATRIP, a gene encoding the obligate partner of ATR, in Seckel Syndrome patients, denoting a novel genetic defect in this condition. Finally, I have explored the role of PLK1 and AurA kinase in ATRdependent G2/M checkpoint control and provided compelling evidence of misregulation of this pathway in various MPD-patient derived cell lines. Collectively these data provide important functional insights into the genetic defects that cause MPD disorders and further explore the link between defective ATR-dependent damage response signalling and microcephaly.

We have developed a targeted method to quantify all combinations of methylation on an H3 peptide containing lysines 27 and 36 (H3K27-K36). By using stable isotopes that separately label the histone backbone and its methylations, we tracked the rates of methylation and demethylation in myeloma cells expressing high vs. low levels of the methyltransferase MMSET/WHSC1/NSD2. Following quantification of 99 labeled H3K27-K36 methylation states across time, a kinetic model converged to yield 44 effective rate constants qualifying each methylation and demethylation step as a function of the methylation state on the neighboring lysine. We call this approach MS-based measurement and modeling of histone methylation kinetics (M4K). M4K revealed that, when dimethylation states are reached on H3K27 or H3K36, rates of further methylation on the other site are reduced as much as 100-fold. Overall, cells with high MMSET have as much as 33-fold increases in the effective rate constants for formation of H3K36 mono- and dimethylation. At H3K27, cells with high MMSET have elevated formation of K27me1, but even higher increases in the effective rate constants for its reversal by demethylation. These quantitative studies lay bare a bidirectional antagonism between H3K27 and H3K36 that controls the writing and erasing of these methylation marks. Additionally, the integrated kinetic model was used to correctly predict observed abundances of H3K27-K36 methylation states within 5% of that actually established in perturbed cells. Such predictive power for how histone methylations are established should have major value as this family of methyltransferases matures as drug targets.

Cytoplasmic dynein is a retrograde motor protein complex that carries cargo such as organelles and growth factors along microtubules from the cell periphery towards the peri-nuclear region.

The cytoplasmic dynein complex is centred around two homodimerised heavy chains, within which multiple mutations have been identified in human neurological diseases.

The ‘Legs at odd angles’ (Loa) mouse has a missense ‘T’ to ‘A’ point mutation in the cytoplasmic dynein heavy chain gene (Dync1h1), resulting in a phenylalanine to tyrosine substitution at position 580. Mice homozygous for this mutation die within 24 hours of birth whilst heterozygote’s manifest an age-related and progressive neurodegeneration.

Fixed and live-cell microscopy shows aberrant movement of endocytosed growth factors in Loa. Retrograde speed is reduced with a distinct lack of the fastest moving carriers. Moreover, the overall pattern of movement is altered with increased anterograde and side-steps occurring in Loa.

Impaired endosomal trafficking of growth factors for degradation prolongs the activation of extracellular signal related kinases 1 and 2 (ERK 1/2) and increases the expression of the immediate early gene c-Fos in mouse embryonic fibroblasts. Motor neurons also show increased levels c-Fos however this can be induced by starvation, indicating their enhanced susceptibility to stress.

The light chain (KLC) of dynein’s opposing motor - kinesin is one of many genes differentially expressed in Loa compared to wild-type. In addition, associations of KLC with the dynein complex is altered in Loa.

Similarities between human neurological diseases and Loa both at the organism and cellular level make Loa a valuable tool towards understanding cellular mechanisms fundamental to the process of disease. Through understanding comes advancement towards therapeutic targets to improve the lives of thousands of people worldwide.

Yersinia pestis, the aetiological agent of plague, is responsible for a disease that has killed over 200 million people throughout history and generated three pandemics. This bacterium’s terrible success in causing disease is owed greatly to the virulence factors it expresses. Two of these factors are V antigen (LcrV) and F1 antigen (Caf1), both of which are two major antigens which the immune system produces antibodies against. V antigen is already known to have vital roles in Y. pestis gene expression and translocating other virulence factors into the host cells as well as having some immunosuppressive effects while F1 antigen is better known for possessing an antiphagocytic effect. The effects that these two antigens have in modulating the innate immune system of Mono Mac 6 cells were studied, such as modulation of expression of pattern recognition receptors (PRRs), in particular Toll-like receptors (TLRs), activation of NF-κB and secretion of cytokines, particularly those involved in inflammatory responses, as well as localising where in the cell these antigens target to. It was demonstrated that both V and F1 antigens possess immunosuppressive abilities, such as downregulation of TLRs as well as inhibitition of NF-κB activation and suppression of secretion of the cytokines TNF-α, IL-6 and IL-10. Furthermore, stimulation with only either V or F1 antigens can upregulate expression of the scavenger receptor CD36 and are capable of inducing secretion of the anti-inflammatory cytokine IL-10. V and F1 antigens were found to localise in the Golgi apparatus 30 minutes after stimulation and it was also determined that these antigens interfere with the signalling molecule MyD88.

Although most DNA double strand breaks (DSBs) are repaired by DNA nonhomologous
end!joining (NHEJ), DSBs at heterochromatin (HC) regions undergo repair
by homologous recombination (HR) in G2 phase. Repair of DSBs at HC regions
requires ATM-dependent KAP1 phosphorylation and subsequent HC relaxation. The
mediator proteins facilitate DSB repair at HC in G1 phase by retaining ATM and hence
pKAP1 at DSBs until the completion of repair. In this thesis, I investigated the role of
the mediator proteins in enabling DSB repair in G2 phase. I demonstrate that the
mediator proteins are required for the slow component of DSB repair in G2, which
represents HR. They also promote ATM-dependent pKAP1 formation in G2 as in G1.
In addition, I have described a role for MDC1 in Rad51 loading and for RNF8 in DNA
resection. Moreover, I demonstrate that BRCA1 overcomes an inhibitory barrier by
53BP1 to resection by promoting a G2!specific enlargement in 53BP1 foci during HR
that involves 53BP1 repositioning to the foci periphery and vacation from the central
core. RPA foci form in the core devoid of 53BP1. 53BP1 has opposing roles in HR; it
creates a restrictive barrier to resection but promotes pKAP1 and HC relaxation. RAP80
also inhibits resection by binding to ubiquitylated histones at DSBs. I demonstrate that
the DUB enzyme, POH1, is required to overcome the barrier posed to resection by
RAP80 since its depletion leads to deficient 53BP1 vacation of the central core and
deficient resection. BRCA1 and POH1 cooperate during G2 phase to promote resection
and DSB repair by HR. Additionally; I investigated the role(s) of the chromatin
remodelers BAF180 and CHD7 in transcriptional silencing following DSB induction, a
process that requires ATM, RNF8 and RNF168. I demonstrate that deficient
transcriptional silencing leads to a DSB repair defect at early times post IR.

RbpA is a RNA polymerase-binding protein that was identified in Streptomyces coelicolor. It is found in all Actinobacteria, including the pathogenic agent Mycobacterium tuberculosis. Streptomyces strains that have an rbpA mutation grow at a slower rate than the wild-type and are more sensitive to the RNAP-targeting antibiotic, rifampicin. RbpA binds to and activates σHrdB, the principal sigma factor that directs transcription of most housekeeping genes in S. coelicolor. Using bacterial two-hybrid analysis and in vitro pull down assays, RbpA was shown to interact with region 1.2-2.4 of σHrdB. This region forms part of a major interface with core RNA polymerase and is involved in the recognition of, and binding to, the -10 promoter element. Rv2050, the homologue of RbpA in M. tuberculosis, was also shown to interact with the principal sigma factor of this organism, σA. Structural studies on RbpA and Rv2050 revealed that it is composed of two regions, a structured N-terminal β-fold region and an flexible or unstable C-terminal region, which interacts with sigma. Alanine-scanning site-directed mutagenesis on the C-terminal region of RbpA identified important residues involved in σHrdB interaction as well as residues that might be involved in transcriptional activation.

Epstein Barr Virus, a human herpes virus associated with infectious mononucleosis, Burkitt’s lymphoma, Nasopharyngeal Carcinoma and Hodgkin’s disease, can infect B cells and establish latency. Zta, a member of the bZIP transcription factor family, is a viral transcription and replication factor required for activation of lytic cycle.

Zta is able to bind DNA, through specific Zta Response Elements (ZREs). Interestingly, Zta binds in a methylation dependent manner to specific CpGcontaining ZREs, known as Class III ZREs. RpZRE3 is one of the first examples of such a site, and can be found in the promoter of a key viral lytic gene, BRLF1. Through computational analysis and in vitro binding assays, it was found that the core 7mer sequence of RpZRE3 is sufficient for complex formation in vitro, and that the core sequence can be found in a variety of human gene promoter regions.

As more methylation dependent ZREs emerged, a method to predict CpGcontaining ZREs was devised using position frequency matrices, coupled with in vitro binding verification, leading to the discovery of 12 novel CpG containing methylation dependent ZREs. The regulatory regions of both the EBV genome (type 1) and the human genome were mapped for a list of experimentally verified ZRE core sequences (both methylation dependent and independent). This analysis allowed identification of novel viral and host genes, potentially under the control of Zta activation, which were further analysed using existing transcriptome and methylome data.

An investigation into potential host factors using the same methylation dependent mechanism was started, and an unknown protein was seen to bind in a methylation dependent manner to RpZRE3 in the absence of Zta in vitro.

Self-assembling molecules are central to a plethora of processes found in nature, biotechnology
and even disease. The importance of the non-covalent interaction of monomers to the formation
of fibrillar assemblies is evident in the repeated use of this mechanism throughout nature, from
essential cellular processes such as the formation of the cytoskeleton to the production of silk.
Further, it has been recognised in the last two decades that a self-assembly mechanism, that is
the formation of amyloid, underpins the pathology of protein misfolding diseases; it is therefore
essential to dissect these mechanisms.

Despite recent technological and model system developments, self-assembling molecules
remain challenging to investigate. Using combined structural and biophysical characterisations
of penta- and hexa-peptide self-assembling model systems these investigations shed further
light on the structure of amyloid-like fibrils. The elucidation of the structures of these fibrillar
systems not only has implications for disease but also makes them well placed for consideration
for biotechnological applications.

In reflecting upon how cross-ß structural architectures can be organised in the fibrillar state, a
molecular and supramolecular model of fibrils formed by a fragment of !-synuclein is reported.
The fibrils are found to consist of a novel and elaborate cross-ß architecture that leads to a
helical supramolecular assembly spanning length scales previously unobserved for such a
system.

Where self-assembly is a useful route to supramolecular structure formation, the use of low
molecular weight gelator (LMWG) peptides to create fibrillar structures with defined material
properties is also explored. The complex link between molecular structure, self-assembled
architecture, fibril formation, fibril interaction and ultimately bulk material properties is
described. It is found that the determinants of self-assembly are distinct from the determinants
of gelation and so future LMWG design will have to consider both individually.

This work presents methodological advances in the characterisation of self-assembled structures.
The investigations presented here have relevance for disease related processes but also to the
technological use of these systems as materials. Finally, this work emphasises the beauty of the
extravagant, yet elegant connection between molecular interaction and supramolecular selfassembly.

The Elemental Defence Hypothesis, proposed by Boyd and Martens in 1992, suggests that high foliar metal concentrations deter herbivore feeding and this protection from herbivory may be one factor explaining the adaptive value of metal hyperaccumulation. However, lower foliar metal concentrations than those occurring in hyperaccumulators, may also confer this advantage, but the benefits of metal uptake by this group of plants (known as accumulators) has been relatively less-studied. Despite this potential advantage, metal accumulation is a relatively rare phenomenon, suggesting it may have costs as well as benefits.

A field survey of metallicolous populations of the hyperaccumulator Thlaspi caerulescens J. & C. Presl. (recently renamed as Noccaea caerulescens (J. & C. Presl.) F. K. Meyer) (Brassicaceae) and the accumulator Rumex acetosa L. (Polygonaceae), found on zinc (Zn) contaminated mining sites, revealed between-population differences in chewing herbivore damage, in the efficiency with which they uptake soil Zn into their shoots (measured as the concentration factor), and the foliar Zn concentration of T. caerulescens. However, foliar Zn concentration was not correlated with damage within a species.

In a series of pot experiments using two populations of R. acetosa, the foliar Zn concentration was manipulated through the addition of Zn to the soil and through differences in Zn uptake rate between populations. This thesis investigated how these manipulations influenced herbivory by generalist Helix aspersa Müller (Helicidae), and how plant competitive ability (in terms of biomass) was determined by a combination of population identity, soil Zn concentration and presence of herbivores.

When two R. acetosa populations were grown under 1500 and 45,000 mg/kg soil Zn concentrations, population differences were found in shoot biomass and competitive ability. The outcomes of intra- compared with inter-population competition depended on soil Zn concentration. When herbivores were present, shoot damage was low, usually < 15% of foliage removed. Snail preference was dependent upon the interaction between population identity and soil Zn concentration, partially supporting the Elemental Defence Hypothesis.

Poly(ADP-ribose) polymerase 1 (PARP1) is a primary DNA damage sensor whose (ADP-ribose) polymerase activity is acutely regulated by interaction with DNA breaks. Upon activation at sites of DNA damage, PARP1 modifies itself and other proteins by covalent addition of long, branched polymers of ADP-ribose, which in turn recruit downstream DNA repair and chromatin remodeling factors. PARP1 recognizes DNA damage through its N-terminal DNA-binding domain (DBD), which consists of a tandem repeat of an unusual zinc-finger (ZnF) domain. We have determined the crystal structure of the human PARP1-DBD bound to a DNA break. Along with functional analysis of PARP1 recruitment to sites of DNA damage in vivo, the structure reveals a dimeric assembly whereby ZnF1 and ZnF2 domains from separate PARP1 molecules form a strand-break recognition module that helps activate PARP1 by facilitating its dimerization and consequent trans-automodification.

Work was undertaken towards the synthesis of the promising antibiotic lactonamycin (iii). Following the work of Parsons et al. it was proposed that cyclisation of the ene-diyne (i) would give access to advanced pentacyclic intermediate (ii) and that from this a total synthesis of lactonamycin would be achieved (scheme I).

Scheme I : Proposed Parsons, Board, Waters cyclisation to form the pentacycle (iii)(For image refer to pdf).
A synthesis towards the cyclisation precursor (i) was carried out and a route to the key tetrasubstituted phthalide (v) established. Further chemistry was proposed to complete the synthesis of lactonamycin (scheme II).

Scheme II : Formation of a fully substituted benzolactone.(For image refer to pdf).
During attempts to introduce the β-bromoallyl group of key intermediate (v) using a high temperature Claisen rearrangement it was established that the benzodioxin (vii) underwent thermolysis to generate the reactive quinone methide intermediate (viii) and that in the presence of a nucleophilic solvent the adduct (ix) was formed (scheme III). Model studies showed the reaction to be both general and high-yielding.

Scheme III : Novel quinone methide methodology. (For image refer to pdf).

This thesis addresses the concept of atomically precise manufacturing and aims to examine some likely aspects of the necessary infrastructure and knowledge that will be required from a theoretical standpoint.

By way of introduction, I trace the history of Science Fiction’s influence on scientific research and examine some examples that have specifically inspired the thinking behind nanoscience and nanotechnology. More serious speculation, both in favour of and arguing against the possibility of bottom-up manufacturing is also discussed. I look at two schools of thought; directed assembly, typified by the ambition to assemble molecular structures piece by piece and self assembly, where networks of molecules form into arrays on substrates, imparting novel properties.

Various methodologies and tools available to the nanotechnologist are examined. Density functional theory, as employed in the AIMpro code, and Molecular Mechanics are discussed, particularly in respect of their strengths and weaknesses for use in simulating the kind of nanoscale processes appropriate to nanomanufacturing. The theoretical basis behind scanning tunneling microscopes is also examined, with particular attention paid to their potential for upscaling in the future.

Some components found within scanning tunneling microscopes are simulated using Density Functional Theory. Models of pure tungsten tips are studied at various levels of complexity in order to decide upon a reasonable compromise between accuracy and ease of computation. The nature of the interlayer interaction in few layer graphenes is examined and pristine and defected graphitic surfaces, are studied with a view towards their use as nano-workbenches. Their images as produced in scanning tunneling microscopes are simulated. Density Functional Theory is applied to organic molecules self-assembling on metallic substrates. Specifically, tetracene on a clean copper surface and on an oxygen-terminated copper surface is studied.

Finally, I discuss the significance of the results of each section, taken individually and as a whole, and try to put it into perspective regarding the practicality of actually employing this paradigm realistically in the near future.

Coxsackievirus B5 (CBV5) is a positive sense, single-stranded RNA virus belonging to the Enterovirus genus of the Picornaviridae family. It can cause many serious diseases, including viral myocarditis (which can lead on to dilated cardiomyopathy), aseptic meningitis, and pancreatitis. The structure and cell cycle of CBV5 is typical of a picornavirus.

Viral RNA is detected by Toll-like receptors (TLRs) and retinoic acid inducible gene-I (RIG-I)-like receptors (RLRs). The RLR family, consisting of RIG-I, MDA5 (melanoma differentiation-associated gene 5), and LGP2, are pattern recognition receptors that detect a range of different viruses. RIG-I and MDA5 are homologous cytoplasmic proteins containing an N-terminal region with two caspase activation and recruitment domains (CARDs), a central SF2 type DExD/H-box RNA helicase domain, and a C-terminal repressor domain (RD). Once a viral ligand has been detected and bound by RIG-I and MDA5, both signal downstream through their CARDs to activate IRF3/7 and NF-κB indirectly, via the protein intermediate IPS-1 (IFN-β promoter stimulator 1), and initiate an immune response.

RIG-I and MDA5 contribute to antiviral signalling in different ways depending on the virus involved. MDA5 has been shown to be critical for Picornaviridae detection, whilst RIG-I can detect a wide variety of different viruses and pathogen associated molecular patterns.

Results presented here show the expression levels of both are upregulated in response to CBV5 infection in human cardiac cells, with MDA5 expression levels being slightly greater than RIG-I. However, in Huh cells, RIG-I expression levels are greater than those of MDA5, indicating that it plays a role in CBV5 sensing. The presence of both phospho-IκB (corresponding to NF-κB activation) and IRF3 is detected in both cardiac cells and Huh cells in response to CBV5, and IFN-β production is also greatly upregulated. RIG-I and MDA5 colocalise with the adaptor protein IPS-1 in response to CBV5 infection, again indicating the synergistic response by the two RLRs, and both RLRs form homodimers in the cytoplasm. Overall, this suggest that both MDA5 and RIG-I act synergistically to detect CBV5 and initiate a downstream immune response, although MDA5 appears to be the marginally stronger sensor.

DNA replication is a central mechanism to all forms of life. Errors occurring during DNA replication can result in mutagenesis and genome rearrangements, which can cause various diseases. In this work I have investigated the stability of direct tandem repeats (TRs) in the context of replication and replication-associated repair mechanisms. During DNA replication the replication fork encounters many obstacles, such as DNA-protein barriers, secondary DNA structures and DNA lesions. How and if replication resumes or restarts in these circumstances in order to complete genome replication is not well understood and the fidelity of replication in response to such obstacles remains unclear. I have developed TR assays to assess replication errors in the context of replication fork restart and secondary structures. The results suggest that structured DNA (G4) can cause instability of TRs in the context of normal replication and that restarted replication can be intrinsically error-prone. Surprisingly, the mutagenic effect of G4-DNA on TR stability was not elevated in the context of replication fork restart. Therefore, deletions of TRs containing G4-DNA are not more susceptible to the compromised fidelity of a restarted replication fork.

Structures such as stalled replication forks can induce checkpoint responses to maintain genome stability. The stabilisation of replication forks is central in the response to replication stress. These protective mechanisms include the regulation of enzymatic activities. Mus81-Eme1 is a structurespecific endonuclease which is regulated by the DNA replication checkpoint, but has also been shown to be required for replication fork restart in certain circumstances. In collaboration with Professor Neil McDonald I analysed a novel domain identified in Mus81-Eme1. Mutagenesis of key residues deduced from the protein structure and comparison of their genetic analysis to known phenotypes of Mus81-Eme1 suggests distinct requirements for this domain.

Epstein-Barr virus (EBV) establishes a persistent latent infection in B lymphocytes and is associated with the development of numerous human tumors. Epstein-Barr nuclear antigen 3C (EBNA 3C) is essential for B-cell immortalization, has potent cell cycle deregulation capabilities, and functions as a regulator of both viral- and cellular-gene expression. We performed transcription profiling on EBNA 3C-expressing B cells and identified several chemokines and members of integrin receptor-signaling pathways, including CCL3, CCL4, CXCL10, CXCL11, ITGA4, ITGB1, ADAM28, and ADAMDEC1, as cellular target genes that could be repressed by the action of EBNA 3C alone. Chemotaxis assays demonstrated that downregulation of CXCL10 and -11 by EBNA 3C is sufficient to reduce the migration of cells expressing the CXCL10 and -11 receptor CXCR3. Gene repression by EBNA 3C was accompanied by decreased histone H3 lysine 9/14 acetylation and increased histone H3 lysine 27 trimethylation. In an EBV-positive cell line expressing all latent genes, we identified binding sites for EBNA 3C at ITGB1 and ITGA4 and in a distal regulatory region between ADAMDEC1 and ADAM28, providing the first demonstration of EBNA 3C association with cellular-gene control regions. Our data implicate indirect mechanisms in CXCL10 and CXCL11 repression by EBNA 3C. In summary, we have unveiled key cellular pathways repressed by EBNA 3C that are likely to contribute to the ability of EBV-immortalized cells to modulate immune responses, adhesion, and B-lymphocyte migration to facilitate persistence in the host.

We demonstrate a 10- to 25-fold increase in the amount of c-myc protein in several independent cell lines derived from patients with multiple myeloma (MM). This is not accompanied by a corresponding increase in the overall level of the c-myc mRNA. There is, however, a 3.4-fold increase in the amount of c-myc mRNA associated with the polysomes in these cell lines without any detectable change in either the polysome size or the rate of translation elongation, thus suggesting that there is an increase in the extent of mobilisation of c-myc mRNA to the polysomes in MM. Analysis of the 5' untranslated region of c-myc has revealed the presence of a mutation, in all of the MM cell lines examined, in a region which has been implicated previously in the translational control of this mRNA species. These data suggest aberrant translational control of the c-myc gene in cell lines derived from patients with MM, which may contribute towards pathogenesis of the disease.

Previous studies on the regulation of c-myc have focused on the transcriptional control of this proto-oncogene. We have investigated the signalling pathways involved under circumstances in which there is a translational upregulation in the levels of c-myc protein. We have demonstrated an up to tenfold serum-dependent increase of c-myc protein levels in Epstein-Barr virus immortalized B-cell lines 2-4 h after disruption of cellular aggregates, which is not accompanied by an equivalent increase in mRNA. Overall protein synthesis rates only increased threefold suggesting that the c-myc message was being selectively translated. We observed increases in the phosphorylation of p70 and p85 S6 kinases and of initiation factor eIF-4E binding protein 1 (4E-BP1) 1-2 h after stimulation, suggesting activation of the FRAP/TOR signalling pathway. The increased phosphorylation of 4E-BP1 led to a decrease in its association with eIF-4E and an increase in its association with the eIF-4G component of the eIF-4F initiation complex. The signalling inhibitors rapamycin and wortmannin blocked the phosphorylation of 4E-BP1 and abolished the translational component of the c-myc response. Our data suggest that dissociation of eIF-4E from 4E-BP1, leading to an increase in the formation of the eIF-4F initiation complex, relieves the translation repression imposed on the c-myc mRNA by its structured 5'UTR.

EBNA3C is a potent repressor of transcription when bound to DNA as a fusion with the DNA binding domain (DBD) of GALA. A survey of promoters has revealed that the wild-type, unfused EBNA3C can specifically repress expression from reporter plasmids containing the Epstein-Barr virus Cp latency-associated promoter. Repression of Cp activity required amino acids 207 to 368, which encompasses a region resembling a basic DBD adjacent to a leucine zipper DNA binding motif and a site which binds to the cellular factor CBF1/RBP-Jkappa. However, amino acids 207 to 368 are dispensable when the protein is bound to DNA as a fusion with the GAL4 DBD, thus implicating this region in DNA binding. Mutation of the CBF1/RBP-Jkappa binding site in EBNA3C abrogated repression, strongly suggesting that CBF1/RBP-Jkappa is necessary for targeting the viral protein to Cp. Consistent with this result, mutation of the EBNA2 response element (a CBF1/RBP-Jkappa binding site) in Cp also prevented significant repression. In addition, amino acids 346 to 543, which were previously defined as important for the repressor activity of the GAL4-EBNA3C fusion proteins, also appear to be necessary for the repression of Cp. Since repression by these fusions was not observed in all cell types, it seems likely that EBNA3C either depends on a corepressor which may interact with amino acids 346 to 543 or is modified in a cell-specific manner in order to repress. These data are consistent with EBNA3C contributing to the regulation of EBNA expression in latently infected B cells through CBF1/RBP-Jkappa and another factor, but this need not directly involve EBNA2. Finally, although it has been reported that EBNA3C can upregulate CD21 in some B cells, we were unable to demonstrate any effect of EBNA3C on reporter plasmids which contain the CD21 promoter.