Mammalian DNA base excision repair (BER) is accelerated by poly(ADP-ribose) polymerases (PARPs) and the scaffold protein XRCC1. PARPs are sensors that detect single-strand break intermediates, but the critical role of XRCC1 during BER is unknown. Here, we show that protein complexes containing DNA polymerase β and DNA ligase III that are assembled by XRCC1 prevent excessive engagement and activity of PARP1 during BER. As a result, PARP1 becomes “trapped” on BER intermediates in XRCC1-deficient cells in a manner similar to that induced by PARP inhibitors, including in patient fibroblasts from XRCC1-mutated disease. This excessive PARP1 engagement and trapping renders BER intermediates inaccessible to enzymes such as DNA polymerase β and impedes their repair. Consequently, PARP1 deletion rescues BER and resistance to base damage in XRCC1−/− cells. These data reveal excessive PARP1 engagement during BER as a threat to genome integrity and identify XRCC1 as an “anti-trapper” that prevents toxic PARP1 activity.
p53-binding protein 1 (53BP1) regulates both the DNA damage response and p53 signaling. Although 53BP1’s function is well established in DNA double-strand break repair, how its role in p53 signaling is modulated remains poorly understood. Here, we identify the scaffolding protein AHNAK as a G1 phase-enriched interactor of 53BP1. We demonstrate that AHNAK binds to the 53BP1 oligomerization domain and controls its multimerization potential. Loss of AHNAK results in hyper-accumulation of 53BP1 on chromatin and enhanced phase separation, culminating in an elevated p53 response, compromising cell survival in cancer cells but leading to senescence in non-transformed cells. Cancer transcriptome analyses indicate that AHNAK-53BP1 cooperation contributes to the suppression of p53 target gene networks in tumors and that loss of AHNAK sensitizes cells to combinatorial cancer treatments. These findings highlight AHNAK as a rheostat of 53BP1 function, which surveys cell proliferation by preventing an excessive p53 response.
Repetitive DNA is packaged into heterochromatin to maintain its integrity. We use CRISPR/Cas9 to induce DSBs in different mammalian heterochromatin structures. We demonstrate that in pericentric heterochromatin, DSBs are positionally stable in G1 and recruit NHEJ factors. In S/G2, DSBs are resected and relocate to the periphery of heterochromatin, where they are retained by RAD51. This is independent of chromatin relaxation but requires end resection and RAD51 exclusion from the core. DSBs that fail to relocate are engaged by NHEJ or SSA proteins. We propose that the spatial disconnection between end resection and RAD51 binding prevents the activation of mutagenic pathways and illegitimate recombination. Interestingly, in centromeric heterochromatin, DSBs recruit both NHEJ and HR proteins throughout the cell cycle. Our results highlight striking differences in the recruitment of DNA repair factors between pericentric and centromeric heterochromatin and suggest a model in which the commitment to specific DNA repair pathways regulates DSB position.
Topoisomerase II (TOP2) relieves torsional stress by forming transient cleavage complex intermediates (TOP2ccs) that contain TOP2-linked DNA breaks (DSBs). While TOP2ccs are normally reversible, they can be "trapped" by chemotherapeutic drugs such as etoposide and subsequently converted into irreversible TOP2-linked DSBs. Here, we have quantified etoposide-induced trapping of TOP2ccs, their conversion into irreversible TOP2-linked DSBs, and their processing during DNA repair genome-wide, as a function of time. We find that while TOP2 chromatin localization and trapping is independent of transcription, it requires pre-existing binding of cohesin to DNA. In contrast, the conversion of trapped TOP2ccs to irreversible DSBs during DNA repair is accelerated 2-fold at transcribed loci relative to non-transcribed loci. This conversion is dependent on proteasomal degradation and TDP2 phosphodiesterase activity. Quantitative modeling shows that only two features of pre-existing chromatin structure-namely, cohesin binding and transcriptional activity-can be used to predict the kinetics of TOP2-induced DSBs.
The convergence of two DNA replication forks creates unique problems during DNA replication termination. In E. coli and SV40, the release of torsional strain by type II topoisomerases is critical for converging replisomes to complete DNA synthesis, but the pathways that mediate fork convergence in eukaryotes are unknown. We studied the convergence of reconstituted yeast replication forks that include all core replisome components and both type I and type II topoisomerases. We found that most converging forks stall at a very late stage, indicating a role for additional factors. We showed that the Pif1 and Rrm3 DNA helicases promote efficient fork convergence and completion of DNA synthesis, even in the absence of type II topoisomerase. Furthermore, Rrm3 and Pif1 are also important for termination of plasmid DNA replication in vivo. These findings identify a eukaryotic pathway for DNA replication termination that is distinct from previously characterized prokaryotic mechanisms.
In mitosis, cells inactivate DNA double-strand break (DSB) repair pathways to preserve genome stability. However, some early signaling events still occur, such as recruitment of the scaffold protein MDC1 to phosphorylated histone H2AX at DSBs. Yet, it remains unclear whether these events are important for maintaining genome stability during mitosis. Here, we identify a highly conserved protein-interaction surface in MDC1 that is phosphorylated by CK2 and recognized by the DNA-damage response mediator protein TOPBP1. Disruption of MDC1-TOPBP1 binding causes a specific loss of TOPBP1 recruitment to DSBs in mitotic but not interphase cells, accompanied by mitotic radiosensitivity, increased micronuclei, and chromosomal instability. Mechanistically, we find that TOPBP1 forms filamentous structures capable of bridging MDC1 foci in mitosis, indicating that MDC1-TOPBP1 complexes tether DSBs until repair is reactivated in the following G1 phase. Thus, we reveal an important, hitherto-unnoticed cooperation between MDC1 and TOPBP1 in maintaining genome stability during cell division.
Cohesin subunits are frequently mutated in cancer, but how they function as tumor suppressors is unknown. Cohesin mediates sister chromatid cohesion, but this is not always perturbed in cancer cells. Here, we identify a previously unknown role for cohesin. We find that cohesin is required to repress transcription at DNA double-strand breaks (DSBs). Notably, cohesin represses transcription at DSBs throughout interphase, indicating that this is distinct from its known role in mediating DNA repair through sister chromatid cohesion. We identified a cancer-associated SA2 mutation that supports sister chromatid cohesion but is unable to repress transcription at DSBs. We further show that failure to repress transcription at DSBs leads to large-scale genome rearrangements. Cancer samples lacking SA2 display mutational patterns consistent with loss of this pathway. These findings uncover a new function for cohesin that provides insights into its frequent loss in cancer.
To maintain genome stability, cells need to replicate their DNA before dividing. The kinases CDK1 and PLK1 drive mitotic entry and become active when bulk DNA synthesis is completed at the S/G2 transition. Here, we have tested the hypothesis that DNA replication controls activation of mitotic kinases. Using an optimized double-degron system, we find that human cells unable to initiate DNA replication in S-phase promptly activate CDK1 and PLK1 and prematurely enter mitosis. In the presence of DNA replication, inhibition of CHK1 and p38 leads to premature activation of CDK1 and PLK1. While CDK2 activity promotes DNA replication, activation of CDK1 in S-phase induces severe replication stress. We propose that mitotic kinase activation is governed by a CDK2- and DNA replication-dependent feed-forward loop that ensures timely cell division while preserving genome stability. DNA replication thus functions as a break that coordinates cell cycle activities and determines cell cycle duration.
Canonical non-homologous end joining (c-NHEJ) repairs DNA double-strand breaks (DSBs) in G1 cells with biphasic kinetics. We show that DSBs repaired with slow kinetics, including those localizing to heterochromatic regions or harboring additional lesions at the DSB site, undergo resection prior to repair by c-NHEJ and not alt-NHEJ. Resection-dependent c-NHEJ represents an inducible process during which Plk3 phosphorylates CtIP, mediating its interaction with Brca1 and promoting the initiation of resection. Mre11 exonuclease, EXD2, and Exo1 execute resection, and Artemis endonuclease functions to complete the process. If resection does not commence, then repair can ensue by c-NHEJ, but when executed, Artemis is essential to complete resection-dependent c-NHEJ. Additionally, Mre11 endonuclease activity is dispensable for resection in G1. Thus, resection in G1 differs from the process in G2 that leads to homologous recombination. Resection-dependent c-NHEJ significantly contributes to the formation of deletions and translocations in G1, which represent important initiating events in carcinogenesis.
Poly(ADP-ribose) is synthesized by PARP enzymes during the repair of stochastic DNA breaks. Surprisingly, however, we show that most if not all endogenous poly(ADP-ribose) is detected in normal S phase cells at sites of DNA replication. This S phase poly(ADP-ribose) does not result from damaged or misincorporated nucleotides or from DNA replication stress. Rather, perturbation of the DNA replication proteins LIG1 or FEN1 increases S phase poly(ADP-ribose) more than 10-fold, implicating unligated Okazaki fragments as the source of S phase PARP activity. Indeed, S phase PARP activity is ablated by suppressing Okazaki fragment formation with emetine, a DNA replication inhibitor that selectively inhibits lagging strand synthesis. Importantly, PARP activation during DNA replication recruits the single-strand break repair protein XRCC1, and human cells lacking PARP activity and/or XRCC1 are hypersensitive to FEN1 perturbation. Collectively, our data indicate that PARP1 is a sensor of unligated Okazaki fragments during DNA replication and facilitates their repair.
Eukaryotic mRNA degradation often occurs in a process whereby translation initiation is inhibited and the mRNA is targeted for decapping. In yeast cells, Pat1, Scd6, Edc3, and Dhh1 all function to promote decapping by an unknown mechanism(s). We demonstrate that purified Scd6 and a region of Pat1 directly repress translation in vitro by limiting the formation of a stable 48S preinitiation complex. Moreover, while Pat1, Edc3, Dhh1, and Scd6 all bind the decapping enzyme, only Pat1 and Edc3 enhance its activity. We also identify numerous direct interactions between Pat1, Dcp1, Dcp2, Dhh1, Scd6, Edc3, Xrn1, and the Lsm1-7 complex. These observations identify three classes of decapping activators that function to directly repress translation initiation and/or stimulate Dcp1/2. Moreover, Pat1 is identified as critical in mRNA decay by first inhibiting translation initiation, then serving as a scaffold to recruit components of the decapping complex, and finally activating Dcp2.
The recalcitrance of many bacterial infections to antibiotic treatment is thought to be due to the presence of persisters that are non-growing, antibiotic-insensitive cells. Eventually, persisters resume growth, accounting for relapses of infection. Salmonella is an important pathogen that causes disease through its ability to survive inside macrophages. After macrophage phagocytosis, a significant proportion of the Salmonella population forms non-growing persisters through the action of toxin-antitoxin modules. Here we reveal that one such toxin, TacT, is an acetyltransferase that blocks the primary amine group of amino acids on charged tRNA molecules, thereby inhibiting translation and promoting persister formation. Furthermore, we report the crystal structure of TacT and note unique structural features, including two positively charged surface patches that are essential for toxicity. Finally, we identify a detoxifying mechanism in Salmonella wherein peptidyl-tRNA hydrolase counteracts TacT-dependent growth arrest, explaining how bacterial persisters can resume growth.
Sister chromatid cohesion conferred by entrapment
of sister DNAs within a tripartite ring formed between
cohesin’s Scc1, Smc1, and Smc3 subunits is created
during S and destroyed at anaphase through Scc1
cleavage by separase. Cohesin’s association with
chromosomes is controlled by opposing activities:
loading by Scc2/4 complex and release by a separase-
independent releasing activity as well as by
cleavage. Coentrapment of sister DNAs at replication
is accompanied by acetylation of Smc3 by Eco1,
which blocks releasing activity and ensures that sisters
remain connected. Because fusion of Smc3 to
Scc1 prevents release and bypasses the requirement
for Eco1, we suggested that release is mediated
by disengagement of the Smc3/Scc1 interface. We
show that mutations capable of bypassing Eco1 in
Smc1, Smc3, Scc1, Wapl, Pds5, and Scc3 subunits
reduce dissociation of N-terminal cleavage fragments
of Scc1 (NScc1) from Smc3. This process involves
interaction between Smc ATPase heads and
is inhibited by Smc3 acetylation.
G quadruplexes (G4s) can present potent blocks to DNA replication. Accurate and timely replication of G4s in vertebrates requires multiple specialized DNA helicases and polymerases to prevent genetic and epigenetic instability. Here we report that PrimPol, a recently described primase-polymerase (PrimPol), plays a crucial role in the bypass of leading strand G4 structures. While PrimPol is unable to directly replicate G4s, it can bind and reprime downstream of these structures. Disruption of either the catalytic activity or zinc-finger of PrimPol results in extreme G4-dependent epigenetic instability at the BU-1 locus in avian DT40 cells, indicative of extensive uncoupling of the replicative helicase and polymerase. Together, these observations implicate PrimPol in promoting restart of DNA synthesis downstream of, but closely coupled to, G4 replication impediments.
The stability and activity of numerous signaling proteins in both normal and cancer cells depends on the dimeric molecular chaperone heat shock protein 90 (Hsp90). Hsp90's function is coupled to ATP binding and hydrolysis and requires a series of conformational changes that are regulated by cochaperones and numerous posttranslational modifications (PTMs). SUMOylation is one of the least-understood Hsp90 PTMs. Here, we show that asymmetric SUMOylation of a conserved lysine residue in the N domain of both yeast (K178) and human (K191) Hsp90 facilitates both recruitment of the adenosine triphosphatase (ATPase)-activating cochaperone Aha1 and, unexpectedly, the binding of Hsp90 inhibitors, suggesting that these drugs associate preferentially with Hsp90 proteins that are actively engaged in the chaperone cycle. Importantly, cellular transformation is accompanied by elevated steady-state N domain SUMOylation, and increased Hsp90 SUMOylation sensitizes yeast and mammalian cells to Hsp90 inhibitors, providing a mechanism to explain the sensitivity of cancer cells to these drugs. © 2014 Elsevier Inc.
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.
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.
Activation of many protein kinases depends on their interaction with the Hsp90 molecular chaperone system. Recruitment of protein kinase clients to the Hsp90 chaperone system is mediated by the cochaperone adaptor protein Cdc37, which acts as a scaffold, simultaneously binding protein kinases and Hsp90. We have now expressed and purified an Hsp90-Cdc37-Cdk4 complex, defined its stoichiometry, and determined its 3D structure by single-particle electron microscopy. Comparison with the crystal structure of Hsp90 allows us to identify the locations of Cdc37 and Cdk4 in the complex and suggests a mechanism by which conformational changes in the kinase are coupled to the Hsp90 ATPase cycle.
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.
Semliki Forest virus serves as a paradigm for membrane fusion and assembly. Our icosahedral reconstruction combined 5276 particle images from 48 cryo-electron micrographs and determined the virion structure to 9 Å resolution. The improved resolution of this map reveals an N-terminal arm linking capsid subunits and defines the spike-capsid interaction sites. It illustrates the paired helical nature of the transmembrane segments and the elongated structures connecting them to the spike projecting domains. A 10 Å diameter density in the fusion protein lines the cavity at the center of the spike. These clearly visible features combine with the variation in order between the layers to provide a framework for understanding the structural changes during the life cycle of an enveloped virus.
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.
Actively transcribed regions of the genome are vulnerable to genomic instability. Recently, it was discovered that transcription is repressed in response to neighboring DNA double-strand breaks (DSBs). It is not known whether a failure to silence transcription flanking DSBs has any impact on DNA repair efficiency or whether chromatin remodelers contribute to the process. Here, we show that the PBAF remodeling complex is important for DSB-induced transcriptional silencing and promotes repair of a subset of DNA DSBs at early time points, which can be rescued by inhibiting transcription globally. An ATM phosphorylation site on BAF180, a PBAF subunit, is required for both processes. Furthermore, we find that subunits of the PRC1 and PRC2 polycomb group complexes are similarly required for DSB-induced silencing and promoting repair. Cancer-associated BAF180 mutants are unable to restore these functions, suggesting PBAF's role in repressing transcription near DSBs may contribute to its tumor suppressor activity.
MRE11 within the MRE11-RAD50-NBS1 (MRN) complex acts in DNA double-strand break repair (DSBR), detection, and signaling; yet, how its endo- and exonuclease activities regulate DSBR by nonhomologous end-joining (NHEJ) versus homologous recombination (HR) remains enigmatic. Here, we employed structure-based design with a focused chemical library to discover specific MRE11 endo- or exonuclease inhibitors. With these inhibitors, we examined repair pathway choice at DSBs generated in G2 following radiation exposure. While nuclease inhibition impairs radiation-induced replication protein A (RPA) chromatin binding, suggesting diminished resection, the inhibitors surprisingly direct different repair outcomes. Endonuclease inhibition promotes NHEJ in lieu of HR, while exonuclease inhibition confers a repair defect. Collectively, the results describe nuclease-specific MRE11 inhibitors, define distinct nuclease roles in DSB repair, and support a mechanism whereby MRE11 endonuclease initiates resection, thereby licensing HR followed by MRE11 exonuclease and EXO1/BLM bidirectional resection toward and away from the DNA end, which commits to HR.
DNA damage can stall the DNA replication machinery, leading to genomic instability. Thus, numerous mechanisms exist to complete genome duplication in the absence of a pristine DNA template, but identification of the enzymes involved remains incomplete. Here, we establish that Primase-Polymerase (PrimPol; CCDC111), an archaeal-eukaryotic primase (AEP) in eukaryotic cells, is involved in chromosomal DNA replication. PrimPol is required for replication fork progression on ultraviolet (UV) lightdamaged DNA templates, possibly mediated by its ability to catalyze translesion synthesis (TLS) of these lesions. This PrimPol UV lesion bypass pathway is not epistatic with the Pol h-dependent pathway and, as a consequence, protects xeroderma pigmentosum variant (XP-V) patient cells from UV-induced cytotoxicity. In addition, we establish that PrimPol is also required for efficient replication fork progression during an unperturbed S phase. These and other findings indicate that PrimPol is an important player in replication fork progression in eukaryotic cells.
Faithful copying of the genome is essential for life. In
eukaryotes, a single archaeo-eukaryotic primase (AEP), DNA primase, is required for the initiation and progression of DNA replication. Here we have identified additional eukaryotic AEP-like proteins with DNA-dependent primase and/or polymerase activity. Uniquely, the genomes of trypanosomatids, a group of kinetoplastid protozoa of significant medical importance, encode two PrimPol-like (PPL) proteins. In the African trypanosome, PPL2 is a nuclear enzyme present in G2 phase cells. Following PPL2
knockdown, a cell-cycle arrest occurs after the bulk of DNA synthesis, the DNA damage response is activated, and cells fail to recover. Consistent with this phenotype, PPL2 replicates damaged DNA templates in vitro, including templates containing the UV-induced pyrimidine-pyrimidone (6-4) photoproduct. Furthermore, PPL2 accumulates at sites of nuclear DNA damage. Taken together, our results indicate an essential role for PPL2 in postreplication tolerance of endogenous DNA damage, thus allowing completion of genome duplication.
The BRCT-domain protein Rad4(TopBP1) facilitates activation of the DNA damage checkpoint in Schizosaccharomyces pombe by physically coupling the Rad9-Rad1-Hus1 clamp, the Rad3(ATR) -Rad26(ATRIP) kinase complex, and the Crb2(53BP1) mediator. We have now determined crystal structures of the BRCT repeats of Rad4(TopBP1), revealing a distinctive domain architecture, and characterized their phosphorylation-dependent interactions with Rad9 and Crb2(53BP1). We identify a cluster of phosphorylation sites in the N-terminal region of Crb2(53BP1) that mediate interaction with Rad4(TopBP1) and reveal a hierarchical phosphorylation mechanism in which phosphorylation of Crb2(53BP1) residues Thr215 and Thr235 promotes phosphorylation of the noncanonical Thr187 site by scaffolding cyclin-dependent kinase (CDK) recruitment. Finally, we show that the simultaneous interaction of a single Rad4(TopBP1) molecule with both Thr187 phosphorylation sites in a Crb2(53BP1) dimer is essential for establishing the DNA damage checkpoint.
A hallmark of the cellular response to DNA double-strand breaks (DSBs) is histone H2AX phosphorylation in chromatin to generate gamma-H2AX. Here, we demonstrate that gamma-H2AX densities increase transiently along DNA strands as they are broken and repaired in G1 phase cells. The region across which gamma-H2AX forms does not spread as DSBs persist; rather, gamma-H2AX densities equilibrate at distinct levels within a fixed distance from DNA ends. Although both ATM and DNA-PKcs generate gamma-H2AX, only ATM promotes gamma-H2AX formation to maximal distance and maintains gamma-H2AX densities. MDC1 is essential for gamma-H2AX formation at high densities near DSBs, but not for generation of gamma-H2AX over distal sequences. Reduced H2AX levels in chromatin impair the density, but not the distance, of gamma-H2AX formed. Our data suggest that H2AX fuels a gamma-H2AX self-reinforcing mechanism that retains MDC1 and activated ATM in chromatin near DSBs and promotes continued local phosphorylation of H2AX
Histone H3 serine 10 phosphorylation is a hallmark of mitotic chromosomes, but its full function remains to be elucidated. We report here that two SR protein splicing factors, SRp20 and ASF/SF2, associate with interphase chromatin, are released from hyperphosphorylated mitotic chromosomes, but reassociate with chromatin late in M-phase. Inhibition of Aurora B kinase diminished histone H3 serine 10 phosphorylation and increased SRp20 and ASF/SF2 retention on mitotic chromosomes. Unexpectedly, we also found that HP1 proteins interact with ASF/SF2 in mitotic cells. Strikingly, siRNA-mediated knockdown of ASF/SF2 caused retention of HP1 proteins on mitotic chromatin. Finally, ASF/SF2-depleted cells released from a mitotic block displayed delayed G0/G1 entry, suggesting a functional consequence of these interactions. These findings underscore the evolving role of histone H3 phosphorylation and demonstrate a direct, functional, and histone-modification-regulated association of SRp20 and ASF/SF2 with chromatin
Many critical protein kinases rely on the Hsp90 chaperone machinery for stability and function. After initially forming a ternary complex with kinase client and the cochaperone p50(Cdc37), Hsp90 proceeds through a cycle of conformational changes facilitated by ATP binding and hydrolysis. Progression through the chaperone cycle requires release of p50(Cdc37) and recruitment of the ATPase activating cochaperone AHA1, but the molecular regulation of this complex process at the cellular level is poorly understood. We demonstrate that a series of tyrosine phosphorylation events, involving both p50(Cdc37) and Hsp90, are minimally sufficient to provide directionality to the chaperone cycle. p50(Cdc37) phosphorylation on Y4 and Y298 disrupts client-p50(Cdc37) association, while Hsp90 phosphorylation on Y197 dissociates p50(Cdc37) from Hsp90. Hsp90 phosphorylation on Y313 promotes recruitment of AHA1, which stimulates Hsp90 ATPase activity, furthering the chaperoning process. Finally, at completion of the chaperone cycle, Hsp90 Y627 phosphorylation induces dissociation of the client and remaining cochaperones.
In many prokaryotes, a specific DNA primase/polymerase (PolDom) is required for nonhomologous end-joining repair of DNA double-strand breaks. Here, we report the crystal structure of a catalytically active conformation of Mycobacterium tuberculosis PolDom, consisting of a polymerase bound to a DNA end with a 3’ overhang, two metal ions and an incoming nucleotide but, significantly, lacking a primer strand. This structure represents a unique example of a polymerase:DNA complex in a pre-ternary intermediate state. This polymerase intermediate occurs in solution, stabilizing the enzyme on DNA ends and promoting nucleotide extension off short incoming termini. We also demonstrate that an invariant Arg220, contained in a conserved loop (Loop 2), plays an essential role in catalysis by regulating binding of a second metal ion at the active site. We propose that this NHEJ intermediate facilitates extension reactions involving critically short or non-complementary DNA ends thus promoting break repair and minimizing sequence loss during DSB repair
Mammalian polynucleotide kinase 3′ phosphatase (PNK) plays a key role in the repair of DNA damage, functioning as part of both the nonhomologous end-joining (NHEJ) and base excision repair (BER) pathways. Through its two catalytic activities, PNK ensures that DNA termini are compatible with extension and ligation by either removing 3′-phosphates from, or by phosphorylating 5′-hydroxyl groups on, the ribose sugar of the DNA backbone. We have now determined crystal structures of murine PNK with DNA molecules bound to both of its active sites. The structure of ssDNA engaged with the 3′-phosphatase domain suggests a mechanism of substrate interaction that assists DNA end seeking. The structure of dsDNA bound to the 5′-kinase domain reveals a mechanism of DNA bending that facilitates recognition of DNA ends in the context of single-strand and double-strand breaks and suggests a close functional cooperation in substrate recognition between the kinase and phosphatase active sites
Yeast histone H2A is phosphorylated on Ser129 upon DNA damage, an event required for efficient repair. We show that phosphorylation occurs rapidly over a large region around DNA double-strand breaks (DSBs). Histone H4 acetylation is also important for DSB repair, and we found that the NuA4 HAT complex associates specifically with phospho-H2A peptides. A single NuA4 subunit, Arp4, is responsible for the interaction. The NuA4 complex is recruited to a DSB concomitantly with the appearance of H2A P-Ser129 and Arp4 is important for this binding. Arp4 is also a subunit of the Ino80 and Swr1 chromatin remodeling complexes, which also interact with H2A P-Ser129 and are recruited to DSBs. This association again requires Arp4 but also prior NuA4 recruitment and action. Thus, phosphorylation of H2A at DNA damage sites creates a mark recognized by different chromatin modifiers. This interaction leads to stepwise chromatin reconfiguration, allowing efficient DNA repair.
Saccharomyces WEE1 (Swe1), the only "true" tyrosine kinase in budding yeast, is an Hsp90 client protein. Here we show that Swe1(Wee1) phosphorylates a conserved tyrosine residue (Y24 in yeast Hsp90 and Y38 in human Hsp90 alpha) in the N domain of Hsp90. Phosphorylation is cell-cycle associated and modulates the ability of Hsp90 to chaperone a selected clientele, including v-Src and several other kinases. Nonphosphorylatable mutants have normal ATPase activity, support yeast viability, and productively chaperone the Hsp90 client glucocorticoid receptor. Deletion of SWE1 in yeast - increases Hsp90 binding to its inhibitor geldanamycin, and pharmacologic inhibition/silencing of Wee1 sensitizes cancer cells to Hsp90 inhibitor-induced apoptosis. These findings demonstrate that Hsp90 chaperoning of distinct client proteins is differentially regulated by specific posttranslational modification of a unique subcellular pool of the chaperone, and they provide a strategy to increase the cellular potency of Hsp90 inhibitors.
When inappropriate DNA structures arise, they are sensed by DNA structure-dependent checkpoint pathways and subsequently repaired. Recruitment of checkpoint proteins to such structures precedes recruitment of proteins involved in DNA metabolism. Thus, checkpoints can regulate DNA metabolism. We show that fission yeast Rad9, a 9-1-1 heterotrimeric checkpoint-clamp component, is phosphorylated by Hsk1(Cdc7), the Schizosaccharomyces pombe Dbf4-dependent kinase (DDK) homolog, in response to replication-induced DNA damage. Phosphorylation of Rad9 disrupts its interaction with replication protein A (RPA) and is dependent on 9-1-1 chromatin loading, the Rad9-associated protein Rad4/Cut5(TopBP1), and prior phosphorylation by Rad3(ATR). rad9 mutants defective in DDK phosphorylation show wild-type checkpoint responses but abnormal DNA repair protein foci and decreased viability after replication stress. We propose that Rad9 phosphorylation by DDK releases Rad9 from DNA damage sites to facilitate DNA repair.
Ku-dependent nonhomologous end joining (NHEJ) is a double-strand break repair process conserved in all branches of cellular life but has not previously been implicated in the DNA metabolic processes of viruses. We identified Ku homologs in Corndog and Omega, two related mycobacteriophages of Mycobacterium smegmatis. These proteins formed homodimers and bound DNA ends in a manner identical to other Ku's and stimulated joining of ends by the host NHEJ DNA ligase (LigD). Omega and Corndog are unusual in having short 4 base cos ends that would not be expected to self-anneal and would therefore require NHEJ during phage genome circularization. Consistently, M. smegmatis LigD null strains are entirely and selectively unable to support infection by Corndog or Omega, with concomitant failure of genome circularization. These results establish a new paradigm for sequestration of the host cell NHEJ process by bacteriophage and provide a framework for understanding similar transactions in eukaryotic viral infections.
Most types of DNA damage block replication fork progression during DNA synthesis because replicative DNA polymerases are unable to accommodate altered DNA bases in their active sites. To overcome this block, eukaryotic cells employ specialized translesion synthesis (TLS) polymerases, which can insert nucleotides opposite damaged bases. In particular, TLS by DNA polymerase ¿ (pol¿) is the major pathway for bypassing UV photoproducts. How the cell switches from replicative to TLS polymerase at the site of blocked forks is unknown. We show that, in human cells, PCNA becomes monoubiquitinated following UV irradiation of the cells and that this is dependent on the hRad18 protein. Monoubiquitinated PCNA but not unmodified PCNA specifically interacts with pol¿, and we have identified two motifs in pol¿ that are involved in this interaction. Our findings provide an attractive mechanism by which monoubiquitination of PCNA might mediate the polymerase switch.
Hsp90-mediated function of NLR receptors in plant and animal innate immunity depends on the cochaperone Sgt1 and, at least in plants, on a cysteine- and histidine-rich domains (CHORD)-containing protein Rar1. Functionally, CHORD domains are associated with CS domains, either within the same protein, as in the mammalian melusin and Chp1,or in separate but interacting proteins, as in the plant Ran l and Sgt1. Both CHORD and CS domains are independently capable of interacting with the molecular chaperone Hsp90 and can coexist in complexes with Hsp90. We have now determined the structure of an Hsp90-CS-CHORD ternary complex, providing a framework for understanding the dynamic nature of Hsp90-Rar1-Sgt1 complexes. Mutational and biochemical analyses define the architecture of the ternary complex that recruits nucleotide-binding leucine-rich repeat receptors (NLRs) by manipulating the structural elements to control the ATPase-dependent conformational cycle of the chaperone.
Activation of protein kinase clients by the Hsp90 system is mediated by the cochaperone protein Cdc37. Cdc37 requires phosphorylation at Ser13, but little is known about the regulation of this essential posttranslational modification. We show that Ser13 of uncomplexed Cdc37 is phosphorylated in vivo, as well as in binary complex with a kinase (C-K), or in ternary complex with Hsp90 and kinase (H-C-K). Whereas pSer13-Cdc37 in the H-C-K complex is resistant to nonspecific phosphatases, it is efficiently dephosphorylated by the chaperone-targeted protein phosphatase 5 (PP5/Ppt1), which does not affect isolated Cdc37. We show that Cdc37 and PP5/Ppt1 associate in Hsp90 complexes in yeast and in human tumor cells, and that PP5/Ppt1 regulates phosphorylation of Ser13-Cdc37 in vivo, directly affecting activation of protein kinase clients by Hsp90-Cdc37. These data reveal a cyclic regulatory mechanism for Cdc37, in which its constitutive phosphorylation is reversed by targeted dephosphorylation in Hsp90 complexes.
The Y family of DNA polymerases plays crucial roles in carrying out translesion synthesis past damaged bases in DNA. Several recent papers suggest that they might have other roles as well in gene conversion, in nucleotide excision repair (NER), and in DNA replication under stressed conditions.
The basic unit of chromatin in eukaryotes is the nucleosome, comprising 146 bp of DNA wound around two copies of each of four core histones. Chromatin is further condensed by association with linker histones. Saccharomyces cerevisiae Hho1p has sequence homology to other known linker histones and interacts with nucleosomes in vitro. However, disruption of HHO1 results in no significant changes in the phenotypes examined thus far. Here, we show that Hho1p is inhibitory to DNA repair by homologous recombination (HR). We find Hho1p is abundant and associated with the genome, consistent with a global role in DNA repair. Furthermore, we establish that Hho1p is required for a full life span and propose that this is mechanistically linked to its role in HR. Finally, we show that Hho1p is inhibitory to the recombination-dependent mechanism of telomere maintenance. The role of linker histones in genome stability, aging, and tumorigenesis is discussed.
Several studies have suggested that SUMO may participate in the regulation of heterochromatin, but direct evidence is lacking. Here, we present a direct link between sumoylation and heterochromatin stability. SUMO deletion impaired silencing at heterochromatic regions and induced histone H3 Lys4 methylation, a hallmark of active chromatin in fission yeast. Our findings showed that the SUMO-conjugating enzyme Hus5/Ubc9 interacted with the conserved heterochromatin proteins Swi6, Chp2 (a paralog of Swi6), and Clr4 (H3 Lys9 methyltransferase). Moreover, chromatin immunoprecipitation (ChIP) revealed that Hus5 was highly enriched in heterochromatic regions in a heterochromatin-dependent manner, suggesting a direct role of Hus5 in heterochromatin formation. We also found that Swi6, Chp2, and Clr4 themselves can be sumoylated in vivo and defective sumoylation of Swi6 or Chp2 compromised silencing. These results indicate that Hus5 associates with heterochromatin through interactions with heterochromatin proteins and modifies substrates whose sumoylations are required for heterochromatin stability, including heterochromatin proteins themselves.
Rho family GTPases are important cellular switches and control a number of physiological functions. Understanding the molecular basis of interaction of these GTPases with their effectors is crucial in understanding their functions in the cell. Here we present the crystal structure of the complex of Rac2 bound to the split pleckstrin homology (spPH) domain of phospholipase C-gamma(2) (PLC gamma(2)). Based on this structure, we illustrate distinct requirements for PLC gamma(2) activation by Rac and EGF and generate Rac effector mutants that specifically block activation of PLC gamma(2), but not the related PLC beta(2) isoform. Furthermore, in addition to the complex, we report the crystal structures of free spPH and Rac2 bound to GDP and GTP gamma S. These structures illustrate a mechanism of conformational switches that accompany formation of signaling active complexes and highlight the role of effector binding as a common feature of Rac and Cdc42 interactions with a variety of effectors.
The hereditary disorder ataxia telangiectasia (A-T) is associated with striking cellular radiosensitivity that cannot be attributed to the characterized cell cycle checkpoint defects. By epistasis analysis, we show that ataxia telangiectasia mutated protein (ATM) and Artemis, the protein defective in patients with RS-SCID, function in a common double-strand break (DSB) repair pathway that also requires H2AX, 53BP1, Nbs1, Mre11, and DNA-PK. We show that radiation-induced Artemis hyperphosphorylation is ATM dependent. The DSB repair process requires Artemis nuclease activity and rejoins approximately 10% of radiation-induced DSBs. Our findings are consistent with a model in which ATM is required for Artemis-dependent processing of double-stranded ends with damaged termini. We demonstrate that Artemis is a downstream component of the ATM signaling pathway required uniquely for the DSB repair function but dispensable for ATM-dependent cell cycle checkpoint arrest. The significant radiosensitivity of Artemis-deficient cells demonstrates the importance of this component of DSB repair to survival.
The replicative bypass of base damage in DNA (translesion DNA synthesis [TLS]) is a ubiquitous mechanism for relieving arrested DNA replication. The process requires multiple polymerase switching events during which the high-fidelity DNA polymerase in the replication machinery arrested at the primer terminus is replaced by one or more polymerases that are specialized for TLS. When replicative bypass is fully completed, the primer terminus is once again occupied by high-fidelity polymerases in the replicative machinery. This review addresses recent advances in our understanding of DNA polymerase switching during TLS in bacteria such as E. coli and in lower and higher eukaryotes.
For a cancer cell to resist treatment with drugs that trap topoisomerases covalently on the DNA, the topoisomerase must be removed. In this study, we provide evidence that the Schizosaccharomyces pombe Rad32(Mre11) nuclease activity is involved in the removal of both Top2 from 5' DNA ends as well as Top1 from 3' ends in vivo. A ctp1(ctIP) deletion is defective for Top2 removal but overproficient for Top1 removal, suggesting that Ctp1(ctIP) plays distinct roles in removing topoisomerases from 5' and 3' DNA ends. Analysis of separation of function mutants suggests that MRN-dependent topoisomerase removal contributes significantly to resistance against topoisomerase-trapping drugs. This study has important implications for our understanding of the role of the MRN complex and CtIP in resistance of cells to a clinically important group of anticancer drugs.
Ataxia Telangiectasia Mutated (ATM) signaling is essential for the repair of a subset of DNA double-strand breaks (DSBs); however, its precise role is unclear. Here, we show that ≤25% of DSBs require ATM signaling for repair, and this percentage correlates with increased chromatin but not damage complexity. Importantly, we demonstrate that heterochromatic DSBs are generally repaired more slowly than euchromatic DSBs, and ATM signaling is specifically required for DSB repair within heterochromatin. Significantly, knockdown of the transcriptional repressor KAP-1, an ATM substrate, or the heterochromatin-building factors HP1 or HDAC1/2 alleviates the requirement for ATM in DSB repair. We propose that ATM signaling temporarily perturbs heterochromatin via KAP-1, which is critical for DSB repair/processing within otherwise compacted/inflexible chromatin. In support of this, ATM signaling alters KAP-1 affinity for chromatin enriched for heterochromatic factors. These data suggest that the importance of ATM signaling for DSB repair increases as the heterochromatic component of a genome expands.
Ras proteins signal to a number of distinct pathways by interacting with diverse effectors. Studies of ras/effector interactions have focused on three classes, Raf kinases, ral guanylnucleotide-exchange factors, and phosphatidylinositol-3-kinases. Here we describe ras interactions with another effector, the recently identified phospholipase C epsilon (PLCepsilon). We solved structures of PLCepsilon RA domains (RA1 and RA2) by NMR and the structure of the RA2/ras complex by X-ray crystallography. Although the similarity between ubiquitin-like folds of RA1 and RA2 proves that they are homologs, only RA2 can bind ras. Some of the features of the RA2/ras interface are unique to PLCepsilon, while the ability to make contacts with both switch I and II regions of ras is shared only with phosphatidylinositol-3-kinase. Studies of PLCepsilon regulation suggest that, in a cellular context, the RA2 domain, in a mode specific to PLCepsilon, has a role in membrane targeting with further regulatory impact on PLC activity.
The mixed-lineage leukemia protein MLL1 is a transcriptional regulator with an essential role in early development and hematopoiesis. The biological function of MLL1 is mediated by the histone H3K4 methyltransferase activity of the carboxyl-terminal SET domain. We have determined the crystal structure of the MLL1 SET domain in complex with cofactor product AdoHcy and a histone H3 peptide. This structure indicates that, in order to form a well-ordered active site, a highly variable but essential component of the SET domain must be repositioned. To test this idea, we compared the effect of the addition of MLL complex members on methyltransferase activity and show that both RbBP5 and Ash2L but not Wdr5 stimulate activity. Additionally, we have determined the effect of posttranslational modifications on histone H3 residues downstream and upstream from the target lysine and provide a structural explanation for why H3T3 phosphorylation and H3K9 acetylation regulate activity.
Nicotinamide adenine dinucleotides have emerged as key signals of the cellular redox state. Yet the structural basis for allosteric gene regulation by the ratio of reduced NADH to oxidized NAD+ is poorly understood. A key sensor among Gram-positive bacteria, Rex represses alternative respiratory gene expression until a limited oxygen supply elevates the intracellular NADH:NAD+ ratio. Here we investigate the molecular mechanism for NADH/NAD+ sensing among Rex family members by determining structures of Thermus aquaticus Rex bound to (1) NAD+, (2) DNA operator, and (3) without ligand. Comparison with the Rex/NADH complex reveals that NADH releases Rex from the DNA site following a 40° closure between the dimeric subunits. Complementary site-directed mutagenesis experiments implicate highly conserved residues in NAD-responsive DNA-binding activity. These rare views of a redox sensor in action establish a means for slight differences in the nicotinamide charge, pucker, and orientation to signal the redox state of the cell.
ype II topoisomerases are essential for resolving topologically entwined double-stranded DNA. Although anti-topoisomerase 2 (Top2) drugs are clinically important antibiotics and chemotherapies, to our knowledge, the mechanisms of cell killing by Top2 depletion and inactivation have never been directly compared. We show that depletion of Top2 protein from budding yeast cells prevents DNA decatenation during S phase. Cells complete DNA replication and enter the ensuing mitosis on schedule, suffering extensive chromosome missegregation. Cytokinesis through incompletely segregated chromosomes causes lethal DNA damage. By contrast, expression of catalytically inactive Top2 causes a stable G2 arrest requiring an intact DNA damage checkpoint. Checkpoint activation correlates with an inability to complete DNA replication, resulting in hypercatenated, gapped daughter DNA molecules. Thus, Top2 depletion and inactivation kill cells by different mechanisms, which has implications for understanding the nature of the catenation checkpoint, how DNA replication terminates, how anti-Top2 drugs work, and how new drugs might be designed.
Template switching induced by stalled replication forks has recently been proposed to underlie complex genomic rearrangements. However, the resulting models are not supported by robust physical evidence. Here, we analyzed replication and recombination intermediates in a well-defined fission yeast system that blocks replication forks. We show that, in response to fork arrest, chromosomal rearrangements result from Rad52-dependent nascent strand template exchange occurring during fork restart. This template exchange occurs by both Rad51-dependent and -independent mechanisms. We demonstrate that Rqh1, the BLM homolog, limits Rad51-dependent template exchange without affecting fork restart. In contrast, we report that the Srs2 helicase promotes both fork restart and template exchange. Our data demonstrate that template exchange occurs during recombination-dependent fork restart at the expense of genome rearrangements.
Here Budiman et al. (2009) demonstrate that the selective translation of selenocysteine-containing proteins can be regulated by the mutually exclusive binding of eIF4a3 and SECIS binding protein 2 (SBP2) to a cis-acting element in the 3' untranslated region (3'UTR) of the target mRNA.
DNA polymerase eta is a Y family polymerase involved in translesion synthesis (TLS). Its action is initiated by simultaneous interaction between the PIP box in pol eta and PCNA and between the UBZ in pol eta and monoubiquitin attached to PCNA. Whereas monoubiquitination of PCNA is required for its interaction with pol eta during TLS, we now show that monoubiquitination of pol eta inhibits this interaction, preventing its functions in undamaged cells. Identification of monoubiquitination sites within pol eta nuclear localization signal (NLS) led to the discovery that pol eta NLS directly contacts PCNA, forming an extended pol eta-PCNA interaction surface. We name this the PCNA-interacting region (PIR) and show that its monoubiquitination is downregulated by various DNA-damaging agents. We propose that this mechanism ensures optimal availability of nonubiquitinated, TLS-competent pol eta after DNA damage. Our work shows how monoubiquitination can either positively or negatively regulate the assembly of a protein complex, depending on which substrates are targeted by ubiquitin.
The sliding clamp, PCNA, of the archaeon Sulfolobus solfataricus P2 is a heterotrimer of three distinct subunits (PCNA1, 2, and 3) that assembles in a defined manner. The PCNA heterotrimer, but not individual subunits, stimulates the activities of the DNA polymerase, DNA ligase I, and the flap endonuclease (FEN1) of S. solfataricus. Distinct PCNA subunits contact DNA polymerase, DNA ligase, or FEN1, imposing a defined architecture at the lagging strand fork and suggesting the existence of a preformed scanning complex at the fork. This provides a mechanism to tightly couple DNA synthesis and Okazaki fragment maturation. Additionally, unique subunit-specific interactions between components of the clamp loader, RFC, suggest a model for clamp loading of PCNA.
DNA ligase IV functions in DNA nonhomologous end-joining and V(D)J recombination. Four patients with features including immunodeficiency and developmental and growth delay were found to have mutations in the gene encoding DNA ligase IV (LIG4). Their clinical phenotype closely resembles the DNA damage response disorder, Nijmegen breakage syndrome (NBS). Some of the mutations identified in the patients directly disrupt the ligase domain while others impair the interaction between DNA ligase IV and Xrcc-4. Cell lines from the patients show pronounced radiosensitivity. Unlike NBS cell lines, they show normal cell cycle checkpoint responses but impaired DNA double-strand break rejoining. An unexpected V(D)J recombination phenotype is observed involving a small decrease in rejoining frequency coupled with elevated imprecision at signal junctions.
The telomerase enzyme, which synthesizes telomeric DNA repeats, is regulated in cis at individual chromosome ends by the telomeric protein/DNA complex in a manner dependent on telomere repeat-array length. A dynamic interplay between telomerase-inhibiting factors bound at duplex DNA repeats and telomerase-promoting ones bound at single-stranded terminal DNA overhangs appears to modulate telomerase activity and to be directly related to the transient deprotection of telomeres. We discuss recent advances on the mechanism of telomerase regulation at chromosome ends in both yeast and mammalian systems.
Target recognition in RNA silencing is governed by the "seed sequence" of a guide RNA strand associated with the PIWI/MID domain of an Argonaute protein in RISC. Using a reconstituted in vitro target recognition system, we show that a model PIWI/MID domain protein confers position-dependent tightening and loosening of guide-strand-target interactions. Over the seed sequence, the interaction affinity is enhanced up to approximately 300-fold. Enhancement is achieved through a reduced entropy penalty for the interaction. In contrast, interactions 3' of the seed are inhibited. We quantified mismatched target recognition inside and outside the seed, revealing amplified discrimination at the third position in the seed mediated by the PIWI/MID domain. Thus, association of the guide strand with the PIWI/MID domain generates an enhanced affinity anchor site over the seed that can promote fidelity in target recognition and stabilize and guide the assembly of the active silencing complex.
Changes in promoter structure and occupation have been shown to modify the splicing pattern of several genes, evidencing a coupling between transcription and alternative splicing. It has been proposed that the promoter effect involves modulation of RNA pol II elongation rates. The C4 point mutation of the Drosophila pol II largest subunit confers on the enzyme a lower elongation rate. Here we show that expression of a human equivalent to Drosophila's C4 pol II in human cultured cells affects alternative splicing of the fibronectin EDI exon and adenovirus E1a pre-mRNA. Most importantly, resplicing of the Hox gene Ultrabithorax is stimulated in Drosophila embryos mutant for C4, which demonstrates the transcriptional control of alternative splicing on an endogenous gene. These results provide a direct proof for the elongation control of alternative splicing in vivo.
Rad9, Rad1, and Hus1 form a heterotrimeric complex (9-1-1) that is loaded onto DNA at sites of DNA damage. DNA-loaded 9-1-1 activates signaling through the Chk1 arm of the DNA damage checkpoint response via recruitment and stimulation of ATR. Additionally, 9-1-1 may play a direct role in facilitating DNA damage repair via interaction with a number of DNA repair enzymes. We have now determined the crystal structure of the human 9-1-1 complex, revealing a toroidal structure with a similar architecture to the homotrimeric PCNA DNA-binding clamp. The structure explains the formation of a unique heterotrimeric arrangement and reveals significant differences among the three subunits in the sites implicated in binding to the clamp loader and to ligand proteins. Biochemical analysis reveals a single repair enzyme-binding site on 9-1-1 that can be blocked competitively by the PCNA-binding cell-cycle regulator p21
The mechanisms by which the progression of eukaryotic replication forks is controlled after DNA damage are unclear. We have found that fork progression is slowed by cisplatin or UV treatment in intact vertebrate cells and in replication assays in vitro. Fork slowing is reduced or absent in irs1SF CHO cells and XRCC3-/- chicken DT40 cells, indicating that fork slowing is an active process that requires the homologous recombination protein XRCC3. The addition of purified human Rad51C-XRCC3 complex restores fork slowing in permeabilized XRCC3-/- cells. Moreover, the requirement for XRCC3 for fork slowing can be circumvented by addition of human Rad51. These data demonstrate that the recombination proteins XRCC3 and Rad51 cooperatively modulate the progression of replication forks on damaged vertebrate chromosomes.
PARP-3 is a member of the ADP-ribosyl transferase superfamily of unknown function. We show that PARP-3 is stimulated by DNA double-strand breaks (DSBs) in vitro and functions in the same pathway as the poly (ADP-ribose)-binding protein APLF to accelerate chromosomal DNA DSB repair. We implicate PARP-3 in the accumulation of APLF at DSBs and demonstrate that APLF promotes the retention of XRCC4/DNA ligase IV complex in chromatin, suggesting that PARP-3 and APLF accelerate DNA ligation during nonhomologous end-joining (NHEJ). Consistent with this, we show that class switch recombination in Aplf−/− B cells is biased toward microhomology-mediated end-joining, a pathway that operates in the absence of XRCC4/DNA ligase IV, and that the requirement for PARP-3 and APLF for NHEJ is circumvented by overexpression of XRCC4/DNA ligase IV. These data identify molecular roles for PARP-3 and APLF in chromosomal DNA double-strand break repair reactions.
For a cancer cell to resist treatment with drugs that trap topoisomerases covalently on the DNA, the topoisomerase must be removed. In this study, we provide evidence that the Schizosaccharomyces pombe Rad32Mre11 nuclease activity is involved in the removal of both Top2 from 5′ DNA ends as well as Top1 from 3′ ends in vivo. A ctp1CtIP deletion is defective for Top2 removal but overproficient for Top1 removal, suggesting that Ctp1CtIP plays distinct roles in removing topoisomerases from 5′ and 3′ DNA ends. Analysis of separation of function mutants suggests that MRN-dependent topoisomerase removal contributes significantly to resistance against topoisomerase-trapping drugs. This study has important implications for our understanding of the role of the MRN complex and CtIP in resistance of cells to a clinically important group of anticancer drugs.
Nucleotide excision repair (NER) is the most versatile DNA repair system that deals with the major UV photoproducts in DNA, as well as many other DNA adducts. The early steps of NER are well understood, whereas the later steps of repair synthesis and ligation are not. In particular, which polymerases are definitely involved in repair synthesis and how they are recruited to the damaged sites has not yet been established. We report that, in human fibroblasts, approximately half of the repair synthesis requires both polκ and polδ, and both polymerases can be recovered in the same repair complexes. Polκ is recruited to repair sites by ubiquitinated PCNA and XRCC1 and polδ by the classical replication factor complex RFC1-RFC, together with a polymerase accessory factor, p66, and unmodified PCNA. The remaining repair synthesis is dependent on polɛ, recruitment of which is dependent on the alternative clamp loader CTF18-RFC.
The yeast single-strand TG-repeat telomere binding protein Cdc13 and the telomerase accessory protein Est1 play essential roles in chromosome end replication. To determine whether a proposed Cdc13-Est1 interaction recruits telomerase (Est2), we used a simplified system in which telomere formation was monitored at an HO-induced DNA double-strand break (DSB). Tethering of either Cdc13 or Est1 adjacent to a DSB promoted telomere formation, and tethering of Est1, even in the absence of a DSB, resulted in the recruitment of Est2. Est1 association with a DSB containing an adjacent short TG-repeat sequence depended on the Cdc13-Est1 interaction affected by cdc13-2 and est1-60 mutations, whereas Cdc13 association did not. Similarly, Est2 binding to the DSB also required the Cdc13-Est1 interaction, but not synthesis of new TG repeats at the break site. These data demonstrate a critical role for Est1 in recruiting telomerase to its site of action, in cooperation with the telomere binding protein Cdc13.
The hereditary disorder ataxia telangiectasia (A-T) is associated with striking cellular radiosensitivity that cannot be attributed to the characterized cell cycle checkpoint defects. By epistasis analysis, we show that ataxia telangiectasia mutated protein (ATM) and Artemis, the protein defective in patients with RS-SCID, function in a common double-strand break (DSB) repair pathway that also requires H2AX, 53BP1, Nbs1, Mre11, and DNA-PK. We show that radiation-induced Artemis hyperphosphorylation is ATM dependent. The DSB repair process requires Artemis nuclease activity and rejoins approximately 10% of radiation-induced DSBs. Our findings are consistent with a model in which ATM is required for Artemis-dependent processing of double-stranded ends with damaged termini. We demonstrate that Artemis is a downstream component of the ATM signaling pathway required uniquely for the DSB repair function but dispensable for ATM-dependent cell cycle checkpoint arrest. The significant radiosensitivity of Artemis-deficient cells demonstrates the importance of this component of DSB repair to survival.
Most types of DNA damage block replication fork progression during DNA synthesis because replicative DNA polymerases are unable to accommodate altered DNA bases in their active sites. To overcome this block, eukaryotic cells employ specialized translesion synthesis (TLS) polymerases, which can insert nucleotides opposite damaged bases. In particular, TLS by DNA polymerase eta (poleta) is the major pathway for bypassing UV photoproducts. How the cell switches from replicative to TLS polymerase at the site of blocked forks is unknown. We show that, in human cells, PCNA becomes monoubiquitinated following UV irradiation of the cells and that this is dependent on the hRad18 protein. Monoubiquitinated PCNA but not unmodified PCNA specifically interacts with poleta, and we have identified two motifs in poleta that are involved in this interaction. Our findings provide an attractive mechanism by which monoubiquitination of PCNA might mediate the polymerase switch.
The Sleeping Beauty (SB) element is a useful tool to probe transposon-host interactions in vertebrates. We investigated requirements of DNA repair factors for SB transposition in mammalian cells. Factors of nonhomologous end joining (NHEJ), including Ku, DNA-PKcs, and Xrcc4 as well as Xrcc3/Rad51C, a complex that functions during homologous recombination, are required for efficient transposition. NHEJ plays a dominant role in repair of transposon excision sites in somatic cells. Artemis is dispensable for transposition, consistent with the lack of a hairpin structure at excision sites. Ku physically interacts with the SB transposase. DNA-PKcs is a limiting factor for transposition and, in addition to repair, has a function in transposition that is independent from its kinase activity. ATM is involved in excision site repair and affects transposition rates. The overlapping but distinct roles of repair factors in transposition and in V(D)J recombination might influence the outcomes of these mechanistically similar processes.
The sliding clamp, PCNA, of the archaeon Sulfolobus solfataricus P2 is a heterotrimer of three distinct subunits (PCNA1, 2, and 3) that assembles in a defined manner. The PCNA heterotrimer, but not individual subunits, stimulates the activities of the DNA polymerase, DNA ligase I, and the flap endonuclease (FEN1) of S. solfataricus. Distinct PCNA subunits contact DNA polymerase, DNA ligase, or FEN1, imposing a defined architecture at the lagging strand fork and suggesting the existence of a preformed scanning complex at the fork. This provides a mechanism to tightly couple DNA synthesis and Okazaki fragment maturation. Additionally, unique subunit-specific interactions between components of the clamp loader, RFC, suggest a model for clamp loading of PCNA.