An investigation into the function of sumoylation in genomic stability in schizosaccharomyces pombe

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.