Multiple Genome Stability Roles for SIZ-dependent Sumoylation in Saccharomyces cerevisiae

Conjugation of the small ubiquitin-like modifier SUMO is essential for viability in Saccharomyces cerevisiae. About 95% of sumoylation in budding yeast is catalyzed by E3 ligases, Siz1 and Siz2. This thesis analyzes multiple roles for SIZ-dependent sumoylation in genome stability. The siz1Δ siz2Δ mutant is sensitive to UV-irradiation and using genetic analyses I show that SIZ genes can be functionally grouped with genes that operate in the nucleotide excision repair pathway (NER). The NER pathway repairs DNA damage induced by UV irradiation. Biochemical evidence that quantifies removal of UV-dependent DNA damage illustrates that siz1Δ siz2Δ mutants have defects in NER. These defects were specific for two of the three NER subpathways, Rad16-dependent global genome repair and Rpb9-dependent transcription coupled repair (TCR), but not in the Rad26-associated TCR pathway. Furthermore, I provide evidence that demonstrates that several NER proteins, including the yeast XPC homolog Rad4, are sumoylated in a SIZ- and DNA damage-dependent manner. In the second part of this thesis, I investigate the Top1-dependent DNA damage that accumulates in siz1Δ siz2Δ mutants. Genetic analyses show that siz1Δ siz2Δ mutant is viable, but lethal when combined with the homologous recombination (HR) mutant rad52Δ. Triple mutant lethality is suppressed by deletion of the topoisomerase I (TOP1) gene. The goal of this study was to characterize the defective SUMO-dependent process in this mutant. I developed temperature sensitive degron strains that allow conditional induction of lethality. These strains undergo an S-phase dependent mitotic arrest and activate the Rad53 DNA damage checkpoint. Based on these and other data I conclude that Siz-dependent sumoylation has multiple roles in maintaining genome stability specifically in response to UV-induced and Top1-dependent DNA damage