Analysis of genome stability in mutants defective for the SUMO isopeptidase Smt4/Ulp2

Post-translational modification by the s&barbelow;mall u&barbelow;biquitin-like modifier (SUMO) is an important determinant of genome maintenance and transmission. In the budding yeast Saccharomyces cerevisiae, mutants defective for the SUMO isopeptidase Smt4/Ulp2 fail to remove and correctly process SUMO moieties, resulting in the accumulation of aberrant SUMO conjugates. Associated with this biochemical defect, Deltasmt4/ulp2 mutants exhibit a range of genome instability phenotypes---including reduced cell viability. DNA damage sensitivity, and chromosome loss. Smt4/Ulp2-related isopeptidases are found throughout eukaryotic cells, indicating conserved roles for desumoylation in controlling genome integrity.;This dissertation examines mechanisms through which Smt4/Ulp2 acts to maintain genome stability. Based on this work, one function for Smt4/Ulp2 appears to be to maintain high-fidelity DNA replication. Homologous recombination is elevated in Deltasmt4/ulp2 strains and post-replication DNA repair is necessary to sustain Deltasmt4/ulp2 viability. Furthermore. Deltasmt4/ulp2 cells display a high level of spontaneous Rad52 DNA repair foci, and the frequency of these foci is greatly elevated following exposure to DNA replication inhibitors. Importantly, repair foci also persist for extended periods during recovery from replication stress, indicating a second function for Smt4/Ulp2 in terminating DNA recombination. Sumoylation of the non-replicative helicase Sgs1 has been proposed to regulate unwinding of DNA recombination intermediates, raising the possibility that Sgs1 and Smt4/Ulp2 act in a common pathway. In support of this, MMS treated Delta smt4/ulp2 mutants are similar to sgs1 strains in that they accumulate an "X-spike" DNA structure indicative of persistent recombination, and sumoylated forms of Sgs1 accumulate in Delta smt4/ulp2 strains. Thus, Sgs1 may be one SUMO substrate that is targeted by Smt4/Ulp2 to resolve recombination intermediates.;Perturbations to DNA replication forks are often associated with genome instability and gross chromosomal rearrangements (GCRs). Surprisingly, it proved difficult to recover GCRs in Deltasmt4/ulp2 strains. In examining the basis for this, it was found that Deltasmt4/ulp2 cells experience a lethal mitotic catastrophe during recovery from replication stress due to an inability to disjoin and segregate sister chromatids. I propose failure to resolve recombination intermediates linking sister chromatids may preferentially kill those Deltasmt4/ulp2 cells that would give rise to GCRs, suggesting a possible strategy for eliminating genetically unstable cells.