2399

DNA topoisomerase I (Top1p) functions in DNA replication, transcription and recombination by catalyzing changes in DNA topology. The enzyme transiently cleaves one strand of duplex DNA via the formation of a covalent enzyme-DNA intermediate. Camptothecin (CPT) targets Top1 by stabilizing the Top1-DNA complex, inducing S-phase dependent cell death. To define cellular processes that function in the recognition and repair of CPT-induced DNA lesions, conditional yeast mutants exhibiting enhanced sensitivity to Top1 poisons at 36°C were isolated. Two self-poisoning top1 mutants were used in these screens: Top1T722A mimics CPT by inhibiting DNA religation, while Top1N726H exhibits increased rates of DNA cleavage. These enzymes induce distinct DNA lesions, yet, the same Pro123 to Leu mutation in the SUMO conjugating enzyme, Ubc9, was identified in a ubc9-10 mutant isolated in both screens. Ubc9 is a conserved, essential enzyme that acts alone or in concert with E3 ligases to covalently attach the ubiquitin-like protein, SUMO, to lysine residues in target proteins. Sumoylation may alter protein activity, sub-cellular localization or stability. Mutant ubc9-10 cells were hypersensitive to CPT and other DNA damaging agents, such as Hydroxyurea (HU), UV light and MMS, at 36°C without detectable alterations in Top1 protein levels or activity. These phenotypes differ from the contradictory effects that mutating consensus SUMO sites in Top1 had on CPT sensitivity. Mutation of Lys residues in N-terminal SUMO sites in Top1T722A suppressed cell sensitivity to CPT. In wild-type Top1, mutation of the same SUMO sites, or a SUMO site in the linker domain enhanced cell sensitivity to CPT. However, as the Top1-sumo mutants failed to recapitulate the pattern or extent of ubc9-10 cell sensitivity to Top1 poisons, SUMO conjugation affects additional factors that protect cells from Top1 poisons and other genotoxic stress. Indeed, several lines of evidence suggest such a role for the Ulp2, an isopeptidase that catalyzes SUMO deconjugation, and the Rad6 ubiquitin conjugating enzyme. First, the ubc9-10 mutant complemented the essential function of the Ulp2 protease at 36°C, but not cell hypersensitivity to HU and CPT. Moreover, cells deleted for ULP2 were extremely sensitive to Top1 protein levels. Second, although the single mutants are viable in the absence of DNA damage, the ubc9-10,rad6Δ double mutant was inviable at 36°C. This synthetic lethal phenotype indicates a common essential function is abrogated by alterations in Rad6 and Ubc9. rad6Δ cells were also hypersensitive to CPT. As PCNA function in error-free and error-prone postreplicative repair is modulated by Rad6 and Ubc9-dependent modifications, defects in Ubc9 conjugation may alter cell sensitivity to Top1 poisons through direct effects on postreplication repair. Supported by NIH grant CA23099 and ALSAC.

[Proc Amer Assoc Cancer Res, Volume 46, 2005]