Eukaryotic DNA topoisomerase I (Top1p) plays important roles in DNA replication, transcription and recombination, and catalyzes changes in DNA topology through the transient breakage and rejoining of a single DNA strand in duplex DNA. Top1p is also the target of camptothecin (CRT), which reversibly stabilizes a covalent enzyme-DNA intermediate. During S-phase, replication fork collisions with CRT-stabilized complexes produce DNA lesions that signal cell cycle arrest and cell death. However, little is known about the nature of the lesions produced and the cellular processes that resolve Top1p-induced DNA damage. Post-translational protein modifications by ubiquitin and ubiquitin-like proteins (such as SUMO) have emerged as critical regulatory mechanisms governing cell cycle progression, DNA repair, endocytosis, nucleocytoplasmic trafficking, transcription, chromatin structure and cell division. SUMO is attached to target proteins via an isopeptide linkage by the sequential action of an E1, E2 (Ubc9) and, in some cases, E3 enzymes. Sumoylation of human Top1p has been associated with alterations in nucleolar localization and CRT sensitivity. However, the mechanisms regulating SUMO conjugation of Top1p and the functional consequences of sumoylation on cellular pathways required for the effective repair of DNA lesions induced by chemotherapeutic agents that target Top1p have yet to be addressed. The goal of this proposal is to define the role of Ubc9-catalyzed sumoylation in protecting cells from Top1p poisons in the yeast Saccharomyces cerevisiae. Highly conserved mechanisms of Top1p poisoning and SUMO conjugation, in concert with the isolation of a conditional ubc9-10 mutant with enhanced sensitivity to Top1p-induced damage, makes this genetically tractable system particularly suited to the study of sumoylation and Top1p-mediated lethality. Genetic interactions between the SUMO protease, Ulp2p and Top1p in maintaining genomic stability will also be investigated. To accomplish this, integrated biochemical, structural and genetics analyses will assess the defects in Ubc9p substrate specificity, reveal the structural basis for these alterations and define the cellular processes whose function in modulating resistance to Top1p poisons is affected by defects in SUMO conjugation. Human/yeast Ubc9p chimeras will further define residues that dictate substrate specificity. Additional studies will determine if similar mechanisms regulate ulp2Adelta cell sensitivity to Top1p levels and genomic stability.
