Eukaryotic DNA topoisomerase I (Top1p) has important functions in DNA replication, transcription, and recombination. This enzyme also constitutes the cellular target of camptothecin (CPT), which induces S-phase-dependent cytotoxicity. To define cellular pathways that regulate cell sensitivity to Top1p-induced DNA lesions, we described a yeast genetic screen for conditional tah (top1T722A-hypersensitive) mutants with enhanced sensitivity to low levels of the CPT mimetic mutant top1T722A (Reid, R. J., Fiorani, P., Sugawara, M., and Bjornsti, M. A. (1999) Proc. Natl. Acad. Sci. U. S. A. 96, 11440-11445; Fiorani, P., Reid, R. J., Schepis, A., Jacquiau, H. R., Guo, H., Thimmaiah, P., Benedetti, P., and Bjornsti, M. A. (2004) J. Biol. Chem. 279, 21271-21281). Here we report that tah mutant ubc9-10 harbors a hypomorphic allele of UBC9, which encodes the essential SUMO (small ubiquitin-related modifier) E2-conjugating enzyme. The same conditional ubc9P123L mutant was also isolated in an independent screen for enhanced sensitivity to a distinct Top1p poison, Top1N726Hp. The ubc9-10 mutant exhibited a decrease in global protein sumoylation and increased sensitivity to a wide range of DNA-damaging agents independent of Top1p. Deletion of the Ulp2 SUMO protease failed to restore ubc9-10 cell resistance to Top1p poisons or hydroxyurea yet adversely affected wild-type TOP1 cell genetic stability and sensitivity to hydroxyurea. Moreover, although mutation of different consensus SUMO sites in the N terminus and linker region of yeast Top1p failed to recapitulate ubc9-10 mutant phenotypes, they revealed distinct and subtle effects on cell sensitivity to CPT. These results provide insights into the complexities of SUMO conjugation and the confounding effects of SUMO modification on Top1p function and cell sensitivity to genotoxic agents. © 2005 by The American Society for Biochemistry and Molecular Biology, Inc.