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Topoisomerase II (Top2) is the primary target for active anti-cancer agents such as etoposide and doxorubicin. We have developed an efficient approach for identifying inhibitor hypersensitive Top2 mutants. We isolated a panel of mutants conferring hypersensitivity to the intercalator mAMSA. Two mutants in yeast Top2, changing Pro473Leu and Gly737Val, conferred extraordinary hypersensitivity to mAMSA were chosen for further characterization. Both mutations change amino acids that are conserved in all eukaryotic topoisomerases. The mutant proteins were overexpressed, purified, and their biochemical activities were assessed. Both mutants encode enzymes that are highly sensitive to inhibition by mAMSA and other intercalating agents. Both mutant enzymes exhibit elevated levels of mAMSA induced Top2:DNA covalent complexes. While Gly737Val Top2p generated elevated levels of Top2 mediated double strand breaks in vitro, the Pro473Leu mutant protein showed only a modest increase in Top2 mediated double strand breaks, but much higher levels of Top2 mediated single strand breaks. These observations led us to construct alleles of Top2 that could generate enzyme mediated single strand breaks, but not enzyme mediated double strand breaks. We observed that expression in yeast of a Pro473Leu mutant that could only generate single strand breaks was still able to confer hypersensitivity to mAMSA. These results indicate that generation of single strand breaks by Top2 targeting agents can be an important component of cell killing by Top2 targeting drugs. We are currently assessing how cells recognize and process Top2:DNA covalent complexes that contain single strand breaks. These results suggest the importance of exploring the detailed mechanisms of action of different classes of Top2 poisons.

98th AACR Annual Meeting-- Apr 14-18, 2007; Los Angeles, CA