INCREASED DRUG AFFINITY AS THE MECHANISTIC BASIS FOR DRUG HYPERSENSITIVITY OF A MUTANT TYPE-II TOPOISOMERASE

Altered sensitivity of topoisomerase II to anticancer drugs profoundly affects the response of eukaryotic cells to these agents. Therefore, several approaches were employed to elucidate the mechanism of drug hy persensitivity of the mutant yeast type II topoisomerase, top2H1012Y. This mutant, which is similar to 5-fold hypersensitive to ellipticine, formed DNA cleavage complexes more rapidly than the wild-type yeast e nzyme in the presence of the drug. Conversely, no change in the rate o f DNA religation was observed. There was, however, a correlation betwe en increased cleavage rates and enhanced drug binding affinity. The ap parent dissociation constant for ellipticine in the mutant topoisomera se II . drug . DNA ternary complex was similar to 5-fold lower than in the wild-type ternary complex. Furthermore, the apparent K-D value fo r the mutant binary (topoisomerase II . drug) complex was similar to 2 -fold lower than the corresponding wild-type complex, indicating that drug hypersensitivity is intrinsic to the enzyme. These findings stron gly suggest that the enhanced ellipticine binding affinity for topoiso merase II is the mechanistic basis for drug hypersensitivity of top2H1 012Y.