A MUTATION IN YEAST TOP2 HOMOLOGOUS TO A QUINOLONE-RESISTANT MUTATIONIN BACTERIA - MUTATION OF THE AMINO-ACID HOMOLOGOUS TO SER(83) OF ESCHERICHIA-COLI GYRA ALTERS SENSITIVITY TO EUKARYOTIC TOPOISOMERASE INHIBITORS

In prokaryotic type II topoisomerases (DNA gyrases), mutations that re sult in resistance to quinolones frequently occur at Ser(83) or Ser(84 ) of the gyrA subunit. Mutations to Trp, Ala, and Leu have been identi fied, all of which confer high levels of quinolone resistance. Extensi ve segments of DNA gyrase are homologous to eukaryotic topoisomerase I I, and Ser(741) of yeast TOP2 is homologous to Ser(83) of prokaryotic DNA gyrA. Introduction of the Ser(741) --> Trp mutation into yeast TOP 2 confers resistance to 6,8-difluoro 7-(4'-hydroxyphenyl)-1-cyclopropy l-4-quinolone-3 carboxylic acid (CP 115,953), a fluoroquinolone with s ubstantial activity against eukaryotic topoisomerase II, whereas chang ing Ser(741) to either Leu or Ala does not change sensitivity to quino lones. Interestingly, Ser(741) --> Trp in the yeast TOP2 also confers hypersensitivity to etoposide. Sensitivity to intercalating anti topoi somerase II agents such as amsacrine is not changed by any of the thre e mutations. The topoisomerase II protein carrying the Ser(741) --> Tr p mutation was overexpressed and purified. The purified mutant enzyme had enhanced levels of etoposide stabilized covalent complex as compar ed with the wild type enzyme and reduced cleavage with CP-115,953. Unl ike the wild type enzyme, etoposide-stabilized cleavage is not readily reversible by heat. We suggest that Ser(741) is near a binding site f or both quinolones and etoposide and that the Ser(741) --> Trp mutatio n leads to a more stable ternary complex between etoposide, DNA, and t he mutant enzyme.