A MUTATION IN YEAST TOP2 HOMOLOGOUS TO A QUINOLONE-RESISTANT MUTATION IN 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 result in resistance to quinolones frequently occur at Ser(83) or Ser(84) of the gyrA subunit. Mutations to Trp, Ala, and Leu have been identified, all of which confer high levels of quinolone resistance. Extensive segments of DNA gyrase are homologous to eukaryotic topoisomerase II, and Ser(741) of yeast TOP2 is homologous to Ser(83) of prokaryotic DNA gyrA. Introduction of the Ser(741) --> Trp mutation into yeast TOP2 confers resistance to 6,8-difluoro 7-(4'-hydroxyphenyl)-1-cyclopropyl-4-quinolone-3 carboxylic acid (CP 115,953), a fluoroquinolone with substantial activity against eukaryotic topoisomerase II, whereas changing Ser(741) to either Leu or Ala does not change sensitivity to quinolones. Interestingly, Ser(741) --> Trp in the yeast TOP2 also confers hypersensitivity to etoposide. Sensitivity to intercalating anti topoisomerase II agents such as amsacrine is not changed by any of the three mutations. The topoisomerase II protein carrying the Ser(741) --> Trp mutation was overexpressed and purified. The purified mutant enzyme had enhanced levels of etoposide stabilized covalent complex as compared with the wild type enzyme and reduced cleavage with CP-115,953. Unlike the wild type enzyme, etoposide-stabilized cleavage is not readily reversible by heat. We suggest that Ser(741) is near a binding site for both quinolones and etoposide and that the Ser(741) --> Trp mutation leads to a more stable ternary complex between etoposide, DNA, and the mutant enzyme.