A YEAST TYPE-II TOPOISOMERASE SELECTED FOR RESISTANCE TO QUINOLONES -MUTATION OF HISTIDINE-1012 TO TYROSINE CONFERS RESISTANCE TO NONINTERCALATIVE DRUGS BUT HYPERSENSITIVITY TO ELLIPTICINE

A mutant yeast type II topoisomerase was generated by in vitro mutagen esis followed by selection in vivo for resistance to the quinolone CP- 115,953. The resulting mutant enzyme had a single point mutation which converted His(1012) to Tyr (top2H1012Y). top2H1012Y was overexpressed in yeast, purified, and characterized in vitro, The mutant type II to poisomerase was slightly less active than the wild type enzyme, appare ntly due to a decreased affinity for DNA. The affinity of the mutant e nzyme for ATP was similar to that of wild type topoisomerase ZI. As de termined by DNA cleavage assays, top2H1012Y was resistant to CP-115,95 3 and etoposide both prior to and following the DNA strand-passage eve nt. In marked contrast, the mutant enzyme displayed wild type sensitiv ity to amsacrine and was severalfold hypersensitive to ellipticine, A similar pattern of resistance was observed in yeast cells harboring th e top2H1012Y allele. Thus, it appears that the mutant type II topoisom erase can distinguish between nonintercalative and intercalative agent s, Finally, the His(1012) --> Tyr mutation defines a potential new dru g resistance-conferring region on eukaryotic topoisomerase II.