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 YEAST TYPE-II TOPOISOMERASE SELECTED FOR RESISTANCE TO QUINOLONES - MUTATION OF HISTIDINE-1012 TO TYROSINE CONFERS RESISTANCE TO NONINTERCALATIVE DRUGS BUT HYPERSENSITIVITY TO ELLIPTICINE
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DOI：
10.1074/jbc.270.4.1913
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发表时间：
1995-01-27
影响因子：
4.8
通讯作者：
OSHEROFF, N
中科院分区：
文献类型：
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作者：
ELSEA, SH;HSIUNG, YC;OSHEROFF, N
通过体外诱变和体内筛选对喹诺酮类药物CP-115,953的抗性，获得了酵母II型拓扑异构酶突变体。由此产生的突变酶具有单点突变，将His（1012）转化为Tyr （top2H1012Y）。top2H1012Y在酵母中过表达、纯化和体外鉴定，突变型II型拓扑异构酶活性略低于野生型酶，显然是由于对DNA的亲和力降低。突变酶对ATP的亲和力与野生型拓扑异构酶ZI相似。DNA切割实验表明，在DNA链传递事件之前和之后，top2H1012Y对CP-115,953和依托泊苷都具有抗性。与此形成鲜明对比的是，突变酶对amsacrine表现出野生型敏感性，对ellipticine表现出数倍的超敏感性。在含有top2H1012Y等位基因的酵母细胞中也观察到类似的抗性模式。因此，突变型II型拓扑异构酶似乎可以区分非插入性和插入性药物。最后，His(1012) -> Tyr突变在真核拓扑异构酶II上定义了一个潜在的新的耐药区域。
A mutant yeast type II topoisomerase was generated by in vitro mutagenesis 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 topoisomerase was slightly less active than the wild type enzyme, apparently due to a decreased affinity for DNA. The affinity of the mutant enzyme for ATP was similar to that of wild type topoisomerase ZI. As determined by DNA cleavage assays, top2H1012Y was resistant to CP-115,953 and etoposide both prior to and following the DNA strand-passage event. In marked contrast, the mutant enzyme displayed wild type sensitivity to amsacrine and was severalfold hypersensitive to ellipticine, A similar pattern of resistance was observed in yeast cells harboring the top2H1012Y allele. Thus, it appears that the mutant type II topoisomerase can distinguish between nonintercalative and intercalative agents, Finally, the His(1012) --> Tyr mutation defines a potential new drug resistance-conferring region on eukaryotic topoisomerase II.