Knab A M, Fertala J, Bjornsti M A
Department of Biochemistry and Molecular Biology, Thomas Jefferson University, Philadelphia, Pennsylvania 19107.
J Biol Chem. 1995 Mar 17;270(11):6141-8. doi: 10.1074/jbc.270.11.6141.
The cytotoxic plant alkaloid camptothecin promotes DNA topoisomerase I-linked nicks in DNA by stabilizing a covalently bound enzyme-DNA complex. In the yeast Saccharomyces cerevisiae, substitution of Arg and Ala for the amino acid residues immediately N-terminal to the active site tyrosine in the yeast and human DNA topoisomerase I mutants, top1 vac, results in camptothecin resistance. To examine the mechanism of drug resistance, we assessed the sensitivity of these enzymes to several classes of DNA topoisomerase poisons. Yeast cells expressing the camptothecin-resistant top1 vac mutants were resistant to all of the camptothecin derivatives cytotoxic to wild-type TOP1-expressing cells. This correlated with a significant reduction in drug-induced DNA cleavage in vitro. However, the yeast and human mutant enzymes differed in their responses to the minor groove binding ligand netropsin and to saintopin, a DNA intercalator that targets both DNA topoisomerase I and II. The yeast mutant enzyme demonstrated enhanced sensitivity to the action of saintopin but was resistant to the inhibitory effects of netropsin. In contrast, the human Top1 vac enzyme was resistant to saintopin and indistinguishable from the wild-type enzyme in its response to the netropsin. These results are discussed in terms of enzyme function and the different modes of action of these DNA topoisomerase poisons.
细胞毒性植物生物碱喜树碱通过稳定共价结合的酶 - DNA复合物来促进DNA中与拓扑异构酶I相关的切口。在酿酒酵母中，将酵母和人类DNA拓扑异构酶I突变体top1 vac活性位点酪氨酸紧邻的N端氨基酸残基替换为精氨酸和丙氨酸，会导致对喜树碱产生抗性。为了研究耐药机制，我们评估了这些酶对几类DNA拓扑异构酶毒物的敏感性。表达对喜树碱耐药的top1 vac突变体的酵母细胞对所有对表达野生型TOP1的细胞具有细胞毒性的喜树碱衍生物均有抗性。这与体外药物诱导的DNA切割显著减少相关。然而，酵母和人类突变酶对小沟结合配体纺锤菌素以及对圣托品（一种靶向DNA拓扑异构酶I和II的DNA嵌入剂）的反应有所不同。酵母突变酶对圣托品的作用表现出增强的敏感性，但对纺锤菌素的抑制作用具有抗性。相比之下，人类Top1 vac酶对圣托品具有抗性，并且在对纺锤菌素的反应方面与野生型酶没有区别。我们根据酶的功能以及这些DNA拓扑异构酶毒物的不同作用模式对这些结果进行了讨论。
