Wasserman R A, Austin C A, Fisher L M, Wang J C
Department of Biochemistry and Molecular Biology, Harvard University, Cambridge, Massachusetts 02138.
Cancer Res. 1993 Aug 1;53(15):3591-6.
A plasmid was constructed for the expression of human DNA topoisomerase II alpha in yeast from a galactose-inducible promoter of the yeast GAL1 gene. Expression of a recombinant human enzyme, in which the first 28 of the 1531 codons of human DNA topoisomerase II alpha were replaced by the first five codons of yeast DNA topoisomerase II, was shown to rescue the lethal phenotype of thermal sensitive yeast DNA topoisomerase II mutants at 35 degrees C. Purification of the human enzyme overexpressed in yeast yielded a single polypeptide with an apparent mass of 170 kDa, and the properties of the purified recombinant enzyme were found to be the same as those reported for human DNA topoisomerase II alpha purified from HeLa cells. Studies with the anticancer drug amsacrine indicated that the human enzyme, either inside yeast cells or in its purified form, is a target of the drug; inhibition of the purified enzyme by teniposide (VM-26) and merbarone was also demonstrated. These studies demonstrate that yeast strains expressing human DNA topoisomerase II alpha provide a convenient system for studying drugs targeting the enzyme; unlike mammalian systems, potential complications due to the presence of human DNA topoisomerase II beta can be eliminated in this system. Overexpression of human DNA topoisomerase II alpha in yeast also provides a convenient source of the enzyme for in vitro studies.
构建了一种质粒，用于在酵母中从酵母GAL1基因的半乳糖诱导型启动子表达人DNA拓扑异构酶IIα。已证明，重组人酶（其中人DNA拓扑异构酶IIα的1531个密码子中的前28个被酵母DNA拓扑异构酶II的前五个密码子取代）可挽救热敏感酵母DNA拓扑异构酶II突变体在35℃时的致死表型。在酵母中过表达的人酶的纯化产生了一种表观质量为170 kDa的单一多肽，并且发现纯化的重组酶的性质与从HeLa细胞纯化的人DNA拓扑异构酶IIα的性质相同。用抗癌药安吖啶进行的研究表明，人酶无论是在酵母细胞内还是其纯化形式下，都是该药物的作用靶点；也证明了替尼泊苷（VM - 26）和美巴龙对纯化酶的抑制作用。这些研究表明，表达人DNA拓扑异构酶IIα的酵母菌株为研究靶向该酶的药物提供了一个便利的系统；与哺乳动物系统不同，该系统可以消除由于人DNA拓扑异构酶IIβ的存在而导致的潜在并发症。在酵母中过表达人DNA拓扑异构酶IIα也为体外研究提供了一个便利的酶来源。
