EXPRESSION, DOMAIN-STRUCTURE, AND ENZYMATIC-PROPERTIES OF AN ACTIVE RECOMBINANT HUMAN DNA TOPOISOMERASE II-BETA
EXPRESSION, DOMAIN-STRUCTURE, AND ENZYMATIC-PROPERTIES OF AN ACTIVE RECOMBINANT HUMAN DNA TOPOISOMERASE II-BETA
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DOI：
10.1074/jbc.270.26.15739
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发表时间：
1995-06-30
影响因子：
4.8
通讯作者：
FISHER, LM
中科院分区：
文献类型：
--
作者：
AUSTIN, CA;MARSH, KL;FISHER, LM
人类细胞表达两种遗传上不同的 DNA 拓扑异构酶 II 亚型，即 α 和 β，它们催化 ATP 依赖性 DNA 链通道，是重要的抗肿瘤药物靶标。在此，我们首次报道了在半乳糖诱导物的控制下，通过在酵母穿梭载体中克隆拓扑异构酶 II β cDNA，成功地在酵母中过度表达人拓扑异构酶 II β。 将重组人拓扑异构酶 II beta（残基 46-1621 与酵母拓扑异构酶 II 的前 5 个残基融合）纯化至同质，产生足够数量的酶活性多肽，以分析其结构域结构并与重组人拓扑异构酶 II α 进行比较。用胰蛋白酶或蛋白酶 SV8 部分消化 β，产生约 130、90、62 和 45-50 kDa 的片段，这些片段源自蛋白质的三个有限且离散区域（A、B 和 C）的切割，表明存在至少四个结构域。重组人拓扑异构酶 II α 和 β 诱导的 DNA 断裂是由多种 观察到药物诱导的 DNA 断裂的异构体差异。这些对人类拓扑异构酶 II β 与 α 协同的研究应有助于确定它们在癌症化疗中的各自作用，并应有助于细胞毒性抗肿瘤药物的设计、靶向和测试。
Human cells express two genetically distinct isoforms of DNA topoisomerase II, alpha and beta, which catalyze ATP-dependent DNA strand passage and are an important antitumor drug target, Here we report for the first time the successful overexpression of human topoisomerase II beta in yeast by cloning a topoisomerase II beta cDNA in a yeast shuttle vector under the control of a galactose inducible promoter, Recombinant human topoisomerase II beta (residues 46-1621 fused to the first 5 residues of yeast topoisomerase II) was purified to homogeneity, yielding an enzymatically active polypeptide in sufficient quantity to allow analysis of its domain structure and comparison with that of recombinant human topoisomerase II alpha. Partial digestion of beta with either trypsin or protease SV8 generated fragments of approximately 130, 90, 62, and 45-50 kDa, arising from cleavage at three limited and discrete regions of the protein (A, B, and C) indicating the presence of at least four structural domains, Recombinant human topoisomerase II alpha and beta induced DNA breakage which was promoted by a variety of agents, Isoform differences in drug-induced DNA breakage were observed, These studies of human topoisomerase II beta in concert with alpha should aid the determination of their individual roles in cancer chemotherapy and should facilitate the design, targeting, and testing of cytotoxic antitumor agents.