FUNCTION AND BIOLOGY OF EUKARYOTIC DNA TOPOISOMERASES

拓扑异构酶II是一种调节拓扑结构的重要酶 DNA的状态通过一个完整的螺旋通过一个瞬时的双链 打破它在一个单独的螺旋中产生的。这种酶是需要的 适当的染色体结构和分离，并在 DNA复制和重组。超越了它的关键生理学 功能上，拓扑异构酶II是几个最活跃的靶标 目前用于治疗人类恶性肿瘤的抗癌药物。这些药物 通过一种明显不同的机制来诱导它们的细胞毒作用 比其他以酶为靶标的药物更有效。而不是抑制 酶的催化活性、抗癌药物的大幅提高 正常的共价拓扑异构酶II裂解的DNA复合体水平， 但在酶的催化循环中，中间体是转瞬即逝的。因此， 这些药物毒化拓扑异构酶II，并将其从必需的 酶转化为一种生理毒素，在处理过的细胞中产生DNA损伤。 尽管拓扑异构酶II对真核生物的生存能力很重要 对于人类癌症的治疗，细胞之间的相互作用 酶，它的DNA和ATP底物，以及抗癌药物 具有明确的特征。因此，这项建议的最终目标是 是为了进一步描述拓扑异构酶II执行的机制 它的基本反应和抗癌药物改变的机制 酶的催化作用。更具体地说，这样做的目的是 建议1)进一步明确拓扑异构酶的催化机理 2)进一步阐明抗癌药物的作用机制(S) 增加拓扑异构酶II-DNA裂解复合体的水平，以及3) 确定酶产生抗药性的机制(S) 对抗癌药物过敏。 本研究的主要研究模型为黑腹果蝇 和酵母菌(酿酒酵母)。果蝇和酵母菌II型 酶是任何真核生物拓扑异构酶(I)中最具特性的 或ii)和酵母允许一定程度的遗传操作一种拓扑异构酶 II的过度表达是任何其他真核系统所无法比拟的。这个 拟议的研究将利用最近开发的几种分析方法。 拓扑异构酶II的催化机理将通过 分析与其底物的相互作用，并确定它如何 切割DNA，催化DNA链传递，并选择DNA的位置 乳沟。抗癌药物的作用机制将通过 确定酶-药物-DNA三元复合体是如何形成的，以及药物是如何形成的 与拓扑异构酶II相互作用，影响其催化活性。最后， 拓扑异构酶II对抗癌药物敏感性的变化 将通过产生和表征突变酶来进行评估 对这些药剂具有抵抗力或过敏性。

Topoisomerase II is an essential enzyme that modulates the topological state of DNA by passing an intact helix through a transient double-stranded break that it generates in a separate helix. The enzyme is required for proper chromosome structure and segregation and plays important roles in DNA replication and recombination. Beyond its critical physiological functions, topoisomerase II is the target for several of the most active anticancer agents currently used to treat human malignancies. These drugs elicit their cytotoxic effects by a mechanism that is markedly different than those of other enzyme-targeted agents. Rather than inhibiting the catalytic activity of the enzyme, anticancer drugs dramatically increase levels of covalent topoisomerase II-cleaved DNA complexes that are normal, but fleeting, intermediates ina the catalytic cycle of the enzyme. Thus, these agents poison topoisomerase II and convert it from an essential enzyme to a physiological toxin that generates DNA damage in treated cells. Despite the importance of topoisomerase II to the viability of eukaryotic cells and to the treatment of human cancers, interactions between the enzyme, its DNA and ATP substrates, and anticancer drugs have not been definitively characterized. Therefore, the ultimate goal of this proposal is to further delineate the mechanism by which topoisomerase II carries out its fundamental reactions and the mechanism by which anticancer drugs alter the catalytic function of the enzyme. More specifically, the aims of this proposal are 1) to further define the catalytic mechanism of topoisomerase II, 2) to further delineate the mechanism(s) by which anticancer drugs increase levels of topoismerase II-DNA cleavage complexes, and 3) to determine the mechanism(s) by which the enzyme becomes resistant or hypersensitive to anticancer drugs. The primary research models for this study will be Drosophila melanogaster and yeast (Saccharomyces cerevisiae). The Drosophila and yeast type II enzymes are the most well characterized of any eukaryotic topoisomerase (I or II) and yeast allows a degree of genetic manipulation an topoisomerase II overexpression that is unmatched by any other eukaryotic system. The proposed studies will take advantage of several recently developed assays. The catalytic mechanism of topoisomerase II will be characterized by analyzing interactions with its substrates and by determining how it cleaves DNA, catalyzes DNA strand passage, and selected sites of DNA cleavage. The mechanism of anticancer drug action will be addressed by determining how the ternary enzyme-drug-DNA complex is formed and how drugs interact with topoisomerase II and affect its catalytic activity. Finally, alterations in the sensitivity of topoisomerase II toward anticancer drugs will be assessed by generating and characterizing mutant enzymes that are either resistant or hypersensitive to these agents.