FUNCTION AND BIOLOGY OF EUKARYOTIC DNA TOPOISOMERASES

拓扑异构酶II是调节拓扑异构酶的必需酶。 DNA的一种状态，通过一个完整的螺旋通过一个短暂的双链 在一个单独的螺旋中产生的断裂。 酶是必需的 正确的染色体结构和分离，并发挥重要作用， DNA复制和重组。 超越其关键的生理 功能，拓扑异构酶II是几个最活跃的目标 目前用于治疗人类恶性肿瘤的抗癌剂。 这些药物 通过一种明显不同的机制引发它们的细胞毒性作用， 比其他酶靶向药物的效果更好。 而不是抑制 酶的催化活性，抗癌药物大大增加 共价拓扑异构酶II切割的DNA复合物的水平是正常的， 但短暂的中间体在酶的催化循环。 因此，在本发明中， 这些药物毒害拓扑异构酶II并将其从必需的 酶转化为生理毒素，在处理的细胞中产生DNA损伤。 尽管拓扑异构酶II对真核生物的生存能力很重要， 细胞之间的相互作用以及人类癌症的治疗， 酶，其DNA和ATP底物，以及抗癌药物尚未被发现。 明确的特征。 因此，本提案的最终目的 是为了进一步阐明拓扑异构酶II进行 它的基本反应和抗癌药物改变的机制 酶的催化功能。 更具体地说，其目的是 建议1）进一步明确拓扑异构酶的催化机理 II，2）进一步描述抗癌药物 增加拓扑异构酶II-DNA切割复合物水平，和3） 确定酶产生耐药性的机制，或 对抗癌药物过敏。 本研究的主要研究模型将是黑腹果蝇 和酵母（Saccharomyces cerevisiae）。 果蝇和酵母II型 酶是任何真核拓扑异构酶（I）的最好的特征 或II）和酵母允许一定程度的遗传操作， II过表达，这是任何其他真核系统无法比拟的。 的 建议的研究将利用几种最近开发的测定法。 拓扑异构酶II的催化机制将通过以下来表征： 分析其与基质的相互作用，并确定它如何 切割DNA，催化DNA链通过，并选择DNA位点 乳沟 抗癌药物的作用机制将通过 确定三元酶-药物-DNA复合物如何形成以及药物如何 与拓扑异构酶II相互作用并影响其催化活性。 最后， 拓扑异构酶II对抗癌药物敏感性的改变 将通过产生和表征突变酶来评估， 对这些药剂有抗性或过敏。

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.