Biochemical Analysis of Type II DNA Topoisomerases

描述(由申请人提供)： 这项建议的长期目标是从结构上确定II型拓扑异构酶的功能和药物抑制机制。II型拓扑异构酶是一类普遍存在的蛋白质，它利用三磷酸腺苷来主动运输一种DNA双链通过另一种DNA双链。这一反应维持了适当的DNA超螺旋水平，并解决了细胞中致命的染色体缠结。真核细胞的II型拓扑异构酶也被某些抑制物所利用，这些抑制剂是癌症的一线临床治疗方法。本提案的重点是真核细胞拓扑异构酶II和古生菌拓扑异构酶VI，它们分别是IIA型和LiB型拓扑异构酶的代表成员。 研究的具体目的如下： 1.了解II型拓扑异构酶如何在构象状态之间进行物理转换。 2.明确II型拓扑异构酶如何与反应底物相互作用和利用。 3.确定某些抗癌药物如何与真核细胞拓扑异构酶II结合并抑制 虽然已经有了一个了解II型拓扑异构酶功能的一般框架，但仍然存在显著的差距。对II型拓扑异构酶机制的详细结构描述加上生化验证，将阐明这些酶功能的关键方面。这里概述的研究将定义II型拓扑异构酶催化一个DNA片段通过另一个依赖于ATP的DNA片段的物理事件，以及抗癌抑制剂通过这些事件颠覆酶的功能。这些努力产生的数据广泛影响了许多重要的科学研究前沿，从理解分子机器如何维持染色体拓扑到剖析各种化疗的分子基础。

DESCRIPTION (provided by applicant): The long-term objective of this proposal is to structurally define type II topoisomerase function and drug inhibition mechanisms. Type II topoisomerases are a ubiquitous class of proteins that use ATP to actively transport one DNA duplex through another. This reaction maintains appropriate levels of DNA supercoiling and resolves lethal chromosome tangles in the cell. Eukaryotic type II topoisomerases are also exploited by certain inhibitors that are frontline clinical therapies for cancer. The focus of this proposal is on eukaryotic topoisomerase II and archaeal topoisomerase VI, two representative members of type IIA and liB topoisomerases, respectively. The specific aims of the research are as follows: 1. To understand how type II topoisomerases physically switch between conformational states. 2. To define how type II topoisomerases interact with and utilize reaction substrates. 3. To determine how certain anticancer agents bind to and inhibit eukaryotic topoisomerase II Although a general framework exists for understanding type II topoisomerase function, significant gaps remain. A detailed structural description of type II topoisomerase mechanism coupled with biochemical validation, will illuminate critical aspects of these enzymes' function. The studies outlined here will define the physical events by which type II topoisomerases catalyze the ATP-dependent passage of one DNA segment through another and by which anticancer inhibitors subvert enzyme function. Data resulting from such efforts broadly impact a number of important scientific research fronts, from understanding how a molecular machine maintains chromosome topology to dissecting the molecular basis of various chemotherapeutic treatments.