Biochemical Analyses of Type II DNA Topoisomerases

描述(申请人提供)：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个DNA双链通过另一个DNA双链。这种反应对于细胞分裂前的超螺旋稳态和解决细胞毒性染色体缠结是必不可少的。II型拓扑异构酶也是治疗癌症和细菌感染的一线临床治疗药物的靶点。 这项建议的长期目标是研究II型拓扑异构酶的分子基础。 功能和药物抑制。尽管这些酶的催化循环的大致框架已经到位，但围绕着II型拓扑异构酶区分DNA的不同拓扑状态、经历变构转换以驱动DNA运输以及被刺激DNA切割的小分子毒物抑制的机制，仍然存在许多关键问题。 使用结构、生化和生物物理方法的组合，我们的目标是通过以下方式填补这些空白：1) 确定“中毒”的II型拓扑异构酶/DNA复合体的结构，2)确定细菌II型拓扑异构酶中新的DNA结合和弯曲结构域如何控制底物选择性和功能输出，以及3)确定DNA变形和拓扑异构酶催化循环中关键结构重排的分子机制和动力学。 我们拟议的研究将定义II型拓扑异构酶促进 通过一个DNA片段通过另一个片段来全局控制DNA拓扑，抗癌和抗菌抑制剂通过这个过程颠覆酶的功能。这些努力产生的数据广泛影响了许多重要的科学前沿，从理解依赖于ATP的分子机器的动力学和染色体超结构的调节，到确定抗拓扑异构酶治疗的物理作用和帮助药物开发。 项目简介：II型拓扑异构酶是解开DNA的分子机器，也是一线抗菌和抗癌治疗的有效靶点。这项建议旨在了解拓扑异构酶反应的生物化学和机制，并确定一些最广泛使用的抗拓扑异构酶药物如何阻断酶的功能。

DESCRIPTION (provided by applicant): Type II topoisomerases are a ubiquitous class of proteins that use ATP to actively transport one DNA duplex through another. This reaction is essential for supercoiling homeostasis and resolving cytotoxic chromosome tangles prior to cell division. Type II topoisomerases are also targeted by drugs that serve as frontline clinical therapies for cancer and bacterial infections. The long-term objective of this proposal is to investigate the molecular basis of type II topoisomerase function and drug inhibition. Although rough framework for the catalytic cycle of these enzymes is in place, there remain many critical questions surrounding the mechanisms by which type II topoisomerases discriminate between different topological states of DNA, undergo allosteric transitions to drive DNA transport, and are inhibited by small molecule "poisons" that stimulate DNA cleavage. Using a combination of structural, biochemical, and biophysical methods we aim to fill these gaps by: 1) Determining the structure of a "poisoned" type II topoisomerase/DNA complex, 2) Establishing how a novel DNA binding and bending domain in bacterial type II topoisomerases controls substrate selectivity and functional output, and 3) Defining the molecular mechanisms and kinetics of DNA deformations and key structural rearrangements in the topoisomerase catalytic cycle. Our proposed studies will define the physical events by which type II topoisomerases facilitate the passage of one DNA segment through another to globally control DNA topology, and by which anticancer and antibacterial inhibitors subvert enzyme function. Data resulting from such efforts broadly impact a number of important scientific fronts, from understanding the dynamics of ATP-dependent molecular machines and regulation of chromosome superstructure, to defining the physical action of anti-topoisomerase therapies and aiding drug development. PROJECT NARRATIVE: Type II topoisomerases are molecular machines that disentangle DNA, as well as validated targets for frontline antibacterial and anticancer therapies. This proposal aims to understand the biochemistry and mechanics of the topoisomerase reaction, and to determine how some of the most widely-used anti-topoisomerase drugs block enzyme function.