Biochemical Analyses of Type II DNA Topoisomerases

II 型 DNA 拓扑异构酶的生化分析

基本信息

批准号：
7909236
负责人：
JAMES M BERGER
金额：
$ 9.42万
依托单位：
UNIVERSITY OF CALIFORNIA BERKELEY
依托单位国家：
美国
项目类别：
财政年份：
2009
资助国家：
美国
起止时间：
2009-08-01 至 2010-11-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/7909236
关键词：
Address Affinity Anti-Bacterial Agents Architecture Bacteria Bacterial Infections Bacterial Typing Binding Biochemical Biochemistry Biology C-terminal Calculi Cell Death Cell Survival Cell division Cell physiology Chemicals Chromosomes Cleaved cell Clinical Communicable Diseases Complex Coupled DNA DNA Binding DNA Double Strand Break DNA Sequence Rearrangement DNA Topoisomerase IV DNA biosynthesis Data Discrimination Drug Delivery Systems Enzymes Event Exhibits Genetic Transcription Homeostasis Image Kinetics Malignant Neoplasms Mechanics Methods Microfluidics Molecular Molecular Machines Mutagenesis Neurofibrillary Tangles Output Pharmaceutical Preparations Poison Poisoning Problem Solving Protein Isoforms Proteins Reaction Regulation Research Structure Structure-Activity Relationship Superhelical DNA Testing Time Topoisomerase Topoisomerase II Topoisomerase Inhibitors Treatment Efficacy Work base cancer therapy cell killing cytotoxic dimer drug development improved in vivo inhibitor/antagonist innovation inorganic phosphate insight interest novel paralogous gene segregation small molecule

项目摘要

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.

描述（由申请人提供）：II 型拓扑异构酶是一类普遍存在的蛋白质，它们使用 ATP 主动将一个 DNA 双链体转运到另一个 DNA 双链体。该反应对于超螺旋稳态和细胞分裂前解决细胞毒性染色体缠结至关重要。 II 型拓扑异构酶也是癌症和细菌感染一线临床治疗药物的靶点。该提案的长期目标是研究 II 型拓扑异构酶的分子基础功能和药物抑制作用。尽管这些酶的催化循环的粗略框架已经就位，但围绕 II 型拓扑异构酶区分 DNA 不同拓扑状态、经历变构转变以驱动 DNA 运输以及被小分子抑制的机制，仍然存在许多关键问题。刺激DNA裂解的毒物”。我们结合使用结构、生物化学和生物物理方法，旨在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 复合物的结构，2) 确定细菌 II 型拓扑异构酶中新型 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) 定义 DNA 变形的分子机制和动力学以及拓扑异构酶催化循环中的关键结构重排。我们提出的研究将定义 II 型拓扑异构酶促进一个 DNA 片段穿过另一个片段以全局控制 DNA 拓扑结构，并通过抗癌和抗菌抑制剂破坏酶的功能。这些努力产生的数据广泛影响了许多重要的科学前沿，从了解 ATP 依赖性分子机器的动力学和染色体超结构的调节，到定义抗拓扑异构酶疗法的物理作用和帮助药物开发。项目叙述：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经过验证的靶标。该提案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功能。