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无处不在的蛋白质类，它们使用ATP积极地通过另一个DNA双链体积极地运输一种DNA。该反应对于细胞分裂之前的超凝固稳态和解决细胞毒性染色体缠结至关重要。 II型拓扑异构酶还受到用作癌症和细菌感染前线临床疗法的药物的目标。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。尽管已经到位这些酶的催化周期的粗糙框架，但围绕II型II型拓扑异构酶区分DNA的不同拓扑状态的机制仍然存在许多关键问题，从而驱动DNA传输的不同拓扑状态，并受到小分子的抑制，并被小分子抑制“刺激DNA裂解的毒药”。使用结构，生化和生物物理方法的结合，我们旨在填补这些空白：1）确定“中毒” II型拓扑异构酶/DNA复合物的结构，2）确定在细菌II型II型拓扑异构酶中的新型DNA结合和弯曲结构群如何控制底物的选择性和功能输出，以及3）定义分子机制和钥匙结构重新培训的分子机制和动力学的培养基，并键入孔培养孔。我们提出的研究将定义II型拓扑异构酶促进的物理事件一个DNA段通过另一个段通过一个全球控制DNA拓扑，抗癌和抗菌抑制剂颠覆了酶的功能。从理解依赖ATP的分子机的动力学和染色体上层建筑的调节到定义抗昆虫异构酶疗法的物理作用和有助于药物开发的物理作用，从理解依赖ATP的分子机器的动力学和调节染色体的动态，产生的数据广泛影响了许多重要的科学方面。项目叙述：II型拓扑异构酶是分解DNA的分子机器，也是验证的前线抗菌和抗癌疗法的靶标。该建议旨在了解拓扑异构酶反应的生物化学和力学，并确定一些最广泛使用的抗昆虫异构酶药物如何阻止酶功能。