MECHANISM OF ENERGY COUPLING BY DNA TOPOISOMERASE II

DNA 拓扑异构酶 II 的能量耦合机制

• 批准号: 2459536
• 负责人: JANET E. LINDSLEY
• 金额: $ 20.38万
• 依托单位: UNIVERSITY OF UTAH
• 依托单位国家: 美国
• 财政年份: 1994
• 资助国家: 美国
• 起止时间: 1994-08-01 至 1999-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2459536
• 关键词: DNA; DNA gyrase; adenosine triphosphate; adenosinetriphosphatase; biological transport; chemical binding; chemical kinetics; conformation; crosslink; enzyme mechanism; fluorescence spectrometry; hydrolysis; mutant; oxidative phosphorylation

The long-term objective of the work in this laboratory is to understand the detailed mechanisms of enzymes that use the chemical energy of nucleotide triphosphates to effect structural changes or movements of enzyme-bound DNA. This proposal addresses the mechanism of the type II DNA topoisomerase. More specifically, these studies are designed to understand how this enzyme utilizes ATP binding/hydrolysis to promote the transport of one DNA duplex through a transient, enzyme-mediated break in another. Apart from being mechanistically challenging and fascinating enzymes, type II topoisomerases are biologically and clinically important. These enzymes are essential in all known organisms and are thought to be required for chromatin condensation and the separation of intertwined chromosomes during both mitosis and meiosis. Through regulating the supercoiling of chromosomal DNA, their activity also affects cell growth and gene expression. They are the targets of a long and growing list of antitumor, antibiotic and anti-fungal drugs, and yet their mechanism of action is largely unknown. The proposed approach is to use a combination of rapid kinetic techniques, mutagenesis and protein chemistry. The single-turnover rates of ATP binding and hydrolysis, as well as DNA transport will be measured under a number of reaction conditions by rapid quench-flow. The mechanism by which phosphorylation of the enzyme increases the steady-state reaction rates will be determined. The comformational changes in the protein and protein-DNA complex that accompany ATP binding, ATP hydrolysis and product dissociation will be followed by stopped-flow fluorescence. Two different types of protein cross-linking experiments will be used to determine whether DNA is transported all the way through the enzyme. Studies on mutant/wild type topoisomerase heterodimers will indicate how the protein conformational changes drive DNA transport. Results from these studies will increase our knowledge of how proteins can couple chemical energy to mechanical work, how topoisomerases may be regulated in the cell, and how to better design topoisomerase II-targeting drugs.

该实验室工作的长期目标是了解 利用化学能的酶的详细机制 三磷酸核苷酸影响结构变化或运动 酶结合 DNA。 该提案涉及 II 类机制 DNA拓扑异构酶。更具体地说，这些研究旨在 了解这种酶如何利用 ATP 结合/水解来促进 通过短暂的、酶介导的断裂来运输一个 DNA 双链体 其他。 除了机械上的挑战和迷人之外 II 型拓扑异构酶在生物学和临床上都很重要。 这些酶在所有已知的生物体中都是必需的，并且被认为是 染色质凝聚和相互缠绕的分离所需的 有丝分裂和减数分裂期间的染色体。 通过调节 染色体DNA的超螺旋，其活性也会影响细胞生长 和基因表达。他们是一长串且不断增加的目标 抗肿瘤、抗生素和抗真菌药物及其作用机制 行动很大程度上是未知的。 所提出的方法是结合使用快速动力学技术， 诱变和蛋白质化学。 ATP单周转率 结合和水解以及 DNA 运输将在 快速骤冷流的反应条件数。其机制 酶的磷酸化提高了稳态反应速率 将被确定。 蛋白质的构象变化和 伴随 ATP 结合、ATP 水解和产物的蛋白质-DNA 复合物 解离后将出现停流荧光。 两种不同的 蛋白质交联实验的类型将用于确定 DNA 是否一路通过酶进行运输。 研究 突变型/野生型拓扑异构酶异二聚体将表明蛋白质如何 构象变化驱动 DNA 运输。这些研究的结果 将增加我们对蛋白质如何将化学能耦合到 机械功，如何在细胞中调节拓扑异构酶，以及如何 更好地设计拓扑异构酶 II 靶向药物。