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: 2189543
• 负责人: JANET E. LINDSLEY
• 金额: $ 17.91万
• 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/2189543
• 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; 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 使用化学能的详细机制 核苷酸三磷酸盐以实现结构变化或运动 酶结合的DNA。 该提案解决了II型的机制 DNA拓扑异构酶。更具体地说，这些研究旨在 了解该酶如何利用ATP结合/水解来促进 将一个DNA双链体通过瞬态，酶介导的破裂运输 其他。 除了具有挑战性和迷人 酶，II型拓扑异构酶在生物学上和临床上都很重要。 这些酶在所有已知生物中都是必不可少的，被认为是 需要染色质凝结和交织在一起的分离 有丝分裂和减数分裂过程中的染色体。 通过调节 染色体DNA的超螺旋，它们的活性也会影响细胞生长 和基因表达。它们是漫长而又不断增长的列表的目标 抗肿瘤，抗生素和抗真菌药物，但它们的机制 动作在很大程度上是未知的。 建议的方法是结合快速动力学技术， 诱变和蛋白质化学。 ATP的单位转变率 结合和水解以及DNA转运将在A下测量 快速淬灭流的反应条件数量。该机制 酶的磷酸化增加了稳态反应速率 将确定。 蛋白质和 伴随ATP结合，ATP水解和产物的蛋白DNA复合物 离解会之后停止流量荧光。 两个不同 蛋白质交联实验的类型将用于确定 DNA是否一直通过酶运输。 研究 突变/野生型拓扑异构酶异二聚体将指示蛋白质如何 构象变化驱动DNA传输。这些研究的结果 将增加我们对蛋白质如何将化学能夫妇搭配到的了解 机械工作，如何在细胞中调节拓扑异构酶以及如何调节 更好地设计拓扑异构酶II靶向药物。