HELICASE CATALYZED DNA UNWINDING

解旋酶催化 DNA 解旋

• 批准号: 6525636
• 负责人: Timothy M Lohman
• 金额: $ 37.36万
• 依托单位: WASHINGTON UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1991
• 资助国家: 美国
• 起止时间: 1991-08-01 至 2003-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6525636
• 关键词: DNA; DNA repair; DNA replication; X ray crystallography; adenosine triphosphate; adenosinetriphosphatase; bioenergetics; biophysics; chemical binding; chemical kinetics; dimer; enzyme mechanism; enzyme model; enzyme structure; enzyme substrate; fluorescence resonance energy transfer; helicase; hydrolysis; intermolecular interaction; molecular assembly /self assembly; site directed mutagenesis; stop flow technique; thermodynamics

DESCRIPTION: (From applicant's abstract)DNA helicases are ATP-dependent motor proteins that unwind duplex DNA to form the single stranded (ss) DNA intermediates required for replication, recombination and repair in all organisms. The principal investigator and his group propose to continue studies of two E. coli DNA helicases, Rep and UvrD (Helicase II), both of which appear to function as homo-dimers and function in replication and repair, respectively. The overall goal is to obtain a molecular understanding of the mechanism(s) by which these DNA helicases unwind duplex DNA and translocate along DNA and how these processes are coupled to ATP binding and hydrolysis. Quantitative biochemical and biophysical approaches will be used to examine the equilibria and kinetics of the interactions that are functionally important for DNA unwinding, such as DNA and nucleotide binding, ATP hydrolysis and protein self-assembly. This requires investigators to understand the molecular details of the known allosteric interactions that are key to the function of these multisubunit enzymes. The principal investigator and his group have proposed a "subunit switching" model for how the Rep dimer translocates and unwinds duplex DNA that makes a number of testable predictions; many of the proposed studies are focused on testing this and other models. Dr. Lohman and his group will use transient kinetic approaches (stopped-flow fluorescence and chemical quenched-flow) to examine the pre-steady state kinetics and mechanism of ATP binding and hydrolysis by Rep and UvrD dimers in various DNA ligation states, some of which are proposed intermediates in DNA unwinding reactions. The thermodynamics, kinetics and mechanism of DNA binding will also be studied. In parallel, they will examine Rep and UvrD catalyzed unwinding of synthetic DNA substrates with the goal of developing a full kinetic mechanism for unwinding of synthetic DNA substrates with goal of developing a full kinetic mechanism for unwinding. Their recent x-ray crystal structure of Rep-ssDNA complexes (in collaboration with G. Waksman) has provided important structural insight and will aid the design of Rep and UrvD mutants to test the functional importance of different domains of the proteins for DNA and ATP binding, ATP hydrolysis, dimerization and DNA unwinding.

描述：（从申请人的摘要中）DNA解旋酶是ATP依赖电机 放开双链DNA的蛋白质形成单个链（SS）DNA 所有人需要复制，重组和维修所需的中间体 有机体。首席研究员及其小组提议继续学习 两个大肠杆菌DNA解旋酶，REP和UVRD（Helicase II），两者均出现 为了充当同型二聚体并在复制和维修中起作用， 分别。总体目标是获得对 这些DNA解旋酶放松双链DNA并转运的机制 沿着DNA以及如何将这些过程与ATP结合和水解耦合。 定量生化和生物物理方法将用于检查 相互作用的平衡和动力学在功能上很重要 DNA解开，例如DNA和核苷酸结合，ATP水解和蛋白 自组装。这要求研究人员了解分子细节 已知的变构相互作用是这些功能的关键 多亚基酶。主要调查员及其小组提出了一个 代表二聚体如何易位和解开双工的“亚基切换”模型 做出许多可测试预测的DNA；许多拟议的研究 专注于测试此模型和其他模型。 Lohman博士和他的小组将使用短暂的动力学方法（停止流程 荧光和化学淬灭流）检查稳态的状态 ATP结合的动力学和机制，由REP和UVRD二聚体在 各种DNA连接状态，其中一些是在DNA中提出的中间体 放松反应。 DNA结合的热力学，动力学和机制 也将研究。同时，他们将检查REP并催化UVRD 放松合成DNA底物，目的是开发完整的 动力学机制，用于取消目标的合成DNA底物 开发完整的动力学机制以放松。他们最近的X射线水晶 Rep-Ssdna复合体的结构（与G. Waksman合作） 提供了重要的结构见解，并将有助于REP和URVD的设计 突变体测试蛋白质不同结构域的功能重要性 对于DNA和ATP结合，ATP水解，二聚化和DNA放松。