Helicase Catalyzed DNA Unwinding

解旋酶催化 DNA 解旋

• 批准号: 7118526
• 负责人: Timothy M Lohman
• 金额: $ 46.37万
• 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 2008-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7118526
• 关键词: DNA binding protein; X ray crystallography; chemical kinetics; enzyme activity; helicase; hydrolysis; membrane transport proteins; molecular assembly /self assembly; molecular dynamics; monomer; mutant; nucleic acid structure; stop flow technique; thermodynamics

DESCRIPTION (provided by applicant): DNA helicases are ATP-dependent motor proteins that unwind duplex DNA to form the single stranded (ss)DNA intermediates required for DNA metabolism in all organisms. We are studying the kinetic mechanisms of DNA unwinding and DNA translocation of three non-hexameric SF1 DNA helicases from E. coil, Rep,UvrD, and RecBCD, which function in replication, repair, and recombination, respectively. RecBCD is a hetero-trimeric complex containing two SF1 helicases (B and D). The overall goal is to obtain a molecular understanding of the kinetic mechanism(s) by which these DNA helicases unwind duplex DNA, translocate along DNA and how these processes are coupled to ATP binding and hydrolysis. Our pre-steady state kinetic studies indicate that Rep and UvrD helicases function as dimers, even though monomers of these enzymes can translocate efficiently along ss-DNA, hence the relationship between ss-DNA translocation by monomers and DNA unwinding by dimers will be investigated. We will use transient kinetic approaches(stopped-flow fluorescence and chemical quenched-flow) to examine the pre-steady state kinetics and mechanism of ss-DNA translocation, ATP binding and hydrolysis by Rep and UvrD monomers during ss-DNA translocation, and compare these to the kinetic mechanism for DNA unwinding by the functional helicase dimers, with the goal of developing a full kinetic mechanism for unwinding. DNA binding, ATP hydrolysis and DNA unwinding by RecBCD and RecBC helicases will also be examined mechanistically. Thermodynamic studies will be performed to understand the energetics of the binding of these helicases to DNA, and how DNA binding energy is used in DNA unwinding. We will also examine mutants of these enzymes, including constitutive dimers of Rep, and the mechanism by which MutL stimulates DNA unwinding by UvrD. These ensemble studies will be complemented by single molecule studies of DNA binding and unwinding and structural studies by x-ray crystallography.

描述（由申请人提供）： DNA 解旋酶是 ATP 依赖性马达蛋白，可解旋双链 DNA，形成所有生物体 DNA 代谢所需的单链 (ss)DNA 中间体。我们正在研究来自大肠杆菌、Rep、UvrD 和 RecBCD 的三种非六聚体 SF1 DNA 解旋酶的 DNA 解旋和 DNA 易位的动力学机制，它们分别在复制、修复和重组中发挥作用。 RecBCD 是包含两个 SF1 解旋酶（B 和 D）的异源三聚体复合物。总体目标是从分子角度了解这些 DNA 解旋酶解旋双链 DNA、沿 DNA 易位的动力学机制，以及这些过程如何与 ATP 结合和水解耦合。我们的稳态前动力学研究表明，Rep 和 UvrD 解旋酶以二聚体的形式发挥作用，尽管这些酶的单体可以沿着 ss-DNA 有效地易位，因此将研究单体的 ss-DNA 易位与二聚体的 DNA 解旋之间的关系。我们将使用瞬态动力学方法（停流荧光和化学猝灭流）来研究单链DNA转位过程中Rep和UvrD单体的ATP结合和水解的前稳态动力学和机制，并进行比较这些与功能性解旋酶二聚体解旋 DNA 的动力学机制相关，目标是开发完整的解旋动力学机制。还将对 RecBCD 和 RecBC 解旋酶的 DNA 结合、ATP 水解和 DNA 解旋进行机械检查。我们将进行热力学研究，以了解这些解旋酶与 DNA 结合的能量，以及 DNA 结合能如何用于 DNA 解旋。我们还将研究这些酶的突变体，包括 Rep 的组成型二聚体，以及 MutL 通过 UvrD 刺激 DNA 解旋的机制。 DNA 结合和解旋的单分子研究以及 X 射线晶体学的结构研究将补充这些整体研究。