HELICASE CATALYZED DNA UNWINDING

解旋酶催化 DNA 解旋

• 批准号: 2183542
• 负责人: Timothy M Lohman
• 金额: $ 23.6万
• 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 1999-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2183542
• 关键词: DNA; DNA binding protein; X ray crystallography; adenosine triphosphate; adenosinetriphosphatase; analytical ultracentrifugation; bioenergetics; chemical binding; chemical kinetics; computer simulation; conformation; dimer; enzyme mechanism; enzyme model; enzyme structure; enzyme substrate; fluorescence spectrometry; helicase; mutant; nucleic acid sequence; solutions; stoichiometry; stop flow technique; synthetic nucleotide; thermodynamics; DNA; DNA binding protein; X ray crystallography; adenosine triphosphate; adenosinetriphosphatase; analytical ultracentrifugation; bioenergetics; chemical binding; chemical kinetics; computer simulation; conformation; dimer; enzyme mechanism; enzyme model; enzyme structure; enzyme substrate; fluorescence spectrometry; helicase; mutant; nucleic acid sequence; solutions; stoichiometry; stop flow technique; synthetic nucleotide; thermodynamics

DNA helicases are DNA-stimulated ATPases that unwind duplex DNA to produce the single-stranded (ss) DNA intermediates required for replication, recombination and repair in all organisms. Our studies focus on two DNA helicases from E. coli, Rep and UvrD (Helicase H), and are designed to obtain a molecular understanding of the mechanism(s) by which DNA helicases unwind duplex DNA and translocate along DNA in reactions coupled to ATP binding and hydrolysis. Rep and UvrD function independently as helicases, but also interact to form hetero-dimers in vitro. 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 and protein self-assembly (the oligomeric nature of helicases appears to be essential for DNA unwinding). Our previous DNA binding studies indicate that Rep dimerizes upon binding 55- or duplex (ds-) DNA and that nucleotides affect these interactions allosterically. An active "rolling" model for how the Rep dimer translocates and unwinds duplex DNA has been proposed that makes a number of testable predictions; many of the proposed studies are focused on testing this model. A major emphasis is on transient kinetic approaches (stopped-flow fluorescence and chemical quench-flow) to examine the kinetics and mechanism of DNA binding to and ATP binding and hydrolysis by the five Rep dimer species that differ in their DNA (ss and ds) ligation state, a subset of which appears to be intermediates in the DNA unwinding reaction. The kinetics and mechanism of nucleotide (ADP, ATP, AMPPNP and fluorescent analogs) binding, ATP hydrolysis and DNA binding will be studied using fluorescence stopped-flow and quench-flow. In parallel, we will examine Rep and UvrD-catalyzed unwinding of synthetic DNA substrates with the goal of developing a full kinetic mechanism for unwinding. Both rapid quench- flow and a novel fluorescence stopped-flow method will be used to study DNA unwinding (effects of ss-DNA tail length, ds-DNA length, sequence and base composition as well as solution conditions). The efficiency of ATP hydrolysis during unwinding and the number of base pairs unwound in a single catalytic event ("step size") will be estimated. "Passive" vs. "active" models of DNA unwinding will be tested using novel, non-natural DNA substrates. A major goal is also to determine the. active oligomeric form of the UvrD helicase and to study its interactions with DNA and nucleotides. The overall goal of these studies is to obtain basic information about the mechanism of DNA unwinding and translocation by this important class of motor proteins. However, the mechanistic information obtained should facilitate studies of other DNA and RNA helicases. Since DNA replication and repair are fundamental to cell growth in all organisms, an understanding of such a basic process as enzyme-catalyzed DNA unwinding will undoubtedly have an impact on our understanding of some cancers that result from defects in replication or repair.

DNA解旋酶是DNA刺激的ATP酶，解脱双链DNA产生 复制所需的单链（SS）DNA中间体， 所有生物的重组和修复。我们的研究集中于两个DNA 大肠杆菌，rep和uvrd（解旋酶H）的解旋酶，被设计为 获得对DNA的机制的分子理解 解旋酶放松双链DNA并沿着DNA转移反应耦合 进行ATP结合和水解。 REP和UVRD独立函数为 解旋酶，但也相互作用以在体外形成异质二聚体。生化 和生物物理方法将用于检查平衡和 对DNA功能上重要的相互作用的动力学 放松，例如DNA和核苷酸结合和蛋白质自组装 （解旋酶的寡聚性质似乎对于DNA至关重要 放松）。我们以前的DNA结合研究表明REP二聚 结合55或双链（DS-）DNA后，核苷酸会影响这些 互动变构。一个主动的“滚动”模型，用于如何代表 已经提出了二聚体易位和休息的双链DNA 可测试的预测数；许多拟议的研究集中于 在测试此模型时。 重点是瞬态动力学方法（停止流动 荧光和化学淬灭流）检查动力学和 DNA与五个REP结合和ATP结合和水解的机理 其DNA（SS和DS）连接状态不同的二聚体物种，一个 其子集似乎是DNA解开反应中的中间体。 核苷酸的动力学和机制（ADP，ATP，AMPPNP和荧光 类似物）结合，将研究使用ATP水解和DNA结合 荧光停止流量和淬火流。同时，我们将检查 REP和UVRD催化的合成DNA底物的放松 开发完整的动力学机制。都快速淬火 流量和一种新型的荧光停止方法将用于研究 DNA解开（SS-DNA尾巴长度，DS-DNA长度，序列和序列的影响 基础组成以及溶液条件）。 ATP的效率 放松过程中的水解和基本对的数量在 将估计单个催化事件（“步长”）。 “被动”与 将使用新颖的非天然测试DNA的“主动”模型 DNA底物。一个主要目标也是确定。活性寡聚 UVRD解旋酶的形式，并研究其与DNA和DNA的相互作用 核苷酸。 这些研究的总体目标是获得有关 DNA的机制放松和易位。 运动蛋白。但是，获得的机械信息应 促进对其他DNA和RNA解旋酶的研究。自DNA复制以来 维修对于所有生物的细胞生长都是基础的 理解酶催化的DNA的基本过程 无疑会影响我们对某些癌症的理解 复制或修复缺陷导致。