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 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，其解旋双链DNA以产生 复制所需的单链（ss）DNA中间体， 重组和修复。我们的研究集中在两个DNA 解旋酶来自E.大肠杆菌、Rep和UvrD（解旋酶H），并被设计为 从分子水平理解DNA 解旋酶解链双链体DNA并在偶联反应中沿沿着DNA移位 ATP结合和水解。Rep和UvrD独立运行， 解旋酶，而且在体外相互作用形成异源二聚体。生化 生物物理学方法将被用来检查平衡， 对DNA功能重要的相互作用的动力学 解旋，如DNA和核苷酸结合以及蛋白质自组装 (the解旋酶的寡聚性质似乎是DNA 解旋）。我们以前的DNA结合研究表明，Rep二聚化 结合55-或双链体（ds-）DNA后，核苷酸会影响这些 变构相互作用。一个积极的“滚动”模型， 二聚体转位和解旋双链体DNA已被提出， 许多可测试的预测;许多拟议的研究集中在 测试这个模型。 一个主要的重点是瞬态动力学方法（停流 荧光和化学猝灭流）来检查动力学， 五种Rep的DNA结合和ATP结合及水解机制 二聚体种类在它们的DNA（ss和ds）连接状态上不同， 其子集似乎是DNA解旋反应中的中间体。 研究了核苷酸（ADP、ATP、AMPPNP和荧光素）的动力学和作用机理 类似物）结合，ATP水解和DNA结合将使用 荧光停止流和猝灭流。与此同时，我们将研究 Rep和UvrD催化的合成DNA底物解旋， 发展出一个完整的解旋动力学机制快速淬火- 流动和一种新的荧光停流方法将被用来研究 DNA解旋（ss-DNA尾长、ds-DNA长度、序列和 基础组成以及溶液条件）。ATP的效率 解旋过程中的水解和解旋的碱基对的数量， 将估计单个催化事件（“步长”）。“被动”与 DNA解旋的“主动”模型将使用新的、非天然的 DNA底物。一个主要的目标也是确定。活性低聚物 UvrD解旋酶的形式，并研究其与DNA的相互作用， 个核苷酸 这些研究的总体目标是获得关于 DNA解旋和易位的机制，这类重要的 马达蛋白然而，所获得的机械信息应 促进其他DNA和RNA解旋酶的研究。由于DNA复制 和修复是所有生物体细胞生长的基础， 对酶催化DNA解旋这样一个基本过程的理解 无疑会对我们对某些癌症的理解产生影响， 由复制或修复中的缺陷引起。