HELICASE CATALYZED DNA UNWINDING

解旋酶催化 DNA 解旋

• 批准号: 2459425
• 负责人: Timothy M Lohman
• 金额: $ 26.65万
• 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/2459425
• 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，可解旋双链 DNA 以产生 复制所需的单链 (ss) DNA 中间体， 所有生物体中的重组和修复。我们的研究重点是两种 DNA 来自大肠杆菌、Rep 和 UvrD（解旋酶 H）的解旋酶，旨在 获得对 DNA 作用机制的分子理解 解旋酶解旋双链 DNA 并在偶联反应中沿着 DNA 易位 ATP 结合和水解。 Rep 和 UvrD 独立发挥作用 解旋酶，而且在体外相互作用形成异源二聚体。生化 和生物物理方法将用于检查平衡和 对 DNA 具有重要功能的相互作用的动力学 解旋，例如 DNA 和核苷酸结合以及蛋白质自组装 （解旋酶的寡聚性质似乎对于 DNA 放松）。我们之前的 DNA 结合研究表明 Rep 二聚化 结合 55- 或双链 (ds-) DNA 后，核苷酸会影响这些 变构相互作用。一个活跃的“滚动”模型，说明销售代表如何 已提出二聚体易位并解开双链 DNA，从而使 可测试预测的数量；许多拟议的研究都集中在 测试这个模型。 主要强调瞬态动力学方法（停流 荧光和化学猝灭流）来检查动力学和 五种 Rep 与 DNA 结合以及 ATP 结合和水解的机制 DNA（单链和双链）连接状态不同的二聚体种类， 其中的子集似乎是 DNA 解旋反应的中间体。 核苷酸（ADP、ATP、AMPPNP 和荧光）的动力学和机制 类似物）结合、ATP 水解和 DNA 结合将使用 荧光停流和猝灭流。同时，我们将检查 Rep 和 UvrD 催化合成 DNA 底物的解旋，目标是 开发完整的放松动力机制。两者均快速淬火 流动和一种新颖的荧光停流方法将用于研究 DNA 解旋（单链 DNA 尾长、双链 DNA 长度、序列和 基础成分以及溶液条件）。 ATP的效率 解旋过程中的水解和解旋碱基对的数量 将估计单个催化事件（“步长”）。 “被动”VS“被动” DNA 解旋的“主动”模型将使用新颖的非天然材料进行测试 DNA 底物。一个主要目标也是确定。活性寡聚体 UvrD 解旋酶的形式并研究其与 DNA 的相互作用 核苷酸。 这些研究的总体目标是获得有关 这一类重要的 DNA 解旋和易位机制 运动蛋白。然而，获得的机械信息应该 促进其他 DNA 和 RNA 解旋酶的研究。自DNA复制以来 和修复是所有生物体细胞生长的基础 了解酶催化 DNA 解旋等基本过程 无疑会影响我们对某些癌症的理解 复制或修复过程中的缺陷造成的。