Dynamics of processivity clamp proteins in bacterial DNA replication

细菌 DNA 复制中持续钳蛋白的动力学

• 批准号: 10245024
• 负责人: PENNY J BEUNING
• 金额: $ 29.82万
• 依托单位: NORTHEASTERN UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2017
• 资助国家: 美国
• 起止时间: 2017-09-30 至 2023-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10245024
• 关键词: Affinity; Antibiotic Resistance; Antibiotics; Bacterial DNA; Binding; Biochemical; Biological Process; Bypass; Cells; Closure by clamp; Complex; DNA; DNA Damage; DNA biosynthesis; DNA replication fork; DNA-Directed DNA Polymerase; Data; Deuterium; Development; Engineering; Escherichia coli; Event; Goals; Hydrogen; Hydrolysis; Knowledge; Laboratories; Life; Mass Spectrum Analysis; Measurement; Methodology; Methods; Microbiological Techniques; Mission; Molecular; Molecular Conformation; Molecular Weight; Motion; Mutation; NMR Spectroscopy; Nuclear Magnetic Resonance; Organism; Play; Process; Protein Dynamics; Proteins; Public Health; Regulation; Relaxation; Research; Resolution; Role; Sampling; Testing; Time; United States National Institutes of Health; Variant; Work; biophysical techniques; conformer; design; dimer; human disease; insight; molecular dynamics; monomer; protein protein interaction; response

Project Summary Accurate and efficient DNA replication is critical for the survival of all organisms. The overall goal of this project is to understand how the molecular dynamics of the processivity clamp regulate its interactions that are central to efficient DNA replication. The focus of this proposal is on the Escherichia coli beta clamp, which is a ring- shaped dimeric protein that encircles DNA and provides a binding platform for other proteins to access DNA. Processivity clamps also increase the efficiency of DNA replication by tethering DNA polymerases to DNA and increasing their processivity. Appropriate loading of the beta clamp is critical for regulation of events at the replication fork and for efficient DNA replication. Our central hypothesis, which is supported by our preliminary data, is that trapping of specific transiently sampled beta clamp conformations by the clamp loader underlies loading of beta onto DNA and determines the efficiency of this process. To test this hypothesis, in aim 1, we will design variants of the beta clamp with altered stability or asymmetry and determine the thermal stability and oligomeric state of these altered clamps. We will also probe the interactions of the altered clamps with the clamp loader. In aim 2, we will determine the dynamics of the beta clamp alone and in complex with clamp loader by Transverse Relaxation Optimized SpectroscopY (TROSY) NMR relaxation and hydrogen exchange measurements. In aim 3, we will determine the biological functions of the designed clamp protein variants, including their ability to interact with and be loaded onto DNA by the clamp loader, and to function in processive DNA replication. Correlating the dynamics of the beta clamp with its proficiency in these different activities will provide important insights into the relationship between clamp dynamics and function. Moreover, this work will provide fundamental insights into the roles of protein dynamics in regulating protein-protein interactions.

项目概要 准确有效的 DNA 复制对于所有生物体的生存至关重要。本项目的总体目标 是了解持续钳的分子动力学如何调节其核心相互作用 以实现高效的 DNA 复制。该提案的重点是大肠杆菌β钳，它是一个环- 形状二聚体蛋白质，环绕 DNA 并为其他蛋白质访问 DNA 提供结合平台。 持续钳还通过将 DNA 聚合酶束缚在 DNA 上来提高 DNA 复制的效率。 提高他们的持续生产能力。 β 钳的适当加载对于调节事件至关重要 复制叉和有效的 DNA 复制。我们的中心假设得到了我们初步的支持 数据，是钳加载器捕获特定瞬时采样的β钳构象的基础 将 beta 加载到 DNA 上并确定该过程的效率。为了检验这个假设，在目标 1 中，我们 将设计具有改变稳定性或不对称性的 β 钳变体，并确定热稳定性 和这些改变的夹子的寡聚状态。我们还将探讨改变后的夹具与 夹具装载机。在目标 2 中，我们将确定单独的 β 钳以及与钳的复合体的动力学 通过横向弛豫优化光谱 (TROSY) NMR 弛豫和氢交换加载 测量。在目标 3 中，我们将确定设计的钳蛋白变体的生物学功能， 包括它们与 DNA 相互作用并通过夹具加载器加载到 DNA 上的能力，以及在 进行性 DNA 复制。将 β 钳的动态与其在这些不同方面的熟练程度相关联 活动将为钳位动力学和功能之间的关系提供重要的见解。而且， 这项工作将为蛋白质动力学在调节蛋白质-蛋白质中的作用提供基本见解 互动。