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.

项目摘要 准确和高效的DNA复制对所有生物体的生存至关重要。这个项目的总体目标是 是为了了解过程钳的分子动力学如何调节它的核心相互作用 到高效的DNA复制。这项提案的重点是大肠杆菌的Beta夹，这是一个环- 围绕DNA的形状二聚体蛋白，为其他蛋白质提供访问DNA的结合平台。 加工性钳夹还通过将DNA聚合酶与DNA和 提高他们的处理能力。适当加载Beta钳位对于调节 复制分叉和高效的DNA复制。我们的中心假设，这得到了我们初步的支持 数据是由钳制加载器捕获特定的瞬时采样的β钳制构象 将β加载到DNA上，并决定这一过程的效率。为了验证这一假设，在目标1中，我们 将设计具有改变的稳定性或不对称性的Beta夹具的变体，并确定热稳定性 以及这些改变的钳子的低聚状态。我们还将探讨更改后的夹具与 夹具装载机。在目标2中，我们将确定单独的Beta钳位和与钳位复合的情况下的动力学 横向弛豫优化谱(TROSY)加载器的核磁共振弛豫和氢交换 测量。在目标3中，我们将确定设计的夹蛋白变体的生物学功能， 包括它们与DNA相互作用和被夹子加载器加载到DNA上的能力，以及在 进行性DNA复制。将Beta钳位的动态与其在这些不同的 这些活动将对钳夹动力学和功能之间的关系提供重要的见解。此外， 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的见解。 互动。