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: 10005418
• 负责人: PENNY J BEUNING
• 金额: $ 31.41万
• 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 2022-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10005418
• 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 准确和有效的DNA复制对所有生物体的生存至关重要。本项目的总体目标 是了解持续合成钳的分子动力学是如何调节它的相互作用的， 高效的DNA复制这项提案的重点是大肠杆菌β夹，这是一个环- 包围DNA并为其他蛋白质提供接近DNA的结合平台的成形二聚体蛋白质。 持续合成能力钳还通过将DNA聚合酶系到DNA上来提高DNA复制的效率， 增加了它们的持续性。β钳的适当加载对于在细胞周期中调节事件是至关重要的。 复制叉和有效的DNA复制。我们的中心假设，这是支持我们的初步 数据，是特定的瞬时采样β钳构象的钳加载器的基础捕获 将β加载到DNA上并决定该过程的效率。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β钳的变体， 和寡聚体状态。我们还将探索改变的钳与 夹钳装载机在目标2中，我们将确定单独的β钳和与钳复合的β钳的动力学 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，我们将确定所设计的钳蛋白变体的生物学功能， 包括它们与DNA相互作用并被钳加载器加载到DNA上的能力，以及它们在 进行性DNA复制将β钳夹的动力学与其在这些不同的 活动将提供重要的见解之间的关系钳动力学和功能。此外，委员会认为， 这项工作将为蛋白质动力学在调节蛋白质-蛋白质之间的作用提供基本的见解。 交互.