Mechanisms of regulation of DNA repair helicases

DNA 修复解旋酶的调控机制

• 批准号: 9158768
• 负责人: Yann R. Chemla
• 金额: $ 27.95万
• 依托单位: UNIVERSITY OF ILLINOIS AT URBANA-CHAMPAIGN
• 依托单位国家: 美国
• 财政年份: 2016
• 资助国家: 美国
• 起止时间: 2016-08-01 至 2021-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9158768
• 关键词: Adopted; Affect; Archaea; Bacteria; Base Pairing; Binding Proteins; Biochemical; Biological Models; Cell physiology; Cells; Complex; DNA; DNA Damage; DNA Repair; DNA Repair Gene; DNA Repair Pathway; DNA-Binding Proteins; Disease; Enzymes; Eukaryota; Exhibits; Fluorescence; Fluorescence Microscopy; Genetic Recombination; Genome; Genome Components; Homologous Gene; Human; In Vitro; Investigation; Link; Maintenance; Measurement; Measures; Methods; Microfluidics; Modeling; Molecular; Molecular Conformation; Molecular Motors; Nucleic Acids; Outcome; Pathway interactions; Play; Process; Prokaryotic Cells; Property; Proteins; RNA; Regulation; Resolution; Role; SS DNA BP; Stretching; System; Techniques; Testing; Time; Training; Virus; Work; biophysical techniques; dimer; helicase; insight; instrumentation; laser tweezer; member; monomer; novel strategies; optical traps; protein protein interaction; prototype; repaired; self assembly; single molecule

PROJECT SUMMARY / ABSTRACT Helicases are a ubiquitous and highly diverse group of enzymes that separate the strands of nucleic acids and are found in bacteria, eukaryotes, archaea, and many viruses. They are essential components of the genome maintenance machinery. Their importance is highlighted in the many human disorders associated with defective helicase function. Many helicases have been shown to carry out multiple, distinct functions in the cell. Often, these processes place very different requirements on the helicase; for instance, one helicase may be tasked with unwinding for short distances, long distances, or not at all, depending on context. How these different functions are defined and regulated remains poorly understood. This project will focus on two proteins, UvrD and XPD, which serve as models for DNA repair helicases in prokaryotes and eukaryotes, respectively. Although they are primarily involved in DNA repair pathways, both helicases also participate in other cellular processes. UvrD and XPD are also prototypes for the two largest structural classes of helicases known, and insights gained on their mechanisms are likely to extend to a number of homologous systems. Prior studies have shown that helicase activity is strongly influenced by oligomeric and conformational state. A monomer can exhibit low or no unwinding activity, but multiple molecules unwind processively; helicases can unwind duplexes in one conformation but displace DNA-bound proteins in another. Helicase roles have thus been proposed to be defined in the cell by protein partners controlling their oligomeric and/or conformational state. These models remain speculative or have not been quantified adequately. In this project, we will investigate the mechanisms by which helicase activity is regulated; first by understanding the factors that limit activity in helicase monomers (Aim 1), next by measuring helicase oligomerization and quantifying how it enhances unwinding activity (Aim 2), and lastly by studying helicase unwinding together with selected protein partners to determine if they exploit the above strategies to regulate helicase activity (Aim 3). These aims will be achieved using a synthesis of single-molecule biophysical techniques—optical tweezers, fluorescence microscopy, and microfluidics—together with traditional biochemical methods. These novel approaches, which exploit the PIs' expertise, will be used to detect the unwinding of helicases at the single molecule level, in real time, and at high resolution, while simultaneously measuring their oligomeric and conformational state. Moreover, these techniques will enable the controlled assembly of multi-component complexes. Beyond providing insights on helicase mechanism and the DNA repair pathways in which they participate, our studies will advance biophysical methods for investigating the dynamics of biomolecular complexes.

项目概要/摘要 解旋酶是一组普遍存在且高度多样化的酶，可分离核酸链和 存在于细菌、真核生物、古细菌和许多病毒中。它们是基因组的重要组成部分 维修机械。它们的重要性在许多与缺陷相关的人类疾病中得到了凸显。 解旋酶功能。许多解旋酶已被证明在细胞中执行多种不同的功能。经常， 这些过程对解旋酶提出了截然不同的要求；例如，一个解旋酶的任务可能是 短距离放松、长距离放松或根本不放松，具体取决于具体情况。这些不同的功能如何 的定义和监管仍然知之甚少。 该项目将重点研究两种蛋白质，UvrD 和 XPD，它们作为 DNA 修复解旋酶的模型 分别是原核生物和真核生物。虽然它们主要参与 DNA 修复途径，但 解旋酶还参与其他细胞过程。 UvrD 和 XPD 也是两个最大的原型 已知解旋酶的结构类别，以及对其机制的了解可能会扩展到许多 的同源系统。先前的研究表明，解旋酶活性受到寡聚体和寡聚体的强烈影响。 构象状态。单体可以表现出低的解旋活性或没有解旋活性，但多个分子可以解旋 逐步地；解旋酶可以解开一种构象中的双链体，但置换另一种构象中的 DNA 结合蛋白。 因此，解旋酶的作用被认为是由控制其寡聚体的蛋白质伴侣在细胞中定义的。 和/或构象状态。 这些模型仍然是推测性的或尚未得到充分量化。在这个项目中，我们将调查 解旋酶活性的调节机制；首先了解限制活动的因素 解旋酶单体（目标 1），接下来通过测量解旋酶寡聚化并量化其增强方式 解旋活性（目标 2），最后通过与选定的蛋白质伙伴一起研究解旋酶解旋来 确定他们是否利用上述策略来调节解旋酶活性（目标 3）。 这些目标将通过综合单分子生物物理技术来实现——光镊、 荧光显微镜、微流体技术以及传统的生化方法。这些小说 利用 PI 专业知识的方法将用于检测单个解旋酶的解旋情况 实时、高分辨率地分子水平，同时测量它们的寡聚物和 构象状态。此外，这些技术将使多组件的受控组装成为可能 复合物。除了提供有关解旋酶机制及其 DNA 修复途径的见解之外 参与，我们的研究将推进研究生物分子动力学的生物物理方法 复合物。