Mechanisms of regulation of DNA repair helicases

项目摘要/摘要 解旋酶是一组普遍存在的高度多样化的酶，它将核酸链和 存在于细菌、真核生物、古生菌和许多病毒中。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突显。 解旋酶功能。许多解旋酶已经被证明在细胞中执行多种不同的功能。通常， 这些过程对解旋酶提出了非常不同的要求；例如，一个解旋酶可能需要 根据环境的不同，可以短距离、长距离或根本不放松。这些不同的功能是如何实现的 被定义和监管的人仍然知之甚少。 该项目将重点研究两种蛋白质，UvrD和XPD，它们可以作为DNA修复解旋酶的模型 原核生物和真核生物。虽然它们主要参与DNA修复途径，但两者 解旋酶还参与其他细胞过程。UvrD和XPD也是两个最大的 已知的解旋酶的结构类别以及对其机制的深入了解可能会扩展到许多 同源系统。先前的研究表明，解旋酶的活性受到寡聚体和 构象状态。一种单体可以表现出很低的解离活性或没有解离活性，但多个分子可以解离。 解旋酶可以解开一种构象中的双链，但可以取代另一种构象中的DNA结合蛋白。 因此，解旋酶的作用已被提议由控制其寡聚体的蛋白质伙伴在细胞中定义 和/或构象状态。 这些模型要么仍然是投机性的，要么是没有得到充分的量化。在这个项目中，我们将调查 解旋酶活性被调节的机制；第一，通过了解限制解旋酶活性的因素 解旋酶单体(目标1)，接下来通过测量解旋酶齐聚并量化其如何增强 解离活性(目标2)，最后通过与选定的蛋白质伙伴一起研究解旋酶来 确定它们是否利用上述策略来调节解旋酶活性(目标3)。 这些目标将使用单分子生物物理技术的合成来实现--光学镊子， 荧光显微镜、微流控技术--以及传统的生化方法。这些小说 利用PI的专业知识的方法将被用来检测解旋酶在单个 分子水平，实时和高分辨率，同时测量它们的低聚物和 构象状态。此外，这些技术将使多组件的受控组装成为可能 复合体。除了提供关于解旋酶机制和DNA修复途径的见解外，它们还 参与进来，我们的研究将推进研究生物分子动力学的生物物理方法 复合体。

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.