Mechanisms of regulation of DNA repair helicases

项目总结/摘要 解旋酶是一种普遍存在且高度多样化的酶，其分离核酸链， 存在于细菌、真核生物、古生菌和许多病毒中。它们是基因组的基本组成部分 维修机械。它们的重要性在许多与缺陷型糖尿病相关的人类疾病中凸显出来。 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。很多时候， 这些过程对解旋酶提出了非常不同的要求;例如，一个解旋酶可能负责 短距离、长距离或根本不展开，这取决于上下文。这些不同的功能 定义和监管仍然知之甚少。 该项目将重点关注两种蛋白质，UvrD和XPD，它们作为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， 解旋酶也参与其它细胞过程。UvrD和XPD也是两个最大的 已知的解旋酶的结构类别，以及对其机制的了解可能会扩展到许多 同源系统。先前的研究已经表明，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或没有解旋活性，但多个分子解旋 解旋酶可以解旋一种构象的双链体，但置换另一种构象的DNA结合蛋白。 因此，有人提出解旋酶的作用在细胞中由控制其寡聚化的蛋白质配偶体来定义。 和/或构象状态。 这些模型仍然是推测性的，或者没有得到充分的量化。在这个项目中，我们将调查 解旋酶活性调节的机制;首先通过了解限制解旋酶活性的因素， 解旋酶单体（目标1），然后通过测量解旋酶寡聚化和量化它如何增强 解旋活性（目标2），最后通过研究解旋酶解旋与选定的蛋白质伴侣， 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-光镊， 荧光显微镜和微流控技术，以及传统的生物化学方法。这些新颖 方法，利用PI的专业知识，将用于检测解旋酶的解旋在单一的 分子水平，在真实的时间，并在高分辨率，同时测量其低聚和 构象状态此外，这些技术将使多部件的受控组装成为可能。 配合物除了提供解旋酶机制和DNA修复途径的见解， 参与，我们的研究将推进生物物理方法，用于研究生物分子的动力学 配合物

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.