Fluorescent probes to monitor Rad51 nucleoprotein dynamics

 描述（由申请方提供）：细胞中的双链DNA断裂主要通过同源重组（HR）系统修复。有缺陷的或不受调节的HR导致染色体重排，导致基因组不稳定性，这反过来又导致各种癌症和遗传性疾病。单链DNA（ssDNA）上Rad 51细丝的形成是HR途径中的关键事件之一，并且受到促重组介体蛋白和抗重组介体蛋白的严格调控。我们的长期目标是精确地确定Rad 51核蛋白丝的形成和拆卸的动力学ssDNA和各种亲和抗HR介体蛋白改变这些动力学的影响HR的机制。技术限制阻碍了Rad 51核蛋白丝形成单链DNA（ssDNA）的研究。我们建议通过开发荧光Rad 51和RPA探针来克服这些障碍，当它们与ssDNA结合时，荧光会发生变化。我们将利用非天然氨基酸掺入策略来开发这些探针，这些探针将与停流动力学测定相结合，以实时监测核蛋白丝的动态。由于我们的探针是Rad 51/RPA缔合/解离的直接报告者，我们将利用这种荧光工具包来研究Rad 51和RPA在多蛋白实验装置中同时存在时如何与ssDNA结合。研究Rad 51丝形成的ssDNA和dsDNA基板上的意义是突出的，我们最近的研究结果，Rad 51丝的结合DNA上下文直径控制Srs 2的活性。Srs 2是从ssDNA清除Rad 51核蛋白丝的抗重组酶，并且作为解旋酶能够解旋dsDNA。Rad 51和Srs 2之间的物理相互作用是拆除核蛋白丝所必需的，并且严格在ssDNA上观察到。当Rad 51结合在dsDNA上时，它通过非常相同的物理相互作用界面抑制Srs 2的DNA解旋活性。具体目标是：（1）开发一种基于荧光的实时检测ssDNA上核蛋白丝动态的工具包。(2)确定Rad 51对Srs 2活性的DNA背景依赖性调节背后的机制。我们将获得核蛋白组装和拆卸过程中各个步骤的动力学参数，并揭示Srs 2和Rad 51之间相互作用的分子细节。这些研究将使我们能够建立一个精确的机制模型，Rad 51核蛋白丝是如何形成的，以及介体蛋白如何改变其动力学。这些信息对于理解介体蛋白在HR相关缺陷中的作用，以及揭示为什么这些蛋白质中的突变会导致基因组不稳定性，癌症和相关遗传疾病至关重要。除了解决这些研究问题，我们将开发多个研究项目，非常适合从事生物医学相关的研究在犹他州州立大学的本科生。

 DESCRIPTION (provided by applicant): Double strand DNA breaks in the cell are repaired primarily by the Homologous Recombination (HR) system. Defective or unregulated HR results in chromosome rearrangements leading to genomic instability, which in turn results in a variety of cancers and hereditary disorders. Formation of the Rad51 filament on single- stranded DNA (ssDNA) is one of the key events in the HR pathway and is under tight regulation by both pro- and anti-recombinogenic mediator proteins. Our long-term goal is to precisely determine the dynamics of Rad51 nucleoprotein filament formation and its disassembly on ssDNA and the mechanism by which various pro- and anti-HR mediator proteins alter these dynamics to influence HR. Technical limitations have hindered the study of Rad51 nucleoprotein filament formation on single-stranded DNA (ssDNA). We propose to overcome these obstacles through the development of fluorescent Rad51 and RPA probes which, when bound to ssDNA will undergo a change in fluorescence. We will be utilizing the unnatural amino acid incorporation strategy to develop these probes which will be combined with stopped flow kinetic assays to monitor the dynamics of the nucleoprotein filament in real-time. Since our probes are direct reporters of Rad51/RPA association/dissociation, we will utilize this fluorescent tool-kit to investigate how Rad51 and RPA bind to ssDNA when they are both present in a multi-protein experimental setup. Significance of studying Rad51 filament formation on both ssDNA and dsDNA substrates is highlighted by our recent findings that the bound-DNA context of a Rad51 filament diametrically controls the activity of Srs2. Srs2 is an anti-recombinase that clears Rad51 nucleoprotein filaments from ssDNA and as a helicase is capable of unwinding dsDNA. Physical interaction between Rad51 and Srs2 is required to dismantle the nucleoprotein filament and is observed strictly on ssDNA. When Rad51 is bound on dsDNA, it inhibits the DNA unwinding activity of Srs2 through the very same physical interaction interface. The Specific Aims are to: (1) Develop a real-time fluorescence-based tool kit to measure nucleoprotein filament dynamics on ssDNA. (2) Determine the mechanism behind the DNA-context dependent regulation of Srs2 activity by Rad51. We will obtain kinetic parameters for the various steps in the nucleoprotein assembly and disassembly process and uncover the molecular details of the interaction between Srs2 and Rad51. These studies will enable us to build a precise mechanistic model of how Rad51 nucleoprotein filaments are formed and how mediator proteins alter its dynamics. This information is critical in understanding the role of mediator proteins in HR associated defects, and to uncover why mutations in these proteins lead to genomic instability, cancers and associated genetic disorders. In addition to addressing these research questions, we will develop multiple research projects well suited to engage undergraduate students in bio-medically relevant research at Utah State University.