Mechanisms of RPA, Recombinases, and Mediators in Homologous Recombination

项目总结 同源重组(HR)是维持基因组稳定性的关键，其功能是 消除DNA双链断裂和染色体损伤。协调HR结果的蛋白质突变 在各种癌症和遗传性疾病中。当双链DNA断裂是 核裂解切除以产生单链DNA(SsDNA)突出物，这些突出物很容易被包被和 受复制蛋白A(RPA)保护。然后RPA被RAD51重组酶取代，形成Long 核蛋白细丝催化下游的链交换反应，促进HR。许多支持和反对的人 抗HR介体蛋白控制RAD51核蛋白细丝在RPA包被的单链DNA上的组装； 从而决定了人力资源的时间和空间的建立。介质蛋白的突变/缺陷，如 BRCA2导致HR严重失控，导致基因组不稳定和相关癌症。就像许多人一样 与DNA、多种蛋白质和DNA的复制、修复和重塑相关的代谢过程 结合能力在相同的DNA模板上共同发挥作用。研究一种单一的 在这样一个多蛋白质环境中，酶的功能在技术上是具有挑战性的。我们应对这一挑战的方法 使用感兴趣的蛋白质的特定部位荧光版本，该版本在 与单链DNA特异性结合。我们利用了非规范氨基酸(NCAA)和菌株促进 环加成生成荧光版本的RPA(RPAf)，其准确地报告其在单链DNA上的动力学， 使我们能够研究RAD51核蛋白细丝是如何在RPA涂层基质上形成的。使用这个 方法，我们已经揭示了RPA中的每个结构域如何与单链DNA结合/解离，并提出了一个新的 RPA功能的范例。我们将利用这一技术进步来确定RPA如何 在HR过程中位于DNA上及其DNA结合区在RPA交换过程中如何发生微解离 (目标1)。我们将破译前HR介体蛋白如何取代RPA，同时促进RAD51的形成 心率期间的核蛋白细丝(目标2)。将使用基于NCAA的方法来实现这些目标 产生荧光蛋白质以及集合和单分子生物物理方法的组合 获取各个步骤的作用机制的动力学参数。除了提供机械性的 对HR机制的深入了解，我们的研究将为研究多蛋白质的动力学提供技术支持 DNA反应。

PROJECT SUMMARY Homologous recombination (HR) is critical for the maintenance of genomic stability, and functions to eliminate DNA double-strand breaks and chromosomal lesions. Mutations in proteins that coordinate HR result in a variety of cancers and hereditary disorders. HR is initiated when a double-stranded DNA break is nucleolytically resected to generate single-stranded DNA (ssDNA) overhangs, which are readily coated and protected by Replication Protein A (RPA). RPA is then replaced by the RAD51 recombinase, which forms long nucleoprotein filaments to catalyze downstream strand exchange reactions to promote HR. A host of pro- and anti-HR mediator proteins control the assembly of the RAD51 nucleoprotein filament on RPA-coated ssDNA; thereby dictating the temporal and spatial establishment of HR. Mutations/defects in mediator proteins such as BRCA2 result in severe deregulation of HR causing genomic instability and associated cancers. As in many metabolic processes associated with replication, repair and remodeling of DNA, multiple proteins with DNA binding capacity function together on the same DNA template. Investigating the mechanism of how a single enzyme functions in such a multi-protein milieu is technically challenging. Our approach to address this challenge uses site-specific fluorescent versions of a protein of interest that produces a change in fluorescence upon specifically binding to ssDNA. We have utilized non-canonical amino acids (ncAA) and strain-promoted cycloaddition to generate fluorescent versions of RPA (RPAf) that accurately reports its dynamics on ssDNA, enabling us to investigate how RAD51 nucleoprotein filaments are formed on RPA coated substrates. Using this methodology, we have uncovered how each domain within RPA binds/dissociates on ssDNA and present a new paradigm for RPA function. We will capitalize on this technological advancement to determine how RPA is situated on DNA during HR and how its DNA binding domains undergo micro-dissociation during RPA-exchange (Aim 1). We will decipher how pro-HR mediator proteins displace RPA while promoting the formation of RAD51 nucleoprotein filaments during HR (Aim 2). These aims will be achieved using ncAA-based approaches to generate fluorescent proteins and a combination of ensemble and single molecule biophysical methodologies to obtain kinetic parameters for the individual steps in their mechanism of action. Beyond providing mechanistic insights on mechanism of HR, our studies will advance technology for investigating the dynamics of multi-protein DNA reactions.