RPA-Directed DNA Repair Mechanisms

项目摘要 复制蛋白A（RPA）是几乎所有DNA修复和复制过程所必需的。RPA结合单个- 链DNA和相互作用的几十个蛋白质在网站的DNA维护。我们感兴趣的是 RPA和其他蛋白质在执行特定DNA修复任务时形成的蛋白质复合物。的 目前的项目研究RPA在尿嘧啶碱基切除修复中作为中心支架的作用。我们的做法是 操纵RPA与尿嘧啶DNA糖基化酶（UNG 2）的结合，以检查它们与尿嘧啶DNA糖基化酶（UNG 2）的相关性。 互动我们已经开发出一种策略，将RPA和UNG 2共价连接在一起形成微型DNA 修复复合物，类似于两种蛋白质在细胞中相互作用时的结构。我们的初步 数据表明我们在细胞和重组体中成功形成RPA-UNG 2蛋白复合物 系统.这使我们能够明确地确定RPA如何作为UNG 2蛋白复合物的一部分发挥作用， 而补充研究则考察了削弱它们之间的联系所产生的影响。具体而言，目标1使用 纯化的RPA和UNG 2构建体以检查单独的蛋白质或作为复合物的一部分的蛋白质的活性。 使用合成DNA底物的结合和酶促实验将确定它们的结构性质。 体内底物。我们特别关注RPA和UNG 2在ssDNA-dsDNA连接处的作用， 已知的底物，通过制备尿嘧啶叉样DNA结构，其类似于在DNA中发现的复制叉。 原子核目的2检测尿嘧啶条件下人结直肠细胞中RPA-UNG 2蛋白复合物 由常用化疗剂诱导的应激。卢旺达爱国军与 UNG 2将受到控制，并与UNG 2在复制叉处的尿嘧啶切除效率相关。最后， 将鉴定与RPA和UNG 2相关的其它细胞蛋白。这些蛋白质的功能 将在尿嘧啶碱基切除修复过程中形成的多蛋白复合物的背景下进行检查。的 本项目的目标范围检查RPA在碱基切除修复将有助于我们的方法 开发，它将广泛适用于检查其他DNA中含有RPA的蛋白质复合物 修复过程。

Project Summary Replication protein A (RPA) is required for nearly every DNA repair and replication process. RPA binds single- stranded DNA and interacts with dozens of proteins at sites of DNA maintenance. We are interested in the protein complexes that form between RPA and other proteins when performing specific DNA repair tasks. The current project examines RPA’s role as a central scaffold in uracil base excision repair. Our approach is to manipulate the binding of RPA to Uracil DNA Glycosylase (UNG2) to examine the relevance of their interaction. We have developed a strategy to covalently tether together RPA and UNG2 to form mini DNA repair complexes that resemble the architecture of the two proteins when they interact in cells. Our Preliminary Data demonstrates our success at forming RPA-UNG2 protein complexes in both cellular and recombinant systems. This allows us to definitively determine how RPA functions as part of a protein complex with UNG2, and complementary studies examine the effects of weakening their association. Specifically, Aim 1 uses purified RPA and UNG2 constructs to examine the activity of the proteins alone or as part of a complex. Binding and enzymatic experiments using synthetic DNA substrates will determine the structural nature of their substrates in vivo. We focus specifically on the action of RPA and UNG2 at ssDNA-dsDNA junctions, their known substrates, by preparing uracilated fork-like DNA structures that resemble replication forks found in the nucleus. Aim 2 examines the RPA-UNG2 protein complex in human colorectal cells under conditions of uracil stress that are induced by commonly used chemotherapeutic agents. The strength of RPA’s interaction with UNG2 will be controlled and correlated with UNG2’s uracil excision efficiency at the replication fork. Finally, additional cellular proteins that associate with RPA and UNG2 will be identified. The function of these proteins will be examined in the context of multi-protein complexes that form during uracil base excision repair. The targeted scope of this project examining RPA in base excision repair will facilitate our methodological development, which will be widely adaptable for examining RPA-containing protein complexes in other DNA repair processes.