RPA-Directed DNA Repair Mechanisms

项目摘要 几乎每个DNA修复和复制过程都需要复制蛋白A(RPA)。RPA绑定单个- 滞留的DNA，并在DNA维护位置与数十种蛋白质相互作用。我们感兴趣的是 在执行特定的DNA修复任务时，RPA和其他蛋白质之间形成的蛋白质复合体。这个 目前的项目考察了RPA在尿嘧啶碱基切除修复中作为中心支架的作用。我们的方法是 操纵RPA与尿嘧啶DNA糖基酶(UNG2)的结合，以检查它们之间的相关性 互动。我们开发了一种策略，将RPA和UNG2共价捆绑在一起，以形成迷你DNA 当两种蛋白质在细胞中相互作用时，修复类似于两种蛋白质结构的复合体。我们的预赛 数据表明我们成功地在细胞和重组中形成了RPA-UNG2蛋白复合体 系统。这使我们能够确定RPA作为与UNG2的蛋白质复合体的一部分是如何发挥作用的， 而互补性研究则考察了削弱它们之间联系的效果。具体地说，目标1使用 纯化的RPA和UNG2构建来单独或作为复合体的一部分来检测蛋白质的活性。 使用合成DNA底物的结合和酶实验将确定它们的结构性质 体内的底物。我们特别关注RPA和UNG2在单链DNA-双链DNA连接中的作用，他们的 已知的底物，通过制备类似于在 原子核。目的2检测尿嘧啶条件下人结肠癌细胞中RPA-UNG2蛋白复合体的表达 由常用化疗药物引起的应激反应。RPA与 在复制分叉处，UNG2将被控制并与UNG2的S尿嘧啶切除效率相关。最后， 与RPA和UNG2相关的其他细胞蛋白将被确定。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体的背景下进行研究。这个 本项目的目标范围是检查碱基切除修复中的RPA，这将有助于我们的方法 这将广泛适用于检测其他DNA中含有RPA的蛋白质复合体 维修流程。

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.