COBRE: UNE MED CTR: P8:MRN AND RPA PROTEIN-PROTEIN INTERACTIONS IN DNA DAMAGE

这个子项目是许多研究子项目中的一个 由NIH/NCRR资助的中心赠款提供的资源。子项目和 研究者（PI）可能从另一个NIH来源获得了主要资金， 因此可以在其他CRISP条目中表示。所列机构为 中心，不一定是研究者的机构。 这项研究的长期目标是了解细胞中基因组的完整性是如何保持的。高度复杂的监视机制，包括DMA修复，DMA复制和检查点蛋白，已经发展到维持基因组的完整性。这些机制的功能障碍可导致各种临床症状，包括癌症风险增加。DNA损伤后激活的下游效应蛋白导致细胞周期阻滞和DNA损伤的研究进展 修复已经完成，但检测和发出信号的DNA损伤机制仍然难以捉摸。因此，我们的目标是定义的复制应激反应途径的传感器组件，并确定它们如何协同工作，稳定停滞的复制叉在S期的细胞周期。 被认为参与这种反应的蛋白质包括MRN复合物（由MRE 11、RAD 50和NBS 1组成）和RPA（RPA，真核细胞中主要的单链DNA结合蛋白，由三个亚基p70、p34、p14组成），这些蛋白质复杂地参与DNA代谢和基因组稳定性的维持。我们最近已经确定了MRN复合物和RPA之间的蛋白质/蛋白质相互作用。我们相信，MRN复合体和RPA一起工作，以应对DNA损伤和停滞的复制叉。但是，MRN复合体和RPA如何感知并启动对停止的复制分叉的响应仍未定义。这项提案将测试的假设，停滞的复制叉刺激MRN复杂的RPA依赖的方式，以稳定和修复损坏的停滞复制的所有网站。为了检验这一假设，提出了以下目标： 目的1：研究RPA与MRN复合物的直接蛋白质-蛋白质相互作用。 目的2：定义RPA在M/R/N复合物招募到停滞复制位点中的作用。 目的3：表征RPA和磷酸化在MRN复合物束缚DNA中的作用。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. The long-term goal of the proposed research is to understand how genomic integrity is preserved in cells. Highly complex surveillance mechanisms, consisting of DMA repair, DMA replication and checkpoint proteins, have developed to maintain genomic integrity. Dysfunction of these mechanisms can lead to a variety of clinical symptoms including an increased risk of cancer. Progress in our understanding of the downstream effectors proteins activated in response to DMA damage that lead to cell cycle arrest and DNA repair have been made but the mechanisms by which DNA damage is detected and signaled remain elusive. Therefore, we aim to define the sensor components of the replication stress response pathway and determine how they work cooperatively in stabilizing stalled replication forks during S-phase of the cell cycle. Among the proteins thought to be involved in this response include the MRN complex (composed of MRE11, RAD50 and NBS1), and RPA, (RPA, the major single-stranded DNA binding protein in eukaryotic cells composed of three subunits p70, p34, p14), proteins that are intricately involved in DNA metabolism and maintenance of genomic stability. We have recently identified a protein/protein interaction between the MRN complex and RPA. We believe that the MRN complex and RPA work together in response to DNA damage and stalled replication forks. However, how the MRN complex and RPA sense and initiate a response to stalled replication forks remains undefined. This proposal will test the hypothesis that stalled replication forks stimulate the MRN complex in an RPA-dependent manner to stabilize and repair damage at all sites of stalled replication. To test this hypothesis the following aims are proposed: Aim 1: To characterize the direct protein-protein interactions of RPA and the MRN complex. Aim 2: Define the role of RPA in the recruitment of the M/R/N complex to sites of stalled replication. Aim 3: Characterize the role of RPA and phosphorylation in the tethering of DNA by the MRN complex.