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

本子项目是利用由NIH/NCRR资助的中心赠款提供的资源的众多研究子项目之一。子项目和研究者（PI）可能已经从另一个NIH来源获得了主要资金，因此可以在其他CRISP条目中表示。列出的机构是中心的，不一定是研究者的机构。这项研究的长期目标是了解基因组完整性是如何在细胞中保存的。高度复杂的监测机制，包括DMA修复，DMA复制和检查点蛋白，已经发展到维持基因组完整性。这些机制的功能障碍可导致各种临床症状，包括增加患癌症的风险。我们对DMA损伤激活的下游效应蛋白的理解取得了进展，这些效应蛋白导致细胞周期阻滞和DNA修复，但DNA损伤检测和信号传递的机制仍然难以捉摸。因此，我们的目标是定义复制应激反应途径的传感器组件，并确定它们如何在细胞周期的s期合作稳定停滞的复制分叉。被认为参与这种反应的蛋白质包括MRN复合物（由MRE11、RAD50和NBS1组成）和RPA （RPA是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。我们最近发现了MRN复合物和RPA之间的蛋白质/蛋白质相互作用。我们认为MRN复合体和RPA共同应对DNA损伤和停滞的复制分叉。然而，MRN复合体和RPA如何感知并启动对停滞复制分叉的响应仍未明确。这一提议将验证一种假设，即停滞复制分叉以依赖于rpa的方式刺激MRN复合体，以稳定和修复所有停滞复制位点的损伤。为了验证这一假设，提出了以下目标：目标1：表征RPA和MRN复合物的直接蛋白质-蛋白质相互作用。目标2：定义RPA在招募M/R/N复合体到停滞复制位点中的作用。目的3：表征RPA和磷酸化在MRN复合物拴住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.