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利用 资源由NIH/NCRR资助的中心拨款提供。子项目和 调查员(PI)可能从NIH的另一个来源获得了主要资金， 并因此可以在其他清晰的条目中表示。列出的机构是 该中心不一定是调查人员的机构。 这项拟议研究的长期目标是了解基因组完整性是如何在细胞中保持的。由DMA修复、DMA复制和检查点蛋白组成的高度复杂的监测机制已经发展起来，以维持基因组的完整性。这些机制的功能障碍会导致各种临床症状，包括增加患癌症的风险。DNA损伤导致细胞周期停滞和DNA损伤所激活的下游效应蛋白的研究进展 修复工作已经完成，但检测和发出DNA损伤信号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通路的传感器组件，并确定它们如何在细胞周期的S阶段稳定停滞的复制叉子中协同工作。 被认为参与这一反应的蛋白质包括MRN复合体(由Mre11、Rad50和NBS1组成)和RPA(RPA，真核细胞中主要的单链DNA结合蛋白，由p70、p34、p14三个亚基组成)，它们复杂地参与DNA代谢和维持基因组稳定。我们最近发现了MRN复合体和RPA之间的蛋白质/蛋白质相互作用。我们认为，MRN复合体和RPA共同作用，以应对DNA损伤和复制分叉停滞。然而，MRN复合体和RPA如何感知并启动对停滞的复制分叉的响应仍未定义。这一建议将检验这样的假设，即停滞的复制叉以RPA依赖的方式刺激MRN复合体，以稳定和修复停滞复制的所有部位的损伤。为了验证这一假设，提出了以下目标： 目的1：研究RPA与MRN复合体之间的直接相互作用。 目的2：确定RPA在将M/R/N复合体招募到停滞复制位点中的作用。 目的3：研究RPA和磷酸化在MRN复合体连接DNA过程中的作用。

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.