Define redundant functions of H2AX and NBS1 in DNA repair

项目摘要 破译基因组不稳定性和肿瘤发生的分子机制是长期的 实验室的目标。这一建议的广泛目标反映了我们寻求全面 了解参与DNA修复的网络，并确定这些蛋白质和途径 交叉、互动、沟通、协调和协作以维护基因组。短期目标是 对几种DNA损伤信号传导和修复途径进行详细的机制研究， 为实现我们开发DNA修复网络用于癌症治疗的长期目标提供基础。 这项提议将定义两个重叠的DNA损伤信号通路，它们共同对 细胞存活我们和其他研究人员已经构建了一个精细的信号通路， 并调节许多DNA损伤修复蛋白在DNA损伤位点的募集和积累。 休息.这种H2 AX依赖性通路由H2 AX、MDC 1、RNF 8和RNF 168组成。然而，修复 在H2 AX-、MDC 1-、RNF 8-或RNF 168-缺陷细胞或小鼠中观察到的缺陷是轻微的， 有一种H2 AX非依赖性机制参与了这些下游修复的募集， proteins.我们认为这种H2 AX非依赖性途径是由NBS 1控制的。 根据我们以前的研究和本提案中提出的初步数据，我们假设， H2 AX-和NBS 1-依赖性途径参与DNA损伤反应，并且对细胞 生存我们认为这两条通路具有冗余功能，特别是在促进同源 重组修复然而，它们并不是完全分开的，因为它们在多个点相交。这 这使得我们很难描述这两个冗余通路的功能。是 我们是否能够阐明单一途径对不断增长的网络的贡献是未知的，即，可以 我们解开网络，了解不同途径交叉和贡献的机制， 到生物过程？我们将在本申请中解决这个问题，并将进一步研究 H2 AX和NBS 1依赖性途径共同作用以确保细胞存活的机制， 完成DNA修复。我们提出了以下具体目标：1）确定NBS 1是否起作用 与已建立的H2 AX-MDC 1-RNF 8-RNF 168途径冗余，以确保细胞存活; 2）描述 NBS 1依赖性途径; 3）探索NBS 1和H2 AX导致细胞致死的潜在机制 共同耗竭这些研究不仅使我们能够了解H2 AX和NBS 1在细胞中的冗余功能， 但也将揭示如何研究蛋白质的功能和途径，在今天的复杂信号 网络.此外，这些研究的结果将为利用DNA修复缺陷提供理论基础， 将综合杀伤力概念应用于癌症患者的精准医疗。

Project Summary Deciphering the molecular mechanisms underlying genomic instability and tumorigenesis is the long-term goal of my laboratory. The broad objective, of this proposal, reflects our pursuit to gain a comprehensive understanding of the network involved in DNA repair and to determine how these proteins and pathways intersect, interact, communicate, coordinate, and collaborate for genome maintenance. The short-term goal is to perform detailed mechanistic studies of several DNA damage signaling and repair pathways, which will provide the foundation to achieve our long-term goal of exploiting DNA repair network for cancer therapy. This proposal will define two overlapping DNA damage-signaling pathways that together are essential for cell survival. We and other researchers have constructed an elaborate signaling pathway that acts downstream of H2AX and regulates the recruitment and accumulation of many DNA damage repair proteins at sites of DNA breaks. This H2AX-dependent pathway is composed of H2AX, MDC1, RNF8, and RNF168. However, repair defects observed in H2AX-, MDC1-, RNF8-, or RNF168-deficient cells or mice are mild, raising the possibility that there is an H2AX-independent mechanism involved in the recruitment of these downstream repair proteins. We propose that this H2AX-independent pathway is controlled by NBS1. On the basis of our previous studies and preliminary data presented in this proposal, we hypothesize that the H2AX- and NBS1-dependent pathways are involved in the DNA damage response and are critical for cell survival. We believe that these two pathways have redundant functions, especially in promoting homologous recombination repair. However, they are not completely separate, since they intersect at multiple points. This makes it considerably challenging for us to delineate the functions of these two redundant pathways. It is unknown whether we can elucidate the contribution of a single pathway to the ever-growing network, i.e., can we untangle the network to understand the mechanisms by which different pathways intersect and contribute to biological processes? We will address this question in this application and we will further study the mechanisms by which the H2AX- and NBS1-dependent pathways act together to ensure cell survival and the completion of DNA repair. We propose the following specific aims: 1) determine whether NBS1 acts redundantly with the established H2AX-MDC1-RNF8-RNF168 pathway to ensure cell survival; 2) delineate the NBS1-dependent pathway; and 3) explore the mechanisms underlying cell lethality caused by NBS1 and H2AX co-depletion. These studies will not only allow us to understand the redundant functions of H2AX and NBS1 in vivo but will also reveal ways to investigate the functions of proteins and pathways in today’s complex signaling networks. Moreover, results from these studies will provide the rationale for exploiting DNA repair defect and applying synthetic lethality concept in precision medicine for cancer patients.