The chromatin response in mammalian double strand break repair

描述（由申请人提供）：本提案的长期目标是确定由组蛋白H2AX磷酸化介导的哺乳动物DSB的染色质反应如何调节DSB修复。我们发现H2AX在控制姐妹染色单体重组（SCR）中起关键作用，这是一种同源重组（HR）过程，在细胞周期的S和G2阶段以无错误的方式修复复制相关的DNA损伤。在最近的工作中，我们发现MDC1，一种h2ax结合接头蛋白，是h2ax依赖性HR/SCR的主要介质。我们发现，对哺乳动物DSB的染色质响应编码了额外的DSB修复功能，调节单链退火（SSA）和非同源末端连接（NHEJ）。我们的工作揭示了先前未被怀疑的基于染色质的DSB修复功能的特异性程度，并建立了DSB修复的“组蛋白代码”的存在。在本提案中，我们将通过分析我们最近发现的一种新的mdc1相互作用蛋白复合物，包括TRIP12和核磷蛋白（NPM1），来确定H2AX/ mdc1介导的HR/SCR的生化基础。我们还将分析DSB染色质响应中编码的其他DSB修复功能。我们的具体目标是：1；定义TRIP12和NPM1的DSB修复功能。我们将使用遗传、生化和细胞生物学技术来实现这一目标。定义H2AX和相关蛋白在SSA和NHEJ中的作用。我们已经为SSA和NHEJ开发了新的哺乳动物报告器，提供快速、特异性和定量的流式细胞术测量这些DSB修复结果。我们将利用这些报告蛋白来确定H2AX及其相关蛋白对SSA和NHEJ的作用。3. 确定53BP1介导NHEJ的机制。我们将使用之前开发的NHEJ报告器，以及本文中描述的新报告器，来确定53BP1如何执行NHEJ。我们将结合使用新型多光子激光诱导DSB和实时成像，以及传统的DSB诱导剂在53BP1-/-细胞中研究53BP1如何调节NHEJ。本提案中所述项目的成功将：阐明以染色质为基础的HR/SCR调控途径。2. 确定TRIP12和NPM1是哺乳动物HR的新的染色质相关成分。3. 验证新的，快速的，特定的单链退火（SSA）和NHEJ报告。4. 揭示53BP1如何调控nhej5。验证一种新型的多光子激光和实时成像工具来研究DSB修复动力学。

DESCRIPTION (provided by applicant): The long-term objective of this proposal is to determine how the chromatin response to a mammalian DSB, mediated by the phosphorylation of histone H2AX, regulates DSB repair. We discovered a critical role for H2AX in controlling sister chromatid recombination (SCR), a homologous recombination (HR) process that operates during S and G2 phases of the cell cycle to repair replication-associated DNA damage in an error free manner. In recent work, we discovered that MDC1, an H2AX-binding adaptor protein, is the major mediator of H2AX-dependent HR/SCR. We discovered that the chromatin response to a mammalian DSB encodes additional DSB repair functions, regulating both single strand annealing (SSA) and non-homologous end joining (NHEJ). Our work revealed a previously unsuspected degree of specificity to chromatin-based DSB repair functions and established the existence of a "histone code" for DSB repair. In this proposal, we will determine the biochemical basis of H2AX/MDC1-mediated HR/SCR, by analyzing a new MDC1-interacting protein complex involving TRIP12 and nucleophosmin (NPM1), which we have recently identified. We will also analyze other DSB repair functions encoded within the DSB chromatin response. Our Specific Aims are: 1. Define DSB repair functions of TRIP12 and NPM1. We will use genetic, biochemical and cell biological techniques to accomplish this Aim. 2. Define roles of H2AX and associated proteins in SSA and in NHEJ. We have developed novel mammalian reporters for SSA and NHEJ that deliver rapid, specific and quantitative flow cytometric measurement of these DSB repair outcomes. We will use these reporters to determine the contribution of H2AX and its associated proteins to SSA and NHEJ. 3. Determine the mechanisms by which 53BP1 mediates NHEJ. We will use NHEJ reporters that we developed previously, as well as new reporters described herein, to determine how 53BP1 executes NHEJ. We will combine this with use of a novel multi-photon laser for DSB induction and real time imaging, as well as conventional DSB inducing agents in 53BP1-/- cells to study how 53BP1 regulates NHEJ. Success in the projects described in this proposal will: 1. Elucidate a chromatin-based pathway for regulating HR/SCR. 2. Identify TRIP12 and NPM1 as novel chromatin-associated components of mammalian HR. 3. Validate new, rapid and specific reporters of single strand annealing (SSA) and NHEJ. 4. Reveal how 53BP1 regulates NHEJ 5. Validate a novel multi-photon laser and real time imaging tool to study the kinetics of DSB repair.