The chromatin response in mammalian double strand break repair

描述(由申请人提供)：这项建议的长期目标是确定染色质对哺乳动物DSB的反应是如何通过组蛋白H2AX的磷酸化来调节DSB修复的。我们发现H2AX在控制姐妹染色单体重组(SCR)中起着关键作用，SCR是一种同源重组(HR)过程，运行在细胞周期的S和G2期，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是一种与H_2AX结合的接头蛋白，是H_2AX依赖的HR/SCR的主要介质。我们发现，染色质对哺乳动物DSB的反应编码额外的DSB修复功能，调节单链退火(SSA)和非同源末端连接(NHEJ)。我们的工作揭示了基于染色质的DSB修复功能以前未被怀疑的程度的特异性，并建立了DSB修复的“组蛋白密码”的存在。在这个建议中，我们将通过分析我们最近发现的涉及TRIP12和核磷蛋白(NPM1)的新的MDC1相互作用蛋白复合体来确定H_2AX/MDC1介导的HR/SCR的生化基础。我们还将分析DSB染色质反应中编码的其他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。我们将利用遗传、生化和细胞生物学技术来实现这一目标。2.明确H_2AX及其相关蛋白在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程序，可以快速、特异和定量地测量这些DSB修复结果。我们将使用这些记者来确定H_2AX及其相关蛋白对SSA和NHEJ的贡献。3.确定53BP1介导NHEJ的机制。我们将使用我们之前开发的NHEJ记者以及这里描述的新记者来确定53BP1如何执行NHEJ。我们将结合使用一种新型的多光子激光进行DSB诱导和实时成像，以及在53BP1-/-细胞中使用传统的DSB诱导剂来研究53BP1如何调节NHEJ。本提案中描述的项目的成功将：1.阐明一种基于染色质的调节HR/SCR的途径。2.鉴定TRIP12和NPM1为哺乳动物HR染色质相关的新成分。3.验证了新型、快速、特异的单链退火法(SSA)和NHEJ法。4.揭示53BP1是如何调控NHEJ的。5.验证一种新的多光子激光和实时成像工具来研究DSB修复的动力学。

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.