The chromatin response in mammalian double strand break repair

哺乳动物双链断裂修复中的染色质反应

• 批准号: 8294525
• 负责人: Ralph Scully
• 金额: $ 32.73万
• 依托单位: BETH ISRAEL DEACONESS MEDICAL CENTER
• 依托单位国家: 美国
• 财政年份: 2005
• 资助国家: 美国
• 起止时间: 2005-09-15 至 2015-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8294525
• 关键词: Acute Myelocytic Leukemia; Adaptor Signaling Protein; Affinity Chromatography; BARD1 gene; BRCA1 gene; Binding; Binding Proteins; Biochemical; Biological; C-terminal; Cell Cycle; Cell Nucleus; Cells; Chromatin; Collaborations; Complex; DNA; DNA Damage; DNA Repair; Data; Double Strand Break Repair; Excision; FHA Domain; G2 Phase; G22P1 gene; Genes; Genetic; Genetic Recombination; Genomic Instability; Hereditary Breast and Ovarian Cancer Syndrome; Histone Code; Histone H4; Image; Imaging Device; Immunoglobulin Class Switching; Immunoglobulin Switch Recombination; Kinetics; Lasers; Life; Lysine; Malignant Neoplasms; Mammalian Cell; Mammals; Measurement; Mediating; Mediator of activation protein; Mutate; NBS1 gene; NPM1 gene; Nonhomologous DNA End Joining; Outcome; Pathway interactions; Phosphorylation; Process; Property; Proteins; Reaction; Recruitment Activity; Reporter; Role; S Phase; Serine; Sister Chromatid; Site; Specificity; Susceptibility Gene; System; Systems Biology; Techniques; Time; Work; base; chromatin modification; homologous recombination; human H2AX protein; medical schools; multi-photon; novel; nucleophosmin; prevent; protein complex; protein structure; repaired; research study; response; success; ubiquitin-protein ligase

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.

描述（由申请人提供）：该提案的长期目标是确定组蛋白H2AX磷酸化介导的染色质反应如何调节DSB修复。我们发现了H2AX在控制姐妹染色单体重组（SCR）中的关键作用，这是一种同源重组（HR）过程，该过程在细胞周期的S和G2阶段运行，以以无错误的方式修复与复制相关的DNA损伤。在最近的工作中，我们发现MDC1是H2AX结合衔接蛋白，是H2AX依赖性HR/SCR的主要介体。我们发现对哺乳动物DSB的染色质反应编码其他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对基于染色质DSB修复功能的特异性程度，并确定了用于DSB修复的“组蛋白代码”。在此提案中，我们将通过分析涉及TRIP12和核酚蛋白（NPM1）的新的MDC1相互作用蛋白复合物（NPM1）来确定H2AX/MDC1介导的HR/SCR的生化基础，我们最近已确定。我们还将分析在DSB染色质反应中编码的其他DSB修复功能。我们的具体目的是：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。 2。定义H2AX和相关蛋白在SSA和NHEJ中的作用。我们已经开发了用于SSA和NHEJ的新型哺乳动物记者，这些记者可为这些DSB修复结果提供快速，特定和定量的流式细胞仪测量。我们将使用这些记者来确定H2AX及其相关蛋白对SSA和NHEJ的贡献。 3。确定53BP1介导NHEJ的机制。我们将使用以前开发的NHEJ记者以及本文所述的新记者来确定53BP1如何执行NHEJ。我们将使用新型的多光子激光器进行DSB诱导和实时成像，以及53BP1 - / - 细胞中传统的DSB诱导剂研究53BP1调节NHEJ的传统DSB。在本提案中描述的项目中的成功将：1。阐明基于染色质的途径来调节HR/SCR。 2。将TRIP12和NPM1识别为哺乳动物HR的新型染色质相关成分。 3。验证单链退火（SSA）和NHEJ的新的，快速和特定的记者。 4。揭示53BP1如何调节NHEJ 5。验证一种新型的多光子激光器和实时成像工具来研究DSB修复的动力学。