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: 8720011
• 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/8720011
• 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染色质对哺乳动物 DSB 的反应（由组蛋白 H2AX 磷酸化介导）如何调节 DSB 修复。我们发现 H2AX 在控制姐妹染色单体重组 (SCR) 中发挥着关键作用，SCR 是一种同源重组 (HR) 过程，在细胞周期的 S 期和 G2 期运行，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，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 修复功能的先前未曾怀疑的特异性程度，并确定了 DSB 修复“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涉及 TRIP12 和核磷蛋白 (NPM1) 的新 MDC1 相互作用蛋白复合物，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。我们将利用遗传、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。我们为 SSA 和 NHEJ 开发了新型哺乳动物报告基因，可以对这些 DSB 修复结果进行快速、特异性和定量的流式细胞术测量。我们将使用这些报告基因来确定 H2AX 及其相关蛋白对 SSA 和 NHEJ 的贡献。 3.确定53BP1介导NHEJ的机制。我们将使用我们之前开发的 NHEJ 报告基因以及本文描述的新报告基因来确定 53BP1 如何执行 NHEJ。我们将其与用于 DSB 诱导和实时成像的新型多光子激光以及 53BP1-/- 细胞中的传统 DSB 诱导剂相结合，以研究 53BP1 如何调节 NHEJ。本提案中描述的项目的成功将： 1. 阐明基于染色质的 HR/SCR 调节途径。 2. 将 TRIP12 和 NPM1 鉴定为哺乳动物 HR 的新型染色质相关成分。 3. 验证新的、快速的、特异性的单链退火 (SSA) 和 NHEJ 报告基因。 4. 揭示 53BP1 如何调节 NHEJ 5. 验证新型多光子激光和实时成像工具来研究 DSB 修复动力学。

项目成果

Hijacking the DNA damage response to enhance viral replication: gamma-herpesvirus 68 orf36 phosphorylates histone H2AX.

劫持 DNA 损伤反应以增强病毒复制：γ-疱疹病毒 68 orf36 磷酸化组蛋白 H2AX。

• 发表时间: 2007-07-20
• 影响因子: 16
• 作者: Xie, Anyong;Scully, Ralph
• 通讯作者: Scully, Ralph

PARP3 affects the relative contribution of homologous recombination and nonhomologous end-joining pathways.

PARP3 影响同源重组和非同源末端连接途径的相对贡献。

• 发表时间: 2014-05
• 影响因子: 14.9
• 作者: Beck, Carole;Boehler, Christian;Guirouilh Barbat, Josée;Bonnet, Marie;Illuzzi, Giuditta;Ronde, Philippe;Gauthier, Laurent R;Magroun, Najat;Rajendran, Anbazhagan;Lopez, Bernard S;Scully, Ralph;Boussin, François D;Schreiber, Valérie;Dantz
• 通讯作者: Dantz