Mechanism and regulation of V(D)J repair

V(D)J修复机制及调控

• 批准号: 7727628
• 负责人: PATRICIA CORTES
• 金额: $ 31.73万
• 依托单位: ICAHN SCHOOL OF MEDICINE AT MOUNT SINAI
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-06-15 至 2014-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7727628
• 关键词: Affect; Age; Amino Acids; Antibodies; Antigens; Cells; Cleaved cell; Complex; Cryoelectron Microscopy; Crystallization; Cytoplasm; DNA; DNA Binding Domain; DNA Repair; DNA-PKcs; Data; Data Set; Defect; Development; Disease; G22P1 gene; Gel; Genes; Genetic Recombination; Genome Stability; Genomics; Growth; Growth and Development function; Human; Immune; Immune system; Immunodeficiency and Cancer; Immunologic Deficiency Syndromes; In Vitro; Knowledge; LIG4 gene; Ligase; Link; Lymphocyte; Lymphoid; Mediating; Microcephaly; Molecular; Mutation; N-terminal; Nonhomologous DNA End Joining; Paste substance; Pathway interactions; Patients; Pattern; Peptide Signal Sequences; Phenotype; Play; Post-Translational Protein Processing; Principal Investigator; Process; Property; Protein Fragment; Proteins; RAG1 gene; Recombinant Proteins; Regulation; Role; Specificity; Stem cells; Structure; Syndrome; T-Cell Receptor; T-Lymphocyte; V(D)J Recombination; Work; XRCC4 gene; artemis; base; complex IV; exhaustion; human disease; in vivo; migration; mutant; neural growth; novel; programs; protein complex; protein protein interaction; public health relevance; repaired; research study

DESCRIPTION (provided by applicant): Ligase IV has been shown to play critical roles in Non-Homologous End Joining (NHEJ), V(D)J recombination and development of the immune system, stem cell exhaustion, ageing and neural growth and development, as exemplified by the phenotype of patients with hypomorphic mutations in Ligase IV. Numerous proteins have been shown to modulate Ligase IV activity. Among them, XRCC4 and Cernunnos/XLF are perhaps the best/most conclusively characterized. Both of these factors, like Ligase IV, are part of the core-NHEJ machinery. Human mutations in XRCC4 have not been described yet, but interestingly, Cernunnos/XLF mutations in humans result in growth defects, microcephaly and immunodeficiency, similar to what has been observed for Ligase IV mutations. Our recent findings have identified Artemis, a factor also linked to immunodeficiency in humans, as a protein that directly interacts with Ligase IV, in addition we have observed regulation of XRCC4 by Ligase IV. In this project we propose to analyze the functional relevance of these novel Ligase IV/Artemis complex in genomic stability and V(D)J recombination. The mechanism by which Ligase IV regulates XRCC4 function, and the composition and function of endogenous Ligase IV complexes in lymphocytes will also be investigated. Our preliminary work with Ligase IV recombinant protein and protein fragments has resulted in crystallization of a Ligase IV fragment containing the Ligase IV DNA binding domain. Experiments are proposed to solve the structure of Ligase IV and/or Ligase IV in complex with Artemis. All these are challenging yet very relevant experiments for which we show significant progresses. Furthermore, the impact of mutations identified in patients with LIG4 syndrome on regulation of its function by Artemis and on how these mutant Ligase IV proteins affect XRCC4 function will also be investigated. Information gained from the proposed studies will contribute to a molecular and structural understanding of NHEJ, V(D)J recombination and human diseases linked to defects in both of these processes. PUBLIC HEALTH RELEVANCE: Despite important progress in our understanding of Ligase IV function, its structure remains largely uncharacterized and our preliminary data suggest novel mechanisms of regulation that remain to be unraveled. This project proposes to investigate our preliminary finding on novel Ligase IV interactions and functions by using in vivo and in vitro approaches; we also propose studies to solve the structure of Ligase IV and Ligase IV complexes. By increasing our knowledge of Ligase IV structure, function and regulation, in V(D)J recombination and DNA repair, the proposed work will contribute to our understanding of human disease, specifically, Ligase IV and Artemis mediated, immunodeficiency and cancer and will facilitate the path to find better treatments for this debilitating diseases.

描述（由申请人提供）：连接酶IV已被证明在非同源末端连接（NHEJ）、V(D)J重组和免疫系统的发育、干细胞衰竭、衰老和神经生长和发育中发挥关键作用，连接酶IV的亚型突变患者的表型就是例证。许多蛋白质已被证明可以调节连接酶IV的活性。其中，XRCC4和Cernunnos/XLF可能是表征最好/最明确的。这两种因子，像连接酶IV一样，都是核心nhej机制的一部分。人类XRCC4突变尚未被描述，但有趣的是，人类的Cernunnos/XLF突变导致生长缺陷，小头畸形和免疫缺陷，类似于连接酶IV突变。我们最近的研究发现，与人类免疫缺陷相关的Artemis是一种直接与连接酶IV相互作用的蛋白，此外，我们还观察到连接酶IV对XRCC4的调控。在这个项目中，我们建议分析这些新型连接酶IV/Artemis复合物在基因组稳定性和V(D)J重组中的功能相关性。我们还将研究连接酶IV调控XRCC4功能的机制，以及淋巴细胞内源性连接酶IV复合物的组成和功能。我们对连接酶IV重组蛋白和蛋白质片段的初步研究已经产生了包含连接酶IV DNA结合域的连接酶IV片段的结晶。提出了解决连接酶IV和/或连接酶IV与阿尔忒弥斯复合物的结构的实验。所有这些都是具有挑战性但非常相关的实验，我们在这些实验中取得了重大进展。此外，还将研究在LIG4综合征患者中发现的突变对Artemis调节其功能的影响，以及这些突变的连接酶IV蛋白如何影响XRCC4功能。从拟议的研究中获得的信息将有助于了解NHEJ， V(D)J重组以及与这两个过程中的缺陷相关的人类疾病的分子和结构。公共卫生相关性：尽管我们对连接酶IV功能的理解取得了重要进展，但其结构在很大程度上仍未表征，我们的初步数据表明，新的调节机制仍有待揭示。本项目拟采用体内和体外两种方法研究我们对新型连接酶IV相互作用和功能的初步发现；我们还提出了解决连接酶IV和连接酶IV复合物结构的研究。通过增加我们对连接酶IV在V(D)J重组和DNA修复中的结构、功能和调控的了解，这项工作将有助于我们对人类疾病，特别是连接酶IV和阿尔忒弥斯介导的免疫缺陷和癌症的理解，并将有助于找到更好的治疗这种使人衰弱的疾病的途径。