Mechanism and regulation of V(D)J repair

描述（由申请人提供）：已证明连接酶IV在非同源末端连接（NHEJ）、V（D）J重组和免疫系统发育、干细胞耗竭、衰老和神经生长发育中发挥关键作用，如连接酶IV中具有亚型突变的患者表型所示。许多蛋白质已显示调节连接酶IV活性。其中，XRCC 4和Cernunnos/XLF可能是最好/最具决定性的特征。这两种因子，如连接酶IV，是核心NHEJ机制的一部分。XRCC 4中的人类突变尚未被描述，但有趣的是，人类中的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突变所观察到的。我们最近的研究结果已经确定Artemis是一种与人类免疫缺陷相关的因子，是一种与连接酶IV直接相互作用的蛋白质，此外，我们还观察到连接酶IV对XRCC 4的调节。在这个项目中，我们建议分析这些新的连接酶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。还将研究连接酶IV调节XRCC 4功能的机制，以及淋巴细胞中内源性连接酶IV复合物的组成和功能。我们对连接酶IV重组蛋白和蛋白片段的初步工作已经导致含有连接酶IV DNA结合结构域的连接酶IV片段的结晶。提出实验来解析连接酶IV和/或与Artemis复合的连接酶IV的结构。所有这些都是具有挑战性的，但非常相关的实验，我们表现出显着的进展。此外，还将研究在LIG 4综合征患者中鉴定的突变对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XRCC 4功能。从拟议的研究中获得的信息将有助于对NHEJ，V（D）J重组和与这两个过程中的缺陷相关的人类疾病的分子和结构理解。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被表征，我们的初步数据表明新的调控机制仍有待解开。本项目旨在通过体内和体外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结构、功能和调控的了解，在V（D）J重组和DNA修复中，拟议的工作将有助于我们理解人类疾病，特别是连接酶IV和Artemis介导的免疫缺陷和癌症，并将促进为这种衰弱性疾病找到更好的治疗方法的途径。

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.