Mechanism and regulation of V(D)J repair

项目摘要 连接酶IV在非同源末端连接(NHEJ)、V(D)J重组中起关键作用 免疫系统的发育、干细胞耗竭、衰老以及神经生长和发育，如 连接酶IV亚型突变患者的表型就是例证。 已被证明可以调节Ligase IV的活性。其中，XRCC4和Cernunnos/XLF可能是 最好的/最确凿的特点。这两个因素，像Ligase IV一样，都是核心的一部分-NHEJ 机械设备。XRCC4的人类突变还没有被描述，但有趣的是，Cernunnos/XLF 人类的突变会导致生长缺陷、小头畸形和免疫缺陷，这与过去的情况类似 观察Ligase IV突变。我们最近的发现发现了Artemis，这也是一个与 在人类免疫缺陷中，作为一种直接与Ligase IV相互作用的蛋白质，此外，我们还观察到 Ligase IV对XRCC4的调控。在本项目中，我们建议分析这些基因的功能相关性 新的连接酶IV/Artemis复合体的基因组稳定性和V(D)J重组。这一机制通过它 连接酶IV调节XRCC4的功能以及内源性连接酶IV复合体的组成和功能 淋巴细胞也将被研究。我们对Ligase IV重组蛋白和蛋白的初步研究 片段导致了包含Ligase IV DNA结合域的Ligase IV片段的结晶。 提出了解决连接酶IV和/或连接酶IV在与Artemis的络合物中的结构的实验。全 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， LIG4综合征患者中发现的突变对Artemis和Artemis调节其功能的影响 这些突变的Ligase IV蛋白如何影响XRCC4的功能也将被研究。获得的信息 将有助于了解NHEJ，V(D)J的分子和结构 重组和人类疾病与这两个过程中的缺陷有关。

Project Summary 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.