ATM FUNCTION DURING V(D)J RECOMBINATION

描述（由申请人提供）：编码 ATM 丝氨酸-苏氨酸激酶的基因突变会导致毛细血管扩张共济失调 (A-T)，这是一种以淋巴细胞减少和涉及抗原受体基因座易位的淋巴肿瘤发病率增加为特征的疾病，表明 ATM 在 V(D)J 重组过程中发挥作用。 ATM 响应 DNA 双链断裂 (DSB) 激活细胞周期检查点。然而，在检查点通路存在孤立缺陷的小鼠中，A-T 的淋巴表型并未重现。我们已经证明，ATM 可以修复 V(D)J 重组过程中产生的 DSB，并抑制这些 DSB 作为染色体易位的异常解析。检查点通路和 DSB 修复的联合缺陷解释了 A-T 的一些淋巴表型。鉴于在 ATM 缺陷淋巴细胞中观察到的所有 V(D)J 重组缺陷，我们提出 ATM 的部分功能是在 RAG 介导的 DNA 裂解后维持 DSB 复合物的稳定性，这是一个将在特定目标一中直接检验的假设。尽管 ATM 可以直接在 RAG 介导的 DSB 修复中发挥作用，但我们预计 ATM 可能会磷酸化执行此功能的蛋白质。在这方面，我们表明 MRN 复合物（Mre11、Rad50 和 Nbs1）、53BP1 和 H2AX 都是 ATM 的靶标，在对 RAG 介导的 DSB 的反应中发挥作用。这些蛋白质如何在 RAG-DSB 修复的 ATM 依赖性途径中发挥作用将在具体目标二中阐明。此外，我们将考虑RAG蛋白在V(D)J重组反应的连接步骤中可能具有ATM依赖性功能的可能性。重要的是，我们相信这些不同的蛋白质将以整合的方式在 ATM 依赖性 RAG-DSB 修复途径中发挥作用。最后，我们将研究 ATM 缺陷细胞中 V(D)J 重组期间产生的 DNA DSB 异常解析为染色体易位的机制（具体目标 3）。这些目标的完成将提供以下重要的新信息：1）RAG介导的DSB如何修复； 2) ATM如何在DSB修复和维持基因组稳定性中发挥作用； 3）促进染色体易位形成的机制。公共健康相关性：共济失调毛细血管扩张突变 (ATM) 蛋白是 DNA 损伤反应的关键启动子。我们已经证明，ATM 还参与修复淋巴细胞抗原受体基因组装过程中产生的 RAG 介导的 DSB。在这里，我们建议鉴定 RAG-DSB 修复的 ATM 依赖性途径中的蛋白质并确定它们如何发挥作用。此外，我们将阐明导致 ATM 缺陷细胞中染色体易位导致 DNA 断裂频繁异常解析的机制。

DESCRIPTION (provided by applicant): Mutations in the gene encoding the ATM serine-threonine kinase cause Ataxia Telangiectasia (A-T), a disease marked by lymphopenia and an increased incidence of lymphoid tumors with translocations involving antigen receptor loci, suggesting that ATM functions during V(D)J recombination. ATM activates cell cycle checkpoints in response to DNA double strand breaks (DSBs). However, the lymphoid phenotypes of A-T are not recapitulated in mice with isolated deficiencies in checkpoint pathways. We have demonstrated that ATM functions to repair DSBs generated during V(D)J recombination, and to suppress the aberrant resolution of these DSBs as chromosomal translocations. The combined defect in checkpoint pathways and DSB repair explains some of the lymphoid phenotypes of A-T. Given all of the defects in V(D)J recombination observed in ATM-deficient lymphocytes, we have proposed that ATM functions, in part, to maintain the stability of DSB complexes after RAG-mediated DNA cleavage, which is a hypothesis that will be directly tested in Specific Aim One. Although ATM could function directly in the repair of RAG-mediated DSBs, we expect that ATM will likely phosphorylate proteins that perform this function. In this regard we show that the MRN complex (Mre11, Rad50 and Nbs1), 53BP1 and H2AX, which are all targets of ATM, function in the response to RAG-mediated DSBs. How these proteins function in the ATM-dependent pathway of RAG-DSB repair will be elucidated in Specific Aim Two. In addition, we will consider the possibility that the RAG proteins may have ATM-dependent functions in the joining step of the V(D)J recombination reaction. Importantly, we believe that these different proteins will function in an integrated manner in the ATM-dependent RAG-DSB repair pathway. Finally, we will investigate the mechanisms by which DNA DSBs generated during V(D)J recombination in ATM-deficient cells become aberrantly resolved as chromosomal translocations (Specific Aim 3). The completion of these aims will provide important new information about: 1) how RAG-mediated DSBs are repaired; 2) how ATM functions in DSB repair and in maintaining genomic stability; and 3) the mechanisms that promote the formation of chromosomal translocations. PUBLIC HEALTH RELEVANCE: The ataxia telangiectasia mutated (ATM) protein is a critical initiator of DNA damage responses. We have shown that ATM is also involved in the repair of RAG-mediated DSBs generated during lymphocyte antigen receptor gene assembly. Here we propose to identify the proteins in the ATM-dependent pathway of RAG- DSB repair and determine how they function. Furthermore, we will elucidate the mechanisms that lead to the frequent aberrant resolution of DNA breaks as chromosomal translocations in ATM-deficient cells.