ATM FUNCTION DURING V(D)J RECOMBINATION

描述(由申请人提供)：ATM丝氨酸-苏氨酸激酶编码基因的突变导致毛细血管扩张性共济失调(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激活细胞周期检查点以响应DNA双链断裂(DSB)。然而，在检查点通路孤立缺陷的小鼠中，A-T的淋巴表型不能重现。我们已经证明，ATM能够修复V(D)J重组过程中产生的双链断裂，并抑制这些双链断裂作为染色体易位的异常分辨。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V(D)J重组的所有缺陷，我们提出ATM的部分功能是在RAG介导的DNA切割后维持DSB复合体的稳定性，这是一个将在特定目标1中直接检验的假设。虽然ATM可以直接在RAG介导的DSB的修复中发挥作用，但我们预计ATM可能会磷酸化执行这一功能的蛋白质。在这方面，我们证明了MRN复合体(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。这些蛋白质如何在依赖ATM的RAG-DSB修复途径中发挥作用将在特定目标2中阐明。此外，我们还将考虑RAG蛋白在V(D)J重组反应的加入步骤中可能具有ATM依赖的功能的可能性。重要的是，我们相信这些不同的蛋白质将以一种整合的方式在ATM依赖的RAG-DSB修复途径中发挥作用。最后，我们将研究ATM缺陷细胞在V(D)J重组过程中产生的DNA DSB作为染色体易位而异常分解的机制(特定目标3)。这些目标的完成将为以下方面提供重要的新信息：1)RAG介导的DSB是如何修复的；2)ATM如何在DSB修复和维持基因组稳定中发挥作用；以及3)促进染色体易位形成的机制。公共卫生相关性：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者。我们已经证明，ATM也参与了在淋巴细胞抗原受体基因组装过程中产生的RAG介导的DSB的修复。在这里，我们建议识别依赖ATM的RAG-DSB修复途径中的蛋白质，并确定它们的功能。此外，我们将阐明在ATM缺陷细胞中导致DNA断裂作为染色体易位的频繁异常分解的机制。

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.