ATM FUNCTION DURING V(D)J RECOMBINATION

描述（由申请人提供）：随着发育中的淋巴细胞组装抗原受体基因，每个人每天都会产生超过十亿个 RAG 介导的 DNA 双链断裂 (DSB)。这些 DSB 通过非同源末端连接 (NHEJ) DSB 修复途径进行修复，以完成所有抗原受体基因的第二个外显子。即使 NHEJ 以近乎完美的效率运行，每天仍有数千个 RAG DSB 无法修复。 RAG DSB 在 G1 期发育淋巴细胞中产生，与 G1 期产生的其他 DNA 断裂一样，它们激活 ATM 激酶，从而协调 DSB 反应和修复。 ATM 缺陷会导致正常 RAG DSB 修复部分受阻，并导致 RAG DSB 异常修复显着增加，从而导致潜在致癌染色体易位和缺失。这向我们表明，除了促进正常的 RAG DSB 修复（我们最初提案的重点）之外，ATM 还可能调节新的途径，其主要功能是防止未修复的 RAG DSB 被异常修复。我们推断，这些途径中蛋白质的缺陷会导致基因组不稳定性和癌症的增加，但不会导致 NHEJ 介导的 DSB 修复出现明显缺陷。事实上，组蛋白 H2AX 的缺乏符合这一预期。 H2AX 在位于 DSB（包括 RAG DSB）侧翼的远距离染色质中被 ATM 磷酸化（形成 β-H2AX）。一般 RAG DSB 修复不需要 -H2AX。相反，我们已经证明，α-H2AX 可以防止未修复的 RAG DSB 被 CtIP 异常切除，CtIP 是通过 S-G2 中的同源重组 (HR) 启动 DSB 修复的核酸酶。这些被切除的 DNA 末端不能通过 NHEJ 正常连接，但它们可以通过异常途径连接，这些异常途径利用断裂 DNA 末端的同源性形成染色体缺失和易位。因此，H2AX 是保留断裂 DNA 末端结构（通过 ATM 介导的 α-H2AX 形成）的途径的一部分，直到它们通过 NHEJ 正常连接或激活 p53 介导的细胞死亡。我们将阐明这种 H2AX 依赖性途径的组成部分，并确定它们在 G1 中保留 DNA 末端结构的机制。此外，我们将确定导致异常 RAG DSB 修复的途径，我们认为这是由于 G1 期细胞中 NHEJ 和 HR 途径不适当的共激活造成的。 这些研究的完成将为维护淋巴细胞组装抗原受体基因的基因组稳定性的新途径以及促进异常 RAG DSB 解析（如潜在致癌染色体易位和缺失）的机制提供重要的新见解。由于 RAG DSB 修复的要求与 NHEJ 介导的其他类型 DSB 修复类似，因此我们的研究结果将与多种组织中的 DSB 修复和基因组稳定性相关。

DESCRIPTION (provided by applicant): Each individual generates over one billion RAG-mediated DNA double strand breaks (DSBs) every day as developing lymphocytes assemble antigen receptor genes. These DSBs are repaired by the non-homologous end joining (NHEJ) DSB repair pathway to complete the second exon of all antigen receptor genes. Even if NHEJ functioned with near perfect efficiency, thousands of RAG DSBs could persist un-repaired each day. RAG DSBs are generated in G1-phase developing lymphocytes and like other DNA breaks generated in G1, they activate the ATM kinase, which orchestrates DSB responses and repair. ATM deficiency causes a partial block in normal RAG DSB repair and a significant increase in the aberrant repair of RAG DSBs as potentially oncogenic chromosomal translocations and deletions. This suggested to us that in addition to promoting normal RAG DSB repair (the focus of our original proposal), ATM might also regulate novel pathways that function primarily to prevent un-repaired RAG DSBs from being aberrantly repaired. We reasoned that deficiencies of proteins in these pathways would lead to an increase in genomic instability and cancer without causing overt defects in NHEJ-mediated DSB repair. Indeed, deficiency of the histone protein H2AX conforms to this expectation. H2AX is phosphorylated by ATM (forming ?-H2AX) in chromatin at great distances flanking DSBs including RAG DSBs. ?-H2AX is not required for general RAG DSB repair. Rather, we have shown that ?-H2AX prevents un-repaired RAG DSBs from being aberrantly resected by CtIP, the nuclease that initiates DSB repair by homologous recombination (HR) in S-G2. These resected DNA ends cannot be normally joined by NHEJ, but they can be joined by aberrant pathways that form chromosome deletions and translocations using homologies at the broken DNA ends. Thus, H2AX is part of a pathway that preserves the structure of broken DNA ends (by ATM- mediated ?-H2AX formation) until they are either normally joined by NHEJ or activate p53-mediated cell death. We will elucidate the components of this H2AX-dependent pathway and determine the mechanisms by which they preserve DNA end structure in G1. Moreover, we will identify the pathway responsible for aberrant RAG DSB repair, which we believe results from the inappropriate co- activation of NHEJ and HR pathways in G1-phase cells. Completion of these studies will provide important new insights into novel pathways that preserve genomic stability in lymphocytes assembling antigen receptor genes and into the mechanisms that promote aberrant RAG DSB resolution as potentially oncogenic chromosomal translocations and deletions. As the requirements for RAG DSB repair are similar to the NHEJ-mediated repair of other types of DSBs, our findings will be relevant to DSB repair and genome stability in a broad variety of tissues.