ATM FUNCTION DURING V(D)J RECOMBINATION

描述(由申请人提供)：当发育中的淋巴细胞组装抗原受体基因时，每个人每天产生超过10亿个碎屑介导的DNA双链断裂(DSB)。这些DSB通过非同源末端连接(NHEJ)DSB修复途径修复，完成所有抗原受体基因的第二外显子。即使NHEJ以近乎完美的效率运行，每天也有数以千计的破旧DSB无法修复。RAG DSB是在G1期发育的淋巴细胞中产生的，与G1中产生的其他DNA断裂一样，它们激活ATM激酶，后者协调DSB的反应和修复。ATM缺乏会导致RAG DSB正常修复的部分阻断和RAG DSB的异常修复显着增加，这是潜在的致癌染色体易位和缺失。这向我们暗示，除了促进正常的RAG DSB修复(我们最初提议的重点)外，ATM还可能调节主要用于防止未修复的RAG DSB被异常修复的新途径。我们推测，这些途径中蛋白质的缺失将导致基因组不稳定和癌症的增加，而不会导致NHEJ介导的DSB修复的明显缺陷。事实上，组蛋白蛋白H_2AX的缺失符合这一预期。H_2AX在染色质中被ATM(形成γ-H_2AX)在很远的距离上磷酸化，位于DSB的两侧，包括RAG DSB。一般的RAG DSB修复不需要γ-H2 AX。相反，我们已经证明γ-H_2AX防止未修复的RAG DSB被CtIP异常切除，CtIP是在S-G2中通过同源重组(HR)启动DSB修复的核酸酶。这些被切除的DNA末端不能被NHEJ正常连接，但它们可以通过异常路径连接起来，形成染色体缺失和易位，使用断裂DNA末端的同源。因此，H_2AX是保留断裂末端结构的途径的一部分(通过ATM介导的γ-H_2AX形成)，直到它们被NHEJ正常连接或激活P53介导的细胞死亡。我们将阐明这种依赖于H_2AX的途径的成分，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RAG DSB异常修复的途径，我们认为这是由于NHEJ和HR途径在G1期细胞中不适当地共同激活所致。这些研究的完成将为在组装抗原受体基因的淋巴细胞中保持基因组稳定性的新途径提供重要的新见解，并将促进异常RAG DSB分解为潜在致癌染色体易位和缺失的机制。由于RAG DSB修复的要求类似于NHEJ介导的其他类型DSB的修复，我们的发现将与DSB修复和广泛组织中的基因组稳定性相关。

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 γ-H2AX) in chromatin at great distances flanking DSBs including RAG DSBs. γ-H2AX is not required for general RAG DSB repair. Rather, we have shown that γ-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 γ-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.