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.
We will elucidate the functions of a new DNA break repair pathway that prevents aberrant double strand break repair leading to the formation of potentially oncogenic chromosomal lesions such as translocations and deletions. Moreover, we will elucidate the mechanisms responsible for the formation of these aberrant chromosomal lesions. Although focused on RAG DSBs generated in developing lymphocytes, our proposed studies will be broadly applicable to general DSB repair and genome stability in a broad variety of tissues.
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