Lymphocyte antigen receptor gene assembly occurs through the V(D)J recombination reaction. This reaction is initiated when the RAG-1 and RAG-2 proteins, which form the RAG endonuclease, introduces two DNA double strand breaks (DSBs) at the border of two recombining gene segments and their associated V, D or J gene segments. This results in the formation of a coding end and a signal end at each DSB. The coding ends must be joined to form a coding join and the signal ends joined to form a signal join. Also, as a single individual can make up to 60,000,000 RAG DSBs in an hour in developing B and T cells, pathways must be in place to prevent un-repaired RAG DSBs from being aberrantly repaired, forming potentially transforming chromosomal deletions and translocations. The non-homologous end-joining pathway of DNA DSB repair joins DNA DSBs generated by RAG cleavage. NHEJ relies on four core NHEJ factors, XRCC4, DNA Ligase IV, Ku70 and Ku80. However, there are other non-core NHEJ factors that function during the NHEJ-mediated repair of RAG DSBs. For example, coding join formation relies on the ncNHEJ factor, Artemis, which is endonuclease that opens the hairpin-sealed coding ends generated by RAG cleavage. Artemis is not required for joining signal ends. In addition, there are other ncNHEJ factors that are not themselves required for RAG DSB repair, but that likely effect the efficiency of RAG DSB repair or function to prevent aberrant repair of RAG DSBs that are not joined efficiently. Identifying ncNHEJ factors has been challenging given that their deletion does not often lead to a demonstrable defect in the repair of RAG DSBs. However, recent studies have established that combined deficiencies of putative ncNHEJ factors can lead to demonstrable defects in RAG DSBs repair. This has been a powerful candidate-based approach for identifying several ncNHEJ factors and elucidating their mechanisms of action in NHEJ-mediated DSB repair and DNA damage responses (DDR). Indeed, using this type of approach, we have identified a novel ncNHEJ protein, modulator of retroviral infection (MRI), that binds through its N- and C-terminus to a functionally diverse set of NHEJ and DDR proteins. We believe that MRI functions as an adaptor during several steps of NHEJ-mediated repair of RAG DSBs and during the DDR to RAG DSBs. Here we propose a novel unbiased screening approach to identify the network of ncNHEJ factors that function during RAG DSB repair. We also propose to elucidate the mechanisms of action of these ncNHEJ factors focusing initially on the novel ncNHEJ factor, MRI, that we recently discovered that functions in RAG DSB repair. These studies will reveal novel activities required for the NHEJ-mediated repair of RAG DSBs. These activities will likely be generally relevant to NHEJ in other tissues and to our understanding to DNA repair defects that lead to genome evolution in cancers.
Lymphocyte antigen receptor gene assembly requires the repair of DNA double strand breaks (DSBs) by non- homologous end joining (NHEJ). This repair depends on essential core NHEJ factors, however, it is clear that it also relies on non-core NHEJ factors, many of which are unknown, that promote efficient DSB repair and prevent aberrant DSB resolution. The goal of this project is to identify the network of non-core NHEJ factors that function during lymphocyte antigen receptor gene assembly and to determine their mechanism of action.
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