Nuclear export-dependent functions of RAG2 in the DNA damage response system

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The assembly of immunoglobulin and T cell receptor genes through VDJ recombination is central to lymphocyte development. RAG1 and RAG2 generate DNA double strand breaks (DSBs) in an intermediate step of VDJ recombination. This is a risky step that can lead to genomic instability if not tightly regulated. As the physiologic generation of DNA DSBs occurs throughout lymphocyte development, the proper resolution of these breaks in the presence of ongoing RAG endonucleolytic activity must occur before progression to the next stage of development. Moreover, DNA DSBs unrelated to VDJ recombination that are coincidently present with RAG-mediated breaks must also be appropriately resolved. It is not yet clear how the DDR and the RAG proteins coordinate their respective functions in assembly of the antigen receptor loci while maintaining genomic integrity. In our recent studies, we have shown that DNA DSBs triggers ATM-dependent nuclear export and centrosome targeting of RAG2. Relocalization of RAG2 was transient, as the pre-DNA damage localization of RAG2 was re-established following DNA repair. The central hypothesis of this project is that nuclear export and centrosome targeting of RAG2 reinforces G1-S cell cycle arrest until the damaged DNA has been repaired, resulting in increased cell survival and genomic stability. We propose this occurs through regulation of centrosome duplication, which is tightly coordinated with the onset of DNA replication in the S phase. Using a proteomic approach, we have identified specific centrosomal proteins that interact with RAG2 following DNA damage. Now, we will resolve the properties of these interactions using a combination of state- of-the-art imaging with molecular biology approaches that will identify regions of RAG2 that mediate these interactions, and their site of localization within the centrosome. In addition, to test our hypothesis regarding the role of centrosome-targeting of RAG2 following DNA damage, we will measure cell survival and V(D)J recombination fidelity in cells expressing RAG2 that is proficient versus defective in centrosome targeting. This project will elucidate mechanisms that balance lymphocyte survival with maintenance of genomic integrity during development of the adaptive immune system.