V(D)J recombination, genomic instability, and cancer

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DESCRIPTION (provided by applicant): V(D)J recombination, the process that creates billions of distinct, functional antigen receptor genes from separate gene segments, is a crucial step in lymphocyte development. In its power to create diversity lies opportunity for error, however, and aberrant recombination has been implicated as the cause of many leukemias and lymphomas. V(D)J recombination occurs in two stages: first, the RAG-1 and RAG-2 proteins cleave DNA between the recombination signal sequences and the adjoining coding segments. The resulting double strand break leaves blunt ends (signal ends) and covalently sealed DNA hairpins (coding ends), which must be joined in the second stage of the process. The RAG proteins retain both sets of ends in a post-cleavage complex, perhaps to help orchestrate the coupling of these ends to form signal joints and coding joints, respectively. Joining requires members of the nonhomologous end joining (NHEJ) DNA repair pathway. We recently found that joining also requires the RAG proteins themselves and that the post-cleavage complex is critical for both signal and coding joint formation. Interestingly, mice and cell lines with defective NHEJ components are joining-impaired, accumulate broken DNA intermediates in V(D)J recombination, and have a strong propensity to develop lymphomas. In this application we will delineate the roles of the RAG proteins in joining and test the hypothesis that joining-deficient RAG mutants cause genomic instability in lymphocytes, leading to lymphoid neoplasms. We will pursue the following Specific Aims: 1) Perform large-scale, high-throughput screen to identify joining-deficient RAG mutants. 2) Identify and characterize intragenic suppressors of joining-deficient RAG mutants. 3) Generate transgenic and knock-in mice bearing joining-deficient RAG mutants. 4) Determine the contributions of RAG joining mutants to oncogenesis. Together, these studies will lay the foundation for a robust experimental system that should provide numerous insights into the pathogenesis of common human diseases, and may also allow design of diagnostic tests and new therapeutic approaches.