Lymphoid tumors are the most common malignancies in children, and aberrant chromosomal rearrangements are implicated in the generation of many of these cancers. The long term goals of this project remain to understand the mechanisms underlying genomic plasticity and its control in the developing immune system. The variable regions of antigen receptor polypeptide chains are encoded in separate DNA segments that are brought together during lymphoid development by V(D)J recombination, a form of site-specific DNA rearrangement. The proteins RAG-1 and RAG-2 initiate V(D)J recombination by cleaving participating gene segments at specific recombination signal sequences (RSSs). Completion of recombination employs the cellular machinery for non-homologous DNA end joining (NHEJ), a form of DNA double strand break repair (DSBR). V(D)J recombination is coupled to the cell cycle by the periodic destruction of RAG-2. Coordination of V(D)J recombinase activity with NHEJ is required for the maintenance of genomic integrity;when RAG-induced DNA breaks are improperly sensed and repaired, the resulting genomic rearrangements can give rise to lymphoid tumors. Correspondingly, a growing body of evidence indicates that aberrant V(D)J recombination contributes to the pathogenesis of lymphoid malignancies. Renewed funding is requested for continued study of mechanisms that regulate V(D)J recombination and the contributions of these mechanisms to suppression of tumorigenesis. The proposal has the following aims: (1) to define a detailed mechanism by which V(D)J recombination is linked to the cell cycle, by a combination of biochemical analysis and validation in vivo;(2) to understand the physiologic consequences of uncoupling V(D)J recombination from cell cycle, through the use of mice expressing forms of RAG-2 that escape regulation;(3) to elucidate the mechanism by which V(D)J recombination intermediates are disassembled, through development of a cell-free system;and (4) to build a novel, regulable system to study the feedback control of V(D)J recombination, employing chemical rescue of mutant kinase activites in mouse B cell progenitors.
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