The work described in this proposal is a continuation of our long-term project aimed at a complete description of the mechanism of V(D)J recombination. Essential for proper immune system development, this specialized recombination process generates a diverse set of antigen receptors by assembling the variable regions of immunoglobulin and T cell receptor genes. If recombination is impaired, B and T cell differentiation is arrested at an early precursor stage, leading to a severe combined immunodeficiency (scid). Aberrant V(D)J rearrangements, on the other hand, lead to leukemias and lymphomas. To understand both normal immune system development and the molecular basis of oncogenic, aberrant recombination, we must carefully explore this sophisticated system. V(D)J recombination can be divided into two basic steps: DNA cleavage and rejoining of the broken ends. Efficient cleavages requires that a pair of specific DNA sequences, termed recombination signal sequences (RSS), be recognized, assembled into a synaptic complex, and cleaved by the RAG-1 and RAG-2 proteins. After cleavage, the broken ends remain associated with the RAG proteins in a post-cleavage complex. These ends can then e joined to form normal products of recombination. Recent in vitro work has shown that the ends can also participate in an aberrant reaction, transposition, which has been suggested as a cause of oncogenic chromosome rearrangements. The work proposed here will: 1) Dissect the active sites of the RAG proteins and define the role of these sites in the cleavage reaction. 2) Probe the mechanism of RAG- mediated DNA cleavage and how cleavage is regulated by synapsis. 3) Delineate joining mechanisms in the context of the post-cleavage complex. 4) Determine whether RAG-mediated transposition occurs and examine regulatory mechanisms that limit this potentially dangerous reaction. Clearly, our best hope for therapeutic interventions is either scid or lymphoid malignancies rests on a detailed knowledge of normal immune system development and the molecular basis of oncogenic, aberrant recombination events.
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