Characterization of V(D)J cleavage and repair complexes

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DESCRIPTION (provided by applicant): Diversity in the vertebrate lymphocyte antigen receptor repertoire is largely achieved by a process termed V(D)J recombination that assembles antigen receptor genes from arrays of component gene segments by a series of site-specific DNA recombination events. V(D)J recombination is now understood to involve two distinct phases. In the first phase, two proteins, called RAG-1 and RAG-2, introduce DNA double-strand breaks (DSBs) at recombination signal sequences (RSSs) abutting receptor coding segments undergoing rearrangement. In the second phase, the DSB intermediates generated by the RAG proteins are repaired via a non-homologous end-joining (NHEJ) pathway. Mutations that impair the activity of RAG proteins or the NHEJ factors underlie a subset of immunodeficiency disorders in humans and animals. On the other hand, certain forms of leukemia and lymphoma appear to arise from aberrant V(D)J recombination. Insight into the origins of diseases whose etiology involves the V(D)J rearrangement process necessarily requires a molecular level understanding of the protein-DNA complexes that support both phases of V(D)J recombination. Previous efforts have primarily focused on defining RAG-RSS cleavage complexes. Here, RAG-RSS complexes incorporating DNA bending and/or NHEJ factors, representing the interface between the cleavage and joining phases of V(D)J recombination, will be systematically assembled and characterized in vitro using mobility shift and in-gel enzyme assays, as well as DNA footprinting techniques. Toward this end, the following three specific aims are proposed: (i) to identify determinants of HMG-1 (a DNA bending factor) required to promote RAGmediated synapsis and cleavage of V(D)J recombination signals; (ii) determine what role catalytically "dispensable" portions of RAG-1 and RAG-2 play in vitro in modulating RSS recognition, cleavage and/or association with DSB repair factors; and (iii) identify DSB repair factor association with RAG-RSS complexes (-/+ HMG-1) by mobility shift assay and characterize the composition, DNA interactions and activity of these novel protein-DNA complexes.