BIOCHEMISTRY AND REGULATION OF V(D)J RECOMBINATION

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DESCRIPTION: (Adapted from the applicant's abstract) Genes encoding the two critical types of antigen recognition molecules in the immune system immunoglobulin (Ig) and T cell receptor (TCR), are assembled during lymphocyte development by a novel, highly regulated site-specific DNA recombination reaction known as V(D)J recombination. The combinatorial joining of gene segments allows the immune system to encode near limitless antigen recognition capability with only a modest genetic investment. The developing lymphocyte is faced with the problem, however, of regulating this reaction to produce functional Ig or TCR while avoiding the consequences of the genomic instability inherent in somatic recombination. Several new techniques have been developed to study the details of the V(D)J recombination reaction pathway and the molecular mechanisms which regulate this reaction. The investigator has devised a series of PCR-based assays which allow detection of V(D)J recombination reaction intermediates consisting of specifically broken DNA molecules. These assays will be used to determine the precise structure of broken DNA coding ends, their location within nuclei of cells undergoing gene rearrangement, the accessibility of these ends, and the identities of proteins which are bound to them. The hypothesis that aberrant V(D)J recombination is involved in the etiology of leukemia associated chromosomal translocation will be tested by looking for DNA breaks at cryptic recombination signal sequences (RSSs) in proto-oncogenes. Recently, researchers at the NIH reported the first in vitro system capable of specific recognition and cleavage of rearranging genes. This system will be used to address the question of how targeting of the recombinase is regulated by performing in vitro cleavage assays on nuclei isolated from various lymphoid precursors. The idea that chromatin structure dictates the choice of target loci by the recombinase will also be tested. Finally, to determine whether the minimally active "core" domains of recombinase genes RAG-1 and RAG-2 can rescue B-cell development in either RAG-I or RAG-2 deficient transgenic mice, mice expressing these proteins will be made. Lastly, the investigator will screen for proteins in lymphoid precursors which interact with the unessential domains of the RAGs.