The genes which encode immunoglobulin and T cell receptor molecules are assembled from V, D, and J gene-segments by a novel series of site-specific DNA recombination reactions known as V(D)J recombination. The recombinase consists of the lymphocyte-specific proteins RAG1 and RAG2 and the widely expressed non-homologous end-joining proteins. The recombinase recognizes conserved recombination signal sequences (RSSs) which flank gene segments and introduces dsDNA breaks which are repaired to form coding and signal joints. Recombination at the seven complex antigen-receptor loci in B and T cells is regulated with respect to lineage specificity, order within a lineage, and allelic exclusion. We and others have shown that V(D)J recombination is regulated in part by the accessibility of RSSs within chromatin structure and that germline transcription of unrearranged gene segments either reflects or actually causes chromatin accessibility. The central goal of this proposal is to understand, at the biochemical level, the mechanisms which dictate the biological regulation of V(D)J recombination. This goal will be pursued through pursuit of the following specific aims: 1) to determine the location and structure of nucleosomes across the J-kappa cluster of gene segments and how nucleosomes are disrupted to allow recombinase accessibility;2) to determine whether the V(D)J recombinase nicks DNA in vivo and whether the conversion from nicked to broken DNA is a regulated step in the reaction;3) to determine the catalytic properties in vivo of a core-domain mutant of RAG2;and 4) to define the role of transcriptional regulatory elements in the regulation of V(D)J recombination. This work is significant because of the essential role of antigen receptors in acquired immunity and the potential involvement of the recombinase in generation of chromosomal translocations associated with lymphoid malignancies.
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