The antigen receptors of B and T lymphocytes are encoded in discrete DNA segments that are joined during development by site-specific DNA rearrangements. Antigen receptor gene assembly, or V(D)J rearrangement, is the only known example of site-specific DNA recombination in vertebrates. Aberrant V(D)J recombination is likely to be involved in generating the chromosomal translocations between cellular protooncogenes and antigen receptor loci that are seen in a high proportion of lymphoid malignancies. An understanding of V(D)J recombination and its regulation continues to be a long-term goal of this project. Two proteins, RAG-1 and RAG-2, are necessary and sufficient for activation of V(D)J rearrangement. Our past studies have provided the following evidence that expression of the RAG-2 protein and V(D)J recombination are regulated in the cell cycle: (1) expression of RAG-2 protein is restricted to G0/G1 by a posttranscriptional mechanism; (2) RAG-2 is phosphorylated by a cyclin-dependent kinase (cdk) at a specific site in vitro; (3) phosphorylation of this site in vivo is associated with rapid degradation of RAG-2; and (4) site-specific double-strand DNA breaks at V(D)J recombination signal sequences also accumulate preferentially in G0/G1. Our working hypothesis is that phosphorylation of RAG-2 by one or more cdk's regulates accumulation of RAG-2, in turn regulating V(D)J recombination. Additional observations suggest that the association between RAG-2 phosphorylation and degradation may reflect a more general mechanism. In the next funding period, we wish to examine how RAG-2 expression is coupled to the cell cycle and the relationship of this regulation to the timing of V(D)J recombination. To this end, we propose the following specific aims: (1) to define the structural determinants of RAG-2 instability and cell cycle regulation; (2) to determine the relative contributions of protein synthesis and degradation to regulation of RAG-2 accumulation in the cell cycle; (3) to assess phosphorylation of RAG-2 in the cell cycle and to test whether RAG-2 degradation is targeted by phosphorylation; (4) to determine whether cell cycle-dependent accumulation of V(D)J recombination intermediates is a consequence of regulated RAG-2 expression; (5) to examine the physiologic consequences of unscheduled RAG-2 expression in cultured cells and in transgenic mice; and (6) to develop a system for analysis of RAG-2 degradation in yeast, with the long-term goal of examining this process at the genetic level.
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