A central process in the development of a competent immune system is the series of genomic rearrangement events that produce a mature, functional immunoglobulin or T cell receptor gene. The failure of developing lymphocytes to correctly assemble their antigen receptor genes can lead to immunodeficiency or lymphoid tumors. As we previously demonstrated, the lymphoid-specific RAG1 and RAG2 proteins carry out the first stage of this complex process, known as V(D)J recombination, by cleaving DNA at recombination signal sequences (RSSs) that flank the DNA encoding antigen receptor gene segments. Following cleavage, the RSS-ended DNA is joined together into a """"""""signal joint"""""""" and the two coding ends joined into a """"""""coding joint"""""""". The recombinase itself, though dedicated to carrying out V(D)J recombination in lymphoid cells, is also fully capable of mediating an alternative reaction, transposition, both in vitro and in yeast. In this RAG-mediated reaction, the RAG proteins insert the RSS-ended DNA into another DNA site. But RAG-mediated transposition in lymphoid cells is exceedingly rare. Both the cleavage step and the processing of the cleaved ends are tightly regulated, preventing genomic instability that could be deleterious to the developing lymphocytes. Here we seek to understand the transition between the cleavage stage of the reaction in which the RAG proteins must assemble a cleavage competent complex and cut the DNA and the repair stage in which the cleaved ends completed with RAG proteins must interface with the host repair machinery. How is the active cleavage complex organized? What are the functions of the non-catalytic domain of the RAG2 protein? Do factors that interact with this domain modulate its activity? What controls whether cleaved signal ends become signal joints or instead undergo transposition? How do the RAG proteins hand off the cleaved DNA ends to the cellular repair machinery? To answer these questions we propose to 1) carry out a mechanistic analysis of the role(s) of the RAG2 C-terminal domain; 2) Dissect the organization and composition of the active RAG 1/2 synaptic complex; 3) test whether mammalian nonhomologous end joining factors have evolved specific roles to aid in RAG-mediated reactions; 4) develop a powerful genetic assay in yeast for selecting RAG mutants and dissecting the mechanisms of signal joint formation and transposition. In summary, our goals over the next years are to understand the molecular basis for the control of RAG 1/2 activity and the coordination between the cleavage and repair stages of antigen receptor assembly. ? ?
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