Immunoglobulin and T Cell Receptor Gene Assembly

Yale University, New Haven CT

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Abstract

DESCRIPTION (provided by applicant): V(D)J recombination is the site-specific recombination reaction that assembles the variable portions of immunoglobulin and T cell receptor genes. The reaction is essential for lymphocyte development and in many species is responsible for generating the diverse array of antigen receptor genes necessary for an effective immune response. Errors in the recombination process can lead to chromosomal translocations and hence to the development of human malignancies, particularly childhood leukemias. The causes of such translocations are thought to be improper targeting of the recombination machinery (DNA recognition errors), and the premature release of broken chromosomal ends from the recombination machinery before they have been properly joined (end release errors). Very little is known, however, about how such errors occur or what cellular processes act to prevent them. The proteins encoded by the recombination activating genes, RAG1 and RAG2, play central roles in the targeting of V(D)J recombination and in the efficient rejoining of the broken DNA ends. The RAG proteins are therefore likely to be crucial determinants of genomic stability in developing lymphocytes. The hypothesis underlying this application is that V(D)J recombination occurs within highly organized nucleoprotein structures whose integrity and specificity are in large part determined by RAG1 and RAG2. We propose to investigate the critical protein-protein and protein-DNA interactions involving RAG1 and RAG2 using conventional and recently developed, spectroscopic methods. A particular emphasis will be on understanding interaction surfaces and dynamic aspects of the reaction, especially conformational changes that occur when RAG1 interacts with RAG2 or with DNA. In addition, we will use biochemical and fluorescence methods to investigate the formation and structure of essential higher order "synaptic" complexes formed during V(D)J recombination. Finally, we have identified RAG1 mutants prone to end release errors in vitro, and will target these mutations to the endogenous murine RAG1 locus by homologous recombination. These mice will allow us to test the hypothesis that RAG-mediated stabilization of post-cleavage complexes is important for genome integrity and the prevention of lymphoid tumors. We will test the contribution of "gatekeeper" and "caretaker" genes to the suppression of such tumors and attempt to establish an animal model of lymphomagenesis. Overall, our experiments will address both the targeting and rejoining functions of the RAG proteins, with the long term goal of linking these fundamental activities to chromosomal translocations found in human malignancies.