The generation of a diverse repertoire of antigen binding is the primary function of lymphoid cells. The V(D)J recombination activity is the enzyme system that catalyzes this gene segment assembly process. Though we now know much about this reaction, the primary and central questions are still largely unanswered. Specifically, what are the enzymatic components of this recombinase; how are these components regulated; how many steps are there in the reaction; what steps are affected in congenital defects of the immune system; what steps are the failure points that result in chromosomal translocations; what steps are shared with general DNA repair? In addition to these enzymologic and molecular biologic questions, questions of chromatin structure relate to how some loci are left accessible to the recombinase and others are not. Both the enzymologic aspects and the locus activation (chromatin structure) aspects of V(D)J recombination are relevant to the inception of chromosomal translocations in lymphoid leukemias and lymphomas; these malignancies account for 50 percent of all cancer in children under age 5, and 8 percent of all cancer in adults. In this project, a set of studies is proposed directed at (a) identifying and isolating components of the V(D)J recombination activity, including the murine scid gene; (b) determining how these components function; (c) determining the role of congenital mutations that result in human SCID; (d) determining the components shared between V(D)J recombination and general DNA end joining; (e) determining the steps in coding joint diversification; (f) determining the enzymatic steps in coding end processing; (g) establishing the role of structure-specific endonucleases in DNA end joining; (h) establishing a cell-free system for the biochemical analysis of the steps of the V(D)J recombination reaction; (i) determining the molecular basis for chromatin structure changes that make antigen receptor loci accessible or not; (j) describing the DNA methylation changes at antigen receptor loci that occur as a function of differentiation in primary B cell progenitors derived from bone marrow; and (k) understanding the role of CpG methylation in determining chromatin structure and inaccessibility to the V(D)J recombinase. These studies will be conducted using molecular, genetic and biochemical approaches. Based on these studies and related studies of others in this field, the long-term goal is a biochemical understanding of the catalytic components of the recombinase, the steps of the reaction, and the basis for locus accessibility.
