A central process in the development of a competent immune system is the series of genomic rearrangement events that take place to produce a mature, functional immunoglobulin or T cell receptor gene. This complex process, known as V(D)J recombination, has been studied intensively since its discovery in 1975, but the reaction and enzymatic machinery involved in it remain poorly understood. We have recently identified two genes, RAG-1 and RAG-2, that are capable of inducing this lymphoid specific event in non- lymphoid cells (i.e. fibroblasts) and are extremely likely to encode part of the V(D)J recombinase machinery. These genes provide new tools with which to probe the events surrounding the rearrangement process. Our goal over the next years is to understand the function(s) of the RAG genes and through them to gain a greater understanding of how the rearrangement events are carried out and regulated. First, we will conduct a mutational analysis of RAG-1 and RAG-2 to determine the minimum functional sequence required for recombinase activity and to functionally dissect the RAG proteins. Deletion and point mutations will be analyzed using a number of standard assays for recombinase activity to determine if any of the mutants are defective in particular aspects of the reaction. Second, we will attempt to develop new assays with which to identify partial reaction products of the V(D)J recombination reaction. In developing these assays we will make use of the mutants described above and of our ability to control where and when the RAG genes and recombinase activity are expressed. Third, both RAG-1 and RAG-2 are required for the induction of V(D)J recombination. Thus, we have designed genetic and biochemical experiments to test whether these proteins interact with each other, or if either protein multimerizes. If interactions are detected, we will determine the portions of the protein(s) required for the interactions. Further, we will attempt to define other factors with which RAG-1 and/or RAG-2 interact. The identification of such factors would be important for our understanding of the events involved in V(D)J recombination. Fourth, we will use both genetic and biochemical approaches in order to test RAG-1 and RAG-2 for a number of enzymatic activities (alone and in combination) that are expected properties of the V(D)J recombinase. Furthermore, because we have demonstrated a correlation between RAG-2 expression and gene conversion activity, we will test the hypothesis that there is a causal relationship between the two. In summary, developing lymphocytes must assemble their antigen receptor genes; failure to do so can result in immunodeficiency. The proposed experiments should provide information on the structure and function(s) of RAG-1 and RAG-2, two genes that appear to play a critical role in V(D)J recombination. Further, this work should lead to a better understanding of how this exceedingly important and complex reaction is carried out.
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