MECHANISM OF CODING JOINT FORMATION

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DESCRIPTION (Adapted from Investigator's Abstract): VDJ recombination takes place during lymphocyte development and is the only site specific recombination process thus far identified in vertebrates. Aberrant VDJ recombination events have profound effects in vivo, and have been suggested as the cause of numerous lymphomas and leukemias. Antigen receptor gene assembly is directed by consensus recombination signal sequences (RSS) and the reaction can be separated into four steps: recognition and cleavage of the DNA followed by processing and joining of the cleaved DNA ends. The joining reaction is essential for antigen receptor gene assembly as well as to maintain chromosomal integrity. The early steps of the recognition and cleavage of the recombination signal sequences are mediated by the products of the recombination activating genes 1 and 2 (RAG1 and RAG2). Inactivation of the RAG1 or RAG2 gene in either human or mouse results in severe combined immune deficiency. Despite rapid progress in understanding the recognition and cleavage steps of the VDJ recombination, the molecular mechanisms that govern processing and joining of the ends remain largely obscure. Despite the apparent complexity of the reaction, only four proteins have been implicated in the joining process DNA-PKCs, Ku, XRCC4, and ligase IV. These factors are known to be essential for understanding the molecular mechanism that mediates processing and joining of the DNA ends and VDJ joining. The major stumbling block in understanding the molecular mechanism that mediates processing and joining of the DNA ends during V(D)J recombination was the lack of in vitro systems to study these reactions. We have now developed different cell free systems capable of catalyzing processing of hairpin end intermediates as well as formation and joining of coding ends. We plan to use these newly developed in vitro systems to detail the molecular events that occur during V(D)J recombination by characterizing the intermediates and products of the reaction as well as the proteins involved in each step. As VDJ recombination and DNA double strand break repair are rapidly converging and it is clear that they share a number of factors, our studies will contribute to the understanding of both of these important process.