Our objective is to gain an understanding of the molecular and biochemical mechanisms of lymphoid V(D)J recombination and DNA double strand break (DSB) repair. These two pathways share at least three common factors, SCID (severe combined immune deficiency), XRS-6 (x-ray sensitive-6), XR-1 (x- ray-1) and probably share many others. In particular, in this grant application we describe a novel and powerful strategy using retroviral mutagenesis for the creation of mammalian cell mutants. This strategy has already allowed us to isolate and characterize three mutants that are defective in these two pathways. The use of retroviral insertional mutagenesis to simultaneously mutate and molecularly """"""""tag"""""""" in cis genes will expedite the cloning of these and additional genes involved in recombination and repair. The relatedness of V(D)J recombination and DSB repair is underscored by the existence of several human immune deficiency/chromosome breakage syndromes, such as Blooms' syndrome, ataxia telangiectasia and Fanconi's anemia. Individuals affected by these diseases are characterized by having impaired immune capabilities as well as being cancer prone. We intend to characterize these two important biological pathways using 6 lines of experimentation: 1. Creation and isolation of mammalian cell mutants mutants which are sensitive to x-irradiation (sexi mutants). 2. Characterization of the DNA repair capacity of the sexi mutants. 3. Characterization of the V(D)J recombinational capacity of the sexi mutants. 4. Characterization of homologous and illegitimate recombinational capacities of the sexi mutants. 5. Cloning of the SEXI genes. 6. Molecular characterization of the SEXI genes. The ultimate goal of these studies will be to use the sexi mutations as tools to understand the molecular mechanisms of lymphoid gene rearrangement and DNA repair.
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