The long range goals of the proposed research are to elucidate the molecular mechanisms of DNA double-strand break (DSB) and single-strand break (SSB) induced recombination repair in mammalian cells. Recombination processes are involved in a variety of important biological phenomena, such as the regulation of gene expression and antibody gene assembly. It is likely many of the biological effects of ionizing radiation, such as mutagenesis, induction recombination. The proposed study involves shuttle vector mediated transfection and is also relevant to gene therapy. The following approaches will be used to examine recombination mechanisms in mammalian cells: 1. Defined DSBs and SSBs will be created in plasmids carrying heteroallelic genes to allow characterization of the initiation, intermediates, and products of break-induced genetic recombination, and to test predictions of recombination models. Specifically: (a) the lengths and positions of heteroduplex DNA regions formed during DSB- and SSB- induced recombination will be examined. (b) DSB-DSB and DSB-SSb interactions will be analyzed. (c) conversion tract lengths will be measured. (d) hypotheses concerning the role of transcription in recombination processes will be tested. 2. Specific recombination events will be examined in X-ray sensitive Chinese hamster ovary cell lines in an effort to characterize the molecular defect(s) in these cells. 3. Recombination processes will be investigated in normal human cells from patients with altered DNA damage repair capacity, increased sensitivity to radiation, and increased susceptibility to cancer (Fanconi's anemia, Bloom's syndrome and ataxia telangiectasia). 4. Homologous and non-homologous recombination will be examined in vitro using extracts from each Chinese hamster ovary and human cell line.
