The ultimate goal of this program is to construct an in vitro system composed of defined proteins to study the mechanisms employed by higher eukaryotes to contend with DNA damage. Drosophila will be employed in these studies, because a parallel genetic analysis will be performed to also define the in vivo function of the enzymes under investigation. Since the available mutants and those we expect to isolate are analogues of human repair-related disorders, these studies will provide evidence relevant to human DNA repair and its involvement in mutagenesis, carcinogenesis, and recombination. The isolation and characterization of several key enzymes of DNA metabolism will be undertaken. These enzymes include DNA polymerase Beta, DNA polymerase Gamma, two ATP-dependent deoxyribonucleases, a rec A-like protein, and a damage-specific endonuclease. Following their enzymological characterization, defects in these enzymes will be sought among the available repair-deficient mutants. Selected genes will be cloned either by screening a cDNA expression library or by screening genomic libraries with mixed-sequence oligonucleotide probes. The clones will be employed to study the normal regulation of these genes and to alter that regulation to define their role in DNA repair, recombination and synthesis. The clones will also be employed to guide a genetic study which will further define the in vivo function of the proteins. The recovered clones will provide probes which can potentially be employed to recover analogous genes from the human genome. Mitochondrial DNA metabolism will also be investigated in an effort to develop a model in vitro system.
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