The DNA carrying the genetic information of living cells is subject to potentially harmful modification by inherent instabilities, errors in replication, and natural and manmade environmental factors such as radiation and mutagenic chemicals. In order to correct such alterations, normal cells have developed efficient, enzymologically mediated repair processes. If damage goes uncorrected by being missed during repair, or in repair-deficient cells, the DNA alterations can lead to mutations, developmental abnormalities, cancer, immunological changes, and possibly contribute to aging. In this research the simple eukaryote, Dictyostelium discoideum, will be used as a model system for studying basic molecular mechanisms of DNA repair. One group of experiments will focus on the enzymology of repair in repair-proficient cells and repair-deficient mutants, with special attention to damage-specific glycosylases and DNA endonucleases. These enzymes will be isolated and characterized by column chromatographic approaches, gel electrophoresis, and assay on specifically damaged substrate DNAs (initially, UV-damaged). Developmental regulation of repair-related enzymes will be studied during differentiation of the amoeboid cells to spore and stalk cells. Recently isolated thymidine-requiring strains will be used to achieve greater uptake of radioisotopic and density labels into DNA during repair and hence obtain more definitive results on damage-induced changes in DNA replication than has been possible previously. In a second group of experiments, DNA repair genes will be cloned by various approaches: (1) functional complementation of repair defects in yeast; (2) immunological screening of bacterial clones containing genomic DNA in expression plasmids, using antibodies to purified repair enzymes; (3) selection by sequence homologies to cloned yeast repair genes; and (4) by direct complementation of repair defects in D. discoideum following its transformation by appropriate vectors. To help in the development of a more reproducible genetic transformation system for the organism, we will also clone the thymidylate synthetase gene(s) of D. discoideum using analogous approaches. Characterization of this gene is also of interest because of its central role in DNA precursor metabolism. Cloned repair genes will be used as probes to study the regulation of transcription in repair-proficient and repair-deficient cells during vegetative growth and development. Using in vivo and in vitro approaches, we will also seek to identify the gene products of the cloned genes.
