The DNA of living organisms is subject to modification by inherent instability, errors in replication, and natural and man-made environmental factors such as radiation and mutagenic chemicals. To cope with these changes, cells have developed efficient enzymological processes for repairing damage to their DNA. If the damage goes uncorrected by being missed during repair, or in repair-deficient cells, it can lead to mutations, developmental abnormalities, cancer, immunological changes, and possibly contribute to aging. The simple eukaryote Dictyostelium discoideum will be used as a model system for studying basic mechanisms of DNA repair. Information acquired will aid in the understanding of DNA repair and how it relates to the health problems listed above. Repair genes will be cloned and characterized. These clones will be selected from genomic libraries in D. discoideum-E. coli shuttle vectors by functional complementation of defects in repair mutants. High frequencies of transformation will be achieved by electroporation. Another approach will use the amino acid sequences of peptides isolated from purified repair enzymes. The corresponding oligonucleotides will be used as hybridization probes for selecting the structural genes from cDNA and genomic libraries. The cloned genes will be used to study their regulation under a variety of developmental conditions in wild-type and repair-deficient mutants. Another set of experiments will identify repair-associated proteins by their ability to bind to oligonucleotide sequences bearing specific damage such as cyclobutane pyrimidine dimers or chemical adducts. DNA-binding gel retardation analysis will be used. When such proteins are identified, their genes will be cloned directly by using damaged oligonucleotides as recognition-site probes for the binding proteins synthesized in clones from expression libraries of lambdagt 11. Using specific-gene hybridization approaches, differential gene repair during development will be studied in normal cells and repair mutants. This organism is especially suited for these studies since several developmentally regulated genes have been isolated and characterized. They are transcriptionally regulated by cAMP. This occurs in the absence of semiconservative DNA replication. Rates of repair of dimers and adducts will be determined as a function of the transcriptional state of these genes. In addition, we will extend our current studies of the molecular defects in repair mutants isolated in the thymidine-auxotroph HPS401. These experiments are more readily done in these mutants than those previously available because of the 50-fold higher level of isotopic labeling of their DNA during short periods of DNA replication and repair. Special attention will be given to those strains with differential sensitivities to various specific damaging agents, such as ultraviolet light, bulky chemical adducts, simple alkylations, and gamma rays. These mutants will also be included in the studies outlined above involving regulation, DNA binding proteins, and differential gene repair.
