Our long term goals are to elucidate the genetic and molecular mechanisms of excision repair of DNA damaged by ultraviolet (UV) light irradiation in the yeast Saccharomyces cerevisiae. The overall objective of this proposal is to study the structure, regulation, and function of the RAD7, RAD14, RAD16, RAD23, and MMS19 genes involved in excision of pyrimidine dimers. The RAD14, RAD16, and MMS19 genes will be isolated by complementation of UV or MMS sensitivity of the corresponding mutants. The complementing DNA fragments will be subcloned and the identity of the cloned gene confirmed by mapping it to its location in the yeast genome. The cloned genes will be used for making deletions of the corresponding genes in the yeast genome, and the effects of deletions studies. The size and direction of the RAD14, RAD16, and MMS19 transcripts will be determined by using M13mp18 and M13mp19 phages containing an internal fragment of the RAD or MMS gene. Radioactively labeled single-stranded DNA probes from these recombinant phages will be used in hybridizations to yeast RNA. The nucleotide sequence of the RAD14, RAD16, RAD23, and MMS19 genes will be determined by the Sanger dideoxy method. The 5' and 3' mRNA termini of the RAD7, RAD14, RAD16, RAD23, and MMS19 genes will be located by S1 nuclease mapping. To determine if the RAD7, RAD14, RAD16, RAD23, and MMS19 genes show regulated expression, transcript levels, and Beta-galactosidase levels in yeast strains containing in-frame fusions of these genes with the E. coli lacZ gene, will be examined following UV irradiation. RAD-lacZ fusions will be used for determining the intracellular location of the hybrid proteins by cell fractionation and immunofluorescence. The amino acid sequence determinants in these RAD and MMS proteins affecting their nuclear localization will be identified. The RAD7, RAD14, RAD16, RAD23, and MMS19 genes will be fused to a strong yeast promoter in a multicopy plasmid for overproducing their proteins, and the RAD or MMS gene encoded proteins purified from yeast strains containing these multicopy plasmids. The purified proteins will be examined for DNA binding, DNA unwinding, ATPase, UV endonuclease, exonuclease and nucleosome binding. Human xeroderma pigmentosum patients are defective in excision of UV induced pyrimidine dimers from DNA and show an increased frequency of cancers. The proposed studies should provide a model system for understanding the complex mechanisms of excision repair in eukaryotes, including humans.
Showing the most recent 10 out of 56 publications
