Our overall objective is to clone the RAD1, RAD2, RAD3 and RAD4 gene of the yeast, Saccharomyces cerevisiae, which function in incision of DNA containing ultraviolet light (UV) induced pyrimidine dimers and to study their structure, regulation of expression and protein products. The RAD genes are being cloned either by complementation in yeast or by overlap hybridization. We will verify the identity of the DNA fragments complementing the rad mutants by integrating and mapping them to their chromosomal site in the yeast genome. Fine structure restriction maps of RAD genes will be obtained and each gene subcloned to contain the smallest flanking sequence. We will determine the mRNA sizes of the RAD genes by using RNA gel blots and enquire if there is induction of RAD genes at the transcriptional level following UV irradiation or treatment with other DNA damaging agents. The direction of transcription of the RAD genes will be determined by using the RAD DNA fragments cloned in phage M13 as probes. The 5 foot termini of in vivo RAD transcripts will be located by S1 nuclease mapping. The nucleotide sequence of the RAD genes will be determined by the Sanger dideoxy method. We will check if we can identify the RAD proteins in the E. coli maxicell system or in yeast cells containing the RAD genes on high copy number plasmids. Fusions of RAD genes with the E. coli lacz gene will be constructed to study regulation at the translational level and to purify the RAD proteins. The purified RAD proteins will be characterized for DNA binding, ATPase and UV endonuclease activity. In humans, xeroderma pigmentosum patients are defective in removing UV induced pyrimidine dimers from DNA and show an increased frequency of cancers. Other human genetic diseases are also associated with defective DNA repair and increased carcinogenesis. Therefore, it is important to understand the molecular mechanisms of DNA repair in eukaryotes. The proposed work should provide a model system for understanding the molecular and genetic mechanisms of incision of UV irradiated DNA in eukaryotes, including humans.
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