The specific aim of this proposal is to isolate and characterize the proteins that constitute the damage-specific DNA incising activity involved in nucleotide excision repair in the yeast Saccharomyces cerevisiae. In order to isolate these proteins in the large amounts required for detailed biochemical studies we have cloned 4 of the 5 yeast RAD genes that are believed to code for the damage-specific DNA incising activity. We propose to isolate the remaining RAD gene by screening a yeast genomic library of plasmids containing yeast DNA inserts. We propose to subclone these RAD genes and to sequence all 5 genes of interest using the Maxan-Gilbert technique. We also propose to construct fusion plasmids in which the E. coli Beta galactosidase structural gene is under the control of RAD promoters. These fusion plasmids will be used to monitor induction of any RAD genes by measuring expression of Beta galactosidase following exposure of transformed (with single copy plasmids) cells to DNA damage. We also propose to tailor these fusion plasmids so as to replace the RAD promoters with a strong, regulable exogenous yeast promoter such as the GAL1 promoter, for over-expression of RAD proteins in yeast. The efficiency of the GAl1 promoter will be tested by measuring expression of Beta galactosidase in yeast. Over-expression of yeast RAD proteins will be achieved by deletion of the Beta galactosidase fragment. RAD proteins will be purified from transformed yeast cells and characterized biochemically and biophysically.
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