Molecular cloning and primary structure of the uracil-DNA-glycosylase gene from Saccharomyces cerevisiae

Abstract

The structural gene for the Saccharomyces cerevisiae repair enzyme uracil-DNA-glycosylase (UNG1) was selected from a yeast genomic library in the multicopy vector YEp24 by complementation of the ung1-1 mutant in in vitro enzyme assays. The sequenced gene has an open reading frame which codes for a protein with molecular weight of 40,471. The measured size of the mRNA of 1.25 kb is in agreement with the predicted molecular weight of the protein. The gene product was overproduced about 100-fold in strains carrying an UNG1 gene containing plasmid at 100-200 copies/cell. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of cleared lysates from such an overproducing strain, followed by renaturation of enzyme activity from individual gel slices showed the presence of two enzymatic activities in comparable quantities with Mr values of 39,500 and 33,000, indicating that the full size protein is either readily degraded in vivo or is very sensitive to proteolytic digestion in vitro. The carboxyl-terminal two-thirds of the yeast uracil-DNA-glycosylase is highly homologous to the entire Escherichia coli enzyme (50% amino acid identity). Genetic mapping experiments have localized the UNG1 gene on the left arm of chromosome XIII at 17 cM from the GAL80 locus proximal to the centromer. Deletions of the UNG1 gene are viable.