Primary structure of the RAD52 gene in Saccharomyces cerevisiae

Abstract

The RAD52 gene of Saccharomyces cerevisiae, which is involved in genetic recombination and DNA repair, was cloned by transformation of rad52-1 mutant cells to methyl methanesulfonate resistance with BamHI fragments of Rad+ genomic DNA inserted into the Escherichia coli-S. cerevisiae shuttle vector YRp7. A plasmid carrying a 2.0-kilobase BamHI fragment was found to partially complement methyl methanesulfonate sensitivity of the rad52-1 mutant. By using this fragment as a hybridization probe, a plasmid that fully complemented the methyl methanesulfonate sensitivity of the mutant was isolated, which carries a 3.3-kilobase SalI fragment containing most of the 2.0-kilobase BamHI fragment. Analysis of the nucleotide sequence of the SalI fragment revealed the presence of a large open reading frame of 1,512 nucleotides. The rad52-1 mutant DNA has a single-base change in this reading frame, which leads to an amino acid substitution. Analysis of mRNA synthesized in yeast by the S1 mapping technique disclosed possible transcription initiation and termination points of the RAD52 gene and suggested formation of the gene product without splicing of the transcript.