In the yeast, Saccharomyces cerevisiae, the mating type of a haploid cell is determined by the specific allele, a or Alpha, present at the MAT locus on chromosome III. Two other loci, HML and HMR, are also on chromosome III and contain unexpressed copies of Alpha and a information respectively. Haploid strains that contain the HO gene switch mating type regularly. This has been shown to occur by transposition of a copy of mating type information from an unexpressed locus to MAT. The biochemical steps by which this occurs are unknown. An early event is thought to be the formation of a double strand break in the chromosome at a specific site within the MAT locus. Cleavage is catalyzed by the HO endonuclease, encoded in the HO gene. By genetic and physical means a region in MAT important for switching was localized and the position of the double strand break was mapped. This proposal aims to identify the specific nucleotides in MAT DNA required for switching in vivo. In addition, this proposal aims to characterize the interaction between HO endonuclease and its recognition sequence in vitro. Specific point mutations will be constructed by oligonucleotide directed mutagenesis in a clone of the MATa gene in the region of the double strand break. Each mutation will be assessed for its effect on cleavage of MAT DNA by HO endonuclease in vitro and for its effect on switching in vivo. HO endonuclease will be purified from strains of yeast and E. coli that have been engineered to over produce the HO gene product. The structure and function of the protein will be assessed by alkylation inhibition, active site affinity labeling, and limited proteolysis. HO endonuclease represents one of only a few eukaryotic proteins currently identified that interact with a specific sequence of DNA. Site-specific DNA transposition is a common phenomenon among many organisms. It functions to alter the pattern of gene expression and to combine gene segments in the formation of new proteins. The understanding of the specific DNA/protein interactions in yeast mating type switching may reveal mechanisms common to all systems.
