Rules of donor preference in saccharomyces mating-type gene switching revealed by a competition assay involving two types of recombination.
Mating type (MAT) switching in Saccharomyces cerevisiae is initiated by a double-strand break (DSB) created at MAT by HO endonuclease. MATa cells activate the entire left arm of chromosome III; thus MATa preferentially recombines with the silent donor HML. In contrast, MAT alpha cells inactivate the left arm, including HML, and thus preferentially recombine with HMR, 100 kb to the right of MAT. We present a novel competition assay, in which the DSB at MAT can be repaired either by MAT switching or by single-strand annealing (SSA) between two URA3 genes flanking MAT. With preferred donors, MATa or MAT alpha switching occurs 65-70% of the time in competition with SSA. When HML is deleted, 40% of MATa cells recombine with the "wrong" donor HMR; however, when HMR is deleted, only 18% of MAT alpha cells recombine with HML. In interchromosomal switching, with donors on chromosome III and MAT on chromosome V, MATa retains its strong preference for HML and switching is efficient, when the chromosome III recombination enhancer is present. However, MAT alpha donor preference is lost and interchromosomal switching is very inefficient. These experiments demonstrate the utility of using competition between two outcomes to measure the relative efficiency of recombination.
Wu
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Rosenstiel Center, Brandeis University, Waltham, Massachusetts 02254-9110, USA.
Wu
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Haber
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D002453:Cell Cycle / Q000235:genetics; D015825:Chromosomes, Fungal; D005812:Genes, Switch; D000072235:Mating Factor; D010455:Peptides / Q000235:genetics*; D011995:Recombination, Genetic; D012441:Saccharomyces cerevisiae / Q000235:genetics*
D010455:Peptides; D000072235:Mating Factor