Identification of the heterothallic mutation in HO-endonuclease of S. cerevisiae using HO/ho chimeric genes

HO-endonuclease initiates a mating-type switch in the yeast S. cerevisiae by making a doublestrand cleavage in the DNA of the mating-type gene, MAT. Heterothallic strains of yeast have a stable mating type and contain a recessive ho allele. Here we report the sequence of the ho allele; ho has four point mutations all of which encode for substitute amino acids. The fourth mutation is a leucine to histidine substitution within a presumptive zinc finger. Chimeric HO/ho genes were constructed in vivo by converting different parts of the sequence of the genomic ho allele to the HO sequence by gene conversion. HO activity was assessed by three bioassays: a mating-type switch, extinction of expression of an a-specific reporter gene, and the appearance of Can^r Ade^- papillae resulting from excision of an engineered Ty element containing the HO-endonuclease target site and a SUP4^o gene. We found that the replacement of the fourth point mutation in ho to the HO sequence restored HO activity to the chimeric endonuclease.