Mobile introns and inteins self-propagate by 'homing', a gene conversion process initiated by site-specific homing endonucleases. The VMA intein, which encodes the PI-SceI endonuclease in Saccharomyces cerevisiae, is present in several different yeast strains. Surprisingly, a wild wine yeast (DH1-1A) contains not only the intein(+) allele, but also an inteinless allele that has not undergone gene conversion. To elucidate how these two alleles co-exist, we characterized the endonuclease encoded by the DH1-1A intein(+) allele and the target site in the intein(-) allele. Sequence analysis reveals seven mutations in the 31 bp recognition sequence, none of which occurs at positions that are individually critical for activity. However, binding and cleavage of the sequence by PI-SceI is reduced 10-fold compared to the S.cerevisiae target. The PI-SceI analog encoded by the DH1-1A intein(+) allele contains 11 mutations at residues in the endonuclease and protein splicing domains. None affects protein splicing, but one, a R417Q substitution, accounts for most of the decrease in DNA cleavage and DNA binding activity of the DH1-1A protein. Loss of activity in the DH1-1A endonuclease and target site provides one explanation for co-existence of the intein(+) and intein(-) alleles.

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Journal Article | Research Support, Non-U.S. Gov't

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Gimble FS

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