Genetics, Vol 130, 451-460, Copyright © 1992

INVESTIGATIONS

Site-Specific Recombination Determined by I-SceI, a Mitochondrial Group I Intron-Encoded Endonuclease Expressed in the Yeast Nucleus

A. Plessis, A. Perrin, J. E. Haber and B. Dujon
Unite de genetique moleculaire des levures, URA1149 du CNRS, Departement de Biologie moleculaire, Institut Pasteur, 75724 Paris Cedex 15, France

The Saccharomyces cerevisiae mitochondrial endonuclease I-SceI creates a double-strand break as the initiating step in the gene conversional transfer of the omega(+) intron to omega(-) DNA. We have expressed a galactose-inducible synthetic I-SceI gene in the nucleus of yeast that also carries the I-SceI recognition site on a plasmid substrate. We find that the galactose-induced I-SceI protein can be active in the nucleus and efficiently catalyze recombination. With a target plasmid containing direct repeats of the Escherichia coli lacZ gene, one copy of which is interrupted by a 24-bp cutting site, galactose induction produces both deletions and gene conversions. Both the kinetics and the proportion of deletions and gene conversions are very similar to analogous events initiated by a galactose-inducible HO endonuclease gene. We also find that, in a rad52 mutant strain, the repair of double-strand breaks initiated by I-SceI and by HO are similarly affected: the formation of deletions is reduced, but not eliminated. Altogether, these results suggest either that the two endonucleases act in the same way after double-strand break formation or that the two endonucleases are not involved in subsequent steps.

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