Site-specific recombination determined by I-SceI, a mitochondrial group I intron-encoded endonuclease expressed in the yeast nucleus.
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.
Plessis
A
A
Unité de génétique moléculaire des levures, URA1149 du CNRS, Département de Biologie moléculaire, Institut Pasteur, Paris, France.
Perrin
A
A
Haber
J E
JE
Dujon
B
B
D001483:Base Sequence; D015139:Blotting, Southern; D002467:Cell Nucleus / Q000201:enzymology*; D002872:Chromosome Deletion; D003001:Cloning, Molecular; D004249:DNA Damage; D004260:DNA Repair; D004271:DNA, Fungal / Q000378:metabolism; D015252:Deoxyribonucleases, Type II Site-Specific / Q000235:genetics / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics; D005785:Gene Conversion; D015870:Gene Expression; D007438:Introns; D008928:Mitochondria / Q000201:enzymology*; D008969:Molecular Sequence Data; D009154:Mutation; D010957:Plasmids; D011995:Recombination, Genetic; D012441:Saccharomyces cerevisiae / Q000201:enzymology; D029701:Saccharomyces cerevisiae Proteins
D004271:DNA, Fungal; D029701:Saccharomyces cerevisiae Proteins; C530845:HO protein, S cerevisiae; C037725:SCEI protein, S cerevisiae; D015252:Deoxyribonucleases, Type II Site-Specific