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.

[1]  B. Dujon,et al.  Purification and characterization of the in vitro activity of I-Sce I, a novel and highly specific endonuclease encoded by a group I intron. , 1990, Nucleic acids research.

[2]  Bernard Dujon,et al.  An intron-encoded protein is active in a gene conversion process that spreads an intron into a mitochondrial gene , 1985, Cell.

[3]  M. Resnick.  Genetic control of radiation sensitivity in Saccharomyces cerevisiae. , 1969, Genetics.

[4]  J. Haber,et al.  Characterization of double-strand break-induced recombination: homology requirements and single-stranded DNA formation , 1992, Molecular and cellular biology.

[5]  L. Guarente,et al.  A GAL10-CYC1 hybrid yeast promoter identifies the GAL4 regulatory region as an upstream site. , 1982, Proceedings of the National Academy of Sciences of the United States of America.

[6]  J. Haber,et al.  Homothallic mating type switching generates lethal chromosome breaks in rad52 strains of Saccharomyces cerevisiae , 1981, Molecular and cellular biology.

[7]  G. Fink,et al.  Transformation of yeast. , 1978, Proceedings of the National Academy of Sciences of the United States of America.

[8]  J. Haber,et al.  Two alternative pathways of double-strand break repair that are kinetically separable and independently modulated , 1992, Molecular and cellular biology.

[9]  J. Strathern,et al.  Homothallic switching of yeast mating type cassettes is initiated by a double-stranded cut in the MAT locus , 1982, Cell.

[10]  J. Szostak,et al.  Double-strand breaks at an initiation site for meiotic gene conversion , 1989, Nature.

[11]  K. McEntee,et al.  A rapid and efficient procedure for transformation of intact Saccharomyces cerevisiae by electroporation. , 1989, Biochemical and biophysical research communications.

[12]  A. Riggs,et al.  Genomic sequencing. , 1993, Methods in molecular biology.

[13]  T. Shibata,et al.  A subunit of yeast site-specific endonuclease SceI is a mitochondrial version of the 70-kDa heat shock protein. , 1990, The Journal of biological chemistry.

[14]  E. Maryon,et al.  Characterization of recombination intermediates from DNA injected into Xenopus laevis oocytes: evidence for a nonconservative mechanism of homologous recombination , 1991, Molecular and cellular biology.

[15]  J. Szostak,et al.  Extensive 3′-overhanging, single-stranded DNA associated with the meiosis-specific double-strand breaks at the ARG4 recombination initiation site , 1991, Cell.

[16]  C. Hodgson,et al.  Hybridization probe size control: optimized 'oligolabelling'. , 1987, Nucleic acids research.

[17]  E. Chen,et al.  A 24-base-pair DNA sequence from the MAT locus stimulates intergenic recombination in yeast. , 1986, Proceedings of the National Academy of Sciences of the United States of America.

[18]  J. Haber,et al.  Efficient repair of HO-induced chromosomal breaks in Saccharomyces cerevisiae by recombination between flanking homologous sequences , 1988, Molecular and cellular biology.

[19]  B. Dujon,et al.  Group I introns as mobile genetic elements: facts and mechanistic speculations--a review. , 1989, Gene.

[20]  B. Dujon,et al.  Universal code equivalent of a yeast mitochondrial intron reading frame is expressed into E. coli as a specific double strand endonuclease , 1986, Cell.

[21]  J. Strathern,et al.  A site-specific endonuclease essential for mating-type switching in Saccharomyces cerevisiae , 1983, Cell.

[22]  J. Szostak,et al.  Yeast recombination: the association between double-strand gap repair and crossing-over. , 1983, Proceedings of the National Academy of Sciences of the United States of America.

[23]  R. Mortimer,et al.  A genetic study of x-ray sensitive mutants in yeast. , 1974, Mutation research.

[24]  K. Murata,et al.  Transformation of intact yeast cells treated with alkali cations. , 1984, Journal of bacteriology.

[25]  J. Haber,et al.  Physical monitoring of mating type switching in Saccharomyces cerevisiae , 1988, Molecular and cellular biology.

[26]  N. Sternberg,et al.  Model for homologous recombination during transfer of DNA into mouse L cells: role for DNA ends in the recombination process , 1984, Molecular and cellular biology.

[27]  N. Sternberg,et al.  Intermolecular recombination between DNAs introduced into mouse L cells is mediated by a nonconservative pathway that leads to crossover products , 1990, Molecular and cellular biology.

[28]  M S Meselson,et al.  A general model for genetic recombination. , 1975, Proceedings of the National Academy of Sciences of the United States of America.

[29]  J. Nickoloff,et al.  In vivo analysis of the Saccharomyces cerevisiae HO nuclease recognition site by site-directed mutagenesis , 1990, Molecular and cellular biology.

[30]  B. Ozenberger,et al.  A unique pathway of double-strand break repair operates in tandemly repeated genes , 1991, Molecular and cellular biology.

[31]  M. Resnick,et al.  The Role of Radiation (rad) Genes in Meiotic Recombination in Yeast. , 1980, Genetics.