Induction of homologous recombination in mammalian chromosomes by using the I-SceI system of Saccharomyces cerevisiae.
The mitochondrial intron-encoded endonuclease I-SceI of Saccharomyces cerevisiae has an 18-bp recognition sequence and, therefore, has a very low probability of cutting DNA, even within large genomes. We demonstrate that double-strand breaks can be initiated by the I-SceI endonuclease at a predetermined location in the mouse genome and that the breaks can be repaired with a donor molecule homologous regions flanking the breaks. This induced homologous recombination is approximately 2 orders of magnitude more frequent than spontaneous homologous recombination and at least 10 times more frequent than random integration near an active promoter. As a consequence of induced homologous recombination, a heterologous novel sequence can be inserted at the site of the break. This recombination can occur at a variety of chromosomal targets in differentiated and multipotential cells. These results demonstrate homologous recombination involving chromosomal DNA by the double-strand break repair mechanism in mammals and show the usefulness of very rare cutter endonucleases, such as I-SceI, for designing genome rearrangements.
Choulika
A
A
Unité de Biologie moléculaire du Développement, Institut Pasteur, Université Pierre et Marie Curie, Paris, France.
Perrin
A
A
Dujon
B
B
Nicolas
J F
JF
D016475:3T3 Cells; D000818:Animals; D015152:Blotting, Northern; D015139:Blotting, Southern; D002875:Chromosomes / Q000378:metabolism; D004247:DNA / Q000378:metabolism; D004249:DNA Damage; D004260:DNA Repair / Q000235:genetics*; D015252:Deoxyribonucleases, Type II Site-Specific / Q000378:metabolism*; D051379:Mice; D016254:Mutagenesis, Insertional; D011995:Recombination, Genetic / Q000235:genetics*; D012441:Saccharomyces cerevisiae / Q000201:enzymology*; D029701:Saccharomyces cerevisiae Proteins; D013379:Substrate Specificity
D029701:Saccharomyces cerevisiae Proteins; D004247:DNA; C037725:SCEI protein, S cerevisiae; D015252:Deoxyribonucleases, Type II Site-Specific