Repair of site-specific double-strand breaks in a mammalian chromosome by homologous and illegitimate recombination.
In mammalian cells, chromosomal double-strand breaks are efficiently repaired, yet little is known about the relative contributions of homologous recombination and illegitimate recombination in the repair process. In this study, we used a loss-of-function assay to assess the repair of double-strand breaks by homologous and illegitimate recombination. We have used a hamster cell line engineered by gene targeting to contain a tandem duplication of the native adenine phosphoribosyltransferase (APRT) gene with an I-SceI recognition site in the otherwise wild-type APRT+ copy of the gene. Site-specific double-strand breaks were induced by intracellular expression of I-SceI, a rare-cutting endonuclease from the yeast Saccharomyces cerevisiae. I-SceI cleavage stimulated homologous recombination about 100-fold; however, illegitimate recombination was stimulated more than 1,000-fold. These results suggest that illegitimate recombination is an important competing pathway with homologous recombination for chromosomal double-strand break repair in mammalian cells.
Sargent
R G
RG
Verna and Marrs McLean Department of Biochemistry, Baylor College of Medicine, Houston, Texas 77030, USA.
Brenneman
M A
MA
Wilson
J H
JH
D000228:Adenine Phosphoribosyltransferase / Q000235:genetics; D000818:Animals; D001483:Base Sequence; D016466:CHO Cells; D002875:Chromosomes / Q000235:genetics; D006224:Cricetinae; D004249:DNA Damage; D004260:DNA Repair / Q000235:genetics*; D015252:Deoxyribonucleases, Type II Site-Specific; D015321:Gene Rearrangement / Q000235:genetics; D018390:Gene Targeting; D008969:Molecular Sequence Data; D017354:Point Mutation / Q000235:genetics; D011995:Recombination, Genetic / Q000235:genetics*; D012441:Saccharomyces cerevisiae / Q000201:enzymology; D029701:Saccharomyces cerevisiae Proteins; D017422:Sequence Analysis, DNA
D029701:Saccharomyces cerevisiae Proteins; D000228:Adenine Phosphoribosyltransferase; C037725:SCEI protein, S cerevisiae; D015252:Deoxyribonucleases, Type II Site-Specific