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.

[1]  Nikil D. Dutt,et al.  System and architecture-level power reduction of microprocessor-based communication and multi-media applications , 2000, IEEE/ACM International Conference on Computer Aided Design. ICCAD - 2000. IEEE/ACM Digest of Technical Papers (Cat. No.00CH37140).

[2]  J. Wang,et al.  Substrate recognition and induced DNA distortion by the PI-SceI endonuclease, an enzyme generated by protein splicing. , 1996, Journal of molecular biology.

[3]  J. Haber,et al.  Cell cycle and genetic requirements of two pathways of nonhomologous end-joining repair of double-strand breaks in Saccharomyces cerevisiae , 1996, Molecular and cellular biology.

[4]  M. Brenneman,et al.  Stimulation of intrachromosomal homologous recombination in human cells by electroporation with site-specific endonucleases. , 1996, Proceedings of the National Academy of Sciences of the United States of America.

[5]  M. Meuth,et al.  The influence of a (GT)29 microsatellite sequence on homologous recombination in the hamster adenine phosphoribosyltransferase gene. , 1996, Nucleic Acids Research.

[6]  D. Roth,et al.  High-frequency illegitimate integration of transfected DNA at preintegrated target sites in a mammalian genome , 1996, Molecular and cellular biology.

[7]  R. V. Merrihew,et al.  Efficient modification of the APRT gene by FLP/FRT site-specific targeting , 1995, Somatic cell and molecular genetics.

[8]  B. Shafer,et al.  DNA synthesis errors associated with double-strand-break repair. , 1995, Genetics.

[9]  M. Meuth,et al.  Deletion mapping of highly conserved transcribed sequence downstream fromAPRT locus , 1995, Somatic cell and molecular genetics.

[10]  B. Dujon,et al.  Induction of homologous recombination in mammalian chromosomes by using the I-SceI system of Saccharomyces cerevisiae , 1995, Molecular and cellular biology.

[11]  D. Segal,et al.  Endonuclease-induced, targeted homologous extrachromosomal recombination in Xenopus oocytes , 1995, Proceedings of the National Academy of Sciences of the United States of America.

[12]  R. Bollag,et al.  Spontaneous and restriction enzyme-induced chromosomal recombination in mammalian cells. , 1994, Proceedings of the National Academy of Sciences of the United States of America.

[13]  P. Rouet,et al.  Introduction of double-strand breaks into the genome of mouse cells by expression of a rare-cutting endonuclease. , 1994, Molecular and cellular biology.

[14]  J. Yanowitz,et al.  Stringent sequence requirements for the formation of human telomeres. , 1994, Proceedings of the National Academy of Sciences of the United States of America.

[15]  J. Phillips,et al.  Illegitimate recombination induced by DNA double-strand breaks in a mammalian chromosome , 1994, Molecular and cellular biology.

[16]  P. Rouet,et al.  Expression of a site-specific endonuclease stimulates homologous recombination in mammalian cells. , 1994, Proceedings of the National Academy of Sciences of the United States of America.

[17]  J. Haber,et al.  Two different types of double-strand breaks in Saccharomyces cerevisiae are repaired by similar RAD52-independent, nonhomologous recombination events , 1994, Molecular and cellular biology.

[18]  M Lichten,et al.  Meiosis-induced double-strand break sites determined by yeast chromatin structure. , 1994, Science.

[19]  B. Dujon,et al.  Homologous recombination in plant cells is enhanced by in vivo induction of double strand breaks into DNA by a site-specific endonuclease. , 1993, Nucleic acids research.

[20]  B. Dujon,et al.  Asymmetrical recognition and activity of the I‐SceI endonuclease on its site and on intron‐exon junctions. , 1993, The EMBO journal.

[21]  C. Hutchison,et al.  A major difference between the divergence patterns within the lines-1 families in mice and voles. , 1993, Molecular biology and evolution.

[22]  J. Haber.  Exploring the pathways of homologous recombination , 1992, Current Biology.

[23]  J. Little,et al.  X rays induce interallelic homologous recombination at the human thymidine kinase gene , 1992, Molecular and cellular biology.

[24]  B. Dujon,et al.  Site-specific recombination determined by I-SceI, a mitochondrial group I intron-encoded endonuclease expressed in the yeast nucleus. , 1992, Genetics.

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

[26]  R. Bollag,et al.  Direct-repeat analysis of chromatid interactions during intrachromosomal recombination in mouse cells , 1991, Molecular and cellular biology.

[27]  J. H. Wilson,et al.  A novel selection system for recombinational and mutational events within an intron of a eucaryotic gene. , 1990, Nucleic Acids Research.

[28]  D. Roth,et al.  Comparison of filler DNA at immune, nonimmune, and oncogenic rearrangements suggests multiple mechanisms of formation , 1989, Molecular and cellular biology.

[29]  C. Caskey,et al.  Construction of plasmids that express E. coli beta-galactosidase in mammalian cells. , 1989, Nucleic acids research.

[30]  D. Roth,et al.  Relative rates of homologous and nonhomologous recombination in transfected DNA. , 1985, Proceedings of the National Academy of Sciences of the United States of America.

[31]  M. Di Liberto,et al.  Analysis of the 3' end of the human pro-alpha 2(I) collagen gene. Utilization of multiple polyadenylation sites in cultured fibroblasts. , 1983, The Journal of biological chemistry.

[32]  M. Meuth,et al.  Structure of mutant alleles at the aprt locus of Chinese hamster ovary cells. , 1983, Journal of molecular biology.

[33]  A. S. Waldman,et al.  Repair of a specific double-strand break generated within a mammalian chromosome by yeast endonuclease I-SceI. , 1994, Nucleic acids research.

[34]  M. Meuth,et al.  Molecular patterns of aprt gene rearrangements. , 1990, Progress in clinical and biological research.