Extensive Loss of Heterozygosity Is Suppressed during Homologous Repair of Chromosomal Breaks

ABSTRACT Loss of heterozygosity (LOH) is a common genetic alteration in tumors and often extends several megabases to encompass multiple genetic loci or even whole chromosome arms. Based on marker and karyotype analysis of tumor samples, a significant fraction of LOH events appears to arise from mitotic recombination between homologous chromosomes, reminiscent of recombination during meiosis. As DNA double-strand breaks (DSBs) initiate meiotic recombination, a potential mechanism leading to LOH in mitotically dividing cells is DSB repair involving homologous chromosomes. We therefore sought to characterize the extent of LOH arising from DSB-induced recombination between homologous chromosomes in mammalian cells. To this end, a recombination reporter was introduced into a mouse embryonic stem cell line that has nonisogenic maternal and paternal chromosomes, as is the case in human populations, and then a DSB was introduced into one of the chromosomes. Recombinants involving alleles on homologous chromosomes were readily obtained at a frequency of 4.6 × 10−5; however, this frequency was substantially lower than that of DSB repair by nonhomologous end joining or the inferred frequency of homologous repair involving sister chromatids. Strikingly, the majority of recombinants had LOH restricted to the site of the DSB, with a minor class of recombinants having LOH that extended to markers 6 kb from the DSB. Furthermore, we found no evidence of LOH extending to markers 1 centimorgan or more from the DSB. In addition, crossing over, which can lead to LOH of a whole chromosome arm, was not observed, implying that there are key differences between mitotic and meiotic recombination mechanisms. These results indicate that extensive LOH is normally suppressed during DSB-induced allelic recombination in dividing mammalian cells.

[1]  S. Jinks-Robertson,et al.  The role of the mismatch repair machinery in regulating mitotic and meiotic recombination between diverged sequences in yeast. , 1999, Genetics.

[2]  J. Haber,et al.  Double-strand break repair in the absence of RAD51 in yeast: a possible role for break-induced DNA replication. , 1996, Proceedings of the National Academy of Sciences of the United States of America.

[3]  R. Richards.  Fragile and unstable chromosomes in cancer: causes and consequences. , 2001, Trends in genetics : TIG.

[4]  L. Lefebvre,et al.  Selection for transgene homozygosity in embryonic stem cells results in extensive loss of heterozygosity , 2001, Nature Genetics.

[5]  M. Kupiec,et al.  The Relationship between Homology Length and Crossing Over during the Repair of a Broken Chromosome* , 2000, The Journal of Biological Chemistry.

[6]  S. Schwartz,et al.  Covariation of Synaptonemal Complex Length and Mammalian Meiotic Exchange Rates , 2002, Science.

[7]  E. Lander,et al.  A genetic linkage map of the mouse: current applications and future prospects. , 1993, Science.

[8]  M. Jasin.  Double-Strand Break Repair and Homologous Recombination in Mammalian Cells , 2001 .

[9]  G. Gloor,et al.  Efficient copying of nonhomologous sequences from ectopic sites via P-element-induced gap repair , 1994, Molecular and cellular biology.

[10]  Rudolf Jaenisch,et al.  DNA hypomethylation leads to elevated mutation rates , 1998, Nature.

[11]  Peter W. Laird,et al.  THE ROLE OF DNA METHYLATION IN CANCER GENETICS AND EPIGENETICS , 1996 .

[12]  M. Jasin,et al.  Repair of Double-Strand Breaks by Homologous Recombination in Mismatch Repair-Defective Mammalian Cells , 2001, Molecular and Cellular Biology.

[13]  J. Haber,et al.  Genetic Requirements for RAD51- andRAD54-Independent Break-Induced Replication Repair of a Chromosomal Double-Strand Break , 2001, Molecular and Cellular Biology.

[14]  Jack W. Szostak,et al.  The double-strand-break repair model for recombination , 1983, Cell.

[15]  R. Camerini-Otero,et al.  Cloning, characterization, and localization of mouse and human SPO11. , 1999, Genomics.

[16]  W. Cavenee,et al.  Loss of constitutional heterozygosity in human cancer. , 1991, Annual review of genetics.

[17]  J. Murray,et al.  Site-specific recombinases: tools for genome engineering. , 1993, Trends in genetics : TIG.

[18]  M. Jasin,et al.  BRCA2 is required for homology-directed repair of chromosomal breaks. , 2001, Molecular cell.

[19]  N. Copeland,et al.  Efficient Cre-loxP–induced mitotic recombination in mouse embryonic stem cells , 2002, Nature Genetics.

[20]  M. Jasin,et al.  Loss of heterozygosity induced by a chromosomal double-strand break. , 1997, Proceedings of the National Academy of Sciences of the United States of America.

[21]  Rudolf Jaenisch,et al.  Chromosomal deletion complexes in mice by radiation of embryonic stem cells , 1997, Nature Genetics.

[22]  J. Tischfield,et al.  Mitotic recombination is suppressed by chromosomal divergence in hybrids of distantly related mouse strains , 2001, Nature Genetics.

[23]  E. Friedberg,et al.  DNA damage and repair , 2003, Nature.

[24]  P. Cohen,et al.  The time course and chromosomal localization of recombination-related proteins at meiosis in the mouse are compatible with models that can resolve the early DNA-DNA interactions without reciprocal recombination. , 2002, Journal of cell science.

[25]  S. Keeney,et al.  Mechanism and control of meiotic recombination initiation. , 2001, Current topics in developmental biology.

[26]  J. Haber,et al.  HO endonuclease-induced recombination in yeast meiosis resembles Spo11-induced events. , 2000, Proceedings of the National Academy of Sciences of the United States of America.

[27]  N. Ellis,et al.  Ku DNA end-binding protein modulates homologous repair of double-strand breaks in mammalian cells. , 2001, Genes & development.

[28]  Y. Nakamura,et al.  Molecular evidence that homologous recombination occurs in proliferating human somatic cells. , 1990, Proceedings of the National Academy of Sciences of the United States of America.

[29]  W. Bigbee,et al.  Evidence for increased in vivo mutation and somatic recombination in Bloom's syndrome. , 1989, Proceedings of the National Academy of Sciences of the United States of America.

[30]  A. Berns,et al.  Highly efficient gene targeting in embryonic stem cells through homologous recombination with isogenic DNA constructs. , 1992, Proceedings of the National Academy of Sciences of the United States of America.

[31]  N. Kleckner,et al.  A pathway for generation and processing of double-strand breaks during meiotic recombination in S. cerevisiae , 1990, Cell.

[32]  J. Tischfield,et al.  Mitotic recombination produces the majority of recessive fibroblast variants in heterozygous mice. , 1999, Proceedings of the National Academy of Sciences of the United States of America.

[33]  Judith A. Blake,et al.  The Mouse Genome Database (MGD): the model organism database for the laboratory mouse , 2002, Nucleic Acids Res..

[34]  Eric S. Lander,et al.  Large-scale discovery and genotyping of single-nucleotide polymorphisms in the mouse , 2000, Nature Genetics.

[35]  J. Stringer,et al.  Embryonic stem cells and somatic cells differ in mutation frequency and type , 2002, Proceedings of the National Academy of Sciences of the United States of America.

[36]  M. Hultén,et al.  Crossing over analysis at pachytene in man , 1998, European Journal of Human Genetics.

[37]  J. Haber,et al.  Multiple Pathways of Recombination Induced by Double-Strand Breaks in Saccharomyces cerevisiae , 1999, Microbiology and Molecular Biology Reviews.

[38]  K. Kinzler,et al.  Mechanisms underlying losses of heterozygosity in human colorectal cancers , 2001, Proceedings of the National Academy of Sciences of the United States of America.

[39]  D. Leach,et al.  Recombination at double-strand breaks and DNA ends: conserved mechanisms from phage to humans. , 2001, Molecular cell.

[40]  Myron F. Goodman,et al.  The importance of repairing stalled replication forks , 2000, Nature.

[41]  N. Ellis,et al.  Somatic intragenic recombination within the mutated locus BLM can correct the high sister-chromatid exchange phenotype of Bloom syndrome cells. , 1995, American journal of human genetics.

[42]  S. Keeney,et al.  A mouse homolog of the Saccharomyces cerevisiae meiotic recombination DNA transesterase Spo11p. , 1999, Genomics.

[43]  R. Jaenisch,et al.  The role of DNA methylation in cancer genetic and epigenetics. , 1996, Annual review of genetics.

[44]  Christine Richardson,et al.  Frequent chromosomal translocations induced by DNA double-strand breaks , 2000, Nature.

[45]  T. Dryja,et al.  Mitotic recombination map of 13cen-13q14 derived from an investigation of loss of heterozygosity in retinoblastomas. , 1999, Proceedings of the National Academy of Sciences of the United States of America.

[46]  M. Daly,et al.  A map of human genome sequence variation containing 1.42 million single nucleotide polymorphisms , 2001, Nature.

[47]  M. Jasin,et al.  Double-strand break repair by interchromosomal recombination: suppression of chromosomal translocations. , 1998, Genes & development.

[48]  H. Vrieling,et al.  Determination of spontaneous loss of heterozygosity mutations in Aprt heterozygous mice. , 1998, Nucleic acids research.

[49]  M. Jasin,et al.  Sister chromatid gene conversion is a prominent double‐strand break repair pathway in mammalian cells , 2000, The EMBO journal.

[50]  M. Jasin,et al.  Homology-directed dna repair, mitomycin-c resistance, and chromosome stability is restored with correction of a Brca1 mutation. , 2001, Cancer research.

[51]  J. Miyazaki,et al.  Site-specific recombination of a transgene in fertilized eggs by transient expression of Cre recombinase. , 1995, Proceedings of the National Academy of Sciences of the United States of America.

[52]  S. West,et al.  Distribution of the Rad51 recombinase in human and mouse spermatocytes , 1997, The EMBO journal.

[53]  Jeremy M. Stark,et al.  ATP Hydrolysis by Mammalian RAD51 Has a Key Role during Homology-directed DNA Repair* , 2002, The Journal of Biological Chemistry.

[54]  A. Grosovsky,et al.  Interchromosomal gene conversion at an endogenous human cell locus. , 2001, Genetics.

[55]  A. Bradley,et al.  Cancer predisposition caused by elevated mitotic recombination in Bloom mice , 2000, Nature Genetics.