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. 2001 Jun;158(2):757–767. doi: 10.1093/genetics/158.2.757

Interchromosomal gene conversion at an endogenous human cell locus.

P J Quintana 1, E A Neuwirth 1, A J Grosovsky 1
PMCID: PMC1461692  PMID: 11404339

Abstract

To examine the relationship between gene conversion and reciprocal exchange at an endogenous chromosomal locus, we developed a reversion assay in a thymidine kinase deficient mutant, TX545, derived from the human lymphoblastoid cell line TK6. Selectable revertants of TX545 can be generated through interchromosomal gene conversion at the site of inactivating mutations on each tk allele or by reciprocal exchange that alters the linkage relationships of inactivating polymorphisms within the tk locus. Analysis of loss of heterozygosity (LOH) at intragenic polymorphisms and flanking microsatellite markers was used to initially evaluate allelotypes in TK(+) revertants for patterns associated with either gene conversion or crossing over. The linkage pattern in a subset of convertants was then unambiguously established, even in the event of prereplicative recombinational exchanges, by haplotype analysis of flanking microsatellite loci in tk(-/-) LOH mutants collected from the tk(+/-) parental convertant. Some (7/38; 18%) revertants were attributable to easily discriminated nonrecombinational mechanisms, including suppressor mutations within the tk coding sequence. However, all revertants classified as a recombinational event (28/38; 74%) were attributed to localized gene conversion, representing a highly significant preference (P < 0.0001) over gene conversion with associated reciprocal exchange, which was never observed.

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Selected References

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  1. Amundson S. A., Liber H. L. A comparison of induced mutation at homologous alleles of the tk locus in human cells. II. Molecular analysis of mutants. Mutat Res. 1992 May;267(1):89–95. doi: 10.1016/0027-5107(92)90113-g. [DOI] [PubMed] [Google Scholar]
  2. Benjamin M. B., Little J. B. X rays induce interallelic homologous recombination at the human thymidine kinase gene. Mol Cell Biol. 1992 Jun;12(6):2730–2738. doi: 10.1128/mcb.12.6.2730. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Biedermann K. A., Sun J. R., Giaccia A. J., Tosto L. M., Brown J. M. scid mutation in mice confers hypersensitivity to ionizing radiation and a deficiency in DNA double-strand break repair. Proc Natl Acad Sci U S A. 1991 Feb 15;88(4):1394–1397. doi: 10.1073/pnas.88.4.1394. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Bigger C. A., Cheh A., Latif F., Fishel R., Canella K. A., Stafford G. A., Yagi H., Jerina D. M., Dipple A. DNA strand breaks induced by configurationally isomeric hydrocarbon diol epoxides. Drug Metab Rev. 1994;26(1-2):287–299. doi: 10.3109/03602539409029798. [DOI] [PubMed] [Google Scholar]
  5. Bradshaw H. D., Jr, Deininger P. L. Human thymidine kinase gene: molecular cloning and nucleotide sequence of a cDNA expressible in mammalian cells. Mol Cell Biol. 1984 Nov;4(11):2316–2320. doi: 10.1128/mcb.4.11.2316. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Cavenee W. K., Dryja T. P., Phillips R. A., Benedict W. F., Godbout R., Gallie B. L., Murphree A. L., Strong L. C., White R. L. Expression of recessive alleles by chromosomal mechanisms in retinoblastoma. 1983 Oct 27-Nov 2Nature. 305(5937):779–784. doi: 10.1038/305779a0. [DOI] [PubMed] [Google Scholar]
  7. Dobo K. L., Giver C. R., Eastmond D. A., Rumbos H. S., Grosovsky A. J. Extensive loss of heterozygosity accounts for differential mutation rate on chromosome 17q in human lymphoblasts. Mutagenesis. 1995 Jan;10(1):53–58. doi: 10.1093/mutage/10.1.53. [DOI] [PubMed] [Google Scholar]
  8. Donoho G., Jasin M., Berg P. Analysis of gene targeting and intrachromosomal homologous recombination stimulated by genomic double-strand breaks in mouse embryonic stem cells. Mol Cell Biol. 1998 Jul;18(7):4070–4078. doi: 10.1128/mcb.18.7.4070. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Dronkert M. L., Beverloo H. B., Johnson R. D., Hoeijmakers J. H., Jasin M., Kanaar R. Mouse RAD54 affects DNA double-strand break repair and sister chromatid exchange. Mol Cell Biol. 2000 May;20(9):3147–3156. doi: 10.1128/mcb.20.9.3147-3156.2000. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Elliott B., Richardson C., Winderbaum J., Nickoloff J. A., Jasin M. Gene conversion tracts from double-strand break repair in mammalian cells. Mol Cell Biol. 1998 Jan;18(1):93–101. doi: 10.1128/mcb.18.1.93. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Essers J., van Steeg H., de Wit J., Swagemakers S. M., Vermeij M., Hoeijmakers J. H., Kanaar R. Homologous and non-homologous recombination differentially affect DNA damage repair in mice. EMBO J. 2000 Apr 3;19(7):1703–1710. doi: 10.1093/emboj/19.7.1703. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Fain P. R., Solomon E., Ledbetter D. H. Second international workshop on human chromosome 17. Cytogenet Cell Genet. 1991;57(2-3):66–77. [PubMed] [Google Scholar]
  13. Flemington E., Bradshaw H. D., Jr, Traina-Dorge V., Slagel V., Deininger P. L. Sequence, structure and promoter characterization of the human thymidine kinase gene. Gene. 1987;52(2-3):267–277. doi: 10.1016/0378-1119(87)90053-9. [DOI] [PubMed] [Google Scholar]
  14. Fogel S., Hurst D. D. Meiotic gene conversion in yeast tetrads and the theory of recombination. Genetics. 1967 Oct;57(2):455–481. doi: 10.1093/genetics/57.2.455. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Foss H. M., Hillers K. J., Stahl F. W. The conversion gradient at HIS4 of Saccharomyces cerevisiae. II. A role for mismatch repair directed by biased resolution of the recombinational intermediate. Genetics. 1999 Oct;153(2):573–583. doi: 10.1093/genetics/153.2.573. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Fung-Leung W. P., Mak T. W. Embryonic stem cells and homologous recombination. Curr Opin Immunol. 1992 Apr;4(2):189–194. doi: 10.1016/0952-7915(92)90012-4. [DOI] [PubMed] [Google Scholar]
  17. Gao Y., Chaudhuri J., Zhu C., Davidson L., Weaver D. T., Alt F. W. A targeted DNA-PKcs-null mutation reveals DNA-PK-independent functions for KU in V(D)J recombination. Immunity. 1998 Sep;9(3):367–376. doi: 10.1016/s1074-7613(00)80619-6. [DOI] [PubMed] [Google Scholar]
  18. Giver C. R., Grosovsky A. J. Radiation specific patterns of loss of heterozygosity on chromosome 17q. Mutat Res. 2000 May 30;450(1-2):201–209. doi: 10.1016/s0027-5107(00)00026-9. [DOI] [PubMed] [Google Scholar]
  19. Giver C. R., Nelson S. L., Jr, Cha M. Y., Pongsaensook P., Grosovsky A. J. Mutational spectrum of X-ray induced TK- human cell mutants. Carcinogenesis. 1995 Feb;16(2):267–275. doi: 10.1093/carcin/16.2.267. [DOI] [PubMed] [Google Scholar]
  20. Godwin A. R., Liskay R. M. The effects of insertions on mammalian intrachromosomal recombination. Genetics. 1994 Feb;136(2):607–617. doi: 10.1093/genetics/136.2.607. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Grist S. A., McCarron M., Kutlaca A., Turner D. R., Morley A. A. In vivo human somatic mutation: frequency and spectrum with age. Mutat Res. 1992 Apr;266(2):189–196. doi: 10.1016/0027-5107(92)90186-6. [DOI] [PubMed] [Google Scholar]
  22. Grosovsky A. J., Parks K. K., Giver C. R., Nelson S. L. Clonal analysis of delayed karyotypic abnormalities and gene mutations in radiation-induced genetic instability. Mol Cell Biol. 1996 Nov;16(11):6252–6262. doi: 10.1128/mcb.16.11.6252. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Grosovsky A. J., Walter B. N., Giver C. R. DNA-sequence specificity of mutations at the human thymidine kinase locus. Mutat Res. 1993 Oct;289(2):231–243. doi: 10.1016/0027-5107(93)90074-p. [DOI] [PubMed] [Google Scholar]
  24. Hurst D. D., Fogel S., Mortimer R. K. Conversion-associated recombination in yeast (hybrids-meiosis-tetrads-marker loci-models). Proc Natl Acad Sci U S A. 1972 Jan;69(1):101–105. doi: 10.1073/pnas.69.1.101. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Liang F., Han M., Romanienko P. J., Jasin M. Homology-directed repair is a major double-strand break repair pathway in mammalian cells. Proc Natl Acad Sci U S A. 1998 Apr 28;95(9):5172–5177. doi: 10.1073/pnas.95.9.5172. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Liang F., Jasin M. Extrachromosomal assay for DNA double-strand break repair. Methods Mol Biol. 1999;113:487–497. doi: 10.1385/1-59259-675-4:487. [DOI] [PubMed] [Google Scholar]
  27. Liber H. L., Yandell D. W., Little J. B. A comparison of mutation induction at the tk and hprt loci in human lymphoblastoid cells; quantitative differences are due to an additional class of mutations at the autosomal tk locus. Mutat Res. 1989 Feb;216(1):9–17. doi: 10.1016/0165-1161(89)90018-6. [DOI] [PubMed] [Google Scholar]
  28. Lin Y., Lukacsovich T., Waldman A. S. Multiple pathways for repair of DNA double-strand breaks in mammalian chromosomes. Mol Cell Biol. 1999 Dec;19(12):8353–8360. doi: 10.1128/mcb.19.12.8353. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Liskay R. M., Stachelek J. L. Evidence for intrachromosomal gene conversion in cultured mouse cells. Cell. 1983 Nov;35(1):157–165. doi: 10.1016/0092-8674(83)90218-0. [DOI] [PubMed] [Google Scholar]
  30. MacLeod M. C., Evans F. E., Lay J., Chiarelli P., Geacintov N. E., Powell K. L., Daylong A., Luna E., Harvey R. G. Identification of a novel, N7-deoxyguanosine adduct as the major DNA adduct formed by a non-bay-region diol epoxide of benzo[a]pyrene with low mutagenic potential. Biochemistry. 1994 Mar 15;33(10):2977–2987. doi: 10.1021/bi00176a030. [DOI] [PubMed] [Google Scholar]
  31. Marians K. J. Replication and recombination intersect. Curr Opin Genet Dev. 2000 Apr;10(2):151–156. doi: 10.1016/s0959-437x(00)00059-9. [DOI] [PubMed] [Google Scholar]
  32. Meselson M. S., Radding C. M. A general model for genetic recombination. Proc Natl Acad Sci U S A. 1975 Jan;72(1):358–361. doi: 10.1073/pnas.72.1.358. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Milligan J. R., Aguilera J. A., Nguyen T. T., Ward J. F., Kow Y. W., He B., Cunningham R. P. Yield of DNA strand breaks after base oxidation of plasmid DNA. Radiat Res. 1999 Mar;151(3):334–342. [PubMed] [Google Scholar]
  34. Moran M. F., Ebisuzaki K. In vivo benzo[a]pyrene diol epoxide-induced alkali-labile sites are not apurinic sites. Mutat Res. 1991 Feb;262(2):79–84. doi: 10.1016/0165-7992(91)90111-g. [DOI] [PubMed] [Google Scholar]
  35. Moynahan M. E., Jasin M. Loss of heterozygosity induced by a chromosomal double-strand break. Proc Natl Acad Sci U S A. 1997 Aug 19;94(17):8988–8993. doi: 10.1073/pnas.94.17.8988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Nelson S. L., Giver C. R., Grosovsky A. J. Spectrum of X-ray-induced mutations in the human hprt gene. Carcinogenesis. 1994 Mar;15(3):495–502. doi: 10.1093/carcin/15.3.495. [DOI] [PubMed] [Google Scholar]
  37. Rathjen P. D., Lake J., Whyatt L. M., Bettess M. D., Rathjen J. Properties and uses of embryonic stem cells: prospects for application to human biology and gene therapy. Reprod Fertil Dev. 1998;10(1):31–47. doi: 10.1071/r98041. [DOI] [PubMed] [Google Scholar]
  38. Rousseau-Merck M. F., Versteege I., Legrand I., Couturier J., Mairal A., Delattre O., Aurias A. hSNF5/INI1 inactivation is mainly associated with homozygous deletions and mitotic recombinations in rhabdoid tumors. Cancer Res. 1999 Jul 1;59(13):3152–3156. [PubMed] [Google Scholar]
  39. Sargent R. G., Brenneman M. A., Wilson J. H. Repair of site-specific double-strand breaks in a mammalian chromosome by homologous and illegitimate recombination. Mol Cell Biol. 1997 Jan;17(1):267–277. doi: 10.1128/mcb.17.1.267. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Sharma R. C., Schimke R. T. The propensity for gene amplification: a comparison of protocols, cell lines, and selection agents. Mutat Res. 1994 Jan 16;304(2):243–260. doi: 10.1016/0027-5107(94)90217-8. [DOI] [PubMed] [Google Scholar]
  41. Shulman M. J., Collins C., Connor A., Read L. R., Baker M. D. Interchromosomal recombination is suppressed in mammalian somatic cells. EMBO J. 1995 Aug 15;14(16):4102–4107. doi: 10.1002/j.1460-2075.1995.tb00082.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Skopek T. R., Liber H. L., Penman B. W., Thilly W. G. Isolation of a human lymphoblastoid line heterozygous at the thymidine kinase locus: possibility for a rapid human cell mutation assay. Biochem Biophys Res Commun. 1978 Sep 29;84(2):411–416. doi: 10.1016/0006-291x(78)90185-7. [DOI] [PubMed] [Google Scholar]
  43. Strathern J. N., Shafer B. K., McGill C. B. DNA synthesis errors associated with double-strand-break repair. Genetics. 1995 Jul;140(3):965–972. doi: 10.1093/genetics/140.3.965. [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. Szostak J. W., Orr-Weaver T. L., Rothstein R. J., Stahl F. W. The double-strand-break repair model for recombination. Cell. 1983 May;33(1):25–35. doi: 10.1016/0092-8674(83)90331-8. [DOI] [PubMed] [Google Scholar]
  45. Taghian D. G., Nickoloff J. A. Chromosomal double-strand breaks induce gene conversion at high frequency in mammalian cells. Mol Cell Biol. 1997 Nov;17(11):6386–6393. doi: 10.1128/mcb.17.11.6386. [DOI] [PMC free article] [PubMed] [Google Scholar]
  46. Thompson L. H., Schild D. The contribution of homologous recombination in preserving genome integrity in mammalian cells. Biochimie. 1999 Jan-Feb;81(1-2):87–105. doi: 10.1016/s0300-9084(99)80042-x. [DOI] [PubMed] [Google Scholar]
  47. Ward J. F. Radiation mutagenesis: the initial DNA lesions responsible. Radiat Res. 1995 Jun;142(3):362–368. [PubMed] [Google Scholar]
  48. Ward J. F. The yield of DNA double-strand breaks produced intracellularly by ionizing radiation: a review. Int J Radiat Biol. 1990 Jun;57(6):1141–1150. doi: 10.1080/09553009014551251. [DOI] [PubMed] [Google Scholar]
  49. Willis K. K., Klein H. L. Intrachromosomal recombination in Saccharomyces cerevisiae: reciprocal exchange in an inverted repeat and associated gene conversion. Genetics. 1987 Dec;117(4):633–643. doi: 10.1093/genetics/117.4.633. [DOI] [PMC free article] [PubMed] [Google Scholar]
  50. Xia F., Amundson S. A., Nickoloff J. A., Liber H. L. Different capacities for recombination in closely related human lymphoblastoid cell lines with different mutational responses to X-irradiation. Mol Cell Biol. 1994 Sep;14(9):5850–5857. doi: 10.1128/mcb.14.9.5850. [DOI] [PMC free article] [PubMed] [Google Scholar]
  51. Yang D., Waldman A. S. Fine-resolution analysis of products of intrachromosomal homeologous recombination in mammalian cells. Mol Cell Biol. 1997 Jul;17(7):3614–3628. doi: 10.1128/mcb.17.7.3614. [DOI] [PMC free article] [PubMed] [Google Scholar]
  52. te Riele H., Maandag E. R., Berns A. Highly efficient gene targeting in embryonic stem cells through homologous recombination with isogenic DNA constructs. Proc Natl Acad Sci U S A. 1992 Jun 1;89(11):5128–5132. doi: 10.1073/pnas.89.11.5128. [DOI] [PMC free article] [PubMed] [Google Scholar]

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