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. 1997 Oct;147(2):743–753. doi: 10.1093/genetics/147.2.743

Mismatch Repair by Efficient Nick-Directed, and Less Efficient Mismatch-Specific, Mechanisms in Homologous Recombination Intermediates in Chinese Hamster Ovary Cells

E M Miller 1, H L Hough 1, J W Cho 1, J A Nickoloff 1
PMCID: PMC1208194  PMID: 9335609

Abstract

Repair of single-base mismatches formed in recombination intermediates in vivo was investigated in Chinese hamster ovary cells. Extrachromosomal recombination was stimulated by double-strand breaks (DSBs) introduced into regions of shared homology in pairs of plasmid substrates heteroallelic at 11 phenotypically silent mutations. Recombination was expected to occur primarily by single-strand annealing, yielding predicted heteroduplex DNA (hDNA) regions with three to nine mismatches. Product spectra were consistent with hDNA only occurring between DSBs. Nicks were predicted on opposite strands flanking hDNA at positions corresponding to original DSB sites. Most products had continuous marker patterns, and observed conversion gradients closely matched predicted gradients for repair initiated at nicks, consistent with an efficient nick-directed, excision-based mismatch repair system. Discontinuous patterns, seen in ~10% of products, and deviations from predicted gradients provided evidence for less efficient mismatch-specific repair, including G-A -> G-C specific repair that may reflect processing by a homologue of Escherichia coli MutY. Mismatch repair was >80% efficient, which is higher than seen previously with covalently closed, artificial hDNA substrates. Products were found in which all mismatches were repaired in a single tract initiated from one or the other nick. We also observed products resulting from two tracts of intermediate length initiated from two nicks.

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Selected References

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  1. Aaltonen L. A., Peltomäki P., Leach F. S., Sistonen P., Pylkkänen L., Mecklin J. P., Järvinen H., Powell S. M., Jen J., Hamilton S. R. Clues to the pathogenesis of familial colorectal cancer. Science. 1993 May 7;260(5109):812–816. doi: 10.1126/science.8484121. [DOI] [PubMed] [Google Scholar]
  2. Bishop D. K., Kolodner R. D. Repair of heteroduplex plasmid DNA after transformation into Saccharomyces cerevisiae. Mol Cell Biol. 1986 Oct;6(10):3401–3409. doi: 10.1128/mcb.6.10.3401. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Borts R. H., Haber J. E. Meiotic recombination in yeast: alteration by multiple heterozygosities. Science. 1987 Sep 18;237(4821):1459–1465. doi: 10.1126/science.2820060. [DOI] [PubMed] [Google Scholar]
  4. Boyer J. C., Umar A., Risinger J. I., Lipford J. R., Kane M., Yin S., Barrett J. C., Kolodner R. D., Kunkel T. A. Microsatellite instability, mismatch repair deficiency, and genetic defects in human cancer cell lines. Cancer Res. 1995 Dec 15;55(24):6063–6070. [PubMed] [Google Scholar]
  5. Brown T. C., Jiricny J. Different base/base mispairs are corrected with different efficiencies and specificities in monkey kidney cells. Cell. 1988 Aug 26;54(5):705–711. doi: 10.1016/s0092-8674(88)80015-1. [DOI] [PubMed] [Google Scholar]
  6. Brown T. C., Jiricny J. Repair of base-base mismatches in simian and human cells. Genome. 1989;31(2):578–583. doi: 10.1139/g89-107. [DOI] [PubMed] [Google Scholar]
  7. Carroll D., Lehman C. W., Jeong-Yu S., Dohrmann P., Dawson R. J., Trautman J. K. Distribution of exchanges upon homologous recombination of exogenous DNA in Xenopus laevis oocytes. Genetics. 1994 Oct;138(2):445–457. doi: 10.1093/genetics/138.2.445. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Conley E. C., Saunders J. R. Recombination-dependent recircularization of linearized pBR322 plasmid DNA following transformation of Escherichia coli. Mol Gen Genet. 1984;194(1-2):211–218. doi: 10.1007/BF00383519. [DOI] [PubMed] [Google Scholar]
  9. Deng W. P., Nickoloff J. A. Mismatch repair of heteroduplex DNA intermediates of extrachromosomal recombination in mammalian cells. Mol Cell Biol. 1994 Jan;14(1):400–406. doi: 10.1128/mcb.14.1.400. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Deng W. P., Nickoloff J. A. Site-directed mutagenesis of virtually any plasmid by eliminating a unique site. Anal Biochem. 1992 Jan;200(1):81–88. doi: 10.1016/0003-2697(92)90280-k. [DOI] [PubMed] [Google Scholar]
  11. Desautels L., Brouillette S., Wallenburg J., Belmaaza A., Gusew N., Trudel P., Chartrand P. Characterization of nonconservative homologous junctions in mammalian cells. Mol Cell Biol. 1990 Dec;10(12):6613–6618. doi: 10.1128/mcb.10.12.6613. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Fang W. H., Modrich P. Human strand-specific mismatch repair occurs by a bidirectional mechanism similar to that of the bacterial reaction. J Biol Chem. 1993 Jun 5;268(16):11838–11844. [PubMed] [Google Scholar]
  13. Grilley M., Holmes J., Yashar B., Modrich P. Mechanisms of DNA-mismatch correction. Mutat Res. 1990 Sep-Nov;236(2-3):253–267. doi: 10.1016/0921-8777(90)90009-t. [DOI] [PubMed] [Google Scholar]
  14. Grimberg J., Nawoschik S., Belluscio L., McKee R., Turck A., Eisenberg A. A simple and efficient non-organic procedure for the isolation of genomic DNA from blood. Nucleic Acids Res. 1989 Oct 25;17(20):8390–8390. doi: 10.1093/nar/17.20.8390. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Hare J. T., Taylor J. H. One role for DNA methylation in vertebrate cells is strand discrimination in mismatch repair. Proc Natl Acad Sci U S A. 1985 Nov;82(21):7350–7354. doi: 10.1073/pnas.82.21.7350. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Heywood L. A., Burke J. F. Repair of single nucleotide DNA mismatches transfected into mammalian cells can occur by short-patch excision. Mutat Res. 1990 Jul;236(1):59–66. doi: 10.1016/0921-8777(90)90033-2. [DOI] [PubMed] [Google Scholar]
  17. Holmes J., Jr, Clark S., Modrich P. Strand-specific mismatch correction in nuclear extracts of human and Drosophila melanogaster cell lines. Proc Natl Acad Sci U S A. 1990 Aug;87(15):5837–5841. doi: 10.1073/pnas.87.15.5837. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Kolodner R. D. Mismatch repair: mechanisms and relationship to cancer susceptibility. Trends Biochem Sci. 1995 Oct;20(10):397–401. doi: 10.1016/s0968-0004(00)89087-8. [DOI] [PubMed] [Google Scholar]
  19. Kunkel T. A. Nucleotide repeats. Slippery DNA and diseases. Nature. 1993 Sep 16;365(6443):207–208. doi: 10.1038/365207a0. [DOI] [PubMed] [Google Scholar]
  20. Lahue R. S., Au K. G., Modrich P. DNA mismatch correction in a defined system. Science. 1989 Jul 14;245(4914):160–164. doi: 10.1126/science.2665076. [DOI] [PubMed] [Google Scholar]
  21. Lehman C. W., Jeong-Yu S., Trautman J. K., Carroll D. Repair of heteroduplex DNA in Xenopus laevis oocytes. Genetics. 1994 Oct;138(2):459–470. doi: 10.1093/genetics/138.2.459. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Lin F. L., Sperle K., Sternberg N. Repair of double-stranded DNA breaks by homologous DNA fragments during transfer of DNA into mouse L cells. Mol Cell Biol. 1990 Jan;10(1):113–119. doi: 10.1128/mcb.10.1.113. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Miller E. M., Nickoloff J. A. Escherichia coli electrotransformation. Methods Mol Biol. 1995;47:105–113. doi: 10.1385/0-89603-310-4:105. [DOI] [PubMed] [Google Scholar]
  24. Modrich P. Mechanisms and biological effects of mismatch repair. Annu Rev Genet. 1991;25:229–253. doi: 10.1146/annurev.ge.25.120191.001305. [DOI] [PubMed] [Google Scholar]
  25. Neddermann P., Jiricny J. The purification of a mismatch-specific thymine-DNA glycosylase from HeLa cells. J Biol Chem. 1993 Oct 5;268(28):21218–21224. [PubMed] [Google Scholar]
  26. Nickoloff J. A. Sepharose spin column chromatography. A fast, nontoxic replacement for phenol:chloroform extraction/ethanol precipitation. Mol Biotechnol. 1994 Feb;1(1):105–108. doi: 10.1007/BF02821513. [DOI] [PubMed] [Google Scholar]
  27. Ray F. A., Miller E. M., Nickoloff J. A. Efficient marker rescue and domain replacement without fragment subcloning. Anal Biochem. 1995 Jan 1;224(1):440–443. doi: 10.1006/abio.1995.1066. [DOI] [PubMed] [Google Scholar]
  28. Southern P. J., Berg P. Transformation of mammalian cells to antibiotic resistance with a bacterial gene under control of the SV40 early region promoter. J Mol Appl Genet. 1982;1(4):327–341. [PubMed] [Google Scholar]
  29. Steeg C. M., Ellis J., Bernstein A. Introduction of specific point mutations into RNA polymerase II by gene targeting in mouse embryonic stem cells: evidence for a DNA mismatch repair mechanism. Proc Natl Acad Sci U S A. 1990 Jun;87(12):4680–4684. doi: 10.1073/pnas.87.12.4680. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Sweetser D. B., Hough H., Whelden J. F., Arbuckle M., Nickoloff J. A. Fine-resolution mapping of spontaneous and double-strand break-induced gene conversion tracts in Saccharomyces cerevisiae reveals reversible mitotic conversion polarity. Mol Cell Biol. 1994 Jun;14(6):3863–3875. doi: 10.1128/mcb.14.6.3863. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Umar A., Boyer J. C., Kunkel T. A. DNA loop repair by human cell extracts. Science. 1994 Nov 4;266(5186):814–816. doi: 10.1126/science.7973637. [DOI] [PubMed] [Google Scholar]
  32. Varlet I., Radman M., Brooks P. DNA mismatch repair in Xenopus egg extracts: repair efficiency and DNA repair synthesis for all single base-pair mismatches. Proc Natl Acad Sci U S A. 1990 Oct;87(20):7883–7887. doi: 10.1073/pnas.87.20.7883. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Wang Z., Rossman T. G. Large-scale supercoiled plasmid preparation by acidic phenol extraction. Biotechniques. 1994 Mar;16(3):460–463. [PubMed] [Google Scholar]
  34. Wiebauer K., Jiricny J. In vitro correction of G.T mispairs to G.C pairs in nuclear extracts from human cells. Nature. 1989 May 18;339(6221):234–236. doi: 10.1038/339234a0. [DOI] [PubMed] [Google Scholar]
  35. Yang D., Waldman A. S. An examination of the effects of double-strand breaks on extrachromosomal recombination in mammalian cells. Genetics. 1992 Dec;132(4):1081–1093. doi: 10.1093/genetics/132.4.1081. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Yeh Y. C., Chang D. Y., Masin J., Lu A. L. Two nicking enzyme systems specific for mismatch-containing DNA in nuclear extracts from human cells. J Biol Chem. 1991 Apr 5;266(10):6480–6484. [PubMed] [Google Scholar]

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