Skip to main content
NCBI home page
Genetics logoLink to Genetics
. 1998 Jan;148(1):59–70. doi: 10.1093/genetics/148.1.59

Evidence for independent mismatch repair processing on opposite sides of a double-strand break in Saccharomyces cerevisiae.

Y S Weng 1, J A Nickoloff 1
PMCID: PMC1459773  PMID: 9475721

Abstract

Double-strand break (DSB) induced gene conversion in Saccharomyces cerevisiae during meiosis and MAT switching is mediated primarily by mismatch repair of heteroduplex DNA (hDNA). We used nontandem ura3 duplications containing palindromic frameshift insertion mutations near an HO nuclease recognition site to test whether mismatch repair also mediates DSB-induced mitotic gene conversion at a non-MAT locus. Palindromic insertions included in hDNA are expected to produce a stem-loop mismatch, escape repair, and segregate to produce a sectored (Ura+/-) colony. If conversion occurs by gap repair, the insertion should be removed on both strands, and converted colonies will not be sectored. For both a 14-bp palindrome, and a 37-bp near-palindrome, approximately 75% of recombinant colonies were sectored, indicating that most DSB-induced mitotic gene conversion involves mismatch repair of hDNA. We also investigated mismatch repair of well-repaired markers flanking an unrepaired palindrome. As seen in previous studies, these additional markers increased loop repair (likely reflecting corepair). Among sectored products, few had additional segregating markers, indicating that the lack of repair at one marker is not associated with inefficient repair at nearby markers. Clear evidence was obtained for low levels of short tract mismatch repair. As seen with full gene conversions, donor alleles in sectored products were not altered. Markers on the same side of the DSB as the palindrome were involved in hDNA less often among sectored products than nonsectored products, but markers on the opposite side of the DSB showed similar hDNA involvement among both product classes. These results can be explained in terms of corepair, and they suggest that mismatch repair on opposite sides of a DSB involves distinct repair tracts.

Full Text

The Full Text of this article is available as a PDF (140.1 KB).

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Aguilera A., Klein H. L. Yeast intrachromosomal recombination: long gene conversion tracts are preferentially associated with reciprocal exchange and require the RAD1 and RAD3 gene products. Genetics. 1989 Dec;123(4):683–694. doi: 10.1093/genetics/123.4.683. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Ahn B. Y., Livingston D. M. Mitotic gene conversion lengths, coconversion patterns, and the incidence of reciprocal recombination in a Saccharomyces cerevisiae plasmid system. Mol Cell Biol. 1986 Nov;6(11):3685–3693. doi: 10.1128/mcb.6.11.3685. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Alani E., Chi N. W., Kolodner R. The Saccharomyces cerevisiae Msh2 protein specifically binds to duplex oligonucleotides containing mismatched DNA base pairs and insertions. Genes Dev. 1995 Jan 15;9(2):234–247. doi: 10.1101/gad.9.2.234. [DOI] [PubMed] [Google Scholar]
  4. Alani E. The Saccharomyces cerevisiae Msh2 and Msh6 proteins form a complex that specifically binds to duplex oligonucleotides containing mismatched DNA base pairs. Mol Cell Biol. 1996 Oct;16(10):5604–5615. doi: 10.1128/mcb.16.10.5604. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Belmaaza A., Chartrand P. One-sided invasion events in homologous recombination at double-strand breaks. Mutat Res. 1994 May;314(3):199–208. doi: 10.1016/0921-8777(94)90065-5. [DOI] [PubMed] [Google Scholar]
  6. Bianchi M. E., Radding C. M. Insertions, deletions and mismatches in heteroduplex DNA made by recA protein. Cell. 1983 Dec;35(2 Pt 1):511–520. doi: 10.1016/0092-8674(83)90185-x. [DOI] [PubMed] [Google Scholar]
  7. Borts R. H., Haber J. E. Length and distribution of meiotic gene conversion tracts and crossovers in Saccharomyces cerevisiae. Genetics. 1989 Sep;123(1):69–80. doi: 10.1093/genetics/123.1.69. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. 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]
  9. Cao L., Alani E., Kleckner N. A pathway for generation and processing of double-strand breaks during meiotic recombination in S. cerevisiae. Cell. 1990 Jun 15;61(6):1089–1101. doi: 10.1016/0092-8674(90)90072-m. [DOI] [PubMed] [Google Scholar]
  10. Detloff P., Petes T. D. Measurements of excision repair tracts formed during meiotic recombination in Saccharomyces cerevisiae. Mol Cell Biol. 1992 Apr;12(4):1805–1814. doi: 10.1128/mcb.12.4.1805. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Detloff P., White M. A., Petes T. D. Analysis of a gene conversion gradient at the HIS4 locus in Saccharomyces cerevisiae. Genetics. 1992 Sep;132(1):113–123. doi: 10.1093/genetics/132.1.113. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Esposito M. S. Evidence that spontaneous mitotic recombination occurs at the two-strand stage. Proc Natl Acad Sci U S A. 1978 Sep;75(9):4436–4440. doi: 10.1073/pnas.75.9.4436. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Esposito M. S., Ramirez R. M., Bruschi C. V. Recombinators, recombinases and recombination genes of yeasts. Curr Genet. 1994 Jan;25(1):1–11. doi: 10.1007/BF00712959. [DOI] [PubMed] [Google Scholar]
  14. Fishman-Lobell J., Haber J. E. Removal of nonhomologous DNA ends in double-strand break recombination: the role of the yeast ultraviolet repair gene RAD1. Science. 1992 Oct 16;258(5081):480–484. doi: 10.1126/science.1411547. [DOI] [PubMed] [Google Scholar]
  15. Gilbertson L. A., Stahl F. W. A test of the double-strand break repair model for meiotic recombination in Saccharomyces cerevisiae. Genetics. 1996 Sep;144(1):27–41. doi: 10.1093/genetics/144.1.27. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Gunn L., Whelden J., Nickoloff J. A. Transfer of episomal and integrated plasmids from Saccharomyces cerevisiae to Escherichia coli by electroporation. Methods Mol Biol. 1995;47:55–66. doi: 10.1385/0-89603-310-4:55. [DOI] [PubMed] [Google Scholar]
  17. Haber J. E., Ray B. L., Kolb J. M., White C. I. Rapid kinetics of mismatch repair of heteroduplex DNA that is formed during recombination in yeast. Proc Natl Acad Sci U S A. 1993 Apr 15;90(8):3363–3367. doi: 10.1073/pnas.90.8.3363. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Holbeck S. L., Smith G. R. Chi enhances heteroduplex DNA levels during recombination. Genetics. 1992 Dec;132(4):879–891. doi: 10.1093/genetics/132.4.879. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Judd S. R., Petes T. D. Physical lengths of meiotic and mitotic gene conversion tracts in Saccharomyces cerevisiae. Genetics. 1988 Mar;118(3):401–410. doi: 10.1093/genetics/118.3.401. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Kadyk L. C., Hartwell L. H. Sister chromatids are preferred over homologs as substrates for recombinational repair in Saccharomyces cerevisiae. Genetics. 1992 Oct;132(2):387–402. doi: 10.1093/genetics/132.2.387. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Kirkpatrick D. T., Petes T. D. Repair of DNA loops involves DNA-mismatch and nucleotide-excision repair proteins. Nature. 1997 Jun 26;387(6636):929–931. doi: 10.1038/43225. [DOI] [PubMed] [Google Scholar]
  22. Lichten M., Fox M. S. Evidence for inclusion of regions of nonhomology in heteroduplex products of bacteriophage lambda recombination. Proc Natl Acad Sci U S A. 1984 Nov;81(22):7180–7184. doi: 10.1073/pnas.81.22.7180. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Lichten M., Goyon C., Schultes N. P., Treco D., Szostak J. W., Haber J. E., Nicolas A. Detection of heteroduplex DNA molecules among the products of Saccharomyces cerevisiae meiosis. Proc Natl Acad Sci U S A. 1990 Oct;87(19):7653–7657. doi: 10.1073/pnas.87.19.7653. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Lieb M. Spontaneous mutation at a 5-methylcytosine hotspot is prevented by very short patch (VSP) mismatch repair. Genetics. 1991 May;128(1):23–27. doi: 10.1093/genetics/128.1.23. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Lovett S. T., Drapkin P. T., Sutera V. A., Jr, Gluckman-Peskind T. J. A sister-strand exchange mechanism for recA-independent deletion of repeated DNA sequences in Escherichia coli. Genetics. 1993 Nov;135(3):631–642. doi: 10.1093/genetics/135.3.631. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Manivasakam P., Rosenberg S. M., Hastings P. J. Poorly repaired mismatches in heteroduplex DNA are hyper-recombinagenic in Saccharomyces cerevisiae. Genetics. 1996 Feb;142(2):407–416. doi: 10.1093/genetics/142.2.407. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. McGill C., Shafer B., Strathern J. Coconversion of flanking sequences with homothallic switching. Cell. 1989 May 5;57(3):459–467. doi: 10.1016/0092-8674(89)90921-5. [DOI] [PubMed] [Google Scholar]
  28. 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]
  29. 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]
  30. Moore C. W., Hampsey D. M., Ernst J. F., Sherman F. Differential mismatch repair can explain the disproportionalities between physical distances and recombination frequencies of cyc1 mutations in yeast. Genetics. 1988 May;119(1):21–34. doi: 10.1093/genetics/119.1.21. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Nag D. K., Petes T. D. Meiotic recombination between dispersed repeated genes is associated with heteroduplex formation. Mol Cell Biol. 1990 Aug;10(8):4420–4423. doi: 10.1128/mcb.10.8.4420. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Nag D. K., Petes T. D. Physical detection of heteroduplexes during meiotic recombination in the yeast Saccharomyces cerevisiae. Mol Cell Biol. 1993 Apr;13(4):2324–2331. doi: 10.1128/mcb.13.4.2324. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Nag D. K., Petes T. D. Seven-base-pair inverted repeats in DNA form stable hairpins in vivo in Saccharomyces cerevisiae. Genetics. 1991 Nov;129(3):669–673. doi: 10.1093/genetics/129.3.669. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Nag D. K., White M. A., Petes T. D. Palindromic sequences in heteroduplex DNA inhibit mismatch repair in yeast. Nature. 1989 Jul 27;340(6231):318–320. doi: 10.1038/340318a0. [DOI] [PubMed] [Google Scholar]
  35. Nassif N., Penney J., Pal S., Engels W. R., Gloor G. B. Efficient copying of nonhomologous sequences from ectopic sites via P-element-induced gap repair. Mol Cell Biol. 1994 Mar;14(3):1613–1625. doi: 10.1128/mcb.14.3.1613. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Neddermann P., Jiricny J. Efficient removal of uracil from G.U mispairs by the mismatch-specific thymine DNA glycosylase from HeLa cells. Proc Natl Acad Sci U S A. 1994 Mar 1;91(5):1642–1646. doi: 10.1073/pnas.91.5.1642. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Negritto M. T., Wu X., Kuo T., Chu S., Bailis A. M. Influence of DNA sequence identity on efficiency of targeted gene replacement. Mol Cell Biol. 1997 Jan;17(1):278–286. doi: 10.1128/mcb.17.1.278. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Nelson H. H., Sweetser D. B., Nickoloff J. A. Effects of terminal nonhomology and homeology on double-strand-break-induced gene conversion tract directionality. Mol Cell Biol. 1996 Jun;16(6):2951–2957. doi: 10.1128/mcb.16.6.2951. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Nickoloff J. A., Chen E. Y., Heffron F. A 24-base-pair DNA sequence from the MAT locus stimulates intergenic recombination in yeast. Proc Natl Acad Sci U S A. 1986 Oct;83(20):7831–7835. doi: 10.1073/pnas.83.20.7831. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Nickoloff J. A., Reynolds R. J. Subcloning with new ampicillin- and kanamycin-resistant analogs of pUC19. Biotechniques. 1991 Apr;10(4):469-70, 472. [PubMed] [Google Scholar]
  41. Nickoloff J. A., Singer J. D., Heffron F. In vivo analysis of the Saccharomyces cerevisiae HO nuclease recognition site by site-directed mutagenesis. Mol Cell Biol. 1990 Mar;10(3):1174–1179. doi: 10.1128/mcb.10.3.1174. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Nickoloff J. A., Singer J. D., Hoekstra M. F., Heffron F. Double-strand breaks stimulate alternative mechanisms of recombination repair. J Mol Biol. 1989 Jun 5;207(3):527–541. doi: 10.1016/0022-2836(89)90462-2. [DOI] [PubMed] [Google Scholar]
  43. Orr-Weaver T. L., Szostak J. W., Rothstein R. J. Yeast transformation: a model system for the study of recombination. Proc Natl Acad Sci U S A. 1981 Oct;78(10):6354–6358. doi: 10.1073/pnas.78.10.6354. [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. Priebe S. D., Westmoreland J., Nilsson-Tillgren T., Resnick M. A. Induction of recombination between homologous and diverged DNAs by double-strand gaps and breaks and role of mismatch repair. Mol Cell Biol. 1994 Jul;14(7):4802–4814. doi: 10.1128/mcb.14.7.4802. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. Ray A., Siddiqi I., Kolodkin A. L., Stahl F. W. Intra-chromosomal gene conversion induced by a DNA double-strand break in Saccharomyces cerevisiae. J Mol Biol. 1988 May 20;201(2):247–260. doi: 10.1016/0022-2836(88)90136-2. [DOI] [PubMed] [Google Scholar]
  46. Ray B. L., White C. I., Haber J. E. Heteroduplex formation and mismatch repair of the "stuck" mutation during mating-type switching in Saccharomyces cerevisiae. Mol Cell Biol. 1991 Oct;11(10):5372–5380. doi: 10.1128/mcb.11.10.5372. [DOI] [PMC free article] [PubMed] [Google Scholar]
  47. 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]
  48. Ronne H., Rothstein R. Mitotic sectored colonies: evidence of heteroduplex DNA formation during direct repeat recombination. Proc Natl Acad Sci U S A. 1988 Apr;85(8):2696–2700. doi: 10.1073/pnas.85.8.2696. [DOI] [PMC free article] [PubMed] [Google Scholar]
  49. Schär P., Kohli J. Marker effects of G to C transversions on intragenic recombination and mismatch repair in Schizosaccharomyces pombe. Genetics. 1993 Apr;133(4):825–835. doi: 10.1093/genetics/133.4.825. [DOI] [PMC free article] [PubMed] [Google Scholar]
  50. Sun H., Treco D., Schultes N. P., Szostak J. W. Double-strand breaks at an initiation site for meiotic gene conversion. Nature. 1989 Mar 2;338(6210):87–90. doi: 10.1038/338087a0. [DOI] [PubMed] [Google Scholar]
  51. Sun H., Treco D., Szostak J. W. Extensive 3'-overhanging, single-stranded DNA associated with the meiosis-specific double-strand breaks at the ARG4 recombination initiation site. Cell. 1991 Mar 22;64(6):1155–1161. doi: 10.1016/0092-8674(91)90270-9. [DOI] [PubMed] [Google Scholar]
  52. 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]
  53. Symington L. S., Petes T. D. Expansions and contractions of the genetic map relative to the physical map of yeast chromosome III. Mol Cell Biol. 1988 Feb;8(2):595–604. doi: 10.1128/mcb.8.2.595. [DOI] [PMC free article] [PubMed] [Google Scholar]
  54. 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]
  55. Weng Y. S., Nickoloff J. A. Nonselective URA3 colony-color assay in yeast ade1 or ade2 mutants. Biotechniques. 1997 Aug;23(2):237–241. doi: 10.2144/97232bm13. [DOI] [PubMed] [Google Scholar]
  56. Weng Y. S., Whelden J., Gunn L., Nickoloff J. A. Double-strand break-induced mitotic gene conversion: examination of tract polarity and products of multiple recombinational repair events. Curr Genet. 1996 Mar;29(4):335–343. doi: 10.1007/BF02208614. [DOI] [PubMed] [Google Scholar]
  57. White C. I., Haber J. E. Intermediates of recombination during mating type switching in Saccharomyces cerevisiae. EMBO J. 1990 Mar;9(3):663–673. doi: 10.1002/j.1460-2075.1990.tb08158.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  58. 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]
  59. Worth L., Jr, Clark S., Radman M., Modrich P. Mismatch repair proteins MutS and MutL inhibit RecA-catalyzed strand transfer between diverged DNAs. Proc Natl Acad Sci U S A. 1994 Apr 12;91(8):3238–3241. doi: 10.1073/pnas.91.8.3238. [DOI] [PMC free article] [PubMed] [Google Scholar]
  60. Wu T. C., Lichten M. Meiosis-induced double-strand break sites determined by yeast chromatin structure. Science. 1994 Jan 28;263(5146):515–518. doi: 10.1126/science.8290959. [DOI] [PubMed] [Google Scholar]
  61. de Massy B., Rocco V., Nicolas A. The nucleotide mapping of DNA double-strand breaks at the CYS3 initiation site of meiotic recombination in Saccharomyces cerevisiae. EMBO J. 1995 Sep 15;14(18):4589–4598. doi: 10.1002/j.1460-2075.1995.tb00138.x. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from Genetics are provided here courtesy of Oxford University Press

RESOURCES