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. 2001 Apr;157(4):1481–1491. doi: 10.1093/genetics/157.4.1481

Efficient incorporation of large (>2 kb) heterologies into heteroduplex DNA: Pms1/Msh2-dependent and -independent large loop mismatch repair in Saccharomyces cerevisiae.

J A Clikeman 1, S L Wheeler 1, J A Nickoloff 1
PMCID: PMC1461601  PMID: 11290705

Abstract

DNA double-strand break (DSB) repair in yeast is effected primarily by gene conversion. Conversion can conceivably result from gap repair or from mismatch repair of heteroduplex DNA (hDNA) in recombination intermediates. Mismatch repair is normally very efficient, but unrepaired mismatches segregate in the next cell division, producing sectored colonies. Conversion of small heterologies (single-base differences or insertions <15 bp) in meiosis and mitosis involves mismatch repair of hDNA. The repair of larger loop mismatches in plasmid substrates or arising by replication slippage is inefficient and/or independent of Pms1p/Msh2p-dependent mismatch repair. However, large insertions convert readily (without sectoring) during meiotic recombination, raising the question of whether large insertions convert by repair of large loop mismatches or by gap repair. We show that insertions of 2.2 and 2.6 kbp convert efficiently during DSB-induced mitotic recombination, primarily by Msh2p- and Pms1p-dependent repair of large loop mismatches. These results support models in which Rad51p readily incorporates large heterologies into hDNA. We also show that large heterologies convert more frequently than small heterologies located the same distance from an initiating DSB and propose that this reflects Msh2-independent large loop-specific mismatch repair biased toward loop loss.

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Selected References

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

  1. Adams D. E., West S. C. Bypass of DNA heterologies during RuvAB-mediated three- and four-strand branch migration. J Mol Biol. 1996 Nov 8;263(4):582–596. doi: 10.1006/jmbi.1996.0600. [DOI] [PubMed] [Google Scholar]
  2. 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]
  3. Bishop D. K., Andersen J., Kolodner R. D. Specificity of mismatch repair following transformation of Saccharomyces cerevisiae with heteroduplex plasmid DNA. Proc Natl Acad Sci U S A. 1989 May;86(10):3713–3717. doi: 10.1073/pnas.86.10.3713. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. 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]
  5. Bishop D. K., Williamson M. S., Fogel S., Kolodner R. D. The role of heteroduplex correction in gene conversion in Saccharomyces cerevisiae. Nature. 1987 Jul 23;328(6128):362–364. doi: 10.1038/328362a0. [DOI] [PubMed] [Google Scholar]
  6. Carraway M., Marinus M. G. Repair of heteroduplex DNA molecules with multibase loops in Escherichia coli. J Bacteriol. 1993 Jul;175(13):3972–3980. doi: 10.1128/jb.175.13.3972-3980.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Chen J., Silver D. P., Walpita D., Cantor S. B., Gazdar A. F., Tomlinson G., Couch F. J., Weber B. L., Ashley T., Livingston D. M. Stable interaction between the products of the BRCA1 and BRCA2 tumor suppressor genes in mitotic and meiotic cells. Mol Cell. 1998 Sep;2(3):317–328. doi: 10.1016/s1097-2765(00)80276-2. [DOI] [PubMed] [Google Scholar]
  8. Chen W., Jinks-Robertson S. The role of the mismatch repair machinery in regulating mitotic and meiotic recombination between diverged sequences in yeast. Genetics. 1999 Apr;151(4):1299–1313. doi: 10.1093/genetics/151.4.1299. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Cho J. W., Khalsa G. J., Nickoloff J. A. Gene-conversion tract directionality is influenced by the chromosome environment. Curr Genet. 1998 Oct;34(4):269–279. doi: 10.1007/s002940050396. [DOI] [PubMed] [Google Scholar]
  10. Corrette-Bennett S. E., Parker B. O., Mohlman N. L., Lahue R. S. Correction of large mispaired DNA loops by extracts of Saccharomyces cerevisiae. J Biol Chem. 1999 Jun 18;274(25):17605–17611. doi: 10.1074/jbc.274.25.17605. [DOI] [PubMed] [Google Scholar]
  11. DasGupta C., Radding C. M. Polar branch migration promoted by recA protein: effect of mismatched base pairs. Proc Natl Acad Sci U S A. 1982 Feb;79(3):762–766. doi: 10.1073/pnas.79.3.762. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. 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]
  13. Gonda D. K., Radding C. M. By searching processively RecA protein pairs DNA molecules that share a limited stretch of homology. Cell. 1983 Sep;34(2):647–654. doi: 10.1016/0092-8674(83)90397-5. [DOI] [PubMed] [Google Scholar]
  14. Harfe B. D., Jinks-Robertson S. Removal of frameshift intermediates by mismatch repair proteins in Saccharomyces cerevisiae. Mol Cell Biol. 1999 Jul;19(7):4766–4773. doi: 10.1128/mcb.19.7.4766. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Harfe B. D., Minesinger B. K., Jinks-Robertson S. Discrete in vivo roles for the MutL homologs Mlh2p and Mlh3p in the removal of frameshift intermediates in budding yeast. Curr Biol. 2000 Feb 10;10(3):145–148. doi: 10.1016/s0960-9822(00)00314-6. [DOI] [PubMed] [Google Scholar]
  16. Iype L. E., Wood E. A., Inman R. B., Cox M. M. RuvA and RuvB proteins facilitate the bypass of heterologous DNA insertions during RecA protein-mediated DNA strand exchange. J Biol Chem. 1994 Oct 7;269(40):24967–24978. [PubMed] [Google Scholar]
  17. Karmazyn M., Horrobin D. F., Oka M., Manku M. S., Ally A. I., Karmali R. A., Morgan R. O., Cunnane S. C. Changes in coronary vascular resistance associated with prolonged hypoxia in isolated rat hearts: a possible role of prostaglandins. Life Sci. 1979 Dec 3;25(23):1991–1999. doi: 10.1016/0024-3205(79)90603-9. [DOI] [PubMed] [Google Scholar]
  18. Kim J. I., Cox M. M., Inman R. B. On the role of ATP hydrolysis in RecA protein-mediated DNA strand exchange. I. Bypassing a short heterologous insert in one DNA substrate. J Biol Chem. 1992 Aug 15;267(23):16438–16443. [PubMed] [Google Scholar]
  19. 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]
  20. Kramer B., Kramer W., Williamson M. S., Fogel S. Heteroduplex DNA correction in Saccharomyces cerevisiae is mismatch specific and requires functional PMS genes. Mol Cell Biol. 1989 Oct;9(10):4432–4440. doi: 10.1128/mcb.9.10.4432. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. 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]
  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. Littman S. J., Fang W. H., Modrich P. Repair of large insertion/deletion heterologies in human nuclear extracts is directed by a 5' single-strand break and is independent of the mismatch repair system. J Biol Chem. 1999 Mar 12;274(11):7474–7481. doi: 10.1074/jbc.274.11.7474. [DOI] [PubMed] [Google Scholar]
  24. Lühr B., Scheller J., Meyer P., Kramer W. Analysis of in vivo correction of defined mismatches in the DNA mismatch repair mutants msh2, msh3 and msh6 of Saccharomyces cerevisiae. Mol Gen Genet. 1998 Feb;257(3):362–367. doi: 10.1007/s004380050658. [DOI] [PubMed] [Google Scholar]
  25. 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]
  26. Myers T. A., Nickoloff J. A. Nonselective colony-color assays for HIS3, LEU2, LYS2, TRP1 and URA3 in ade2 yeast strains using media with limiting nutrients. Biotechniques. 1999 May;26(5):850–854. doi: 10.2144/99265bm10. [DOI] [PubMed] [Google Scholar]
  27. 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]
  28. 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]
  29. 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]
  30. Nickoloff J. A., Sweetser D. B., Clikeman J. A., Khalsa G. J., Wheeler S. L. Multiple heterologies increase mitotic double-strand break-induced allelic gene conversion tract lengths in yeast. Genetics. 1999 Oct;153(2):665–679. doi: 10.1093/genetics/153.2.665. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Nicolas A., Petes T. D. Polarity of meiotic gene conversion in fungi: contrasting views. Experientia. 1994 Mar 15;50(3):242–252. doi: 10.1007/BF01924007. [DOI] [PubMed] [Google Scholar]
  32. Ogawa T., Yu X., Shinohara A., Egelman E. H. Similarity of the yeast RAD51 filament to the bacterial RecA filament. Science. 1993 Mar 26;259(5103):1896–1899. doi: 10.1126/science.8456314. [DOI] [PubMed] [Google Scholar]
  33. Pâques F., Haber J. E. Multiple pathways of recombination induced by double-strand breaks in Saccharomyces cerevisiae. Microbiol Mol Biol Rev. 1999 Jun;63(2):349–404. doi: 10.1128/mmbr.63.2.349-404.1999. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Schultes N. P., Szostak J. W. Decreasing gradients of gene conversion on both sides of the initiation site for meiotic recombination at the ARG4 locus in yeast. Genetics. 1990 Dec;126(4):813–822. doi: 10.1093/genetics/126.4.813. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Sugawara N., Pâques F., Colaiácovo M., Haber J. E. Role of Saccharomyces cerevisiae Msh2 and Msh3 repair proteins in double-strand break-induced recombination. Proc Natl Acad Sci U S A. 1997 Aug 19;94(17):9214–9219. doi: 10.1073/pnas.94.17.9214. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Sung P., Stratton S. A. Yeast Rad51 recombinase mediates polar DNA strand exchange in the absence of ATP hydrolysis. J Biol Chem. 1996 Nov 8;271(45):27983–27986. doi: 10.1074/jbc.271.45.27983. [DOI] [PubMed] [Google Scholar]
  37. Sung P. Yeast Rad55 and Rad57 proteins form a heterodimer that functions with replication protein A to promote DNA strand exchange by Rad51 recombinase. Genes Dev. 1997 May 1;11(9):1111–1121. doi: 10.1101/gad.11.9.1111. [DOI] [PubMed] [Google Scholar]
  38. 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]
  39. 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]
  40. 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]
  41. Tran H. T., Gordenin D. A., Resnick M. A. The prevention of repeat-associated deletions in Saccharomyces cerevisiae by mismatch repair depends on size and origin of deletions. Genetics. 1996 Aug;143(4):1579–1587. doi: 10.1093/genetics/143.4.1579. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Vedel M., Nicolas A. CYS3, a hotspot of meiotic recombination in Saccharomyces cerevisiae. Effects of heterozygosity and mismatch repair functions on gene conversion and recombination intermediates. Genetics. 1999 Apr;151(4):1245–1259. doi: 10.1093/genetics/151.4.1245. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Wang Y., Cortez D., Yazdi P., Neff N., Elledge S. J., Qin J. BASC, a super complex of BRCA1-associated proteins involved in the recognition and repair of aberrant DNA structures. Genes Dev. 2000 Apr 15;14(8):927–939. [PMC free article] [PubMed] [Google Scholar]
  44. Watnick T. J., Gandolph M. A., Weber H., Neumann H. P., Germino G. G. Gene conversion is a likely cause of mutation in PKD1. Hum Mol Genet. 1998 Aug;7(8):1239–1243. doi: 10.1093/hmg/7.8.1239. [DOI] [PubMed] [Google Scholar]
  45. Weiss U., Wilson J. H. Repair of single-stranded loops in heteroduplex DNA transfected into mammalian cells. Proc Natl Acad Sci U S A. 1987 Mar;84(6):1619–1623. doi: 10.1073/pnas.84.6.1619. [DOI] [PMC free article] [PubMed] [Google Scholar]
  46. Weng Y. S., Nickoloff J. A. Evidence for independent mismatch repair processing on opposite sides of a double-strand break in Saccharomyces cerevisiae. Genetics. 1998 Jan;148(1):59–70. doi: 10.1093/genetics/148.1.59. [DOI] [PMC free article] [PubMed] [Google Scholar]
  47. 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]
  48. Weng Y. S., Xing D., Clikeman J. A., Nickoloff J. A. Transcriptional effects on double-strand break-induced gene conversion tracts. Mutat Res. 2000 Oct 16;461(2):119–132. doi: 10.1016/s0921-8777(00)00043-4. [DOI] [PubMed] [Google Scholar]
  49. Wong A. K., Pero R., Ormonde P. A., Tavtigian S. V., Bartel P. L. RAD51 interacts with the evolutionarily conserved BRC motifs in the human breast cancer susceptibility gene brca2. J Biol Chem. 1997 Dec 19;272(51):31941–31944. doi: 10.1074/jbc.272.51.31941. [DOI] [PubMed] [Google Scholar]

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