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. 1996 May;16(5):2164–2173. doi: 10.1128/mcb.16.5.2164

Cell cycle and genetic requirements of two pathways of nonhomologous end-joining repair of double-strand breaks in Saccharomyces cerevisiae.

J K Moore 1, J E Haber 1
PMCID: PMC231204  PMID: 8628283

Abstract

In Saccharomyces cerevisiae, an HO endonuclease-induced double-strand break can be repaired by at least two pathways of nonhomologous end joining (NHEJ) that closely resemble events in mammalian cells. In one pathway the chromosome ends are degraded to yield deletions with different sizes whose endpoints have 1 to 6 bp of homology. Alternatively, the 4-bp overhanging 3' ends of HO-cut DNA (5'-AACA-3') are not degraded but can be base paired in misalignment to produce +CA and +ACA insertions. When HO was expressed throughout the cell cycle, the efficiency of NHEJ repair was 30 times higher than when HO was expressed only in G1. The types of repair events were also very different when HO was expressed throughout the cell cycle; 78% of survivors had small insertions, while almost none had large deletions. When HO expression was confined to the G1 phase, only 21% were insertions and 38% had large deletions. These results suggest that there are distinct mechanisms of NHEJ repair producing either insertions or deletions and that these two pathways are differently affected by the time in the cell cycle when HO is expressed. The frequency of NHEJ is unaltered in strains from which RAD1, RAD2, RAD51, RAD52, RAD54, or RAD57 is deleted; however, deletions of RAD50, XRS2, or MRE11 reduced NHEJ by more than 70-fold when HO was not cell cycle regulated. Moreover, mutations in these three genes markedly reduced +CA insertions, while significantly increasing the proportion of both small (-ACA) and larger deletion events. In contrast, the rad5O mutation had little effect on the viability of G1-induced cells but significantly reduced the frequency of both +CA insertions and -ACA deletions in favor of larger deletions. Thus, RAD50 (and by extension XRS2 and MRE11) exerts a much more important role in the insertion-producing pathway of NHEJ repair found in S and/or G2 than in the less frequent deletion events that predominate when HO is expressed only in G1.

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Selected References

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  1. Ajimura M., Leem S. H., Ogawa H. Identification of new genes required for meiotic recombination in Saccharomyces cerevisiae. Genetics. 1993 Jan;133(1):51–66. doi: 10.1093/genetics/133.1.51. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Alani E., Cao L., Kleckner N. A method for gene disruption that allows repeated use of URA3 selection in the construction of multiply disrupted yeast strains. Genetics. 1987 Aug;116(4):541–545. doi: 10.1534/genetics.112.541.test. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Alani E., Subbiah S., Kleckner N. The yeast RAD50 gene encodes a predicted 153-kD protein containing a purine nucleotide-binding domain and two large heptad-repeat regions. Genetics. 1989 May;122(1):47–57. doi: 10.1093/genetics/122.1.47. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Bardwell A. J., Bardwell L., Tomkinson A. E., Friedberg E. C. Specific cleavage of model recombination and repair intermediates by the yeast Rad1-Rad10 DNA endonuclease. Science. 1994 Sep 30;265(5181):2082–2085. doi: 10.1126/science.8091230. [DOI] [PubMed] [Google Scholar]
  5. Basile G., Aker M., Mortimer R. K. Nucleotide sequence and transcriptional regulation of the yeast recombinational repair gene RAD51. Mol Cell Biol. 1992 Jul;12(7):3235–3246. doi: 10.1128/mcb.12.7.3235. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Boeke J. D., LaCroute F., Fink G. R. A positive selection for mutants lacking orotidine-5'-phosphate decarboxylase activity in yeast: 5-fluoro-orotic acid resistance. Mol Gen Genet. 1984;197(2):345–346. doi: 10.1007/BF00330984. [DOI] [PubMed] [Google Scholar]
  7. Conley E. C., Saunders V. A., Saunders J. R. Deletion and rearrangement of plasmid DNA during transformation of Escherichia coli with linear plasmid molecules. Nucleic Acids Res. 1986 Nov 25;14(22):8905–8917. doi: 10.1093/nar/14.22.8905. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Connolly B., White C. I., Haber J. E. Physical monitoring of mating type switching in Saccharomyces cerevisiae. Mol Cell Biol. 1988 Jun;8(6):2342–2349. doi: 10.1128/mcb.8.6.2342. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Fabre F., Boulet A., Roman H. Gene conversion at different points in the mitotic cycle of Saccharomyces cerevisiae. Mol Gen Genet. 1984;195(1-2):139–143. doi: 10.1007/BF00332736. [DOI] [PubMed] [Google Scholar]
  10. 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]
  11. Fishman-Lobell J., Rudin N., Haber J. E. Two alternative pathways of double-strand break repair that are kinetically separable and independently modulated. Mol Cell Biol. 1992 Mar;12(3):1292–1303. doi: 10.1128/mcb.12.3.1292. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Game J. C., Mortimer R. K. A genetic study of x-ray sensitive mutants in yeast. Mutat Res. 1974 Sep;24(3):281–292. doi: 10.1016/0027-5107(74)90176-6. [DOI] [PubMed] [Google Scholar]
  13. Goedecke W., Pfeiffer P., Vielmetter W. Nonhomologous DNA end joining in Schizosaccharomyces pombe efficiently eliminates DNA double-strand-breaks from haploid sequences. Nucleic Acids Res. 1994 Jun 11;22(11):2094–2101. doi: 10.1093/nar/22.11.2094. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Goedecke W., Vielmetter W., Pfeiffer P. Activation of a system for the joining of nonhomologous DNA ends during Xenopus egg maturation. Mol Cell Biol. 1992 Feb;12(2):811–816. doi: 10.1128/mcb.12.2.811. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Haber J. E. Mating-type gene switching in Saccharomyces cerevisiae. Trends Genet. 1992 Dec;8(12):446–452. doi: 10.1016/0168-9525(92)90329-3. [DOI] [PubMed] [Google Scholar]
  16. Habraken Y., Sung P., Prakash L., Prakash S. A conserved 5' to 3' exonuclease activity in the yeast and human nucleotide excision repair proteins RAD2 and XPG. J Biol Chem. 1994 Dec 16;269(50):31342–31345. [PubMed] [Google Scholar]
  17. Hartwell L. H., Kastan M. B. Cell cycle control and cancer. Science. 1994 Dec 16;266(5192):1821–1828. doi: 10.1126/science.7997877. [DOI] [PubMed] [Google Scholar]
  18. Herskowitz I. A regulatory hierarchy for cell specialization in yeast. Nature. 1989 Dec 14;342(6251):749–757. doi: 10.1038/342749a0. [DOI] [PubMed] [Google Scholar]
  19. Huxley C., Green E. D., Dunham I. Rapid assessment of S. cerevisiae mating type by PCR. Trends Genet. 1990 Aug;6(8):236–236. doi: 10.1016/0168-9525(90)90190-h. [DOI] [PubMed] [Google Scholar]
  20. Ivanov E. L., Haber J. E. RAD1 and RAD10, but not other excision repair genes, are required for double-strand break-induced recombination in Saccharomyces cerevisiae. Mol Cell Biol. 1995 Apr;15(4):2245–2251. doi: 10.1128/mcb.15.4.2245. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Ivanov E. L., Korolev V. G., Fabre F. XRS2, a DNA repair gene of Saccharomyces cerevisiae, is needed for meiotic recombination. Genetics. 1992 Nov;132(3):651–664. doi: 10.1093/genetics/132.3.651. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Ivanov E. L., Sugawara N., White C. I., Fabre F., Haber J. E. Mutations in XRS2 and RAD50 delay but do not prevent mating-type switching in Saccharomyces cerevisiae. Mol Cell Biol. 1994 May;14(5):3414–3425. doi: 10.1128/mcb.14.5.3414. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Jensen R. E., Herskowitz I. Directionality and regulation of cassette substitution in yeast. Cold Spring Harb Symp Quant Biol. 1984;49:97–104. doi: 10.1101/sqb.1984.049.01.013. [DOI] [PubMed] [Google Scholar]
  24. Jeong-Yu S. J., Carroll D. Test of the double-strand-break repair model of recombination in Xenopus laevis oocytes. Mol Cell Biol. 1992 Jan;12(1):112–119. doi: 10.1128/mcb.12.1.112. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Johzuka K., Ogawa H. Interaction of Mre11 and Rad50: two proteins required for DNA repair and meiosis-specific double-strand break formation in Saccharomyces cerevisiae. Genetics. 1995 Apr;139(4):1521–1532. doi: 10.1093/genetics/139.4.1521. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. 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]
  27. King J. S., Valcarcel E. R., Rufer J. T., Phillips J. W., Morgan W. F. Noncomplementary DNA double-strand-break rejoining in bacterial and human cells. Nucleic Acids Res. 1993 Mar 11;21(5):1055–1059. doi: 10.1093/nar/21.5.1055. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Klar A. J., Strathern J. N., Abraham J. A. Involvement of double-strand chromosomal breaks for mating-type switching in Saccharomyces cerevisiae. Cold Spring Harb Symp Quant Biol. 1984;49:77–88. doi: 10.1101/sqb.1984.049.01.011. [DOI] [PubMed] [Google Scholar]
  29. Kramer K. M., Brock J. A., Bloom K., Moore J. K., Haber J. E. Two different types of double-strand breaks in Saccharomyces cerevisiae are repaired by similar RAD52-independent, nonhomologous recombination events. Mol Cell Biol. 1994 Feb;14(2):1293–1301. doi: 10.1128/mcb.14.2.1293. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Lehman C. W., Clemens M., Worthylake D. K., Trautman J. K., Carroll D. Homologous and illegitimate recombination in developing Xenopus oocytes and eggs. Mol Cell Biol. 1993 Nov;13(11):6897–6906. doi: 10.1128/mcb.13.11.6897. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Lin F. L., Sperle K., Sternberg N. Model for homologous recombination during transfer of DNA into mouse L cells: role for DNA ends in the recombination process. Mol Cell Biol. 1984 Jun;4(6):1020–1034. doi: 10.1128/mcb.4.6.1020. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Maryon E., Carroll D. Characterization of recombination intermediates from DNA injected into Xenopus laevis oocytes: evidence for a nonconservative mechanism of homologous recombination. Mol Cell Biol. 1991 Jun;11(6):3278–3287. doi: 10.1128/mcb.11.6.3278. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Mezard C., Nicolas A. Homologous, homeologous, and illegitimate repair of double-strand breaks during transformation of a wild-type strain and a rad52 mutant strain of Saccharomyces cerevisiae. Mol Cell Biol. 1994 Feb;14(2):1278–1292. doi: 10.1128/mcb.14.2.1278. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Miyazaki W. Y., Orr-Weaver T. L. Sister-chromatid cohesion in mitosis and meiosis. Annu Rev Genet. 1994;28:167–187. doi: 10.1146/annurev.ge.28.120194.001123. [DOI] [PubMed] [Google Scholar]
  35. Mézard C., Pompon D., Nicolas A. Recombination between similar but not identical DNA sequences during yeast transformation occurs within short stretches of identity. Cell. 1992 Aug 21;70(4):659–670. doi: 10.1016/0092-8674(92)90434-e. [DOI] [PubMed] [Google Scholar]
  36. Nagley P., Farrell L. B., Gearing D. P., Nero D., Meltzer S., Devenish R. J. Assembly of functional proton-translocating ATPase complex in yeast mitochondria with cytoplasmically synthesized subunit 8, a polypeptide normally encoded within the organelle. Proc Natl Acad Sci U S A. 1988 Apr;85(7):2091–2095. doi: 10.1073/pnas.85.7.2091. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Nasmyth K. The determination of mother cell-specific mating type switching in yeast by a specific regulator of HO transcription. EMBO J. 1987 Jan;6(1):243–248. doi: 10.1002/j.1460-2075.1987.tb04745.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. 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]
  39. Nicolás A. L., Munz P. L., Young C. S. A modified single-strand annealing model best explains the joining of DNA double-strand breaks mammalian cells and cell extracts. Nucleic Acids Res. 1995 Mar 25;23(6):1036–1043. doi: 10.1093/nar/23.6.1036. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Ogawa H., Johzuka K., Nakagawa T., Leem S. H., Hagihara A. H. Functions of the yeast meiotic recombination genes, MRE11 and MRE2. Adv Biophys. 1995;31:67–76. doi: 10.1016/0065-227x(95)99383-z. [DOI] [PubMed] [Google Scholar]
  41. Pfeiffer P., Thode S., Hancke J., Vielmetter W. Mechanisms of overlap formation in nonhomologous DNA end joining. Mol Cell Biol. 1994 Feb;14(2):888–895. doi: 10.1128/mcb.14.2.888. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Rathmell W. K., Chu G. Involvement of the Ku autoantigen in the cellular response to DNA double-strand breaks. Proc Natl Acad Sci U S A. 1994 Aug 2;91(16):7623–7627. doi: 10.1073/pnas.91.16.7623. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Rattray A. J., Symington L. S. Multiple pathways for homologous recombination in Saccharomyces cerevisiae. Genetics. 1995 Jan;139(1):45–56. doi: 10.1093/genetics/139.1.45. [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. 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]
  45. Ray B. L., White C. I., Haber J. E. The TSM1 gene of Saccharomyces cerevisiae overlaps the MAT locus. Curr Genet. 1991 Jul;20(1-2):25–31. doi: 10.1007/BF00312761. [DOI] [PubMed] [Google Scholar]
  46. Roth D. B., Wilson J. H. Nonhomologous recombination in mammalian cells: role for short sequence homologies in the joining reaction. Mol Cell Biol. 1986 Dec;6(12):4295–4304. doi: 10.1128/mcb.6.12.4295. [DOI] [PMC free article] [PubMed] [Google Scholar]
  47. Rothstein R. J. One-step gene disruption in yeast. Methods Enzymol. 1983;101:202–211. doi: 10.1016/0076-6879(83)01015-0. [DOI] [PubMed] [Google Scholar]
  48. Rudin N., Haber J. E. Efficient repair of HO-induced chromosomal breaks in Saccharomyces cerevisiae by recombination between flanking homologous sequences. Mol Cell Biol. 1988 Sep;8(9):3918–3928. doi: 10.1128/mcb.8.9.3918. [DOI] [PMC free article] [PubMed] [Google Scholar]
  49. Scherer S., Davis R. W. Replacement of chromosome segments with altered DNA sequences constructed in vitro. Proc Natl Acad Sci U S A. 1979 Oct;76(10):4951–4955. doi: 10.1073/pnas.76.10.4951. [DOI] [PMC free article] [PubMed] [Google Scholar]
  50. Schiestl R. H., Gietz R. D. High efficiency transformation of intact yeast cells using single stranded nucleic acids as a carrier. Curr Genet. 1989 Dec;16(5-6):339–346. doi: 10.1007/BF00340712. [DOI] [PubMed] [Google Scholar]
  51. Schiestl R. H., Petes T. D. Integration of DNA fragments by illegitimate recombination in Saccharomyces cerevisiae. Proc Natl Acad Sci U S A. 1991 Sep 1;88(17):7585–7589. doi: 10.1073/pnas.88.17.7585. [DOI] [PMC free article] [PubMed] [Google Scholar]
  52. Schiestl R. H., Zhu J., Petes T. D. Effect of mutations in genes affecting homologous recombination on restriction enzyme-mediated and illegitimate recombination in Saccharomyces cerevisiae. Mol Cell Biol. 1994 Jul;14(7):4493–4500. doi: 10.1128/mcb.14.7.4493. [DOI] [PMC free article] [PubMed] [Google Scholar]
  53. Sugawara N., Haber J. E. Characterization of double-strand break-induced recombination: homology requirements and single-stranded DNA formation. Mol Cell Biol. 1992 Feb;12(2):563–575. doi: 10.1128/mcb.12.2.563. [DOI] [PMC free article] [PubMed] [Google Scholar]
  54. Sugawara N., Ivanov E. L., Fishman-Lobell J., Ray B. L., Wu X., Haber J. E. DNA structure-dependent requirements for yeast RAD genes in gene conversion. Nature. 1995 Jan 5;373(6509):84–86. doi: 10.1038/373084a0. [DOI] [PubMed] [Google Scholar]
  55. Taccioli G. E., Gottlieb T. M., Blunt T., Priestley A., Demengeot J., Mizuta R., Lehmann A. R., Alt F. W., Jackson S. P., Jeggo P. A. Ku80: product of the XRCC5 gene and its role in DNA repair and V(D)J recombination. Science. 1994 Sep 2;265(5177):1442–1445. doi: 10.1126/science.8073286. [DOI] [PubMed] [Google Scholar]
  56. Tatebayashi K., Kato J., Ikeda H. Structural analyses of DNA fragments integrated by illegitimate recombination in Schizosaccharomyces pombe. Mol Gen Genet. 1994 Jul 25;244(2):111–119. doi: 10.1007/BF00283511. [DOI] [PubMed] [Google Scholar]
  57. Weiffenbach B., Haber J. E. Homothallic mating type switching generates lethal chromosome breaks in rad52 strains of Saccharomyces cerevisiae. Mol Cell Biol. 1981 Jun;1(6):522–534. doi: 10.1128/mcb.1.6.522. [DOI] [PMC free article] [PubMed] [Google Scholar]
  58. Weiffenbach B., Rogers D. T., Haber J. E., Zoller M., Russell D. W., Smith M. Deletions and single base pair changes in the yeast mating type locus that prevent homothallic mating type conversions. Proc Natl Acad Sci U S A. 1983 Jun;80(11):3401–3405. doi: 10.1073/pnas.80.11.3401. [DOI] [PMC free article] [PubMed] [Google Scholar]
  59. 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]
  60. Wu X., Moore J. K., Haber J. E. Mechanism of MAT alpha donor preference during mating-type switching of Saccharomyces cerevisiae. Mol Cell Biol. 1996 Feb;16(2):657–668. doi: 10.1128/mcb.16.2.657. [DOI] [PMC free article] [PubMed] [Google Scholar]

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