Skip to main content
PMC home page
Nucleic Acids Research logoLink to Nucleic Acids Research
. 1994 Jun 11;22(11):2094–2101. doi: 10.1093/nar/22.11.2094

Nonhomologous DNA end joining in Schizosaccharomyces pombe efficiently eliminates DNA double-strand-breaks from haploid sequences.

W Goedecke 1, P Pfeiffer 1, W Vielmetter 1
PMCID: PMC308126  PMID: 8029017

Abstract

Cells of higher eucaryotes are known to possess mechanisms of illegitimate recombination which promote the joining between nonhomologous ends of broken DNA and thus may serve as basic tools of double-strand-break (DSB) repair. Here we show that cells of the fission yeast Schizosaccharomyces pombe also contain activities of nonhomologous DNA end joining resembling the ones found in higher eucaryotes. Nonhomologous end joining activities were detected by transformation of linearized self-replicating plasmids in yeast cells employing a selection procedure which only propagates transformants carrying recircularized plasmid molecules. Linear plasmid substrates were generated by duplicate restriction cuts carrying either blunt ends or 3' or 5' protruding single strands (PSS) of 4 nt which were efficiently joined in any tested combination. Sequence analysis of joined products revealed that junctional sequences were shortened by 1 to 14 nt. Two mechanisms may account for junction formation (i) loss of terminal nucleotides from PSS tails to produce blunt ends which can be joined to abutting ends and (ii) interactions of DNA termini at patches of sequence homologies (1-4 bp) by formation of overlap intermediates which are subsequently processed. A general feature of the yeast joining system is that end joining can only be detected in the absence of sequence homology between the linear substrate and host genome. In the presence of homology, nonhomologous DNA end joining is efficiently competed by activities of homologous recombination.

Full text

PDF
2094

Images in this article

2099Image
on p.2099

Selected References

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

  1. Brinster R. L., Chen H. Y., Trumbauer M. E., Yagle M. K., Palmiter R. D. Factors affecting the efficiency of introducing foreign DNA into mice by microinjecting eggs. Proc Natl Acad Sci U S A. 1985 Jul;82(13):4438–4442. doi: 10.1073/pnas.82.13.4438. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Clark J. M. Novel non-templated nucleotide addition reactions catalyzed by procaryotic and eucaryotic DNA polymerases. Nucleic Acids Res. 1988 Oct 25;16(20):9677–9686. doi: 10.1093/nar/16.20.9677. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. 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]
  4. Folger K. R., Wong E. A., Wahl G., Capecchi M. R. Patterns of integration of DNA microinjected into cultured mammalian cells: evidence for homologous recombination between injected plasmid DNA molecules. Mol Cell Biol. 1982 Nov;2(11):1372–1387. doi: 10.1128/mcb.2.11.1372. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Gahlmann R., Doerfler W. Integration of viral DNA into the genome of the adenovirus type 2-transformed hamster cell line HE5 without loss or alteration of cellular nucleotides. Nucleic Acids Res. 1983 Nov 11;11(21):7347–7361. doi: 10.1093/nar/11.21.7347. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. 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]
  7. Grimm C., Kohli J., Murray J., Maundrell K. Genetic engineering of Schizosaccharomyces pombe: a system for gene disruption and replacement using the ura4 gene as a selectable marker. Mol Gen Genet. 1988 Dec;215(1):81–86. doi: 10.1007/BF00331307. [DOI] [PubMed] [Google Scholar]
  8. Grimm C., Kohli J. Observations on integrative transformation in Schizosaccharomyces pombe. Mol Gen Genet. 1988 Dec;215(1):87–93. doi: 10.1007/BF00331308. [DOI] [PubMed] [Google Scholar]
  9. Gu H., Förster I., Rajewsky K. Sequence homologies, N sequence insertion and JH gene utilization in VHDJH joining: implications for the joining mechanism and the ontogenetic timing of Ly1 B cell and B-CLL progenitor generation. EMBO J. 1990 Jul;9(7):2133–2140. doi: 10.1002/j.1460-2075.1990.tb07382.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Heyer W. D., Sipiczki M., Kohli J. Replicating plasmids in Schizosaccharomyces pombe: improvement of symmetric segregation by a new genetic element. Mol Cell Biol. 1986 Jan;6(1):80–89. doi: 10.1128/mcb.6.1.80. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. 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]
  12. Kunes S., Botstein D., Fox M. S. Synapsis-mediated fusion of free DNA ends forms inverted dimer plasmids in yeast. Genetics. 1990 Jan;124(1):67–80. doi: 10.1093/genetics/124.1.67. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Kunes S., Botstein D., Fox M. S. Transformation of yeast with linearized plasmid DNA. Formation of inverted dimers and recombinant plasmid products. J Mol Biol. 1985 Aug 5;184(3):375–387. doi: 10.1016/0022-2836(85)90288-8. [DOI] [PubMed] [Google Scholar]
  14. Lehman C. W., Carroll D. Homologous recombination catalyzed by a nuclear extract from Xenopus oocytes. Proc Natl Acad Sci U S A. 1991 Dec 1;88(23):10840–10844. doi: 10.1073/pnas.88.23.10840. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. 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]
  16. Lieber M. R. The mechanism of V(D)J recombination: a balance of diversity, specificity, and stability. Cell. 1992 Sep 18;70(6):873–876. doi: 10.1016/0092-8674(92)90237-7. [DOI] [PubMed] [Google Scholar]
  17. Maundrell K., Hutchison A., Shall S. Sequence analysis of ARS elements in fission yeast. EMBO J. 1988 Jul;7(7):2203–2209. doi: 10.1002/j.1460-2075.1988.tb03059.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Munz P. L., Young C. S. End-joining of DNA fragments in adenovirus transfection of human cells. Virology. 1991 Jul;183(1):160–169. doi: 10.1016/0042-6822(91)90129-y. [DOI] [PubMed] [Google Scholar]
  19. North P., Ganesh A., Thacker J. The rejoining of double-strand breaks in DNA by human cell extracts. Nucleic Acids Res. 1990 Nov 11;18(21):6205–6210. doi: 10.1093/nar/18.21.6205. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Orr-Weaver T. L., Szostak J. W., Rothstein R. J. Genetic applications of yeast transformation with linear and gapped plasmids. Methods Enzymol. 1983;101:228–245. doi: 10.1016/0076-6879(83)01017-4. [DOI] [PubMed] [Google Scholar]
  21. Orr-Weaver T. L., Szostak J. W. Yeast recombination: the association between double-strand gap repair and crossing-over. Proc Natl Acad Sci U S A. 1983 Jul;80(14):4417–4421. doi: 10.1073/pnas.80.14.4417. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Pfeiffer P., Vielmetter W. Joining of nonhomologous DNA double strand breaks in vitro. Nucleic Acids Res. 1988 Feb 11;16(3):907–924. doi: 10.1093/nar/16.3.907. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Resnick M. A., Martin P. The repair of double-strand breaks in the nuclear DNA of Saccharomyces cerevisiae and its genetic control. Mol Gen Genet. 1976 Jan 16;143(2):119–129. doi: 10.1007/BF00266917. [DOI] [PubMed] [Google Scholar]
  24. Robins D. M., Ripley S., Henderson A. S., Axel R. Transforming DNA integrates into the host chromosome. Cell. 1981 Jan;23(1):29–39. doi: 10.1016/0092-8674(81)90267-1. [DOI] [PubMed] [Google Scholar]
  25. Roth D. B., Chang X. B., Wilson J. H. Comparison of filler DNA at immune, nonimmune, and oncogenic rearrangements suggests multiple mechanisms of formation. Mol Cell Biol. 1989 Jul;9(7):3049–3057. doi: 10.1128/mcb.9.7.3049. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Roth D. B., Menetski J. P., Nakajima P. B., Bosma M. J., Gellert M. V(D)J recombination: broken DNA molecules with covalently sealed (hairpin) coding ends in scid mouse thymocytes. Cell. 1992 Sep 18;70(6):983–991. doi: 10.1016/0092-8674(92)90248-b. [DOI] [PubMed] [Google Scholar]
  27. Roth D. B., Porter T. N., Wilson J. H. Mechanisms of nonhomologous recombination in mammalian cells. Mol Cell Biol. 1985 Oct;5(10):2599–2607. doi: 10.1128/mcb.5.10.2599. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. 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]
  29. Schiestl R. H., Dominska M., Petes T. D. Transformation of Saccharomyces cerevisiae with nonhomologous DNA: illegitimate integration of transforming DNA into yeast chromosomes and in vivo ligation of transforming DNA to mitochondrial DNA sequences. Mol Cell Biol. 1993 May;13(5):2697–2705. doi: 10.1128/mcb.13.5.2697. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. 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]
  31. Suzuki K., Imai Y., Yamashita I., Fukui S. In vivo ligation of linear DNA molecules to circular forms in the yeast Saccharomyces cerevisiae. J Bacteriol. 1983 Aug;155(2):747–754. doi: 10.1128/jb.155.2.747-754.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. 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]
  33. Thacker J., Chalk J., Ganesh A., North P. A mechanism for deletion formation in DNA by human cell extracts: the involvement of short sequence repeats. Nucleic Acids Res. 1992 Dec 11;20(23):6183–6188. doi: 10.1093/nar/20.23.6183. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Thode S., Schäfer A., Pfeiffer P., Vielmetter W. A novel pathway of DNA end-to-end joining. Cell. 1990 Mar 23;60(6):921–928. doi: 10.1016/0092-8674(90)90340-k. [DOI] [PubMed] [Google Scholar]
  35. Winegar R. A., Lutze L. H., Rufer J. T., Morgan W. F. Spectrum of mutations produced by specific types of restriction enzyme-induced double-strand breaks. Mutagenesis. 1992 Nov;7(6):439–445. doi: 10.1093/mutage/7.6.439. [DOI] [PubMed] [Google Scholar]

Articles from Nucleic Acids Research are provided here courtesy of Oxford University Press

RESOURCES