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. 1994 May 11;22(9):1643–1650. doi: 10.1093/nar/22.9.1643

Nonhomologous DNA end joining of synthetic hairpin substrates in Xenopus laevis egg extracts.

N Beyert 1, S Reichenberger 1, M Peters 1, M Hartung 1, B Göttlich 1, W Goedecke 1, W Vielmetter 1, P Pfeiffer 1
PMCID: PMC308043  PMID: 8202366

Abstract

Processes of DNA end joining are assumed to play a major role in the elimination of DNA double-strand breaks (DSB) in higher eucaryotic cells. Linear plasmid molecules terminated by nonhomologous restriction ends are the typical substrates used in the analysis of joining mechanisms. However, due to their limited structural variability, DSB ends generated by restriction cleavage cover probably only part of the total spectrum of naturally occurring DSB termini. We therefore devised novel DNA substrates consisting of synthetic hairpin-shaped oligonucleotides which permit the construction of blunt ends and 5'- or 3'-protruding single-strands (PSS) of arbitrary sequence and length. These substrates were tested in extracts of Xenopus laevis eggs known to efficiently join linear plasmids bearing nonhomologous restriction termini (Pfeiffer and Vielmetter, 1988). Sequences of hairpin junctions indicate that the short hairpins are joined by the same mechanisms as the plasmid substrates. However, the bimolecular DNA end joining reaction was only detectable when both hairpin partners had a minimal duplex stem length of 27bp and their PSS-tails did not exceed 10nt.

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Selected References

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  1. Bennett C. B., Lewis A. L., Baldwin K. K., Resnick M. A. Lethality induced by a single site-specific double-strand break in a dispensable yeast plasmid. Proc Natl Acad Sci U S A. 1993 Jun 15;90(12):5613–5617. doi: 10.1073/pnas.90.12.5613. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Bradley M. O., Kohn K. W. X-ray induced DNA double strand break production and repair in mammalian cells as measured by neutral filter elution. Nucleic Acids Res. 1979 Oct 10;7(3):793–804. doi: 10.1093/nar/7.3.793. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Derbyshire M. K., Epstein L. H., Young C. S., Munz P. L., Fishel R. Nonhomologous recombination in human cells. Mol Cell Biol. 1994 Jan;14(1):156–169. doi: 10.1128/mcb.14.1.156. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Fairman M. P., Johnson A. P., Thacker J. Multiple components are involved in the efficient joining of double stranded DNA breaks in human cell extracts. Nucleic Acids Res. 1992 Aug 25;20(16):4145–4152. doi: 10.1093/nar/20.16.4145. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. 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]
  6. Frankenberg D., Frankenberg-Schwager M., Harbich R. Interpretation of the shape of survival curves in terms of induction and repair/misrepair of DNA double-strand breaks. Br J Cancer Suppl. 1984;6:233–238. [PMC free article] [PubMed] [Google Scholar]
  7. Friedberg E. C. Deoxyribonucleic acid repair in the yeast Saccharomyces cerevisiae. Microbiol Rev. 1988 Mar;52(1):70–102. doi: 10.1128/mr.52.1.70-102.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Jeggo P. A. Studies on mammalian mutants defective in rejoining double-strand breaks in DNA. Mutat Res. 1990 Jul;239(1):1–16. doi: 10.1016/0165-1110(90)90028-a. [DOI] [PubMed] [Google Scholar]
  9. 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]
  10. 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]
  11. 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]
  12. Nicolás A. L., Young C. S. Characterization of DNA end joining in a mammalian cell nuclear extract: junction formation is accompanied by nucleotide loss, which is limited and uniform but not site specific. Mol Cell Biol. 1994 Jan;14(1):170–180. doi: 10.1128/mcb.14.1.170. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. 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]
  14. 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]
  15. 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]
  16. 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]
  17. 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]
  18. Roth D. B., Proctor G. N., Stewart L. K., Wilson J. H. Oligonucleotide capture during end joining in mammalian cells. Nucleic Acids Res. 1991 Dec;19(25):7201–7205. doi: 10.1093/nar/19.25.7201. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. 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]
  20. 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]
  21. 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]
  22. 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]
  23. 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]
  24. Wake C. T., Vernaleone F., Wilson J. H. Topological requirements for homologous recombination among DNA molecules transfected into mammalian cells. Mol Cell Biol. 1985 Aug;5(8):2080–2089. doi: 10.1128/mcb.5.8.2080. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. 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]

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