Skip to main content
PMC home page
Nucleic Acids Research logoLink to Nucleic Acids Research
. 1996 Dec 15;24(24):4946–4953. doi: 10.1093/nar/24.24.4946

The joining of non-complementary DNA double-strand breaks by mammalian extracts.

R M Mason 1, J Thacker 1, M P Fairman 1
PMCID: PMC146356  PMID: 9016665

Abstract

We have developed a high efficiency system in which mammalian extracts join DNA double-strand breaks with non-complementary termini. This system has been used to obtain a large number of junction sequences from a range of different break-end combinations, allowing the elucidation of the joining mechanisms. Using an extract of calf thymus it was found that the major mechanism of joining was by blunt-end ligation following removal or fill-in of the single-stranded bases. However, some break-end combinations were joined through an efficient mechanism using short repeat sequences and we have succeeded in separating this mechanism from blunt-end joining by the biochemical fractionation of extracts. Characterization of activities and sequence data in an extensively purified fraction that will join ends by the repeat mechanism led to a model where joining is initiated by 3' strand invasion followed by pairing to short repeat sequences close to the break site. Thus the joining of double-strand breaks by mammalian extracts is achieved by several mechanisms and this system will allow the purification of the factors involved in each by the judicial choice of the non-complementary ends used in the assay.

Full Text

The Full Text of this article is available as a PDF (251.5 KB).

Selected References

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

  1. Bennett C. B., Westmoreland T. J., Snipe J. R., Resnick M. A. A double-strand break within a yeast artificial chromosome (YAC) containing human DNA can result in YAC loss, deletion or cell lethality. Mol Cell Biol. 1996 Aug;16(8):4414–4425. doi: 10.1128/mcb.16.8.4414. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Bradford M. M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem. 1976 May 7;72:248–254. doi: 10.1006/abio.1976.9999. [DOI] [PubMed] [Google Scholar]
  3. Dar M. E., Jorgensen T. J. Deletions at short direct repeats and base substitutions are characteristic mutations for bleomycin-induced double- and single-strand breaks, respectively, in a human shuttle vector system. Nucleic Acids Res. 1995 Aug 25;23(16):3224–3230. doi: 10.1093/nar/23.16.3224. [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. Frankenberg D., Frankenberg-Schwager M., Blöcher D., Harbich R. Evidence for DNA double-strand breaks as the critical lesions in yeast cells irradiated with sparsely or densely ionizing radiation under oxic or anoxic conditions. Radiat Res. 1981 Dec;88(3):524–532. [PubMed] [Google Scholar]
  6. Harrington J. J., Lieber M. R. Functional domains within FEN-1 and RAD2 define a family of structure-specific endonucleases: implications for nucleotide excision repair. Genes Dev. 1994 Jun 1;8(11):1344–1355. doi: 10.1101/gad.8.11.1344. [DOI] [PubMed] [Google Scholar]
  7. Jeggo P. A., Taccioli G. E., Jackson S. P. Menage à trois: double strand break repair, V(D)J recombination and DNA-PK. Bioessays. 1995 Nov;17(11):949–957. doi: 10.1002/bies.950171108. [DOI] [PubMed] [Google Scholar]
  8. Jessberger R., Podust V., Hübscher U., Berg P. A mammalian protein complex that repairs double-strand breaks and deletions by recombination. J Biol Chem. 1993 Jul 15;268(20):15070–15079. [PubMed] [Google Scholar]
  9. Jessberger R., Riwar B., Rolink A., Rodewald H. R. Stimulation of defective DNA transfer activity in recombination deficient SCID cell extracts by a 72-kDa protein from wild-type thymocytes. J Biol Chem. 1995 Mar 24;270(12):6788–6797. doi: 10.1074/jbc.270.12.6788. [DOI] [PubMed] [Google Scholar]
  10. 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]
  11. Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
  12. Laskey R. A., Mills A. D., Morris N. R. Assembly of SV40 chromatin in a cell-free system from Xenopus eggs. Cell. 1977 Feb;10(2):237–243. doi: 10.1016/0092-8674(77)90217-3. [DOI] [PubMed] [Google Scholar]
  13. Lehman C. W., Trautman J. K., Carroll D. Illegitimate recombination in Xenopus: characterization of end-joined junctions. Nucleic Acids Res. 1994 Feb 11;22(3):434–442. doi: 10.1093/nar/22.3.434. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Miles C., Sargent G., Phear G., Meuth M. DNA sequence analysis of gamma radiation-induced deletions and insertions at the APRT locus of hamster cells. Mol Carcinog. 1990;3(4):233–242. doi: 10.1002/mc.2940030411. [DOI] [PubMed] [Google Scholar]
  15. Munz P. L., Young C. S. The creation of adenovirus genomes with viable, stable, internal redundancies centered about the E2b region. Virology. 1987 May;158(1):52–60. doi: 10.1016/0042-6822(87)90237-6. [DOI] [PubMed] [Google Scholar]
  16. 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]
  17. 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]
  18. 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]
  19. Pfeiffer P., Thode S., Hancke J., Keohavong P., Thilly W. G. Resolution and conservation of mismatches in DNA end joining. Mutagenesis. 1994 Nov;9(6):527–535. doi: 10.1093/mutage/9.6.527. [DOI] [PubMed] [Google Scholar]
  20. 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]
  21. 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]
  22. Reeves R., Gorman C. M., Howard B. Minichromosome assembly of non-integrated plasmid DNA transfected into mammalian cells. Nucleic Acids Res. 1985 May 24;13(10):3599–3615. doi: 10.1093/nar/13.10.3599. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. 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]
  24. 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]
  25. Simpson P. J., Savage J. R. Estimating the true frequency of X-ray-induced complex chromosome exchanges using fluorescence in situ hybridization. Int J Radiat Biol. 1995 Jan;67(1):37–45. doi: 10.1080/09553009514550051. [DOI] [PubMed] [Google Scholar]
  26. Stary A., Sarasin A. Molecular analysis of DNA junctions produced by illegitimate recombination in human cells. Nucleic Acids Res. 1992 Aug 25;20(16):4269–4274. doi: 10.1093/nar/20.16.4269. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. 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]
  28. 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]
  29. 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]
  30. 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]
  31. Wist E. The role of DNA polymerases alpha, beta and gamma in nuclear DNA synthesis. Biochim Biophys Acta. 1979 Mar 28;562(1):62–69. doi: 10.1016/0005-2787(79)90126-6. [DOI] [PubMed] [Google Scholar]

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

RESOURCES