Skip to main content
PMC home page
Nucleic Acids Research logoLink to Nucleic Acids Research
. 1995 Mar 25;23(6):1036–1043. doi: 10.1093/nar/23.6.1036

A modified single-strand annealing model best explains the joining of DNA double-strand breaks mammalian cells and cell extracts.

A L Nicolás 1, P L Munz 1, C S Young 1
PMCID: PMC306802  PMID: 7731791

Abstract

The joining of DNA double-strand breaks in vivo is frequently accompanied by the loss of a few nucleotides at the junction between the interacting partners. In vitro systems mimic this loss and, on detailed analysis, have suggested two models for the mechanism of end-joining. One invokes the use of extensive homologous side-by-side alignment of the partners prior to joining, while the other proposes the use of small regions of homology located at or near the terminus of the interacting molecules. to discriminate between these two models, assays were conducted both in vitro and in vivo with specially designed substrates. In vitro, molecules with limited terminal homology were capable of joining, but analysis of the junctions suggested that the mechanism employed the limited homology available. In vivo, the substrates with no extensive homology end-joined with equal efficiency to those with extensive homology in two different topological arrangements. Taken together, these results suggest that extensive homology is not a prerequisite for efficient end-joining, but that small homologies close to the terminus are used preferentially, as predicted by the modified single-strand annealing model.

Full text

PDF
1036

Images in this article

1038Image
on p.1038

Selected References

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

  1. Bett A. J., Prevec L., Graham F. L. Packaging capacity and stability of human adenovirus type 5 vectors. J Virol. 1993 Oct;67(10):5911–5921. doi: 10.1128/jvi.67.10.5911-5921.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Capecchi M. R. Altering the genome by homologous recombination. Science. 1989 Jun 16;244(4910):1288–1292. doi: 10.1126/science.2660260. [DOI] [PubMed] [Google Scholar]
  3. Chinnadurai G., Chinnadurai S., Brusca J. Physical mapping of a large-plaque mutation of adenovirus type 2. J Virol. 1979 Nov;32(2):623–628. doi: 10.1128/jvi.32.2.623-628.1979. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Chroboczek J., Bieber F., Jacrot B. The sequence of the genome of adenovirus type 5 and its comparison with the genome of adenovirus type 2. Virology. 1992 Jan;186(1):280–285. doi: 10.1016/0042-6822(92)90082-z. [DOI] [PubMed] [Google Scholar]
  5. 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]
  6. 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]
  7. 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]
  8. Getts R. C., Stamato T. D. Absence of a Ku-like DNA end binding activity in the xrs double-strand DNA repair-deficient mutant. J Biol Chem. 1994 Jun 10;269(23):15981–15984. [PubMed] [Google Scholar]
  9. 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]
  10. Kelly T. J., Jr, Lewis A. M., Jr Use of nondefective adenovirus-simian virus 40 hybrids for mapping the simian virus 40 genome. J Virol. 1973 Sep;12(3):643–652. doi: 10.1128/jvi.12.3.643-652.1973. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Leach D. R., Stahl F. W. Viability of lambda phages carrying a perfect palindrome in the absence of recombination nucleases. 1983 Sep 29-Oct 5Nature. 305(5933):448–451. doi: 10.1038/305448a0. [DOI] [PubMed] [Google Scholar]
  12. 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]
  13. 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]
  14. Maryon E., Carroll D. Involvement of single-stranded tails in homologous recombination of DNA injected into Xenopus laevis oocyte nuclei. Mol Cell Biol. 1991 Jun;11(6):3268–3277. doi: 10.1128/mcb.11.6.3268. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. McClintock B. The Behavior in Successive Nuclear Divisions of a Chromosome Broken at Meiosis. Proc Natl Acad Sci U S A. 1939 Aug;25(8):405–416. doi: 10.1073/pnas.25.8.405. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. 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]
  17. 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]
  18. Munz P. L., Young C., Young C. S. The genetic analysis of adenovirus recombination in triparental and superinfection crosses. Virology. 1983 Apr 30;126(2):576–586. doi: 10.1016/s0042-6822(83)80014-2. [DOI] [PubMed] [Google Scholar]
  19. 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]
  20. 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]
  21. Ozenberger B. A., Roeder G. S. A unique pathway of double-strand break repair operates in tandemly repeated genes. Mol Cell Biol. 1991 Mar;11(3):1222–1231. doi: 10.1128/mcb.11.3.1222. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Peterson C. L., Orth K., Calame K. L. Binding in vitro of multiple cellular proteins to immunoglobulin heavy-chain enhancer DNA. Mol Cell Biol. 1986 Dec;6(12):4168–4178. doi: 10.1128/mcb.6.12.4168. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. 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]
  24. 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]
  25. Phillips J. W., Morgan W. F. Illegitimate recombination induced by DNA double-strand breaks in a mammalian chromosome. Mol Cell Biol. 1994 Sep;14(9):5794–5803. doi: 10.1128/mcb.14.9.5794. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Rathmell W. K., Chu G. A DNA end-binding factor involved in double-strand break repair and V(D)J recombination. Mol Cell Biol. 1994 Jul;14(7):4741–4748. doi: 10.1128/mcb.14.7.4741. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. 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]
  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. Rouet P., Smih F., Jasin M. Introduction of double-strand breaks into the genome of mouse cells by expression of a rare-cutting endonuclease. Mol Cell Biol. 1994 Dec;14(12):8096–8106. doi: 10.1128/mcb.14.12.8096. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Rudin N., Sugarman E., Haber J. E. Genetic and physical analysis of double-strand break repair and recombination in Saccharomyces cerevisiae. Genetics. 1989 Jul;122(3):519–534. doi: 10.1093/genetics/122.3.519. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. 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]
  32. Suwa A., Hirakata M., Takeda Y., Jesch S. A., Mimori T., Hardin J. A. DNA-dependent protein kinase (Ku protein-p350 complex) assembles on double-stranded DNA. Proc Natl Acad Sci U S A. 1994 Jul 19;91(15):6904–6908. doi: 10.1073/pnas.91.15.6904. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. 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]
  34. Tatzelt J., Fechteler K., Langenbach P., Doerfler W. Fractionated nuclear extracts from hamster cells catalyze cell-free recombination at selective sequences between adenovirus DNA and a hamster preinsertion site. Proc Natl Acad Sci U S A. 1993 Aug 1;90(15):7356–7360. doi: 10.1073/pnas.90.15.7356. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. 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]
  36. 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]
  37. Volkert F. C., Young C. S. The genetic analysis of recombination using adenovirus overlapping terminal DNA fragments. Virology. 1983 Feb;125(1):175–193. doi: 10.1016/0042-6822(83)90072-7. [DOI] [PubMed] [Google Scholar]
  38. 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]

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

RESOURCES