Skip to main content
NCBI home page
Molecular and Cellular Biology logoLink to Molecular and Cellular Biology
. 1994 Jan;14(1):156–169. doi: 10.1128/mcb.14.1.156

Nonhomologous recombination in human cells.

M K Derbyshire 1, L H Epstein 1, C S Young 1, P L Munz 1, R Fishel 1
PMCID: PMC358366  PMID: 8264583

Abstract

Nonhomologous recombination (NHR) is a major pathway for the repair of chromosomal double-strand breaks in the DNA of somatic cells. In this study, a comparison was made between the nonhomologous end joining of transfected adenovirus DNA fragments in vivo and the ability of purified human proteins to catalyze nonhomologous end joining in vitro. Adenovirus DNA fragments were shown to be efficiently joined in human cells regardless of the structure of the ends. Sequence analysis of these junctions revealed that the two participating ends frequently lost nucleotides from the 3' strands at the site of the joint. To examine the biochemical basis of the end joining, nuclear extracts were prepared from a wide variety of mammalian cell lines and tested for their ability to join test plasmid substrates. Efficient ligation of the linear substrate DNA was observed, the in vitro products being similar to the in vivo products with respect to the loss of 3' nucleotides at the junction. Substantial purification of the end-joining activity was carried out with the human immature T-cell-line HPB-ALL. The protein preparation was found to join all types of linear DNA substrates containing heterologous ends with closely equivalent efficiencies. The in vitro system for end joining does not appear to contain any of the three known DNA ligases, on the basis of a number of criteria, and has been termed the NHR ligase. The enriched activity resides in a high-molecular-weight recombination complex that appears to include and require the human homologous pairing protein HPP-1 as well as the NHR ligase. Characterization of the product molecules of the NHR ligase reaction suggests that they are linear oligomers of the monomer substrate joined nonrandomly head-to-head and/or tail-to-tail. The joined ends of the products were found to be modified by a 3' exonuclease prior to ligation, and no circular DNA molecules were detected. These types of products are similar to those required for the breakage-fusion-bridge cycle, a major NHR pathway for chromosome double-strand break repair.

Full text

PDF
156

Images in this article

160Image
on p.160
161Image
on p.161
164Image
on p.164
164Image
on p.164
165Image
on p.165

Selected References

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

  1. Alt F. W., Baltimore D. Joining of immunoglobulin heavy chain gene segments: implications from a chromosome with evidence of three D-JH fusions. Proc Natl Acad Sci U S A. 1982 Jul;79(13):4118–4122. doi: 10.1073/pnas.79.13.4118. [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: 10.1016/0003-2697(76)90527-3. [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. Dignam J. D., Martin P. L., Shastry B. S., Roeder R. G. Eukaryotic gene transcription with purified components. Methods Enzymol. 1983;101:582–598. doi: 10.1016/0076-6879(83)01039-3. [DOI] [PubMed] [Google Scholar]
  5. Duesberg P. H. Cancer genes: rare recombinants instead of activated oncogenes (a review). Proc Natl Acad Sci U S A. 1987 Apr;84(8):2117–2124. doi: 10.1073/pnas.84.8.2117. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Dugaiczyk A., Boyer H. W., Goodman H. M. Ligation of EcoRI endonuclease-generated DNA fragments into linear and circular structures. J Mol Biol. 1975 Jul 25;96(1):171–184. doi: 10.1016/0022-2836(75)90189-8. [DOI] [PubMed] [Google Scholar]
  7. 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]
  8. Fishel R. A., Detmer K., Rich A. Identification of homologous pairing and strand-exchange activity from a human tumor cell line based on Z-DNA affinity chromatography. Proc Natl Acad Sci U S A. 1988 Jan;85(1):36–40. doi: 10.1073/pnas.85.1.36. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Fishel R. A., Warner R. C. Novel dimeric configurations from bacteriophage G4 replicative form DNA. Virology. 1986 Jan 15;148(1):198–209. doi: 10.1016/0042-6822(86)90415-0. [DOI] [PubMed] [Google Scholar]
  10. Fishel R., Derbyshire M. K., Moore S. P., Young C. S. Biochemical studies of homologous and nonhomologous recombination in human cells. Biochimie. 1991 Feb-Mar;73(2-3):257–267. doi: 10.1016/0300-9084(91)90211-i. [DOI] [PubMed] [Google Scholar]
  11. Fishel R., Kolodner R. Gene conversion in Escherichia coli: the recF pathway for resolution of heteroduplex DNA. J Bacteriol. 1989 Jun;171(6):3046–3052. doi: 10.1128/jb.171.6.3046-3052.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. 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]
  13. Gale K. C., Osheroff N. Intrinsic intermolecular DNA ligation activity of eukaryotic topoisomerase II. Potential roles in recombination. J Biol Chem. 1992 Jun 15;267(17):12090–12097. [PubMed] [Google Scholar]
  14. 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]
  15. Gijbels M. J., Visser J. W., Solleveld H. A., Broerse J. J., Zurcher C. Flow cytometric DNA measurement and cytomorphometric analysis of formalin fixed rat mammary tumours. Br J Cancer. 1991 Sep;64(3):523–527. doi: 10.1038/bjc.1991.342. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Graham F. L., van der Eb A. J. A new technique for the assay of infectivity of human adenovirus 5 DNA. Virology. 1973 Apr;52(2):456–467. doi: 10.1016/0042-6822(73)90341-3. [DOI] [PubMed] [Google Scholar]
  17. Hammer R. E., Pursel V. G., Rexroad C. E., Jr, Wall R. J., Bolt D. J., Ebert K. M., Palmiter R. D., Brinster R. L. Production of transgenic rabbits, sheep and pigs by microinjection. Nature. 1985 Jun 20;315(6021):680–683. doi: 10.1038/315680a0. [DOI] [PubMed] [Google Scholar]
  18. Hartwell L. Defects in a cell cycle checkpoint may be responsible for the genomic instability of cancer cells. Cell. 1992 Nov 13;71(4):543–546. doi: 10.1016/0092-8674(92)90586-2. [DOI] [PubMed] [Google Scholar]
  19. Honigberg S. M., Gonda D. K., Flory J., Radding C. M. The pairing activity of stable nucleoprotein filaments made from recA protein, single-stranded DNA, and adenosine 5'-(gamma-thio)triphosphate. J Biol Chem. 1985 Sep 25;260(21):11845–11851. [PubMed] [Google Scholar]
  20. Honigberg S. M., Rao B. J., Radding C. M. Ability of RecA protein to promote a search for rare sequences in duplex DNA. Proc Natl Acad Sci U S A. 1986 Dec;83(24):9586–9590. doi: 10.1073/pnas.83.24.9586. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Hu W. S., Temin H. M. Effect of gamma radiation on retroviral recombination. J Virol. 1992 Jul;66(7):4457–4463. doi: 10.1128/jvi.66.7.4457-4463.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Hu W. S., Temin H. M. Retroviral recombination and reverse transcription. Science. 1990 Nov 30;250(4985):1227–1233. doi: 10.1126/science.1700865. [DOI] [PubMed] [Google Scholar]
  23. Jachymczyk W. J., von Borstel R. C., Mowat M. R., Hastings P. J. Repair of interstrand cross-links in DNA of Saccharomyces cerevisiae requires two systems for DNA repair: the RAD3 system and the RAD51 system. Mol Gen Genet. 1981;182(2):196–205. doi: 10.1007/BF00269658. [DOI] [PubMed] [Google Scholar]
  24. Kolodner R., Evans D. H., Morrison P. T. Purification and characterization of an activity from Saccharomyces cerevisiae that catalyzes homologous pairing and strand exchange. Proc Natl Acad Sci U S A. 1987 Aug;84(16):5560–5564. doi: 10.1073/pnas.84.16.5560. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Krasin F., Hutchinson F. Repair of DNA double-strand breaks in Escherichia coli, which requires recA function and the presence of a duplicate genome. J Mol Biol. 1977 Oct 15;116(1):81–98. doi: 10.1016/0022-2836(77)90120-6. [DOI] [PubMed] [Google Scholar]
  26. 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]
  27. 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]
  28. Lichter P., Cremer T., Borden J., Manuelidis L., Ward D. C. Delineation of individual human chromosomes in metaphase and interphase cells by in situ suppression hybridization using recombinant DNA libraries. Hum Genet. 1988 Nov;80(3):224–234. doi: 10.1007/BF01790090. [DOI] [PubMed] [Google Scholar]
  29. Lindahl T., Barnes D. E. Mammalian DNA ligases. Annu Rev Biochem. 1992;61:251–281. doi: 10.1146/annurev.bi.61.070192.001343. [DOI] [PubMed] [Google Scholar]
  30. Lopez P., Espinosa M., Stassi D. L., Lacks S. A. Facilitation of plasmid transfer in Streptococcus pneumoniae by chromosomal homology. J Bacteriol. 1982 May;150(2):692–701. doi: 10.1128/jb.150.2.692-701.1982. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Marsh J. L., Erfle M., Wykes E. J. The pIC plasmid and phage vectors with versatile cloning sites for recombinant selection by insertional inactivation. Gene. 1984 Dec;32(3):481–485. doi: 10.1016/0378-1119(84)90022-2. [DOI] [PubMed] [Google Scholar]
  32. McCLINTOCK B. Chromosome organization and genic expression. Cold Spring Harb Symp Quant Biol. 1951;16:13–47. doi: 10.1101/sqb.1951.016.01.004. [DOI] [PubMed] [Google Scholar]
  33. McClintock B. The Fusion of Broken Ends of Chromosomes Following Nuclear Fusion. Proc Natl Acad Sci U S A. 1942 Nov;28(11):458–463. doi: 10.1073/pnas.28.11.458. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. McClintock B. The Stability of Broken Ends of Chromosomes in Zea Mays. Genetics. 1941 Mar;26(2):234–282. doi: 10.1093/genetics/26.2.234. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Miller C. K., Temin H. M. High-efficiency ligation and recombination of DNA fragments by vertebrate cells. Science. 1983 May 6;220(4597):606–609. doi: 10.1126/science.6301012. [DOI] [PubMed] [Google Scholar]
  36. Moore S. P., Erdile L., Kelly T., Fishel R. The human homologous pairing protein HPP-1 is specifically stimulated by the cognate single-stranded binding protein hRP-A. Proc Natl Acad Sci U S A. 1991 Oct 15;88(20):9067–9071. doi: 10.1073/pnas.88.20.9067. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Moore S. P., Fishel R. Purification and characterization of a protein from human cells which promotes homologous pairing of DNA. J Biol Chem. 1990 Jul 5;265(19):11108–11117. [PubMed] [Google Scholar]
  38. 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]
  39. 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]
  40. 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]
  41. 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]
  42. Orr-Weaver T. L., Szostak J. W., Rothstein R. J. Yeast transformation: a model system for the study of recombination. Proc Natl Acad Sci U S A. 1981 Oct;78(10):6354–6358. doi: 10.1073/pnas.78.10.6354. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. 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]
  44. 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]
  45. 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]
  46. Resnick M. A. The repair of double-strand breaks in DNA; a model involving recombination. J Theor Biol. 1976 Jun;59(1):97–106. doi: 10.1016/s0022-5193(76)80025-2. [DOI] [PubMed] [Google Scholar]
  47. 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]
  48. 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]
  49. Roth D. B., Wilson J. H. Relative rates of homologous and nonhomologous recombination in transfected DNA. Proc Natl Acad Sci U S A. 1985 May;82(10):3355–3359. doi: 10.1073/pnas.82.10.3355. [DOI] [PMC free article] [PubMed] [Google Scholar]
  50. Sikorav J. L., Church G. M. Complementary recognition in condensed DNA: accelerated DNA renaturation. J Mol Biol. 1991 Dec 20;222(4):1085–1108. doi: 10.1016/0022-2836(91)90595-w. [DOI] [PubMed] [Google Scholar]
  51. Symington L. S. Double-strand-break repair and recombination catalyzed by a nuclear extract of Saccharomyces cerevisiae. EMBO J. 1991 Apr;10(4):987–996. doi: 10.1002/j.1460-2075.1991.tb08033.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  52. 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]
  53. Tait R. C., Rodriguez R. L., West R. W., Jr The rapid purification of T4 DNA ligase from a lambda T4 lig lysogen. J Biol Chem. 1980 Feb 10;255(3):813–815. [PubMed] [Google Scholar]
  54. Teraoka H., Tsukada K. Eukaryotic DNA ligase. Purification and properties of the enzyme from bovine thymus, and immunochemical studies of the enzyme from animal tissues. J Biol Chem. 1982 May 10;257(9):4758–4763. [PubMed] [Google Scholar]
  55. 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]
  56. Tomkinson A. E., Roberts E., Daly G., Totty N. F., Lindahl T. Three distinct DNA ligases in mammalian cells. J Biol Chem. 1991 Nov 15;266(32):21728–21735. [PubMed] [Google Scholar]
  57. Visscher D. W., Zarbo R. J., Greenawald K. A., Crissman J. D. Prognostic significance of morphological parameters and flow cytometric DNA analysis in carcinoma of the breast. Pathol Annu. 1990;25(Pt 1):171–210. [PubMed] [Google Scholar]
  58. 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]
  59. Wang T. C., Smith K. C. Mechanisms for recF-dependent and recB-dependent pathways of postreplication repair in UV-irradiated Escherichia coli uvrB. J Bacteriol. 1983 Dec;156(3):1093–1098. doi: 10.1128/jb.156.3.1093-1098.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  60. Wiaderkiewicz R., Ruiz-Carrillo A. Mismatch and blunt to protruding-end joining by DNA ligases. Nucleic Acids Res. 1987 Oct 12;15(19):7831–7848. doi: 10.1093/nar/15.19.7831. [DOI] [PMC free article] [PubMed] [Google Scholar]
  61. Wilkie T. M., Braun R. E., Ehrman W. J., Palmiter R. D., Hammer R. E. Germ-line intrachromosomal recombination restores fertility in transgenic MyK-103 male mice. Genes Dev. 1991 Jan;5(1):38–48. doi: 10.1101/gad.5.1.38. [DOI] [PubMed] [Google Scholar]
  62. Wilkie T. M., Palmiter R. D. Analysis of the integrant in MyK-103 transgenic mice in which males fail to transmit the integrant. Mol Cell Biol. 1987 May;7(5):1646–1655. doi: 10.1128/mcb.7.5.1646. [DOI] [PMC free article] [PubMed] [Google Scholar]
  63. Wilson J. H., Berget P. B., Pipas J. M. Somatic cells efficiently join unrelated DNA segments end-to-end. Mol Cell Biol. 1982 Oct;2(10):1258–1269. doi: 10.1128/mcb.2.10.1258. [DOI] [PMC free article] [PubMed] [Google Scholar]
  64. Yanisch-Perron C., Vieira J., Messing J. Improved M13 phage cloning vectors and host strains: nucleotide sequences of the M13mp18 and pUC19 vectors. Gene. 1985;33(1):103–119. doi: 10.1016/0378-1119(85)90120-9. [DOI] [PubMed] [Google Scholar]
  65. Zimmerman S. B., Pheiffer B. H. Macromolecular crowding allows blunt-end ligation by DNA ligases from rat liver or Escherichia coli. Proc Natl Acad Sci U S A. 1983 Oct;80(19):5852–5856. doi: 10.1073/pnas.80.19.5852. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from Molecular and Cellular Biology are provided here courtesy of Taylor & Francis

RESOURCES