Use of I-Sce I to Induce DNA Double-Strand Breaks in Nicotiana

  • Holger Puchta
Part of the Methods in Molecular Biology™ book series (MIMB, volume 113)


Double strand-breaks (DSBs) are key intermediates in DNA recombination reactions. The possibility of inducing DSBs at specific sites in the genome by the expression of rare-cutting endonucleases has resulted in a tremendous increase in our knowledge on the mechanisms of DSB repair, especially in yeast (1; see  Chapter 32) and recently also in higher eukaryotes (2; see  Chapter 37). In addition to its importance to the study of basic mechanisms of recombination, DSB induction leads to a dramatic enhancement of recombination frequencies and, therefore, has a great potential to be used as means for controlled genomic change. DSBs are repaired by two different recombination pathways: illegitimate or homologous. In somatic plant cells, homologous recombination is only used as a minor repair pathway. Consequently, an effective gene-targeting technique has not yet been established (3). However, DSB induction via the expression of I-Sce I or HO endonuclease increases homologous recombination frequencies one to two orders of magnitude for extrachromosomal (4) and intrachromosomal (5) recombination and for homologous integration (6). I-Sce I expression also induces homologous recombination between ectopic sites in the tobacco genome (H. Puchta, unpublished results). Synthesis-dependent strand annealing and one-sided invasion have been identified as the primary recombination mechanisms operating to repair DSBs in somatic plant cells (6,7). Recently, we have developed an assay using I-Sce I to study the repair of genomic DSBs by illegitimate recombination (8).


Homologous Integration Illegitimate Recombination Homologous Recombination Frequency MgSO4 Solution Somatic Plant Cell 
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  1. 1.
    Haber, J. E. (1995) In vivo biochemistry: Physical monitoring of recombination induced by site-specific endonucleases. BioEssays 17, 609–620.PubMedCrossRefGoogle Scholar
  2. 2.
    Jasin, M. (1996) Genetic manipulation of genomes with rare-cutting endonucleases. Trends Genet. 12, 224–228.PubMedCrossRefGoogle Scholar
  3. 3.
    Puchta, H. and Hohn, B. (1996) From centiMorgans to basepairs: homologous recombination in plants. Trends Plant Sci. 1, 340–348.Google Scholar
  4. 4.
    Puchta, H., Dujon, B. and Hohn, B. (1993) Homologous recombination in plant cells is enhanced by in vivo induction of double-strand breaks into DNA by a site-specific endonuclease. Nucleic Acids Res. 21, 5034–5040.PubMedCrossRefGoogle Scholar
  5. 5.
    Chiurazzi, M., Ray, A., Viret. J.-F., Perera, R., Wang, X.-H., Lloyd, A., et al. (1996) Enhancement of somatic intrachromosomal homologous recombination in Arabidopsis by HO-endonuclease. Plant Cell 8, 2057–2066.PubMedCrossRefGoogle Scholar
  6. 6.
    Puchta, H., Dujon, B., and Hohn, B. (1996) Two different but related mechanisms are used in plants for the repair of genomic double-strand breaks by homologous recombination. Proc. Natl. Acad. Sci. USA 93, 5055–5060.PubMedCrossRefGoogle Scholar
  7. 7.
    Puchta, H. (1998) Repair of genomic double-strand breaks in somatic plant cells by one-sided invasion of homologous sequences. Plant J. 13, 331–339.CrossRefGoogle Scholar
  8. 8.
    Salomon, S. and Puchta, H. (1998) Capture of genomic and T-DNA sequences during double-strand break repair in somatic plant cells. EMBO J. 17, 6086–6095.PubMedCrossRefGoogle Scholar
  9. 9.
    Potrykus, I. and Spangenberg, G. (eds.) (1995) Gene Transfer to Plants. Springer, Berlin.Google Scholar
  10. 10.
    Rossi, L., Escudero, J., Hohn, B. and Tinland, B. (1993) Efficient and sensitive assay for T-DNA-dependent transient gene expression. Plant Mol. Biol. Rep. 11, 220–229.CrossRefGoogle Scholar
  11. 11.
    Murashige, T. and Skoog, F. (1962) A revised medium for rapid growth and bioassays with tobacco tissue culture. Physiol. Plant. 15, 473–497.CrossRefGoogle Scholar
  12. 12.
    Fulton T. M., Chunwongse, J., and Tanksley, S. D. (1995) Microprep protocol for extraction of DNA from tomato and other herbaceous plants. Plant Mol. Biol. Rep. 13, 207–209.CrossRefGoogle Scholar
  13. 13.
    Puchta, H., Swoboda, P., Gal, S., Blot, M., and Hohn, B. (1995) Intrachromosomal homologous recombination events in populations of plant siblings. Plant Mol. Biol. 28, 281–292.PubMedCrossRefGoogle Scholar
  14. 14.
    Perrin, A., Buckle, M., and Dujon, B. (1993) Asymmetrical recognition and activity of the I-Sce I endonuclease on its site and on intron-exon junctions. EMBO J. 12, 2939–2947.PubMedGoogle Scholar

Copyright information

© Humana Press Inc. 1999

Authors and Affiliations

  • Holger Puchta
    • 1
  1. 1.Institut fur Pflanzengenetik und KulturpflanzenforschungGaterslebenGermany

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