Protocol | Published:

Assessment of protein dynamics and DNA repair following generation of DNA double-strand breaks at defined genomic sites

Nature Protocols volume 3, pages 915922 (2008) | Download Citation



The formation of protein aggregates (foci) at sites of DNA double-strand breaks (DSBs) is mainly studied by immunostaining and is hence limited by the low resolution of light microscopy and the availability of appropriate and selective antibodies. Here, we describe a system using enzymatic creation of site-specific DNA DSBs within the human genome combined with chromatin immunoprecipitation (ChIP) that enables molecular probing of a DSB. Following induction of the I-PpoI enzyme and generation of DSBs, cellular DNA and proteins are crosslinked and analyzed by ChIP for specific proteins at the site of the break. The system allows the direct detection of protein and chromatin dynamics at the site of the break with high resolution, as well as direct measurement of DNA repair defects in human cells. Starting with fragmented chromatin, results can be achieved in 2–3 d.

Access optionsAccess options

Rent or Buy article

Get time limited or full article access on ReadCube.


All prices are NET prices.


  1. 1.

    & Cell-cycle checkpoints and cancer. Nature 432, 316–323 (2004).

  2. 2.

    , & Roles of ATM and NBS1 in chromatin structure modulation and DNA double-strand break repair. Nat. Cell Biol. 9, 683–690 (2007).

  3. 3.

    et al. Interplay between human DNA repair proteins at a unique double-strand break in vivo. EMBO J. 25, 222–231 (2006).

  4. 4.

    et al. Positional stability of single double-strand breaks in mammalian cells. Nat. Cell Biol. 9, 675–682 (2007).

  5. 5.

    et al. A critical role for histone H2AX in recruitment of repair factors to nuclear foci after DNA damage. Curr. Biol. 10, 886–895 (2000).

  6. 6.

    & Efficient repair of HO-induced chromosomal breaks in Saccharomyces cerevisiae by recombination between flanking homologous sequences. Mol. Cell. Biol. 8, 3918–3928 (1988).

  7. 7.

    & Intermediates of recombination during mating type switching in Saccharomyces cerevisiae. EMBO J. 9, 663–673 (1990).

  8. 8.

    & Frequent chromosomal translocations induced by DNA double-strand breaks. Nature 405, 697–700 (2000).

  9. 9.

    , , & Choreography of the DNA damage response: spatiotemporal relationships among checkpoint and repair proteins. see comment Cell 118, 699–713 (2004).

  10. 10.

    , , , & Chromosomal double-strand break repair in Ku80-deficient cells. Proc. Natl. Acad. Sci. USA 93, 8929–8933 (1996).

  11. 11.

    , & Introduction of double-strand breaks into the genome of mouse cells by expression of a rare-cutting endonuclease. Mol. Cell. Biol. 14, 8096–8106 (1994).

  12. 12.

    , , & Characterization of I-Ppo, an intron-encoded endonuclease that mediates homing of a group I intron in the ribosomal DNA of Physarum polycephalum. Mol. Cell. Biol. 10, 3386–3396 (1990).

  13. 13.

    , , & DNA binding and cleavage by the nuclear intron-encoded homing endonuclease I-PpoI. Nature 394, 96–101 (1998).

  14. 14.

    , & Generation of highly site-specific DNA double-strand breaks in human cells by the homing endonucleases I-PpoI and I-CreI. Biochem. Biophys. Res. Commun. 255, 88–93 (1999).

  15. 15.

    et al. CDC25A phosphatase is a target of E2F and is required for efficient E2F-induced S phase. Mol. Cell. Biol. 19, 6379–6395 (1999).

  16. 16.

    & ATM is a target for positive regulation by E2F-1. Oncogene 22, 161–167 (2003).

  17. 17.

    et al. Distribution and dynamics of chromatin modification induced by a defined DNA double-strand break. Curr. Biol. 14, 1703–1711 (2004).

  18. 18.

    Analysis of the cell cycle and a method employing synchronized cells for study of protein expression at various stages of the cell cycle. Biochemistry (Mosc) 69, 485–496 (2004).

  19. 19.

    & Analysis of cell cycle phases and progression in cultured mammalian cells. Methods (Duluth) 41, 143–150 (2007).

  20. 20.

    & Rapid transfer of DNA from agarose gels to nylon membranes. Nucleic Acids Res. 13, 7207–7221 (1985).

Download references


This work was supported by grants from the NIH (CA71387 and CA21765 to M.B.K. and CA48022 and CA77852 to R.J.M. Jr.) and by the American Lebanese Syrian Associated Charities of the St. Jude Children's Research Hospital.

Author information


  1. Department of Human Molecular Genetics and Biochemistry, Sackler School of Medicine, Tel Aviv University, Tel Aviv 69978, Israel.

    • Elijahu Berkovich
  2. Departments of Pathology and Genome Sciences, University of Washington, Box 357705, Seattle, Washington 98195-7705, USA.

    • Raymond J Monnat Jr
  3. Department of Oncology, St. Jude Children's Research Hospital, 332 North Lauderdale Street, Memphis, Tennessee 38105, USA.

    • Michael B Kastan


  1. Search for Elijahu Berkovich in:

  2. Search for Raymond J Monnat in:

  3. Search for Michael B Kastan in:

Corresponding author

Correspondence to Michael B Kastan.

About this article

Publication history



Further reading


By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.