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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

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Abstract

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.

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Acknowledgements

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.

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Affiliations

  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

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Correspondence to Michael B Kastan.

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https://doi.org/10.1038/nprot.2008.54

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