Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Article
  • Published:

Nucleotide-resolution DNA double-strand break mapping by next-generation sequencing

Nature Methods volume 10, pages 361–365 (2013)Cite this article

Subjects

Abstract

We present a genome-wide approach to map DNA double-strand breaks (DSBs) at nucleotide resolution by a method we termed BLESS (direct in situ breaks labeling, enrichment on streptavidin and next-generation sequencing). We validated and tested BLESS using human and mouse cells and different DSBs-inducing agents and sequencing platforms. BLESS was able to detect telomere ends, Sce endonuclease–induced DSBs and complex genome-wide DSB landscapes. As a proof of principle, we characterized the genomic landscape of sensitivity to replication stress in human cells, and we identified >2,000 nonuniformly distributed aphidicolin-sensitive regions (ASRs) overrepresented in genes and enriched in satellite repeats. ASRs were also enriched in regions rearranged in human cancers, with many cancer-associated genes exhibiting high sensitivity to replication stress. Our method is suitable for genome-wide mapping of DSBs in various cells and experimental conditions, with a specificity and resolution unachievable by current techniques.

This is a preview of subscription content, access via your institution

Access options

Buy this article

  • Purchase on SpringerLink
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

Figure 1: BLESS workflow and specificity.
Figure 2: Example of HeLa breakomes associated with specific treatments.
Figure 3: ASR validation.
Figure 4: Biological characterization of ASRs.

Similar content being viewed by others

Accession codes

Primary accessions

Sequence Read Archive

References

  1. Paigen, K. & Petkov, P. Mammalian recombination hot spots: properties, control and evolution. Nat. Rev. Genet. 11, 221–233 (2010).

    Article  CAS  Google Scholar 

  2. Jackson, S.P. & Bartek, J. The DNA-damage response in human biology and disease. Nature 461, 1071–1078 (2009).

    Article  CAS  Google Scholar 

  3. Branzei, D. & Foiani, M. Maintaining genome stability at the replication fork. Nat. Rev. Mol. Cell Biol. 11, 208–219 (2010).

    Article  CAS  Google Scholar 

  4. Branzei, D. & Foiani, M. The DNA damage response during DNA replication. Curr. Opin. Cell Biol. 17, 568–575 (2005).

    Article  CAS  Google Scholar 

  5. Szilard, R.K. et al. Systematic identification of fragile sites via genome-wide location analysis of gamma-H2AX. Nat. Struct. Mol. Biol. 17, 299–305 (2010).

    Article  CAS  Google Scholar 

  6. Harrigan, J.A. et al. Replication stress induces 53BP1-containing OPT domains in G1 cells. J. Cell Biol. 193, 97–108 (2011).

    Article  CAS  Google Scholar 

  7. Seo, J. et al. Genome-wide profiles of H2AX and -H2AX differentiate endogenous and exogenous DNA damage hotspots in human cells. Nucleic Acids Res. 40, 5965–5974 (2012).

    Article  CAS  Google Scholar 

  8. Marti, T.M., Hefner, E., Feeney, L., Natale, V. & Cleaver, J.E. H2AX phosphorylation within the G1 phase after UV irradiation depends on nucleotide excision repair and not DNA double-strand breaks. Proc. Natl. Acad. Sci. USA 103, 9891–9896 (2006).

    Article  CAS  Google Scholar 

  9. Tuduri, S. et al. Topoisomerase I suppresses genomic instability by preventing interference between replication and transcription. Nat. Cell Biol. 11, 1315–1324 (2009).

    Article  CAS  Google Scholar 

  10. Chadwick, B.P. & Lane, T.F. BRCA1 associates with the inactive X chromosome in late S-phase, coupled with transient H2AX phosphorylation. Chromosoma 114, 432–439 (2005).

    Article  CAS  Google Scholar 

  11. Iacovoni, J.S. et al. High-resolution profiling of γH2AX around DNA double strand breaks in the mammalian genome. EMBO J. 29, 1446–1457 (2010).

    Article  CAS  Google Scholar 

  12. Bonner, W.M. et al. γH2AX and cancer. Nat. Rev. Cancer 8, 957–967 (2008).

    Article  CAS  Google Scholar 

  13. Bunting, S.F. et al. 53BP1 inhibits homologous recombination in Brca1-deficient cells by blocking resection of DNA breaks. Cell 141, 243–254 (2010).

    Article  CAS  Google Scholar 

  14. Bothmer, A. et al. 53BP1 regulates DNA resection and the choice between classical and alternative end joining during class switch recombination. J. Exp. Med. 207, 855–865 (2010).

    Article  CAS  Google Scholar 

  15. Hakim, O. et al. DNA damage defines sites of recurrent chromosomal translocations in B lymphocytes. Nature 484, 69–74 (2012).

    Article  CAS  Google Scholar 

  16. Blitzblau, H.G., Bell, G.W., Rodriguez, J., Bell, S.P. & Hochwagen, A. Mapping of meiotic single-stranded DNA reveals double-stranded-break hotspots near centromeres and telomeres. Curr. Biol. 17, 2003–2012 (2007).

    Article  CAS  Google Scholar 

  17. Feng, W. et al. Genomic mapping of single-stranded DNA in hydroxyurea-challenged yeasts identifies origins of replication. Nat. Cell Biol. 8, 148–155 (2006).

    Article  CAS  Google Scholar 

  18. Feng, W., Bachant, J., Collingwood, D., Raghuraman, M.K. & Brewer, B.J. Centromere replication timing determines different forms of genomic instability in Saccharomyces cerevisiae checkpoint mutants during replication stress. Genetics 183, 1249–1260 (2009).

    Article  CAS  Google Scholar 

  19. Leduc, F. et al. Genome-wide mapping of DNA strand breaks. PLoS ONE 6, e17353 (2011).

    Article  CAS  Google Scholar 

  20. Dudley, D.D., Chaudhuri, J., Bassing, C.H. & Alt, F.W. Mechanism and control of V(D)J recombination versus class switch recombination: similarities and differences. Adv. Immunol. 86, 43–112 (2005).

    Article  CAS  Google Scholar 

  21. Sfeir, A.J., Chai, W., Shay, J.W. & Wright, W.E. Telomere-end processing the terminal nucleotides of human chromosomes. Mol. Cell 18, 131–138 (2005).

    Article  CAS  Google Scholar 

  22. Palm, W. & de Lange, T. How shelterin protects mammalian telomeres. Annu. Rev. Genet. 42, 301–334 (2008).

    Article  CAS  Google Scholar 

  23. Casper, A.M., Nghiem, P., Arlt, M.F. & Glover, T.W. ATR regulates fragile site stability. Cell 111, 779–789 (2002).

    Article  CAS  Google Scholar 

  24. Benjamini, Y. & Hochberg, Y. Controlling the false discovery rate: a practical and powerful approach to multiple testing. J. R. Stat. Soc., B 57, 289–300 (1995).

    Google Scholar 

  25. Smit, A. & Hubley, R. RepeatMasker Open v.3.0 〈http://www.repeatmasker.org/〉 (Institute for Systems Biology, Seattle, 1996–2004).

  26. Durkin, S.G. & Glover, T.W. Chromosome fragile sites. Annu. Rev. Genet. 41, 169–192 (2007).

    Article  CAS  Google Scholar 

  27. Zhang, H. & Freudenreich, C.H. An AT-rich sequence in human common fragile site FRA16D causes fork stalling and chromosome breakage in S. cerevisiae. Mol. Cell 27, 367–379 (2007).

    Article  Google Scholar 

  28. Kim, N. & Jinks-Robertson, S. Transcription as a source of genome instability. Nat. Rev. Genet. 13, 204–214 (2012).

    Article  CAS  Google Scholar 

  29. Helmrich, A., Ballarino, M. & Tora, L. Collisions between replication and transcription complexes cause common fragile site instability at the longest human genes. Mol. Cell 44, 966–977 (2011).

    Article  CAS  Google Scholar 

  30. Halazonetis, T.D., Gorgoulis, V.G. & Bartek, J. An oncogene-induced DNA damage model for cancer development. Science 319, 1352–1355 (2008).

    Article  CAS  Google Scholar 

  31. Negrini, S., Gorgoulis, V.G. & Halazonetis, T.D. Genomic instability—an evolving hallmark of cancer. Nat. Rev. Mol. Cell Biol. 11, 220–228 (2010).

    Article  CAS  Google Scholar 

  32. De, S. & Michor, F. DNA replication timing and long-range DNA interactions predict mutational landscapes of cancer genomes. Nat. Biotechnol. 29, 1103–1108 (2011).

    Article  CAS  Google Scholar 

  33. Futreal, P.A. et al. A census of human cancer genes. Nat. Rev. Cancer 4, 177–183 (2004).

    Article  CAS  Google Scholar 

  34. Santarius, T., Shipley, J., Brewer, D., Stratton, M.R. & Cooper, C.S. A census of amplified and overexpressed human cancer genes. Nat. Rev. Cancer 10, 59–64 (2010).

    Article  CAS  Google Scholar 

  35. Ng, S.B. et al. Targeted capture and massively parallel sequencing of 12 human exomes. Nature 461, 272–276 (2009).

    Article  CAS  Google Scholar 

  36. Altshuler, D. et al. An SNP map of the human genome generated by reduced representation shotgun sequencing. Nature 407, 513–516 (2000).

    Article  CAS  Google Scholar 

  37. Chiarle, R. et al. Genome-wide translocation sequencing reveals mechanisms of chromosome breaks and rearrangements in B cells. Cell 147, 107–119 (2011).

    Article  CAS  Google Scholar 

  38. Crosetto, N. et al. Human Wrnip1 is localized in replication factories in a ubiquitin-binding zinc finger-dependent manner. J. Biol. Chem. 283, 35173–35185 (2008).

    Article  CAS  Google Scholar 

  39. Tyteca, S., Vandromme, M., Legube, G., Chevillard-Briet, M. & Trouche, D. Tip60 and p400 are both required for UV-induced apoptosis but play antagonistic roles in cell cycle progression. EMBO J. 25, 1680–1689 (2006).

    Article  CAS  Google Scholar 

  40. Schmittgen, T.D. & Livak, K.J. Analyzing real-time PCR data by the comparative CT method. Nat. Protoc. 3, 1101–1108 (2008).

    Article  CAS  Google Scholar 

  41. Fujita, P.A. et al. The UCSC Genome Browser database: update 2011. Nucleic Acids Res. 39, D876–D882 (2011).

    Article  CAS  Google Scholar 

  42. Beroukhim, R. et al. The landscape of somatic copy-number alteration across human cancers. Nature 463, 899–905 (2010).

    Article  CAS  Google Scholar 

Download references

Acknowledgements

We acknowledge Y. Shiloh (Tel Aviv University) and A.J. Pierce (University of Kentucky) for kindly providing U2OS_DRH-1 cells and I-Sce plasmids. We are grateful to T. Włodarski, A.R. Lehmann, G. Fudenberg and A. Kudlicki for insightful discussions, critical reading of the manuscript and help with data analysis. This work was supported by grants from Deutsche Forschungsgemeinschaft, the Cluster of Excellence “Macromolecular Complexes” of the Goethe University Frankfurt (EXC115), the LOEWE-funded Oncogenic Signaling Frankfurt network, the LOEWE Gene and Cell Therapy Center and the European Research Council (ERC) under the European Union's Seventh Framework Programme (FP7/2007-2013) and ERC grant agreement 250241-LineUb to I.D.; from the Associazione Italiana per la Ricerca sul cancro (AIRC) and the International Association for Cancer Research (AICR) and grant FP7 ERC-2009- StG (proposal 242965–“Lunely”) to R.C.; from the Foundation for Polish Science (TEAM), Polish National Science Centre (2011/02/A/NZ2/00014) and European Regional Development Fund under the Innovative Economy Programme (POIG.02.02.00-14-024/08-00) to K.G.; from Ligue contre le Cancer (équipe labellisée), Agence Nationale de la Recherche (RepliCare) and Institut National du Cancer to P.P.; and by grant UL1TR000071 ITS “Novel Methods” from the National Center for Research Resources, US National Institutes of Health, to M.R. M.B. is a recipient of a Human Frontier Science Program Long-Term Fellowship.

Author information

Author notes

  1. Nicola Crosetto, Magda Bienko & Norbert Dojer

    Present address: Present addresses: Department of Biology, Massachusetts Institute of Technology, Cambridge, Massachusetts, USA (N.C. and M.B.); Institute of Informatics, University of Warsaw, Warsaw, Poland (N.D.; on leave).,

  2. Nicola Crosetto and Abhishek Mitra: These authors contributed equally to this work.

Authors and Affiliations

  1. Institute of Biochemistry II, Goethe University Medical School, Frankfurt, Germany

    Nicola Crosetto, Magda Bienko & Ivan Dikic

  2. Buchmann Institute for Molecular Life Sciences, Goethe University Medical School, Frankfurt, Germany

    Nicola Crosetto, Magda Bienko & Ivan Dikic

  3. Institute for Translational Sciences, University of Texas Medical Branch at Galveston (UTMB), Galveston, Texas, USA

    Abhishek Mitra, Norbert Dojer & Maga Rowicka

  4. Institut de Génétique Humaine (IGH), Centre National de la Recherche Scientifique Unité Propre de Recherche 1142, Montpellier, France

    Maria Joao Silva & Philippe Pasero

  5. Department of Pathology, Children's Hospital, Boston, Massachusetts, USA

    Qi Wang, Elif Karaca & Roberto Chiarle

  6. Harvard Medical School, Boston, Massachusetts, USA

    Qi Wang, Elif Karaca & Roberto Chiarle

  7. Department of Molecular Biotechnology and Health Sciences, University of Torino, Torino, Italy

    Roberto Chiarle

  8. Laboratory of Bioinformatics and Systems Biology, Centre of New Technologies, University of Warsaw, Warsaw, Poland

    Magdalena Skrzypczak & Krzysztof Ginalski

  9. Department of Biochemistry and Molecular Biology, UTMB, Galveston, Texas, USA

    Maga Rowicka

  10. Sealy Center for Molecular Medicine, UTMB, Galveston, Texas, USA

    Maga Rowicka

Authors
  1. Nicola Crosetto
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Abhishek Mitra
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Maria Joao Silva
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Magda Bienko
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Norbert Dojer
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Qi Wang
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Elif Karaca
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Roberto Chiarle
    View author publications

    You can also search for this author in PubMed Google Scholar

  9. Magdalena Skrzypczak
    View author publications

    You can also search for this author in PubMed Google Scholar

  10. Krzysztof Ginalski
    View author publications

    You can also search for this author in PubMed Google Scholar

  11. Philippe Pasero
    View author publications

    You can also search for this author in PubMed Google Scholar

  12. Maga Rowicka
    View author publications

    You can also search for this author in PubMed Google Scholar

  13. Ivan Dikic
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

N.C. and I.D. conceived and developed BLESS, coordinated the project and wrote the manuscript. A.M. developed all necessary code and analyzed Illumina data. M.J.S. and P.P. performed ChIP experiments and analysis. M.B. performed microscopy experiments and prepared figures. Q.W., E.K. and R.C. performed Roche 454 experiments and analyzed the data. N.D. contributed to statistical data analysis. M.S. and K.G. performed paired-end Illumina sequencing. M.R. conceived procedures for computational analysis, supervised the analysis and coordinated the project.

Corresponding authors

Correspondence to Nicola Crosetto, Maga Rowicka or Ivan Dikic.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–7 and Supplementary Tables 1–7 (PDF 5109 kb)

Rights and permissions

Reprints and permissions

About this article

Cite this article

Crosetto, N., Mitra, A., Silva, M. et al. Nucleotide-resolution DNA double-strand break mapping by next-generation sequencing. Nat Methods 10, 361–365 (2013). https://doi.org/10.1038/nmeth.2408

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nmeth.2408

This article is cited by

Search

Advanced search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing