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

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.

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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.

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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.

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Authors and Affiliations

Authors

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.

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The authors declare no competing financial interests.

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

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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

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