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      DNA Breaks and End Resection Measured Genome-wide by End Sequencing

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          SUMMARY

          DNA double-strand breaks (DSBs) arise during physiological transcription, DNA replication, and antigen receptor diversification. Mistargeting or misprocessing of DSBs can result in pathological structural variation and mutation. Here we describe a sensitive method (END-seq) to monitor DNA end resection and DSBs genome-wide at base-pair resolution in vivo. We utilized END-seq to determine the frequency and spectrum of restriction-enzyme-, zinc-finger-nuclease-, and RAG-induced DSBs. Beyond sequence preference, chromatin features dictate the repertoire of these genome-modifying enzymes. END-seq can detect at least one DSB per cell among 10,000 cells not harboring DSBs, and we estimate that up to one out of 60 cells contains off-target RAG cleavage. In addition to site-specific cleavage, we detect DSBs distributed over extended regions during immunoglobulin class-switch recombination. Thus, END-seq provides a snapshot of DNA ends genome-wide, which can be utilized for understanding genome-editing specificities and the influence of chromatin on DSB pathway choice.

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          In Brief

          Canela et al. develop a sensitive and quantitative method that provides a landscape of DNA double-strand breaks and end resection in vivo prior to DNA repair. This opens up the possibility for better understanding the causes and consequences of genome instability.

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          Author and article information

          Journal
          9802571
          20730
          Mol Cell
          Mol. Cell
          Molecular cell
          1097-2765
          1097-4164
          29 November 2018
          28 July 2016
          01 September 2016
          19 December 2018
          : 63
          : 5
          : 898-911
          Affiliations
          [1 ]Laboratory of Genome Integrity
          [2 ]Genetics Branch National Cancer Institute, NIH, Bethesda, MD 20892, USA
          [3 ]Department of Pathology and Laboratory Medicine, Weil Cornell Medical College, New York, NY 10065, USA
          Author notes

          AUTHOR CONTRIBUTIONS

          A.C. developed END-seq, A.C. and N.S. performed the experiments, A.T. and B.P.S. constructed the pre-B cell knockout strains, P.M. provided assistance on deep sequencing, A.C. and S.S. analyzed the data, A.C. and A.N. designed the study and wrote the manuscript, and A.N. supervised the project.

          Article
          PMC6299834 PMC6299834 6299834 nihpa998981
          10.1016/j.molcel.2016.06.034
          6299834
          27477910
          e6cc8e63-c7e3-4c9f-8024-5c7bbe361b79
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