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Mol Cell. 2016 Sep 1;63(5):898-911. doi: 10.1016/j.molcel.2016.06.034. Epub 2016 Jul 28.

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

Author information

1
Laboratory of Genome Integrity, National Cancer Institute, NIH, Bethesda, MD 20892, USA.
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.
4
Laboratory of Genome Integrity, National Cancer Institute, NIH, Bethesda, MD 20892, USA. Electronic address: andre_nussenzweig@nih.gov.

Abstract

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.

PMID:
27477910
PMCID:
PMC6299834
DOI:
10.1016/j.molcel.2016.06.034
[Indexed for MEDLINE]
Free PMC Article

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