title: DNA Breaks and End Resection Measured Genome-wide by End Sequencing.
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.
authors.affiliation: Laboratory of Genome Integrity, National Cancer Institute, NIH, Bethesda, MD 20892, USA.
authors.forename: Andres
authors.initials: A
authors.lastname: Canela
authors.affiliation: Laboratory of Genome Integrity, National Cancer Institute, NIH, Bethesda, MD 20892, USA.
authors.forename: Sriram
authors.initials: S
authors.lastname: Sridharan
authors.affiliation: Laboratory of Genome Integrity, National Cancer Institute, NIH, Bethesda, MD 20892, USA.
authors.forename: Nicholas
authors.initials: N
authors.lastname: Sciascia
authors.affiliation: Laboratory of Genome Integrity, National Cancer Institute, NIH, Bethesda, MD 20892, USA.
authors.forename: Anthony
authors.initials: A
authors.lastname: Tubbs
authors.affiliation: Genetics Branch, National Cancer Institute, NIH, Bethesda, MD 20892, USA.
authors.forename: Paul
authors.initials: P
authors.lastname: Meltzer
authors.affiliation: Department of Pathology and Laboratory Medicine, Weil Cornell Medical College, New York, NY 10065, USA.
authors.forename: Barry P
authors.initials: BP
authors.lastname: Sleckman
authors.affiliation: Laboratory of Genome Integrity, National Cancer Institute, NIH, Bethesda, MD 20892, USA. Electronic address: andre_nussenzweig@nih.gov.
authors.forename: André
authors.initials: A
authors.lastname: Nussenzweig
mesh_terms: D000818:Animals; D064007:Ataxia Telangiectasia Mutated Proteins / Q000235:genetics / Q000276:immunology; D001402:B-Lymphocytes / Q000166:cytology / Q000276:immunology; D002843:Chromatin / Q000737:chemistry* / Q000276:immunology; D004247:DNA / Q000235:genetics* / Q000276:immunology; D053903:DNA Breaks, Double-Stranded; D004261:DNA Replication; D004268:DNA-Binding Proteins / Q000235:genetics / Q000276:immunology; D005786:Gene Expression Regulation; D016678:Genome; D059014:High-Throughput Nucleotide Sequencing / Q000379:methods*; D006657:Histones / Q000235:genetics / Q000276:immunology; D017578:Immunoglobulin Class Switching / Q000235:genetics; D051379:Mice; D054448:Precursor Cells, B-Lymphoid / Q000166:cytology / Q000276:immunology; D016693:Receptors, Antigen, T-Cell, alpha-beta / Q000235:genetics / Q000276:immunology; D011995:Recombination, Genetic; D060168:Thymocytes / Q000166:cytology / Q000276:immunology
chemical_list: D002843:Chromatin; D004268:DNA-Binding Proteins; D006657:Histones; C499308:Rag2 protein, mouse; D016693:Receptors, Antigen, T-Cell, alpha-beta; D004247:DNA; D064007:Ataxia Telangiectasia Mutated Proteins; C576325:Atm protein, mouse