title: Development of a novel method to create double-strand break repair fingerprints using next-generation sequencing.
abstract: Efficient DNA double-strand break (DSB) repair is a critical determinant of cell survival in response to DNA damaging agents, and it plays a key role in the maintenance of genomic integrity. Homologous recombination (HR) and non-homologous end-joining (NHEJ) represent the two major pathways by which DSBs are repaired in mammalian cells. We now understand that HR and NHEJ repair are composed of multiple sub-pathways, some of which still remain poorly understood. As such, there is great interest in the development of novel assays to interrogate these key pathways, which could lead to the development of novel therapeutics, and a better understanding of how DSBs are repaired. Furthermore, assays which can measure repair specifically at endogenous chromosomal loci are of particular interest, because of an emerging understanding that chromatin interactions heavily influence DSB repair pathway choice. Here, we present the design and validation of a novel, next-generation sequencing-based approach to study DSB repair at chromosomal loci in cells. We demonstrate that NHEJ repair "fingerprints" can be identified using our assay, which are dependent on the status of key DSB repair proteins. In addition, we have validated that our system can be used to detect dynamic shifts in DSB repair activity in response to specific perturbations. This approach represents a unique alternative to many currently available DSB repair assays, which typical rely on the expression of reporter genes as an indirect read-out for repair. As such, we believe this tool will be useful for DNA repair researchers to study NHEJ repair in a high-throughput and sensitive manner, with the capacity to detect subtle changes in DSB repair patterns that was not possible previously.
authors.affiliation: Department of Internal Medicine, University of Connecticut, Farmington, CT 06030, United States.
authors.forename: Chen-Pang
authors.initials: CP
authors.lastname: Soong
authors.affiliation: Department of Therapeutic Radiology, Yale University School of Medicine, New Haven, CT 06511, United States.
authors.forename: Gregory A
authors.initials: GA
authors.lastname: Breuer
authors.affiliation: Department of Therapeutic Radiology, Yale University School of Medicine, New Haven, CT 06511, United States.
authors.forename: Ryan A
authors.initials: RA
authors.lastname: Hannon
authors.affiliation: Department of Therapeutic Radiology, Yale University School of Medicine, New Haven, CT 06511, United States.
authors.forename: Savina D
authors.initials: SD
authors.lastname: Kim
authors.affiliation: Department of Therapeutic Radiology, Yale University School of Medicine, New Haven, CT 06511, United States.
authors.forename: Ahmed F
authors.initials: AF
authors.lastname: Salem
authors.affiliation: Yale Center for Genomic Analysis (YCGA), Orange, CT 06477, United States.
authors.forename: Guilin
authors.initials: G
authors.lastname: Wang
authors.affiliation: Department of Therapeutic Radiology, Yale University School of Medicine, New Haven, CT 06511, United States.
authors.forename: Ruoxi
authors.initials: R
authors.lastname: Yu
authors.affiliation: Department of Computer Science, Yale University, New Haven, CT 06511, United States.
authors.forename: Nicholas J
authors.initials: NJ
authors.lastname: Carriero
authors.affiliation: Department of Computer Science, Yale University, New Haven, CT 06511, United States.
authors.forename: Robert
authors.initials: R
authors.lastname: Bjornson
authors.affiliation: Department of Therapeutic Radiology, Yale University School of Medicine, New Haven, CT 06511, United States.
authors.forename: Ranjini K
authors.initials: RK
authors.lastname: Sundaram
authors.affiliation: Department of Therapeutic Radiology, Yale University School of Medicine, New Haven, CT 06511, United States. Electronic address: ranjit.bindra@yale.edu.
authors.forename: Ranjit S
authors.initials: RS
authors.lastname: Bindra
mesh_terms: D000818:Animals; D002843:Chromatin / Q000378:metabolism; D004247:DNA / Q000378:metabolism; D053903:DNA Breaks, Double-Stranded; D059766:DNA End-Joining Repair; D004252:DNA Mutational Analysis / Q000379:methods*; D004268:DNA-Binding Proteins / Q000378:metabolism; D056426:Genetic Loci; D059014:High-Throughput Nucleotide Sequencing / Q000379:methods*; D006801:Humans; D054643:INDEL Mutation; D008322:Mammals; D059767:Recombinational DNA Repair
chemical_list: D002843:Chromatin; D004268:DNA-Binding Proteins; D004247:DNA
keywords: DSB repair; HR; I-SceI; NHEJ