Development of a novel method to create double-strand break repair fingerprints using next-generation sequencing |
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| Affiliation: | 1. Department of Internal Medicine, University of Connecticut, Farmington, CT 06030, United States;2. Department of Therapeutic Radiology, Yale University School of Medicine, New Haven, CT 06511, United States;3. Yale Center for Genomic Analysis (YCGA), Orange, CT 06477, United States;4. Department of Computer Science, Yale University, New Haven, CT 06511, United States;1. EaStChem School of Chemistry, University of Edinburgh, The King’s Buildings, Edinburgh EH9 3JJ, UK;2. New England BioLabs, Inc., 240 County Road, Ipswich, MA 01938, USA;1. Department of Medicine, Sri Venkateswara Institute of Medical Sciences, Tirupati, Andhra Pradesh, 517507, India;2. Department of Biotechnology, Sri Venkateswara Institute of Medical Sciences, Tirupati, Andhra Pradesh, 517507, India;3. Department of Statistics, Sri Venkateswara University, Tirupati, Andhra Pradesh, 517502, India;1. Nanobiotechnology Lab, Department of Bioproducts and Biosystems Engineering, Biotechnology Institute, University of Minnesota, St. Paul, MN 55108, USA;2. c-LEcta GmbH, Perlickstraße 5, 04103 Leipzig, Germany;3. Industrial Biotechnology Division, National Institute for Biotechnology and Genetic Engineering (NIBGE), Jhang Road, Faisalabad, Pakistan;4. Department of Bioinformatics and Biotechnology, Government College University Faisalabad (GCUF), Allama Iqbal Road, 38000 Faisalabad, Pakistan;1. Inorganic and Physical Chemistry Division, CSIR-Indian Institute of Chemical Technology, Hyderabad 500007, India;2. Bioengineering and Environmental Centre, CSIR-Indian Institute of Chemical Technology, Hyderabad 500007, India;3. RMIT-IICT Research Centre, CSIR-Indian Institute of Chemical Technology, Hyderabad 500007, India;4. Advanced Materials & Industrial Chemistry Group, School of Applied Sciences, RMIT University, Melbourne, Australia;1. USDA-Agricultural Research Service, Wind Erosion & Water Conservation Research Unit, Lubbock, TX, USA;2. The University of Texas at El Paso, Department of Chemistry, El Paso, TX 79968, USA;3. USDA-ARS, Central Great Plains Research Station, 40335 County Road GG, Akron, CO 80720, USA;4. USDA-Agricultural Research Service, Wind Erosion & Water Conservation Research Unit, Big Spring, TX, USA;5. Department of Soil Science, North Carolina State University, Raleigh, NC 27695-7619, USA;6. Atmospheric Environment Research Centre, Griffith School of Environment, Griffith University, Brisbane, Australia;1. Department of Nanobiotechnology, Vision Research Foundation, Chennai, India;2. Nanomedicine-Laboratory of Immunology and Molecular Biomedical Research (NLIMBR), School of Medicine (SoM), Centre for Molecular and Medical Research (C-MMR), Faculty of Health, Deakin University, Geelong, Pigdons Road, Waurn Ponds, Geelong, Victoria 3217, Australia;3. Bioinformatics Centre, Vision Research Foundation, Chennai, India;4. Graduate Student Deakin University (ID: 213510168), Nanomedicine-Laboratory of Immunology and Molecular Biomedical Research (NLIMBR), School of Medicine, Deakin University, Australia |
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| 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. |
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| Keywords: | NHEJ HR I-SceI DSB repair |
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