title: A rapid, comprehensive system for assaying DNA repair activity and cytotoxic effects of DNA-damaging reagents.
abstract: DNA repair systems protect cells from genomic instability and carcinogenesis. Therefore, assays for measuring DNA repair activity are valuable, not only for clinical diagnoses of DNA repair deficiency disorders but also for basic research and anticancer drug development. Two commonly used assays are UDS (unscheduled DNA synthesis, requiring a precise measurement of an extremely small amount of repair DNA synthesis) and RRS (recovery of RNA synthesis after DNA damage). Both UDS and RRS are major endpoints for assessing the activity of nucleotide excision repair (NER), the most versatile DNA repair process. Conventional UDS and RRS assays are laborious and time-consuming, as they measure the incorporation of radiolabeled nucleosides associated with NER. Here we describe a comprehensive protocol for monitoring nonradioactive UDS and RRS by studying the incorporation of alkyne-conjugated nucleoside analogs followed by a fluorescent azide-coupling click-chemistry reaction. The system is also suitable for quick measurement of cell sensitivity to DNA-damaging reagents and for lentivirus-based complementation assays, which can be used to systematically determine the pathogenic genes associated with DNA repair deficiency disorders. A typical UDS or RRS assay using primary fibroblasts, including a virus complementation test, takes 1 week to complete.
authors.affiliation: 1] Department of Pharmacology and Therapeutic Innovation, Graduate School of Biomedical Sciences, Nagasaki University, Nagasaki, Japan. [2] Nagasaki University Research Centre for Genomic Instability and Carcinogenesis (NRGIC), Nagasaki University, Nagasaki, Japan. [3] Department of Molecular Medicine, Atomic Bomb Disease Institute, Nagasaki University, Nagasaki, Japan.
authors.forename: Nan
authors.initials: N
authors.lastname: Jia
authors.affiliation: 1] Nagasaki University Research Centre for Genomic Instability and Carcinogenesis (NRGIC), Nagasaki University, Nagasaki, Japan. [2] Department of Molecular Medicine, Atomic Bomb Disease Institute, Nagasaki University, Nagasaki, Japan.
authors.forename: Yuka
authors.initials: Y
authors.lastname: Nakazawa
authors.affiliation: 1] Nagasaki University Research Centre for Genomic Instability and Carcinogenesis (NRGIC), Nagasaki University, Nagasaki, Japan. [2] Department of Molecular Medicine, Atomic Bomb Disease Institute, Nagasaki University, Nagasaki, Japan.
authors.forename: Chaowan
authors.initials: C
authors.lastname: Guo
authors.affiliation: 1] Nagasaki University Research Centre for Genomic Instability and Carcinogenesis (NRGIC), Nagasaki University, Nagasaki, Japan. [2] Department of Molecular Medicine, Atomic Bomb Disease Institute, Nagasaki University, Nagasaki, Japan.
authors.forename: Mayuko
authors.initials: M
authors.lastname: Shimada
authors.affiliation: St. John's Institute of Dermatology, Guy's and St. Thomas' Hospital, London, UK.
authors.forename: Mieran
authors.initials: M
authors.lastname: Sethi
authors.affiliation: Innovative Beauty Science Laboratory, Kanebo Cosmetics Inc., Odawara, Japan.
authors.forename: Yoshito
authors.initials: Y
authors.lastname: Takahashi
authors.affiliation: Department of Pharmacology and Therapeutic Innovation, Graduate School of Biomedical Sciences, Nagasaki University, Nagasaki, Japan.
authors.forename: Hiroshi
authors.initials: H
authors.lastname: Ueda
authors.affiliation: Department of Molecular Medicine, Atomic Bomb Disease Institute, Nagasaki University, Nagasaki, Japan.
authors.forename: Yuji
authors.initials: Y
authors.lastname: Nagayama
authors.affiliation: 1] Nagasaki University Research Centre for Genomic Instability and Carcinogenesis (NRGIC), Nagasaki University, Nagasaki, Japan. [2] Department of Molecular Medicine, Atomic Bomb Disease Institute, Nagasaki University, Nagasaki, Japan. [3] Microbial Genetics Laboratory, Genetic Strains Research Center, National Institute of Genetics, Mishima, Japan.
authors.forename: Tomoo
authors.initials: T
authors.lastname: Ogi
mesh_terms: D000480:Alkynes / Q000737:chemistry*; D001386:Azides / Q000737:chemistry*; D001681:Biological Assay / Q000379:methods*; D057930:Click Chemistry / Q000379:methods*; D004247:DNA / Q000096:biosynthesis; D004260:DNA Repair / Q000502:physiology*; D005453:Fluorescence; D005816:Genetic Complementation Test / Q000379:methods; D006801:Humans; D016086:Lentivirus; D009705:Nucleosides / Q000737:chemistry*; D012313:RNA / Q000096:biosynthesis; D012588:Scintillation Counting; D014466:Ultraviolet Rays
chemical_list: D000480:Alkynes; D001386:Azides; D009705:Nucleosides; D012313:RNA; D004247:DNA