title: In Vitro Fluorogenic Real-Time Assay of the Repair of Oxidative DNA Damage.
abstract: The repair of oxidative damage to DNA is essential to avoid mutations that lead to cancer. Oxidized DNA bases, such as 8-oxoguanine, are a main source of these mutations, and the enzyme 8-oxoguanine glycosylase 1 (OGG1) is the chief human enzyme that excises 8-oxoguanine from DNA. The activity of OGG1 has been linked to human inflammation responses and to cancer, and researchers are beginning to search for inhibitors of the enzyme. However, measuring the activity of the enzyme typically requires laborious gel-based measurements of radiolabeled DNAs. Here we report the design and properties of fluorogenic probes that directly report on the activity of OGG1 (and its bacterial homologue Fpg) in real time as the oxidized base is excised. The probes are short, modified DNA oligomers containing fluorescent DNA bases and are designed to utilize 8-oxoguanine itself as a fluorescence quencher. Screening of combinations of fluorophores and 8-oxoguanine revealed two fluorophores, pyrene and tCo, that are strongly quenched by the damaged base. We tested 42 potential probes containing these fluorophores: the optimum probe, OGR1, yields a 60-fold light-up signal in vitro with OGG1 and Fpg. It can report on oxidative repair activity in mammalian cell lysate and with bacterial cells overexpressing a repair enzyme. Such probes might prove useful in quantifying enzyme activity and performing competitive inhibition assays.
authors.affiliation: Department of Chemistry, Stanford University, Stanford, CA 94305 (USA).
authors.forename: Sarah K
authors.initials: SK
authors.lastname: Edwards
authors.affiliation: Department of Chemistry, Stanford University, Stanford, CA 94305 (USA).|Present Address: Department of Chemistry and Biochemistry, Graduate School of Engineering, Center for Molecular Systems (CMS), Kyushu University, 744 Motooka, Nishi-ku, Fukuoka, 819-0395 (Japan).
authors.forename: Toshikazu
authors.initials: T
authors.lastname: Ono
authors.affiliation: Department of Chemistry, Stanford University, Stanford, CA 94305 (USA).
authors.forename: Shenliang
authors.initials: S
authors.lastname: Wang
authors.affiliation: Department of Chemistry, Stanford University, Stanford, CA 94305 (USA).
authors.forename: Wei
authors.initials: W
authors.lastname: Jiang
authors.affiliation: Department of Chemistry, Stanford University, Stanford, CA 94305 (USA).
authors.forename: Raphael M
authors.initials: RM
authors.lastname: Franzini
authors.affiliation: Department of Chemistry, Stanford University, Stanford, CA 94305 (USA).|Present Address: College of Pharmacy, Research Institute of Pharmaceutical Sciences, Kyungpook National University, Daegu, 702-701 (Republic of Korea).
authors.forename: Jong Wha
authors.initials: JW
authors.lastname: Jung
authors.affiliation: Department of Chemistry, Stanford University, Stanford, CA 94305 (USA).
authors.forename: Ke Min
authors.initials: KM
authors.lastname: Chan
authors.affiliation: Department of Chemistry, Stanford University, Stanford, CA 94305 (USA). kool@stanford.edu.
authors.forename: Eric T
authors.initials: ET
authors.lastname: Kool
mesh_terms: D000818:Animals; D004249:DNA Damage; D045647:DNA Glycosylases / Q000037:antagonists & inhibitors / Q000378:metabolism; D004260:DNA Repair / Q000187:drug effects; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005456:Fluorescent Dyes / Q000737:chemistry; D006147:Guanine / Q000031:analogs & derivatives / Q000737:chemistry / Q000378:metabolism; D006801:Humans; D051379:Mice; D010084:Oxidation-Reduction; D013050:Spectrometry, Fluorescence; D013997:Time Factors
chemical_list: D004791:Enzyme Inhibitors; D005456:Fluorescent Dyes; C024829:8-hydroxyguanine; D006147:Guanine; D045647:DNA Glycosylases; C479058:oxoguanine glycosylase 1, human
keywords: DNA damage; Fpg; OGG1; fluorescent probes; oxoguanine