title: Comet-FISH with strand-specific probes reveals transcription-coupled repair of 8-oxoGuanine in human cells.
abstract: Oxidized bases in DNA have been implicated in cancer, aging and neurodegenerative disease. We have developed an approach combining single-cell gel electrophoresis (comet) with fluorescence in situ hybridization (FISH) that enables the comparative quantification of low, physiologically relevant levels of DNA lesions in the respective strands of defined nucleotide sequences and in the genome overall. We have synthesized single-stranded probes targeting the termini of DNA segments of interest using a polymerase chain reaction-based method. These probes facilitate detection of damage at the single-molecule level, as the lesions are converted to DNA strand breaks by lesion-specific endonucleases or glycosylases. To validate our method, we have documented transcription-coupled repair of cyclobutane pyrimidine dimers in the ataxia telangiectasia-mutated (ATM) gene in human fibroblasts irradiated with 254 nm ultraviolet at 0.1 J/m2, a dose ∼100-fold lower than those typically used. The high specificity and sensitivity of our approach revealed that 7,8-dihydro-8-oxoguanine (8-oxoG) at an incidence of approximately three lesions per megabase is preferentially repaired in the transcribed strand of the ATM gene. We have also demonstrated that the hOGG1, XPA, CSB and UVSSA proteins, as well as actively elongating RNA polymerase II, are required for this process, suggesting cross-talk between DNA repair pathways.
authors.affiliation: Department of Biology, Stanford University, 371 Serra Mall, Stanford, CA 94305-5020, USA.
authors.forename: Jia
authors.initials: J
authors.lastname: Guo
authors.forename: Philip C
authors.initials: PC
authors.lastname: Hanawalt
authors.forename: Graciela
authors.initials: G
authors.lastname: Spivak
mesh_terms: D064007:Ataxia Telangiectasia Mutated Proteins; D002352:Carrier Proteins / Q000502:physiology; D018797:Cell Cycle Proteins / Q000096:biosynthesis / Q000235:genetics; D002460:Cell Line; D020552:Comet Assay / Q000379:methods*; D004265:DNA Helicases / Q000502:physiology; D015342:DNA Probes; D004260:DNA Repair; D045643:DNA Repair Enzymes / Q000502:physiology; D004268:DNA-Binding Proteins / Q000096:biosynthesis / Q000235:genetics; D006147:Guanine / Q000031:analogs & derivatives* / Q000378:metabolism; D006801:Humans; D017404:In Situ Hybridization, Fluorescence / Q000379:methods*; D000075223:Poly-ADP-Ribose Binding Proteins; D017346:Protein Serine-Threonine Kinases / Q000096:biosynthesis / Q000235:genetics; D011740:Pyrimidine Dimers / Q000378:metabolism; D012319:RNA Polymerase II / Q000378:metabolism; D014158:Transcription, Genetic; D025521:Tumor Suppressor Proteins / Q000096:biosynthesis / Q000235:genetics
chemical_list: D002352:Carrier Proteins; D018797:Cell Cycle Proteins; D015342:DNA Probes; D004268:DNA-Binding Proteins; D000075223:Poly-ADP-Ribose Binding Proteins; D011740:Pyrimidine Dimers; D025521:Tumor Suppressor Proteins; C573137:UVSSA protein, human; C024829:8-hydroxyguanine; D006147:Guanine; C576324:ATM protein, human; D064007:Ataxia Telangiectasia Mutated Proteins; D017346:Protein Serine-Threonine Kinases; D012319:RNA Polymerase II; D004265:DNA Helicases; C078673:ERCC6 protein, human; D045643:DNA Repair Enzymes