title: The distribution of DNA damage is defined by region-specific susceptibility to DNA damage formation rather than repair differences.
abstract: The cellular genomes are continuously damaged by reactive oxygen species (ROS) from aerobic processes. The impact of DNA damage depends on the specific site as well as the cellular state. The steady-state level of DNA damage is the net result of continuous formation and subsequent repair, but it is unknown to what extent heterogeneous damage distribution is caused by variations in formation or repair of DNA damage. Here, we used a restriction enzyme/qPCR based method to analyze DNA damage in promoter and coding regions of four nuclear genes: the two house-keeping genes Gadph and Tbp, and the Ndufa9 and Ndufs2 genes encoding mitochondrial complex I subunits, as well as mt-Rnr1 encoded by mitochondrial DNA (mtDNA). The distribution of steady-state levels of damage varied in a site-specific manner. Oxidative stress induced damage in nDNA to a similar extent in promoter and coding regions, and more so in mtDNA. The subsequent removal of damage from nDNA was efficient and comparable with recovery times depending on the initial damage load, while repair of mtDNA was delayed with subsequently slower repair rate. The repair was furthermore found to be independent of transcription or the transcription-coupled repair factor CSB, but dependent on cellular ATP. Our results demonstrate that the capacity to repair DNA is sufficient to remove exogenously induced damage. Thus, we conclude that the heterogeneous steady-state level of DNA damage in promoters and coding regions is caused by site-specific DNA damage/modifications that take place under normal metabolism.
authors.affiliation: Department of Medical Biochemistry, Oslo University Hospital, University of Oslo, Norway; Department of Microbiology, Oslo University Hospital, University of Oslo, Norway.
authors.forename: Janne M
authors.initials: JM
authors.lastname: Strand
authors.affiliation: Department of Medical Biochemistry, Oslo University Hospital, University of Oslo, Norway; Department of Microbiology, Oslo University Hospital, University of Oslo, Norway.
authors.forename: Katja
authors.initials: K
authors.lastname: Scheffler
authors.affiliation: Department of Medical Biochemistry, Oslo University Hospital, University of Oslo, Norway; Department of Microbiology, Oslo University Hospital, University of Oslo, Norway.
authors.forename: Magnar
authors.initials: M
authors.lastname: Bjørås
authors.affiliation: Department of Medical Biochemistry, Oslo University Hospital, University of Oslo, Norway. Electronic address: lars.eide@medisin.uio.no.
authors.forename: Lars
authors.initials: L
authors.lastname: Eide
mesh_terms: D000255:Adenosine Triphosphate / Q000378:metabolism*; D053959:Alpha-Amanitin / Q000494:pharmacology; D000818:Animals; D002460:Cell Line; D004247:DNA / Q000032:analysis*; D004249:DNA Damage; D004260:DNA Repair / Q000187:drug effects; D045643:DNA Repair Enzymes / Q000378:metabolism*; D020043:Genes, Essential; D050259:Genes, Mitochondrial; D051379:Mice; D008810:Mice, Inbred C57BL; D016366:Open Reading Frames; D010084:Oxidation-Reduction; D018384:Oxidative Stress; D000075223:Poly-ADP-Ribose Binding Proteins; D011401:Promoter Regions, Genetic; D014158:Transcription, Genetic
chemical_list: D053959:Alpha-Amanitin; D000075223:Poly-ADP-Ribose Binding Proteins; D000255:Adenosine Triphosphate; D004247:DNA; C513075:Ercc6 protein, mouse; D045643:DNA Repair Enzymes
keywords: DNA repair; Genome dynamics; Genome integrity; Mitochondrial ROS; mtDNA damage; nDNA damage