title: Transcription activities at 8-oxoG lesions in DNA.
abstract: 7,8-Dihydro-8-oxoguanine (8-oxoG) is the most frequent mutagenic lesion caused by oxidative stress. Eukaryotic cells use a specific DNA glycosylase, OGG1, to excise 8-oxoG from DNA. The mild phenotype of OGG1 null mice has been attributed to the existence of alternative pathways, including Cockayne syndrome B (CSB)-dependent transcription coupled repair (TCR), for removal of 8-oxoG. We have studied repair and transcription activities at 8-oxoG lesions with a reconstituted transcription system (RTS; RNA polymerase II, TBP, TFIIA, TFIIB, TFIIE, TFIIF and TFIIH), as well as in cellular extracts and in vivo. All measurable repair activity at 8-oxoG lesions takes place in the 3'-direction from the lesion, indicating base excision repair (BER) activity and negligible role of nucleotide excision repair (NER). Although 8-oxoG has been shown to be preferentially removed from the transcribed strand, in vitro experiments with purified transcription factors failed to identify a definite block for RNA polymerase II at the lesion. However, a weak block was observed at the lesion during transcription carried out with RTS as well as with cellular extracts. RNA polymerase II was identified at the site of the lesion on obstructed templates. Wild-type cells, as well as cells carrying targeted mutations of genes required for removal of 8-oxoG, were transfected with a luciferase expression vector containing an 8-oxoG lesion. No significant obstruction at 8-oxoG lesions was observed by this in vivo approach. In control experiments transcription elongation was completely blocked by cisplatin.
authors.affiliation: Centre for Molecular Biology and Neuroscience, Institute of Medical Microbiology, National Hospital, University of Oslo, 0027 Oslo, Norway.
authors.forename: Elisabeth
authors.initials: E
authors.lastname: Larsen
authors.forename: Kyungrim
authors.initials: K
authors.lastname: Kwon
authors.forename: Frédéric
authors.initials: F
authors.lastname: Coin
authors.forename: Jean-Marc
authors.initials: JM
authors.lastname: Egly
authors.forename: Arne
authors.initials: A
authors.lastname: Klungland
mesh_terms: D000818:Animals; D001483:Base Sequence; D003057:Cockayne Syndrome / Q000235:genetics / Q000378:metabolism; D004247:DNA / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D004249:DNA Damage; D004265:DNA Helicases / Q000172:deficiency / Q000235:genetics; D004260:DNA Repair; D045643:DNA Repair Enzymes; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D015870:Gene Expression; D006151:Guanosine / Q000031:analogs & derivatives* / Q000737:chemistry*; D006367:HeLa Cells; D006801:Humans; D066298:In Vitro Techniques; D008156:Luciferases / Q000235:genetics; D051379:Mice; D018345:Mice, Knockout; D008969:Molecular Sequence Data; D018384:Oxidative Stress; D010957:Plasmids / Q000235:genetics; D000075223:Poly-ADP-Ribose Binding Proteins; D014158:Transcription, Genetic
chemical_list: D000075223:Poly-ADP-Ribose Binding Proteins; D006151:Guanosine; C046215:8-hydroxyguanosine; D004247:DNA; D008156:Luciferases; D004265:DNA Helicases; C078673:ERCC6 protein, human; C513075:Ercc6 protein, mouse; D045643:DNA Repair Enzymes