title: 8-Oxoguanine-mediated transcriptional mutagenesis causes Ras activation in mammalian cells.
abstract: 8-Oxoguanine (8OG) is efficiently bypassed by RNA polymerases in vitro and in bacterial cells in vivo, leading to mutant transcripts by directing incorporation of an incorrect nucleotide during transcription. Such transcriptional mutagenesis (TM) may produce a pool of mutant proteins. In contrast, transcription-coupled repair safeguards against DNA damage, contingent upon the ability of lesions to arrest elongating RNA polymerase. In mammalian cells, the Cockayne syndrome B protein (Csb) mediates transcription-coupled repair, and its involvement in the repair of 8OG is controversial. The DNA glycosylase Ogg1 initiates base excision repair of 8OG, but its influence on TM is unknown. We have developed a mammalian system for TM in congenic mouse embryonic fibroblasts (MEFs), either WT or deficient in Ogg1 (ogg(-/-)), Csb (csb(-/-)), or both. This system uses expression of the Ras oncogene in which an 8OG replaces guanine in codon 61. Repair of 8OG restores the WT sequence; however, bypass and misinsertion opposite this lesion during transcription leads to a constitutively active mutant Ras protein and activation of downstream signaling events, including increased phosphorylation of ERK kinase. Upon transfection of MEFs with replication-incompetent 8OG constructs, we observed a marked increase in phospho-ERK in ogg(-/-) and csb(-/-)ogg(-/-) cells at 6 h, indicating persistence of the lesion and the occurrence of TM. This effect is absent in WT and csb(-/-) cells, suggesting rapid repair. These studies provide evidence that 8OG causes TM in mammalian cells, leading to a phenotypic change with important implications for the role of TM in tumorigenesis.
authors.affiliation: Department of Biochemistry, Emory University School of Medicine, Atlanta, GA 30322, USA.
authors.forename: Tina T
authors.initials: TT
authors.lastname: Saxowsky
authors.forename: Kellen L
authors.initials: KL
authors.lastname: Meadows
authors.forename: Arne
authors.initials: A
authors.lastname: Klungland
authors.forename: Paul W
authors.initials: PW
authors.lastname: Doetsch
mesh_terms: D000818:Animals; D002478:Cells, Cultured; D004249:DNA Damage; D045647:DNA Glycosylases / Q000235:genetics / Q000378:metabolism; D004260:DNA Repair; D045643:DNA Repair Enzymes / Q000235:genetics / Q000378:metabolism; D004789:Enzyme Activation; D005347:Fibroblasts / Q000166:cytology / Q000502:physiology; D006147:Guanine / Q000031:analogs & derivatives* / Q000494:pharmacology; D006801:Humans; D051379:Mice; D018345:Mice, Knockout; D016296:Mutagenesis; D000075223:Poly-ADP-Ribose Binding Proteins; D014158:Transcription, Genetic; D018631:ras Proteins / Q000235:genetics / Q000378:metabolism*
chemical_list: D000075223:Poly-ADP-Ribose Binding Proteins; C024829:8-hydroxyguanine; D006147:Guanine; D045647:DNA Glycosylases; C485990:Ogg1 protein, mouse; C513075:Ercc6 protein, mouse; D018631:ras Proteins; D045643:DNA Repair Enzymes