Transcription through 8-oxoguanine in DNA repair-proficient and Csb(-)/Ogg1(-) DNA repair-deficient mouse embryonic fibroblasts is dependent upon promoter strength and sequence context.
Cells from Cockayne syndrome patients are characterized by a deficiency in transcription-coupled repair (TCR) of UV-induced lesions. These cells have also been shown to be sensitive to oxidative stress and defective in TCR of some oxidative lesions. Because some discrepancies about this pathway have been recently reported in the literature, we describe here a system that allows us to analyze the effect of a unique 8-oxoguanine (8-oxoG) lesion on gene transcription in vivo. We have constructed nonreplicative shuttle vectors containing a single 8-oxoG in the transcribed strand of the luciferase reporter gene. We have positioned this unique lesion in different sequence contexts and we have tested the effect of two promoters with different transcriptional strength on the level of transcriptional bypass/pause due to the presence of the lesion. When we transfected DNA repair-deficient mouse cell lines with these shuttle vectors, we found a approximately 50% decrease in relative luciferase activity in Ogg1(-/-) and Csb(-/-) embryonic mouse cell lines. In Csb(-/-)/Ogg1(-/-) cells, this decrease was even more important achieving eventually up to 90% inhibition of luciferase expression depending upon the promoter strength and the position of the lesion. These results show clearly that a unique 8-oxoG exhibits different effect on gene expression depending upon the nucleotidic sequence around it and needs the wild-type activities of Csb and Ogg1 proteins to be fully repaired.
Pastoriza-Gallego
Manuela
M
Laboratory of Genetic Instability and Cancer, FRE 2939 CNRS, Institut Gustave Roussy, 94805 Villejuif, France.
Armier
Jacques
J
Sarasin
Alain
A
D000818:Animals; D001419:Bacteria / Q000187:drug effects / Q000235:genetics; D001483:Base Sequence; D002460:Cell Line; D003057:Cockayne Syndrome / Q000235:genetics; D045647:DNA Glycosylases / Q000235:genetics / Q000502:physiology; D004260:DNA Repair; D045643:DNA Repair Enzymes / Q000235:genetics / Q000502:physiology; D004622:Embryo, Mammalian / Q000166:cytology; D005347:Fibroblasts / Q000378:metabolism; D006147:Guanine / Q000031:analogs & derivatives* / Q000378:metabolism / Q000494:pharmacology; D008156:Luciferases / Q000032:analysis / Q000235:genetics; D051379:Mice; D018345:Mice, Knockout; D016296:Mutagenesis; D009152:Mutagenicity Tests; D000075223:Poly-ADP-Ribose Binding Proteins; D011401:Promoter Regions, Genetic; D014158:Transcription, Genetic / Q000235:genetics*
D000075223:Poly-ADP-Ribose Binding Proteins; C024829:8-hydroxyguanine; D006147:Guanine; D008156:Luciferases; D045647:DNA Glycosylases; C485990:Ogg1 protein, mouse; C513075:Ercc6 protein, mouse; D045643:DNA Repair Enzymes