8-Oxoguanine DNA glycosylase (Ogg1) causes a transcriptional inactivation of damaged DNA in the absence of functional Cockayne syndrome B (Csb) protein

We have analysed the effect of oxidative guanine lesions on the expression of a transfected reporter gene in mouse embryonic fibroblasts deficient in Cockayne syndrome B protein (Csb) and/or the 8-oxoguanine DNA glycosylase (Ogg1). We used a highly sensitive flow cytometry-based approach and quantit...

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Bibliographic Details
Published in:DNA repair 2009-03, Vol.8 (3), p.309-317
Main Authors: Khobta, Andriy, Kitsera, Nataliya, Speckmann, Bodo, Epe, Bernd
Format: Article
Language:English
Subjects:
8-Oxoguanine DNA glycosylase (Ogg1)
Animals
Base excision repair
Cell Line
Csb
DNA Damage
DNA Glycosylases - physiology
DNA Helicases - deficiency
DNA Repair Enzymes - deficiency
Flow Cytometry
Gene Expression
Gene Silencing
Genes, Reporter - drug effects
Genes, Reporter - genetics
Green Fluorescent Proteins
Guanine - analogs & derivatives
Guanine - biosynthesis
Host cell reactivation
Humans
Mice
Photosensitizing Agents - pharmacology
Poly-ADP-Ribose Binding Proteins
Reverse Transcriptase Polymerase Chain Reaction
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Summary:We have analysed the effect of oxidative guanine lesions on the expression of a transfected reporter gene in mouse embryonic fibroblasts deficient in Cockayne syndrome B protein (Csb) and/or the 8-oxoguanine DNA glycosylase (Ogg1). We used a highly sensitive flow cytometry-based approach and quantitative real-time PCR to measure the changes in gene expression caused by the presence of oxidised guanine residues generated by photosensitisation in the vector DNA. In wild-type cells, small numbers (one or three) of oxidised guanines did not affect gene expression at short times after transfections, whereas progressive reduction of the transgene expression was observed at later time points. Although Ogg1 has a major contribution to the repair of oxidised guanine bases, its absence did not have a strong effect on the gene expression. In contrast, the lack of functional Csb protein caused a pronounced inactivation of the damaged reporter gene. Most strikingly, an additional Ogg1 deficiency significantly attenuated this effect. The results indicate that the processing of oxidative guanine modifications by Ogg1 can mediate host cell inactivation rather than reactivation of the damaged genes and that this effect is strongly enhanced in the absence of Csb.
ISSN:1568-7864
1568-7856
DOI:10.1016/j.dnarep.2008.11.006