Oxidized Guanine Lesions as Modulators of Gene Transcription. Altered p50 Binding Affinity and Repair Shielding by 7,8-Dihydro-8-oxo-2‘-deoxyguanosine Lesions in the NF-κB Promoter Element†
A number of common promoter elements that drive transcription of redox sensitive genes have runs of guanines in their transcription factor recognition sequence. A paradox exists insomuch that the same guanine runs necessary for transcription factor recognition are thermodynamically prone to oxidative modification, potentially altering the binding affinity of transcription factors. 7,8-Dihydro-8-oxo-2?-deoxyguanosine (8-oxo-dG) is a common oxidative modification of guanine that is generated by a variety of metals and reactive oxygen species. We have used the p50 subunit of the NF-?B transcription factor to show that oxidation of guanine to 8-oxo-dG at sites critical for protein recognition impacts transcription factor binding affinity differently depending upon the site of oxidation. It can be argued that the impact of such oxidation will be minimal in repair proficient cells. Therefore, we have developed an assay to assess the ability of these lesions to be shielded by transcription factor binding from recognition and repair by base excision repair (BER) enzymes. In this study, 8-oxo-dG was substituted for guanine at sites G1?G4 in the NF-?B sequence 5?-d(AGTTGAG1G2G3G4ACTTTCCCAGCC)-3?. We have observed that substitution of 8-oxo-dG at the G1 site increases p50 binding affinity by ?2.5-fold compared to that of the unmodified DNA sequence, while substitution at G3 reduces the binding affinity by ?4-fold. Substitution of 8-oxo-dG at the G2 and G4 sites had a minimal impact on p50 binding affinity. Both Escherichia coli fapy glycosylase (Fpg) and human 8-oxo-DNA glycosylase (hOGG1) recognized and cleaved 8-oxo-dG at all four sites within the promoter element. The addition of the p50 transcription factor shielded these lesions from cleavage by the glycosylase in a manner that correlated with the binding affinities of p50 for the different modified sites. These data imply that lesion formation in DNA response elements can modulate gene transcription during oxidative events and that protein binding to these modified sites may allow these lesions to persist on a time scale that impacts global cellular gene transcription.