Abstract
The promoter of the Kirsten ras (KRAS) proto-oncogene contains, upstream of the transcription start site, a quadruplex-forming motif called 32R with regulatory functions. As guanine under oxidative stress can be oxidized to 8-oxoguanine (8OG), we investigated the capacity of glycosylases 8-oxoguanine glycosylase (OGG1) and endonuclease VIII-like 1 (Neil1) to excise 8OG from 32R, either in duplex or G-quadruplex (G4) conformation. We found that OGG1 efficiently excised 8OG from oxidized 32R in duplex but not in G4 conformation. By contrast, glycosylase Neil1 showed more activity on the G4 than the duplex conformation. We also found that the excising activity of Neil1 on folded 32R depended on G4 topology. Our data suggest that Neil1, besides being involved in base excision repair pathway (BER), could play a role on KRAS transcription.
Highlights
We found that OGG1 efficiently excised 8OG from oxidized 32R in duplex but not in G4 conformation
This is based on the following experimental observations: (i) guanines in 32R tend to oxidize more than isolated guanines; the guanine at the 50 end of a G-run shows the lowest ionization potential, i.e., the highest tendency for oxidation [41,42,43]; (ii) the Kirsten ras (KRAS) gene is responsive to Reactive oxygen species (ROS), as it is upregulated when the cellular oxidative stress is increased by H2 O2 or by photoactivated porphyrins [39,44]; (iii) pulldown and chromatin immunoprecipitation (ChIP) experiments show that 8OG and
We investigated the capacity of glycosylases OGG1 and Neil1 to excise 8OG from 32R, either in duplex or G4 conformation
Summary
Neil on Oxidized G-Rich Motif in the KRAS Promoter. Int. As cancer cells produce higher amounts of ROS than normal cells [6,7,8], they have developed an efficient mechanism for controlling the redox homeostasis. It involves Nrf, a redox sensor protein that responds to oxidative stress and the thiol status of the cells [9]. Oxidized guanine is recognized in the cells by DNA glycosylases, i.e., enzymes involved in the base excision repair pathway (BER). Since ROS are enhanced in pancreatic cancer cells [6,7,8,24], they express a high level of Nrf to maintain the redox homeostasis [25]. The results of our study may have implications for the mechanism of Kirsten ras (KRAS) transcription
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