The C-terminal αO helix of human Ogg1 is essential for 8-oxoguanine DNA glycosylase activity: the mitochondrial β-Ogg1 lacks this domain and does not have glycosylase activity

• Published in: Nucleic acids research 2004, Vol.32 (18), p.5596-5608
• Main Authors: Hashiguchi, K., Stuart, J. A., de Souza-Pinto, N. C., Bohr, V. A.
• Format: Article
• Language: English
• ISSN: 0305-1048; 1362-4962
• DOI: 10.1093/nar/gkh863

The human Ogg1 glycosylase is responsible for repairing 8-oxo-7,8-dihydroguanine (8-oxoG) in both nuclear and mitochondrial DNA. Two distinct Ogg1 isoforms are present; α-Ogg1, which mainly localizes to the nucleus and β-Ogg1, which localizes only to mitochondria. We recently showed that mitochondria from ρ0 cells, which lack mitochondrial DNA, have similar 8-oxoG DNA glycosylase activity to that of wild-type cells. Here, we show that β-Ogg1 protein levels are ∼80% reduced in ρ0 cells, suggesting β-Ogg1 is not responsible for 8-oxoG incision in mitochondria. Thus, we characterized the biochemical properties of recombinant β-Ogg1. Surprisingly, recombinant β-Ogg1 did not show any significant 8-oxoG DNA glycosylase activity in vitro. Since β-Ogg1 lacks the C-terminal αO helix present in α-Ogg1, we generated mutant proteins with various amino acid substitutions in this domain. Of the seven amino acid positions substituted (317–323), we identified Val-317 as a novel critical residue for 8-oxoG binding and incision. Our results suggest that the αO helix is absolutely necessary for 8-oxoG DNA glycosylase activity, and thus its absence may explain why β-Ogg1 does not catalyze 8-oxoG incision in vitro. Western blot analysis revealed the presence of significant amounts of α-Ogg1 in human mitochondria. Together with previous localization studies in vivo, this suggests that α-Ogg1 protein may provide the 8-oxoG DNA glycosylase activity for the repair of these lesions in human mitochondrial DNA. β-Ogg1 may play a novel role in human mitochondria.