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

ABSTRACT: 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; alpha-Ogg1, which mainly localizes to the nucleus and beta-Ogg1, which localizes only to mitochondria. We recently showed that mitochondria from rho(0) cells, which lack mitochondrial DNA, have similar 8-oxoG DNA glycosylase activity to that of wild-type cells. Here, we show that beta-Ogg1 protein levels are approximately 80% reduced in rho(0) cells, suggesting beta-Ogg1 is not responsible for 8-oxoG incision in mitochondria. Thus, we characterized the biochemical properties of recombinant beta-Ogg1. Surprisingly, recombinant beta-Ogg1 did not show any significant 8-oxoG DNA glycosylase activity in vitro. Since beta-Ogg1 lacks the C-terminal alphaO helix present in alpha-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 alphaO helix is absolutely necessary for 8-oxoG DNA glycosylase activity, and thus its absence may explain why beta-Ogg1 does not catalyze 8-oxoG incision in vitro. Western blot analysis revealed the presence of significant amounts of alpha-Ogg1 in human mitochondria. Together with previous localization studies in vivo, this suggests that alpha-Ogg1 protein may provide the 8-oxoG DNA glycosylase activity for the repair of these lesions in human mitochondrial DNA. beta-Ogg1 may play a novel role in human mitochondria.

SUBMITTER: Hashiguchi K 

PROVIDER: S-EPMC524278 | biostudies-literature | 2004

REPOSITORIES: biostudies-literature