The C-terminal  O helix of human Ogg1 is essential for 8-oxoguanine DNA glycosylase activity: the mitochondrial  -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.

Mitochondria-targeted Ogg1 and Aconitase-2 Prevent Oxidant-induced Mitochondrial DNA Damage in Alveolar Epithelial Cells

“…Compared with nuclear DNA, the mutation and damage rate of mtDNA by oxidative stress is severalfold higher in part because of the close proximity of mtDNA to the electron transport chain-generating ROS, the lack of histone-containing proteins that shield mtDNA, and the limited mtDNA repair systems (1)(2)(3)(4)(5)(6). A novel finding of this study is that mt-hOgg1-Mut, which lacks the crucial amino acids necessary for 8-oxoG DNA repair (25), prevents oxidant-induced AEC mtDNA damage in a manner similar to mt-hOgg1-WT (Fig. 2).…”

“…2). Notably, mt-Ogg1-Mut, which is incapable of DNA repair yet able to block intrinsic AEC apoptosis (11,25), also prevented oxidantinduced AEC mtDNA damage and intrinsic apoptosis similar to mt-hOgg1-WT (Fig. 2).…”

“…The above findings with oxidative stress-induced mtDNA damage are in accord with our earlier study showing that mt-hOgg1-WT, mt-hOgg1-Mut, or Aco-2 overexpression, each, prevent oxidant-induced AEC apoptosis despite high levels of mitochondrial ROS production, an effect that was attributed to a newly described chaperone function of mt-hOgg1-WT or mt-hOgg1-Mut in preventing oxidative degradation of Aco-2 (11). We reason that Aco-2 is necessary for the protective effects of mt-hOgg1-Mut but not mt-hOgg1-WT because the former is incapable of repairing mtDNA damage and Aco-2, and its mtDNA chaperone function is depleted by siACO-2 (11,25). Our findings concur with investigations showing that Ogg1 depletion promotes oxidant-induced apoptosis and extends these studies by demonstrating that Ogg1 and Aco-2 function coordinately to preserve mtDNA in the setting of oxidative stress (28,38).…”

The Recombination Protein RAD52 Cooperates with the Excision Repair Protein OGG1 for the Repair of Oxidative Lesions in Mammalian Cells

“…Purification of full-length, His 6 -tagged human RAD52 has been described previously (2). Purification of recombinant Histagged OGG1-␣ from Sf9 insect cells and His-and glutathione S-transferasetagged OGG1-␤ from E. coli has been described (13). Oligonucleotides were obtained from Midland Certified Reagent Co., and their sequences are shown in Table 1.…”

“…While OGG1-␣ is a bone fide DNA glycosylase (5) and localizes both to nuclei and mitochondria, OGG1-␤ localizes exclusively to mitochondria. We recently showed that the recombinant OGG1-␤ protein has no DNA glycosylase activity (13). The high degree of conservation of repair pathways for 8-oxoG, from bacteria to humans, along with epidemiological data correlating OGG1 polymorphisms and activity with predisposition to some cancers (11,27,33) attest to the biological importance of the repair of 8-oxoGs and other oxidative DNA lesions.…”