8-Oxoguanosine and uracil repair of nuclear and mitochondrial DNA in red and white skeletal muscle of exercise-trained old rats.

Oxoguanine DNA glycosylase (OGG1) and uracil DNA glycosylase (UDG) are two of the most important repair enzymes that are involved in the base excision repair processes to eliminate oxidative damage from mammalian DNA, which accumulates with aging. Red and white skeletal muscle fibers have very different antioxidant enzyme activities and resistance to oxidative stress. In this paper, we demonstrate that the activity of OGG1 is significantly higher in the red type of skeletal muscle compared with white fibers from old rats. Exercise training resulted in increased OGG1 activity in the nuclei of red fibers and decreased activity in nuclei of white fibers and in the mitochondria of both red and white fibers. The activities of UDG were similar in both red and white muscle fibers. Exercise training appears to increase the activity of UDG in the nuclei and mitochondria. However, exercise training affects the activity of OGG1 in nuclei and mitochondria differently, suggesting different regulation of the enzymes. In contrast, UDG showed similar activities in nuclei and mitochondrial extracts of exercise-trained animals. These data provide evidence for differential regulation of UDG and OGG1 in maintaining fidelity of DNA in oxidatively stressed cells.     

Dimerization and opposite base-dependent catalytic impairment of polymorphic S326C OGG1 glycosylase

Human 8-oxoguanine-DNA glycosylase (OGG1) is the major enzyme for repairing 8-oxoguanine (8-oxoG), a mutagenic guanine base lesion produced by reactive oxygen species (ROS). A frequently occurring OGG1 polymorphism in human populations results in the substitution of serine 326 for cysteine (S326C). The 326 C/C genotype is linked to numerous cancers, although the mechanism of carcinogenesis associated with the variant is unclear. We performed detailed enzymatic studies of polymorphic OGG1 and found functional defects in the enzyme. S326C OGG1 excised 8-oxoG from duplex DNA and cleaved abasic sites at rates 2- to 6-fold lower than the wild-type enzyme, depending upon the base opposite the lesion. Binding experiments showed that the polymorphic OGG1 binds DNA damage with significantly less affinity than the wild-type enzyme. Remarkably, gel shift, chemical cross-linking and gel filtration experiments showed that S326C both exists in solution and binds damaged DNA as a dimer. S326C OGG1 enzyme expressed in human cells was also found to have reduced activity and a dimeric conformation. The glycosylase activity of S326C OGG1 was not significantly stimulated by the presence of AP-endonuclease. The altered substrate specificity, lack of stimulation by AP-endonuclease 1 (APE1) and anomalous DNA binding conformation of S326C OGG1 may contribute to its linkage to cancer incidence.     

Expression and differential intracellular localization of two major forms of human 8-oxoguanine DNA glycosylase encoded by alternatively spliced OGG1 mRNAs

We identified seven alternatively spliced forms of human 8-oxoguanine DNA glycosylase (OGG1) mRNAs, classified into two types based on their last exons (type 1 with exon 7: 1a and 1b; type 2 with exon 8: 2a to 2e). Types 1a and 2a mRNAs are major in human tissues. Seven mRNAs are expected to encode different polypeptides (OGG1-1a to 2e) that share their N terminus with the common mitochondrial targeting signal, and each possesses a unique C terminus. A 36-kDa polypeptide, corresponding to OGG1-1a recognized only by antibodies against the region containing helix-hairpin-helix-PVD motif, was copurified from the nuclear extract with an activity introducing a nick into DNA containing 8-oxoguanine. A 40-kDa polypeptide corresponding to a processed form of OGG1-2a was detected in their mitochondria using antibodies against its C terminus. Electron microscopic immunocytochemistry and subfractionation of the mitochondria revealed that OGG1-2a locates on the inner membrane of mitochondria. Deletion mutant analyses revealed that the unique C terminus of OGG1-2a and its mitochondrial targeting signal are essential for mitochondrial localization and that nuclear localization of OGG1-1a depends on the NLS at its C terminus.     

Specificity of stimulation of human 8-oxoguanine-DNA glycosylase by AP endonuclease

Human 8-oxoguanine-DNA glycosylase OGG1 is an enzyme that removes abundant oxidative lesion 8-oxoguanine (8-oxoG) from DNA. Excision of 8-oxoG by OGG1 is inhibited by the abasic DNA reaction product and is stimulated by AP endonuclease APEX1. Besides 8-oxoG, OGG1 shows activity towards several other base lesions. Here we report that APEX1 efficiently stimulates OGG1 on good substrates (8-oxoadenine, 8-oxoinosine, or 6-methoxy-8-oxoguanine opposite to cytosine) but the stimulation is low or absent with poor OGG1 substrates (8-oxoadenine or 8-oxoinosine opposite to thymine; 8-oxoG or 8-aminoguanine opposite to adenine; 8-oxonebularine, 8-metoxyguanine, inosine or guanine opposite to cytosine). APEX1 significantly improves the ability of OGG1 to excise 8-aminoguanine from its naturally occurring pair with cytosine, making it possible that OGG1 repairs this lesion. Overall, APEX1 serves to improve specificity of OGG1 for its biologically relevant substrates.     

Use of a molecular beacon to track the activity of base excision repair protein OGG1 in live cells

An abundant form of DNA damage caused by reactive oxygen species is 8-oxo-7,8-dihydroguanine for which the base excision repair protein 8-oxoguanine-DNA glycosylase 1 (OGG1) is a major repair enzyme. To assess the location and intracellular activity of the OGG1 protein in response to oxidative stress, we have utilised a fluorescence–quench molecular beacon switch containing a 8-oxo-dG:C base pair and a fluorescent and quencher molecule at opposite ends of a hairpin oligonucleotide. Oxidative stress was induced by treatment with potassium bromate. Flow cytometry demonstrated a concentration-dependent increase in the activity of OGG1 that was detected by the fluorescence produced when the oligonucleotide was cleaved in the cells treated with potassium bromate. This signal is highly specific and not detectable in OGG1 knock out cells. Induction of OGG1 activity is not a result of induction of OGG1 gene expression as assessed by qPCR suggesting a role for protein stabilisation or increased OGG1 catalytic activity. High resolution confocal microscopy pinpointed the location of the fluorescent molecular beacon in live cells to perinuclear regions that were identified as mitochondria by co-staining with mitotracker dye. There is no evidence of cut beacon within the nuclear compartment of the cell. Control experiments with a positive control beacon (G:C base pair and lacking the DAB quencher) did not result in mitochondrial localisation of fluorescence signal indicating that the dye does not accumulate in mitochondria independent of OGG1 activity. Furthermore, faint nuclear staining was apparent confirming that the beacon structure is able to enter the nucleus. In conclusion, these data indicate that the mitochondria are the major site for OGG1 repair activity under conditions of oxidative stress.     

Kinetic conformational analysis of human 8-oxoguanine-DNA glycosylase

7,8-dihydro-8-oxoguanine (8-oxoG) is one of the major DNA lesions formed by reactive oxygen species that can result in transversion mutations following replication if left unrepaired. In human cells, the effects of 8-oxoG are counteracted by OGG1, a DNA glycosylase that catalyzes excision of 8-oxoguanine base followed by a much slower beta-elimination reaction at the 3'-side of the resulting abasic site. Many features of OGG1 mechanism, including its low beta-elimination activity and high specificity for a cytosine base opposite the lesion, remain poorly explained despite the availability of structural information. In this study, we analyzed the substrate specificity and the catalytic mechanism of OGG1 acting on various DNA substrates using stopped-flow kinetics with fluorescence detection. Combining data on intrinsic tryptophan fluorescence to detect conformational transitions in the enzyme molecule and 2-aminopurine reporter fluorescence to follow DNA dynamics, we defined three pre-excision steps and assigned them to the processes of (i) initial encounter with eversion of the damaged base, (ii) insertion of several enzyme residues into DNA, and (iii) enzyme isomerization to the catalytically competent form. The individual rate constants were derived for all reaction stages. Of all conformational changes, we identified the insertion step as mostly responsible for the opposite base specificity of OGG1 toward 8-oxoG:C as compared with 8-oxoG:T, 8-oxoG:G, and 8-oxoG:A. We also investigated the kinetic mechanism of OGG1 stimulation by 8-bromoguanine and showed that this compound affects the rate of beta-elimination rather than pre-excision dynamics of DNA and the enzyme.     

Poly(ADP-ribose) polymerase 1 (PARP-1) binds to 8-oxoguanine-DNA glycosylase (OGG1)

Human 8-oxoguanine-DNA glycosylase (OGG1) plays a major role in the base excision repair pathway by removing 8-oxoguanine base lesions generated by reactive oxygen species. Here we report a novel interaction between OGG1 and Poly(ADP-ribose) polymerase 1 (PARP-1), a DNA-damage sensor protein involved in DNA repair and many other cellular processes. We found that OGG1 binds directly to PARP-1 through the N-terminal region of OGG1, and this interaction is enhanced by oxidative stress. Furthermore, OGG1 binds to PARP-1 through its BRCA1 C-terminal (BRCT) domain. OGG1 stimulated the poly(ADP-ribosyl)ation activity of PARP-1, whereas decreased poly(ADP-ribose) levels were observed in OGG1(-/-) cells compared with wild-type cells in response to DNA damage. Importantly, activated PARP-1 inhibits OGG1. Although the OGG1 polymorphic variant proteins R229Q and S326C bind to PARP-1, these proteins were defective in activating PARP-1. Furthermore, OGG1(-/-) cells were more sensitive to PARP inhibitors alone or in combination with a DNA-damaging agent. These findings indicate that OGG1 binding to PARP-1 plays a functional role in the repair of oxidative DNA damage.     

Structural basis for the lack of opposite base specificity of Clostridium acetobutylicum 8-oxoguanine DNA glycosylase

7,8-Dihydro-8-oxoguanine (8-oxoG) is the major oxidative product of guanine and the most prevalent base lesion observed in DNA molecules. Because 8-oxoG has the capability to form a Hoogsteen pair with adenine (8-oxoG:A) in addition to a normal Watson–Crick pair with cytosine (8-oxoG:C), this lesion can lead to a G:C → T:A transversion after replication. However, 8-oxoG is recognized and excised by the 8-oxoguanine DNA glycosylase (Ogg) of the base excision repair pathway. Members of the Ogg1 family usually display a strong preference for a C opposite the lesion. In contrast, the atypical Ogg1 from Clostridium actetobutylicum (CacOgg) can excise 8-oxoG when paired with either one of the four bases, albeit with a preference for C and A. Here we describe the first high-resolution crystal structures of CacOgg in complex with duplex DNA containing the 8-oxoG lesion paired to cytosine and to adenine. A structural comparison with human OGG1 provides a rationale for the lack of opposite base specificity displayed by the bacterial Ogg.     

Acetylation of human 8-oxoguanine-DNA glycosylase by p300 and its role in 8-oxoguanine repair in vivo

The human 8-oxoguanine-DNA glycosylase 1 (OGG1) is the major DNA glycosylase responsible for repair of 7,8-dihydro-8-oxoguanine (8-oxoG) and ring-opened fapyguanine, critical mutagenic DNA lesions that are induced by reactive oxygen species. Here we show that OGG1 is acetylated by p300 in vivo predominantly at Lys338/Lys341. About 20% of OGG1 is present in acetylated form in HeLa cells. Acetylation significantly increases OGG1's activity in vitro in the presence of AP-endonuclease by reducing its affinity for the abasic (AP) site product. The enhanced rate of repair of 8-oxoG in the genome by wild-type OGG1 but not the K338R/K341R mutant, ectopically expressed in oxidatively stressed OGG1-null mouse embryonic fibroblasts, suggests that acetylation increases OGG1 activity in vivo. At the same time, acetylation of OGG1 was increased by about 2.5-fold after oxidative stress with no change at the polypeptide level. OGG1 interacts with class I histone deacetylases, which may be responsible for its deacetylation. Based on these results, we propose a novel regulatory function of OGG1 acetylation in repair of its substrates in oxidatively stressed cells.     

Cardiac overexpression of 8-oxoguanine DNA glycosylase 1 protects mitochondrial DNA and reduces cardiac fibrosis following transaortic constriction

Cardiac failure is associated with increased levels of oxidized DNA, especially mitochondrial (mtDNA). It is not known if oxidized mtDNA contributes to cardiac dysfunction. To test if protection of mtDNA can reduce cardiac injury, we produced transgenic mice with cardiomyocyte-specific overexpression of the DNA repair enzyme 8-oxoguanine DNA glycosylase 1 (OGG1) isoform 2a. In one line of mice, the transgene increased OGG1 activity by 115% in mitochondria and by 28% in nuclei. OGG1 transgenic mice demonstrated significantly lower cardiac mitochondrial levels of the DNA guanine oxidation product 7,8-dihydro-8-oxoguanine (8-oxo-dG) under basal conditions, after doxorubicin administration, or after transaortic constriction (TAC), but the transgene produced no detectable reduction in nuclear 8-oxo-dG content. OGG1 mice were tested for protection from the cardiac effects of TAC 13 wk after surgery. Compared with FVB-TAC mice, hearts from OGG1-TAC mice had lower levels of β-myosin heavy chain mRNA but they did not display significant differences in the ratio of heart weight to tibia length or protection of cardiac function measured by echocardiography. The principle benefit of OGG1 overexpression was a significant decrease in TAC-induced cardiac fibrosis. This protection was indicated by reduced Sirius red staining on OGG1 cardiac sections and by significantly decreased induction of collagen 1 and 3 mRNA expression in OGG1 hearts after TAC surgery. These results provide a new model to assess the damaging cardiac effects of 8-oxo-dG formation and suggest that increased repair of 8-oxo-dG in mtDNA decreases cardiac pathology.     

Phosphorylation of human oxoguanine DNA glycosylase (alpha-OGG1) modulates its function

Oxoguanine DNA glycosylase (OGG1) initiates the repair of 8-oxoguanine (8-oxoG), a major oxidative DNA base modification that has been directly implicated in cancer and aging. OGG1 functions in the base excision repair pathway, for which a molecular hand-off mechanism has been proposed. To date, only one functional and a few physical protein interactions have been reported for OGG1. Using the yeast two-hybrid system and a protein array membrane, we identified two novel protein interactions of OGG1, with two different protein kinases: Cdk4, a serine-threonine kinase, and c-Abl, a tyrosine kinase. We confirmed these interactions in vitro using recombinant proteins and in vivo by co-immunoprecipitation from whole cell extracts. OGG1 is phosphorylated in vitro by Cdk4, resulting in a 2.5-fold increase in the 8-oxoG/C incision activity of OGG1. C-Abl tyrosine phosphorylates OGG1 in vitro; however, this phosphorylation event does not affect OGG1 8-oxoG/C incision activity. These results provide the first evidence that a post-translational modification of OGG1 can affect its catalytic activity. The distinct functional outcomes from serine/threonine or tyrosine phosphorylation may indicate that activation of different signal transduction pathways modulate OGG1 activity in different ways.     

Mitochondrial DNA repair of oxidative damage in mammalian cells

Nuclear and mitochondrial DNA are constantly being exposed to damaging agents, from endogenous and exogenous sources. In particular, reactive oxygen species (ROS) are formed at high levels as by-products of the normal metabolism. Upon oxidative attack of DNA many DNA lesions are formed and oxidized bases are generated with high frequency. Mitochondrial DNA has been shown to accumulate high levels of 8-hydroxy-2'-deoxyguanosine, the product of hydroxylation of guanine at carbon 8, which is a mutagenic lesion. Most of these small base modifications are repaired by the base excision repair (BER) pathway. Despite the initial concept that mitochondria lack DNA repair, experimental evidences now show that mitochondria are very proficient in BER of oxidative DNA damage, and proteins necessary for this pathway have been isolated from mammalian mitochondria. Here, we examine the BER pathway with an emphasis on mtDNA repair. The molecular mechanisms involved in the formation and removal of oxidative damage from mitochondria are discussed. The pivotal role of the OGG1 glycosylase in removal of oxidized guanines from mtDNA will also be examined. Lastly, changes in mtDNA repair during the aging process and possible biological implications are discussed.     

No evidence of mitochondrial respiratory dysfunction in OGG1-null mice deficient in removal of 8-oxodeoxyguanine from mitochondrial DNA

Accumulation of high levels of mutagenic oxidative mitochondrial DNA (mtDNA) lesions like 8-oxodeoxyguanine (8-oxodG) is thought to be involved in the development of mitochondrial dysfunction in aging and in disorders associated with aging. Mice null for oxoguanine DNA glycosylase (OGG1) are deficient in 8-oxodG removal and accumulate 8-oxodG in mtDNA to levels 20-fold higher than in wild-type mice (N.C. Souza-Pinto et al., 2001, Cancer Res. 61, 5378-5381). We have used these animals to investigate the effects on mitochondrial function of accumulating this particular oxidative base modification. Despite the presence of high levels of 8-oxodG, mitochondria isolated from livers and hearts of Ogg1-/- mice were functionally normal. No differences were detected in maximal (chemically uncoupled) respiration rates, ADP phosphorylating respiration rates, or nonphosphorylating rates with glutamate/malate or with succinate/rotenone. Similarly, maximal activities of respiratory complexes I and IV from liver and heart were not different between wild-type and Ogg1-/- mice. In addition, there was no indication of increased oxidative stress in mitochondria from Ogg1-/- mice, as measured by mitochondrial protein carbonyl content. We conclude, therefore, that highly elevated levels of 8-oxodG in mtDNA do not cause mitochondrial respiratory dysfunction in mice.     

Mechanism of interaction between human 8-oxoguanine-DNA glycosylase and AP endonuclease

Human 8-oxoguanine-DNA glycosylase (OGG1) is the main human base excision protein that removes a mutagenic lesion 8-oxoguanine (8-oxoG) from DNA. Since OGG1 has DNA glycosylase and weak abasic site (AP) lyase activities and is characterized by slow product release, turnover of the enzyme acting alone is low. Recently it was shown that human AP endonuclease (APE1) enhances the activity of OGG1. This enhancement was proposed to be passive, resulting from APE1 binding to or cleavage of AP sites after OGG1 dissociation. Here we present evidence that APE1 could actively displace OGG1 from its product, directly increasing the turnover of OGG1. We have observed that APE1 forms an electrophoretically detectable complex with OGG1 cross-linked to DNA by sodium borohydride. Using oligonucleotide substrates with a single 8-oxoG residue located in their 5'-terminal, central or 3'-terminal part, we have demonstrated that OGG1 activity does not increase only for the first of these three substrates, indicating that APE1 interacts with the DNA stretch 5' to the bound OGG1 molecule. In kinetic experiments, APE1 enhanced the product release constant but not the rate constant of base excision by OGG1. Moreover, OGG1 bound to a tetrahydrofuran analog of an abasic site stimulated the activity of APE1 on this substrate. Using a concatemeric DNA substrate, we have shown that APE1 likely displaces OGG1 in a processive mode, with OGG1 remaining on DNA but sliding away in search for a new lesion. Altogether, our data support a model in which APE1 specifically recognizes an OGG1/DNA complex, distorts a stretch of DNA 5' to the OGG1 molecule, and actively displaces the glycosylase from the lesion.     

Age-associated increase in 8-oxo-deoxyguanosine glycosylase/AP lyase activity in rat mitochondria

The mitochondrial theory of aging postulates that organisms age due to the accumulation of DNA damage and mutations in the multiple mitochondrial genomes, leading to mitochondrial dysfunction. Among the wide variety of DNA damage, 8-oxo-deoxyguanosine (8-oxo-dG) has received the most attention due to its mutagenicity and because of the possible correlation between its accumulation and pathological processes like cancer, degenerative diseases and aging. Although still controversial, many studies show that 8-oxo-dG accumulates with age in the mitochondrial (mt) DNA. However, little is known about the processing of this lesion and no study has yet examined whether mtDNA repair changes with age. Here, we report the first study on age-related changes in mtDNA repair, accomplished by assessing the cleavage activity of mitochondrial extracts towards an 8-oxo-dG-containing substrate. In this study, mitochondria obtained from rat heart and liver were used. We find that this enzymatic activity is higher in 12 and 23 month-old rats than in 6 month-old rats, in both liver and heart extracts. These mitochondrial extracts also cleave oligonucleotides containing a U:A mismatch, at the uracil position, reflecting the combined action of mitochondrial uracil DNA glycosylase (mtUDG) and mitochondrial apurinic/apyrimidinic (AP) endonucleases. The mtUDG activity did not change with age in liver mitochondria, but there was a small increase in activity from 6 to 23 months in rat heart extracts, after normalization to citrate synthase activity. Endonuclease G activity, measured by a plasmid relaxation assay, did not show any age-associated change in liver, but there was a significant decrease from 6 to 23 months in heart mitochondria. Our results suggest that the mitochondrial capacity to repair 8-oxo-dG, the main oxidative base damage suggested to accumulate with age in mtDNA, does not decrease, but rather increases with age. The specific increase in 8-oxo-dG endonuclease activity, rather than a general up-regulation of DNA repair in mitochondria, suggests an induction of the 8-oxo-dG-specific repair pathway with age.     

8-Oxoguanine DNA damage: at the crossroad of alternative repair pathways

Radical oxygen species (ROS) generate various modified DNA bases. Among them 8-oxo-7,8-dihydroguanine (8oxoG) is the most abundant and seems to play a major role in mutagenesis and in carcinogenesis. 8oxoG is removed from DNA by the specific glycosylase OGG1. An additional post-replication repair is needed to correct the 8oxoG/A mismatches that are produced by persistent 8oxoG residues. This review is focused on the mechanisms of base excision repair (BER) of this oxidized base. It is shown that, in vitro, efficient and complete repair of 8oxoG/C pairs requires a core of four proteins, namely OGG1, APE1, DNA polymerase (Pol) beta, and DNA ligase I. Repair occurs predominantly by one nucleotide replacement reactions (short-patch BER) and Pol beta is the polymerase of election for the resynthesis step. However, alternative mechanisms can act on 8oxoG residues since Pol beta-null cells are able to repair these lesions. 8oxoG/A mismatches are repaired by human cell extracts via two BER events which occur sequentially on the two strands. The removal of the mismatched adenine is followed by preferential insertion of a cytosine leading to the formation of 8oxoG/C pairs which are then corrected by OGG1-mediated BER. Both repair events are inhibited by aphidicolin, suggesting that a replicative DNA polymerase is involved in the repair synthesis step. We propose that Pol delta/epsilon-mediated BER (long-patch BER) is the mode of repair when lesions persist or are formed at replication. Finally, we address the issues of the relative contribution of the two BER pathways to oxidative damage repair in vivo and the possible role of BER gene variants as cancer susceptibility genes.     

Exercise improves import of 8-oxoguanine DNA glycosylase into the mitochondrial matrix of skeletal muscle and enhances the relative activity

Exercise has been shown to modify the level/activity of the DNA damage repair enzyme 8-oxoguanine-DNA glycosylase (OGG1) in skeletal muscle. We have studied the impact of regular physical training (8 weeks of swimming) and detraining (8 weeks of rest after an 8-week training session) on the activity of OGG1 in the nucleus and mitochondria as well as its targeting to the mitochondrial matrix in skeletal muscle. Neither exercise training nor detraining altered the overall levels of reactive species; however, mitochondrial levels of carbonylated proteins were decreased in the trained group as assessed by electron spin resonance and biochemical approaches. Importantly, nuclear OGG1 activity was increased by daily exercise training, whereas detraining reversed the up-regulating effect of training. Interestingly, training decreased the outer-membrane-associated mitochondrial OGG1 levels, whereas detraining reversed this effect. These results suggest that exercise training improves OGG1 import into the mitochondrial matrix, thereby increasing OGG1-mediated repair of oxidized guanine bases. Taken together, our data suggest that physical inactivity could impair the mitochondrial targeting of OGG1; however, exercise training increases OGG1 levels/activity in the nucleus and specific activity of OGG1 in mitochondrial compartments, thereby augmenting the repair of oxidized nuclear and mitochondrial DNA bases.     

Factors that influence telomeric oxidative base damage and repair by DNA glycosylase OGG1

Telomeres are nucleoprotein complexes at the ends of linear chromosomes in eukaryotes, and are essential in preventing chromosome termini from being recognized as broken DNA ends. Telomere shortening has been linked to cellular senescence and human aging, with oxidative stress as a major contributing factor. 7,8-Dihydro-8-oxogaunine (8-oxodG) is one of the most abundant oxidative guanine lesions, and 8-oxoguanine DNA glycosylase (OGG1) is involved in its removal. In this study, we examined if telomeric DNA is particularly susceptible to oxidative base damage and if telomere-specific factors affect the incision of oxidized guanines by OGG1. We demonstrated that telomeric TTAGGG repeats were more prone to oxidative base damage and repaired less efficiently than non-telomeric TG repeats in vivo. We also showed that the 8-oxodG-incision activity of OGG1 is similar in telomeric and non-telomeric double-stranded substrates. In addition, telomere repeat binding factors TRF1 and TRF2 do not impair OGG1 incision activity. Yet, 8-oxodG in some telomere structures (e.g., fork-opening, 3'-overhang, and D-loop) were less effectively excised by OGG1, depending upon its position in these substrates. Collectively, our data indicate that the sequence context of telomere repeats and certain telomere configurations may contribute to telomere vulnerability to oxidative DNA damage processing.     

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

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.