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.     

8-Oxoguanine DNA glycosylase-1-mediated DNA repair is associated with Rho GTPase activation and α-smooth muscle actin polymerization

Reactive oxygen species (ROS) are activators of cell signaling and modify cellular molecules, including DNA. 8-Oxo-7,8-dihydroguanine (8-oxoG) is one of the prominent lesions in oxidatively damaged DNA, whose accumulation is causally linked to various diseases and aging processes, whereas its etiological relevance is unclear. 8-OxoG is repaired by the 8-oxoguanine DNA glycosylase-1 (OGG1)-initiated DNA base excision repair (BER) pathway. OGG1 binds free 8-oxoG and this complex functions as an activator of Ras family GTPases. Here we examined whether OGG1-initiated BER is associated with the activation of Rho GTPase and mediates changes in the cytoskeleton. To test this possibility, we induced OGG1-initiated BER in cultured cells and mouse lungs and used molecular approaches such as active Rho pull-down assays, siRNA ablation of gene expression, immune blotting, and microscopic imaging. We found that OGG1 physically interacts with Rho GTPase and, in the presence of 8-oxoG base, increases Rho–GTP levels in cultured cells and lungs, which mediates α-smooth muscle actin (α-SMA) polymerization into stress fibers and increases the level of α-SMA in insoluble cellular/tissue fractions. These changes were absent in cells lacking OGG1. These unexpected data and those showing that 8-oxoG repair is a lifetime process suggest that, via Rho GTPase, OGG1 could be involved in the cytoskeletal changes and organ remodeling observed in various chronic diseases.     

Activation of cellular signaling by 8-oxoguanine DNA glycosylase-1-initiated DNA base excision repair

Accumulation of 8-oxo-7,8-dihydroguanine (8-oxoG) in the DNA results in genetic instability and mutagenesis, and is believed to contribute to carcinogenesis, aging processes and various aging-related diseases. 8-OxoG is removed from the DNA via DNA base excision repair (BER), initiated by 8-oxoguanine DNA glycosylase-1 (OGG1). Our recent studies have shown that OGG1 binds its repair product 8-oxoG base with high affinity at a site independent from its DNA lesion-recognizing catalytic site and the OGG1•8-oxoG complex physically interacts with canonical Ras family members. Furthermore, exogenously added 8-oxoG base enters the cells and activates Ras GTPases; however, a link has not yet been established between cell signaling and DNA BER, which is the endogenous source of the 8-oxoG base. In this study, we utilized KG-1 cells expressing a temperature-sensitive mutant OGG1, siRNA ablation of gene expression, and a variety of molecular biological assays to define a link between OGG1-BER and cellular signaling. The results show that due to activation of OGG1-BER, 8-oxoG base is released from the genome in sufficient quantities for activation of Ras GTPase and resulting in phosphorylation of the downstream Ras targets Raf1, MEK1,2 and ERK1,2. These results demonstrate a previously unrecognized mechanism for cellular responses to OGG1-initiated DNA BER.     

The high binding affinity of human ribosomal protein S3 to 7,8-dihydro-8-oxoguanine is abrogated by a single amino acid change

Previous studies have shown that human ribosomal protein S3 (hS3) has a high apparent binding affinity for 7,8-dihydro-8-oxoguanine (8-oxoG) residues in DNA and interacts with the human base excision repair (BER) proteins OGG1 and APE/Ref-1. We used a combination of computational and experimental approaches to understand the role of hS3 in BER and its potential to hinder repair of 8-oxoG lesions by OGG1 and APE/Ref-1. Sequence analysis was employed to identify hS3 residues likely to be involved in binding to 8-oxoG. One putative site, lysine 132 (K132), located in a helix-hairpin-helix DNA binding motif, was mutated to alanine (K132A). The hS3-K132A mutant retained the ability to cleave abasic DNA, but its capacity to bind 8-oxoG was abrogated completely. The ability of OGG1 to cleave an 8-oxoG-oligonucleotide substrate pre-incubated with hS3 or hS3-K132A was also tested. Pre-incubations with wild-type hS3 and 8-oxoG-containing oligonucleotides completely prevented the subsequent removal of 8-oxoG by OGG1. On the other hand, OGG1 incubations combined with hS3-K132A stimulated cleavage of 8-oxoG in excess of two-fold, confirming previous observations that hS3 positively interacts with OGG1, but only under conditions in which the binding of hS3 to 8-oxoG is limited. Overall, the ability of OGG1 to repair 8-oxoG is compromised when hS3 is bound to 8-oxoG sites. Conversely, in the absence of DNA binding, hS3 interacts positively with OGG1 to produce a more robust removal of 8-oxoG residues in DNA.     

A functional OGG1 homologue from Arabidopsis thaliana

One of the major mutagenic base lesions in DNA caused by exposure to reactive oxygen species is 7,8-dihydro-8-oxoguanine (8-oxoG). Genes coding for DNA repair enzymes that recognise 8-oxoG have been reported in bacteria, yeast, mammals and plants. The prokaryotic and eukaryotic genes are functional homologues but differ in their primary sequence. We have cloned, sequenced, and expressed a new Arabidopsis thaliana cDNA that shows sequence homology to the eukaryotic genes coding for 8-oxoG DNA N-glycosylases (OGG1). The 40.3-kDa enzyme it encodes (AtOGG1) introduces a chain break in a double-stranded oligonucleotide specifically at an 8-oxoG residue. In addition, AtOGG1 can form a Schiff base with 8-oxoG in the presence of NaBH₄, suggesting that it is a bifunctional DNA N-glycosylase. Furthermore, expression of AtOGG1 in an Escherichia coli strain that is deficient in the repair of 8-oxoG in DNA suppresses its spontaneous-mutator phenotype. Thus, we have demonstrated that AtOGG1 is not only a structural but also a functional eukaryotic OGG1 homologue.     

Stimulation of DNA glycosylase activity of OGG1 by NEIL1: functional collaboration between two human DNA glycosylases

The eukaryotic 8-oxoguanine-DNA glycosylase 1 (OGG1) provides the major activity for repairing mutagenic 7,8-dihydro-8-oxoguanine (8-oxoG) induced in the genome due to oxidative stress. Earlier in vitro studies showed that, after excising the base lesion, the human OGG1 remains bound to the resulting abasic (AP) site in DNA and does not turn over efficiently. The human AP-endonuclease (APE1), which cleaves the phosphodiester bond 5' to the AP site, in the next step of repair, displaces the bound OGG1 and thus increases its turnover. Here we show that NEIL1, a DNA glycosylase/AP lyase specific for many oxidized bases but with weak 8-oxoG excision activity, stimulates turnover of OGG1 in a fashion similar to that of APE1 and carries out betadelta-elimination at the AP site. This novel collaboration of two DNA glycosylases, which do not stably interact with each other, in stimulating 8-oxoguanine repair is possible because of higher AP site affinity and stronger AP lyase activity of NEIL1 relative to OGG1. Comparable levels of NEIL1 and OGG1 in some human cells raise the possibility that NEIL1 serves as a backup enzyme to APE1 in stimulating 8-oxoG repair in vivo.     

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.     

Human DNA glycosylases of the bacterial Fpg/MutM superfamily: an alternative pathway for the repair of 8-oxoguanine and other oxidation products in DNA

The mild phenotype associated with targeted disruption of the mouse OGG1 and NTH1 genes has been attributed to the existence of back-up activities and/or alternative pathways for the removal of oxidised DNA bases. We have characterised two new genes in human cells that encode DNA glycosylases, homologous to the bacterial Fpg (MutM)/Nei class of enzymes, capable of removing lesions that are substrates for both hOGG1 and hNTH1. One gene, designated HFPG1, showed ubiquitous expression in all tissues examined whereas the second gene, HFPG2, was only expressed at detectable levels in the thymus and testis. Transient transfections of HeLa cells with fusions of the cDNAs to EGFP revealed intracellular sorting to the nucleus with accumulation in the nucleoli for hFPG1, while hFPG2 co-localised with the 30 kDa subunit of RPA. hFPG1 was purified and shown to act on DNA substrates containing 8-oxoguanine, 5-hydroxycytosine and abasic sites. Removal of 8-oxoguanine, but not cleavage at abasic sites, was opposite base-dependent, with 8-oxoG:C being the preferred substrate and negligible activity towards 8-oxoG:A. It thus appears that hFPG1 has properties similar to mammalian OGG1 in preventing mutations arising from misincorporation of A across 8-oxoG and could function as a back-up repair activity for OGG1 in ogg1(-/-) mice.     

Metallothionein-III prevents gamma-ray-induced 8-oxoguanine accumulation in normal and hOGG1-depleted cells

Metallothioneins (MT) play an important biological role in preventing oxidative damage to cells. We have previously demonstrated that the efficiency of the protective effect of MT-III against the DNA degradation from oxidative damage was much higher than that of MT-I/II. As an extension of the latter investigation, this study aimed to assess the ability of MT-III to suppress 8-oxoguanine (8-oxoG), which is one of the major base lesions formed after an oxidative attack to DNA and the mutant frequency of the HPRT gene in human fibroblast GM00637 cells upon exposure to gamma-rays. We found that human MT-III expression decreased the level of 8-oxoG and mutation frequency in the gamma-irradiated cells. Using an 8-oxoguanine DNA glycosylase (OGG1)-specific siRNAs, we also found that MT-III expression resulted in the suppression of the gamma-radiation-induced 8-oxoG accumulation and mutation in the OGG1-depleted cells. Moreover, the down-regulation of MT in human neuroblastoma SKNSH cells induced by MT-specific siRNA led to a significant increase in the 8-oxoG level, after exposure to gamma-irradiation. These results suggest that under the conditions of gamma-ray oxidative stress, MT-III prevents the gamma-radiation-induced 8-oxoG accumulation and mutation in normal and hOGG1-depleted cells, and this suppression might, at least in part, contribute to the anticarcinogenic and neuroprotective role of MT-III.     

Activation of ras signaling pathway by 8-oxoguanine DNA glycosylase bound to its excision product, 8-oxoguanine

8-Oxo-7,8-dihydroguanine (8-oxoG), arguably the most abundant base lesion induced in mammalian genomes by reactive oxygen species, is repaired via the base excision repair pathway that is initiated with the excision of 8-oxoG by OGG1. Here we show that OGG1 binds the 8-oxoG base with high affinity and that the complex then interacts with canonical Ras family GTPases to catalyze replacement of GDP with GTP, thus serving as a guanine nuclear exchange factor. OGG1-mediated activation of Ras leads to phosphorylation of the mitogen-activated kinases MEK1,2/ERK1,2 and increasing downstream gene expression. These studies document for the first time that in addition to its role in repairing oxidized purines, OGG1 has an independent guanine nuclear exchange factor activity when bound to 8-oxoG.     

Redox regulation of human OGG1 activity in response to cellular oxidative stress

8-Oxoguanine (8-oxoG), a common and mutagenic form of oxidized guanine in DNA, is eliminated mainly through base excision repair. In human cells its repair is initiated by human OGG1 (hOGG1), an 8-oxoG DNA glycosylase. We investigated the effects of an acute cadmium exposure of human lymphoblastoid cells on the activity of hOGG1. We show that coinciding with alteration of the redox cellular status, the 8-oxoG DNA glycosylase activity of hOGG1 was nearly completely inhibited. However, the hOGG1 activity returned to normal levels once the redox cellular status was normalized. In vitro, the activity of purified hOGG1 was abolished by cadmium and could not be recovered by EDTA. In cells, however, the reversible inactivation of OGG1 activity by cadmium was strictly associated with reversible oxidation of the protein. Moreover, the 8-oxoG DNA glycosylase activity of purified OGG1 and that from crude extracts were modulated by cysteine-modifying agents. Oxidation of OGG1 by the thiol oxidant diamide led to inhibition of the activity and a protein migration pattern similar to that seen in cadmium-treated cells. These results suggest that cadmium inhibits hOGG1 activity mainly by indirect oxidation of critical cysteine residues and that excretion of the metal from the cells leads to normalization of the redox cell status and restoration of an active hOGG1. The results presented here unveil a novel redox-dependent mechanism for the regulation of OGG1 activity.     

DNA Sequence Context Effects on the Glycosylase Activity of Human 8-Oxoguanine DNA Glycosylase

Human 8-oxoguanine DNA glycosylase (OGG1) is a key enzyme involved in removing 7,8-dihydro-8-oxoguanine (8-oxoG), a highly mutagenic DNA lesion generated by oxidative stress. The removal of 8-oxoG by OGG1 is affected by the local DNA sequence, and this feature most likely contributes to observed mutational hot spots in genomic DNA. To elucidate the influence of local DNA sequence on 8-oxoG excision activity of OGG1, we conducted steady-state, pre-steady-state, and single turnover kinetic evaluation of OGG1 in alternate DNA sequence contexts. The sequence context effect was studied for a mutational hot spot at a CpG dinucleotide. Altering either the global DNA sequence or the 5′-flanking unmodified base pair failed to influence the excision of 8-oxoG. Methylation of the cytosine 5′ to 8-oxoG also did not affect 8-oxoG excision. In contrast, a 5′-neighboring mismatch strongly decreased the rate of 8-oxoG base removal. Substituting the 5′-C in the CpG dinucleotide with T, A, or tetrahydrofuran (i.e. T:G, A:G, and tetrahydrofuran:G mispairs) resulted in a 10-, 13-, and 4-fold decrease in the rate constant for 8-oxoG excision, respectively. A greater loss in activity was observed when T:C or A:C was positioned 5′ of 8-oxoG (59- and 108-fold, respectively). These results indicate that neighboring structural abnormalities 5′ to 8-oxoG deter its repair thereby enhancing its mutagenic potential.     

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.     

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.     

Excision of 8-oxoguanine within clustered damage by the yeast OGG1 protein

Clustered damages are formed in DNA by ionising radiation and radiomimetic anticancer agents and are thought to be biologically severe. 7,8-dihydro-8-oxoguanine (8-oxoG), a major DNA damage resulting from oxidative attack, is highly mutagenic leading to a high level of G·C→T·A transversions if not previously excised by OGG1 DNA glycosylase/AP lyase proteins in eukaryotes. However, 8-oxoG within clustered DNA damage may present a challenge to the repair machinery of the cell. The ability of yeast OGG1 to excise 8-oxoG was determined when another type of damage [dihydrothymine, uracil, 8-oxoG, abasic (AP) site or various types of single-strand breaks (SSBs)] is present on the complementary strand 1, 3 or 5 bases 5′ or 3′ opposite to 8-oxoG. Base damages have little or no influence on the excision of 8-oxoG by yeast OGG1 (yOGG1) whereas an AP site has a strong inhibitory effect. Various types of SSBs, obtained using either oligonucleotides with 3′- and 5′-phosphate termini around a gap or through conversion of an AP site with either endonuclease III or human AP endonuclease 1, strongly inhibit excision of 8-oxoG by yOGG1. Therefore, this large inhibitory effect of an AP site or a SSB may minimise the probability of formation of a double-strand break in the processing of 8-oxoG within clustered damages.     

Efficiency of excision of 8-oxo-guanine within DNA clustered damage by XRS5 nuclear extracts and purified human OGG1 protein

A major DNA lesion is the strongly mutagenic 8-oxo-7,8-dihydroguanine (8-oxoG) base, formed by oxidative attack at guanine and which leads to a high level of G.C-->T.A transversions. Clustered DNA damages are formed in DNA following exposure to ionizing radiation or radiomimetic anticancer agents and are thought to be biologically severe. The presence of 8-oxoG within clustered DNA damage may present a challenge to the repair machinery of the cell, if the OGG1 DNA glycosylase/AP lyase protein, present in eukaryotic cells, does not efficiently excise its substrate, 8-oxoG. In this study, specific oligonucleotide constructs containing an 8-oxoG located in several positions opposite to another damage (5,6-dihydrothymine (DHT), uracil, 8-oxoG, AP site, or various types of single strand breaks) were used to determine the relative efficiency of purified human OGG1 and mammalian XRS5 nuclear extracts to excise 8-oxoG from clustered damages. A base damage (DHT, uracil, and 8-oxoG) on the opposite strand has little or no influence on the rate of excision of 8-oxoG whereas the presence of either an AP site or various types of single strand breaks has a strong inhibitory effect on the formation of a SSB due to the excision of 8-oxoG by both hOGG1 and the nuclear extract. The binding of hOGG1 to 8-oxoG is not significantly affected by the presence of a neighboring lesion.     

Identification and characterization of OGG1 mutations in patients with Alzheimer's disease

Patients with Alzheimer's disease (AD) exhibit higher levels of 8-oxo-guanine (8-oxoG) DNA lesions in their brain, suggesting a reduced or defective 8-oxoG repair. To test this hypothesis, this study investigated 14 AD patients and 10 age-matched controls for mutations of the major 8-oxoG removal gene OGG1. Whereas no alterations were detected in any control samples, four AD patients exhibited mutations in OGG1, two carried a common single base (C₇₉₆) deletion that alters the carboxyl terminal sequence of OGG1, and the other two had nucleotide alterations leading to single amino acid substitutions. In vitro biochemical assays revealed that the protein encoded by the C₇₉₆-deleted OGG1 completely lost its 8-oxoG glycosylase activity, and that the two single residue-substituted OGG1 proteins showed a significant reduction in the glycosylase activity. These results were consistent with the fact that nuclear extracts derived from a limited number of AD patients with OGG1 mutations exhibited greatly reduced 8-oxoG glycosylase activity compared with age-matched controls and AD patients without OGG1 alterations. Our findings suggest that defects in OGG1 may be important in the pathogenesis of AD in a significant fraction of AD patients and provide new insight into the molecular basis for the disease.     

MUTYH prevents OGG1 or APEX1 from inappropriately processing its substrate or reaction product with its C-terminal domain

MutY homolog (MUTYH) excises adenine opposite 8-oxoguanine (8-oxoG) in DNA, thus preventing occurrence of G:C to T:A transversion. In cell-free extract prepared from the thymocytes of wild type but not MUTYH-null mice, adenine opposite 8-oxoG in DNA was excised by MUTYH, however, the generated apurinic (AP) site opposite 8-oxoG mostly remained unincised. Recombinant mouse MUTYH (mMUTYH) efficiently excised adenine opposite 8-oxoG and prevented mouse AP endonuclease (mAPEX1) from incising the generated AP site. In contrast, an AP site opposite 8-oxoG created by uracil DNA glycosylase or tetrahydrofuran opposite 8-oxoG was efficiently incised by mAPEX1 in the presence of an excess amount of mMUTYH. Mutant mMUTYH with R361A or G365D substitution, excised adenine opposite 8-oxoG as efficiently as did wild-type mMUTYH, but failed to prevent mAPEX1 from incising the generated AP site. Wild-type mMUTYH bound duplex oligonucleotides containing A:8-oxoG pair with a lower apparent K_d than that of the mutants, and prevented OGG1 from excising 8-oxoG opposite adenine or the generated AP site. The G365D mutant failed to prevent OGG1 from excising 8-oxoG opposite the generated AP site, thus indicating that the protection of its own product by mMUTYH is an intrinsic function which depends on the C-terminal domain of mMUTYH.