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.     

Excision of 8-oxoguanine from methylated CpG dinucleotides by human 8-oxoguanine DNA glycosylase

CpG dinucleotides are targets for epigenetic methylation, many of them bearing 5-methylcytosine (mCyt) in the human genome. Guanine in this context can be easily oxidized to 8-oxoguanine (oxoGua), which is repaired by 8-oxoguanine-DNA glycosylase (OGG1). We have studied how methylation affects the efficiency of oxoGua excision from damaged CpG dinucleotides. Methylation of the adjacent cytosine moderately decreased the oxoGua excision rate while methylation opposite oxoGua lowered the rate of product release. Cytosine methylation abolished stimulation of OGG1 by repair endonuclease APEX1. The OGG1 S326C polymorphic variant associated with lung cancer showed poorer base excision and lost sensitivity to the opposite-base methylation. The overall repair in the system reconstituted from purified proteins decreased for CpG with mCyt in the damaged strand.     

An OGG1 orthologue encoding a functional 8-oxoguanine DNA glycosylase/lyase in Arabidopsis thaliana

Repair of the ubiquitous mutagenic lesion 7,8-dihydro-8-oxoguanine (8-oxoG) is initiated in eukaryotes by DNA glycosylases/lyases, such as yeast Ogg1, that do not share significant sequence identity with their prokaryotic counterparts, typified by Escherichia coli MutM (Fpg) protein. The unexpected presence of a functional mutM orthologue in the model plant Arabidopsis thaliana has brought into question the existence of functional OGG1 orthologues in plants. We report here the cDNA cloning, expression and functional characterization of AtOGG1, an Arabidopsis thaliana gene widely expressed in different plant tissues which encodes a 40.3 kDa protein with significant sequence identity to yeast and human Ogg1 proteins. Purified AtOgg1 enzyme specifically cleaves duplex DNA containing an 8-OxoG:C mispair, and the repair reaction proceeds through an imine intermediate characteristic of all bifunctional DNA glycosylases/lyases. Consistent with its in vitro activity, expression of AtOGG1 suppresses the mutator phenotype of an E. coli strain deficient in 8-oxoG repair. Our results suggest that AtOgg1 is an structural and functional homologue of Ogg1 and establish the presence of two distinct 8-oxoG repair enzymes in Arabidopsis.     

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.     

Step-by-step mechanism of DNA damage recognition by human 8-oxoguanine DNA glycosylase

Background

Extensive structural studies of human DNA glycosylase hOGG1 have revealed essential conformational changes of the enzyme. However, at present there is little information about the time scale of the rearrangements of the protein structure as well as the dynamic behavior of individual amino acids.

Methods

Using pre-steady-state kinetic analysis with Trp and 2-aminopurine fluorescence detection the conformational dynamics of hOGG1 wild-type (WT) and mutants Y203W, Y203A, H270W, F45W, F319W and K249Q as well as DNA–substrates was examined.

Results

The roles of catalytically important amino acids F45, Y203, K249, H270, and F319 in the hOGG1 enzymatic pathway and their involvement in the step-by-step mechanism of oxidative DNA lesion recognition and catalysis were elucidated.

Conclusions

The results show that Tyr-203 participates in the initial steps of the lesion site recognition. The interaction of the His-270 residue with the oxoG base plays a key role in the insertion of the damaged base into the active site. Lys-249 participates not only in the catalytic stages but also in the processes of local duplex distortion and flipping out of the oxoG residue. Non-damaged DNA does not form a stable complex with hOGG1, although a complex with a flipped out guanine base can be formed transiently.

General significance

The kinetic data obtained in this study significantly improves our understanding of the molecular mechanism of lesion recognition by hOGG1.     

Accumulation of 8-oxoguanine in the cellular DNA and the alteration of the OGG1 expression during ischemia-reperfusion injury in the rat kidney

During ischemia-reperfusion (I/R) injury in the rat kidney, apoptosis was observed in the distal tubules of the cortico-medullary region and outer medulla (OM) while severe necrosis was seen in the proximal straight tubules of the OM. The majority of these changes disappeared within 2 weeks. We examined the contents of 8-oxo-2'-deoxyguanosine (8-oxo-dG), which is a major type of oxidative damage in DNA, in the rat kidney during I/R injury, and also investigated the expression level of the OGG1 gene encoding the 8-oxoguanine DNA glycosylase. High-performance liquid chromatography with an MS/MS analysis of the nuclear DNA revealed an immediate accumulation of 8-oxo-dG in the nuclear DNA prepared from the cortex and OM of the kidney 1h after I/R, and an immunohistochemical analysis demonstrated the immediate accumulation of 8-oxo-dG in the nuclei of renal tubular cells both in the cortex and OM. A delayed increase of cytoplasmic staining with anti-8-oxo-dG was observed only in the cortico-medulla and OM, where the cytoplasmic staining in the proximal tubular cells is higher than in the distal tubular cells. The level of cytoplasmic staining representing 8-oxo-dG in mitochondrial DNA, peaked at 6h after I/R and preceded the necrosis of proximal tubular cells in the OM. An RNase protection assay showed a high level of OGG1 mRNA in the normal kidney, and the level decreased within 3h only in the OM, and increased thereafter 1-7 days of I/R both in the cortex and OM. In situ hybridization showed higher levels of OGG1 mRNA expression in the renal tubules in the OM than in the cortex of the normal kidney, which decreased rapidly within 3h of I/R. Thus, the accumulation of 8-oxo-dG in the mitochondrial DNA rather than in nuclear DNA is likely to be involved in the pathogenic responses such as necrosis of renal tubular cells during I/R injury of the kidney, together with an altered level of OGG1 expression.     

Role of OGG1 Ser326Cys polymorphism and 8-oxoguanine DNA damage in risk assessment of squamous cell carcinoma of head and neck in North Indian population

Squamous cell carcinoma of head and neck (SCCHN), one of the leading cancers worldwide, is most prevalent in Indian sub-continent. The major risk factors involved are smoking and consumption of alcohol, since they provide high free radical generating environment. We studied 8-oxoguanine DNA-glycosylase (OGG1) Ser326Cys polymorphism in 278 SCCHN cases and 278 matched controls by PCR-RFLP and observed that the variant genotype Ser/Cys exhibited an enhanced risk of ～1.7 folds (OR=1.71, 95% CI=1.20-2.93) and Cys/Cys ～2.5 folds (OR=2.55, 95% CI=1.29-5.00). Furthermore, we found a significant increase in salivary cell 8-OHdG with respect to Ser/Cys and Cys/Cys genotypes of OGG1 in SCCHN cases, when compared to Ser/Ser and Ser/Cys genotypes of the control population. Our results demonstrate that Ser326Cys variant genotype is associated with an increased risk of SCCHN in north India. Ser326Cys variant genotype was found to accumulate more of 8-OHdG, which may serve as a biomarker for early diagnosis of SCCHN.     

Binding capacity of human YB-1 protein for RNA containing 8-oxoguanine

8-oxoguanine (8-oxo-7,8-dihydroguanine) is generated in the cellular nucleotide pool as well as in nucleic acids, by the action of oxygen radicals produced in cells. 8-oxoguanine has the potential to pair with both cytosine and adenine, and thus, the persistence of this base in messenger RNA would cause translational errors. To prevent such an outcome, organisms should have mechanisms for preventing the misincorporation of 8-oxoguanine-containing nucleotide into RNA and for removing 8-oxoguanine-containing RNA from processes of translation. We now report that mammalian Y box-binding protein 1 (YB-1 protein) possesses the activity to bind specifically to RNA containing 8-oxoguanine. On incubation with a purified preparation of YB-1 protein, 8-oxoguanine-containing RNA forms stable complexes with the protein while normal RNA scarcely forms such a complex. Using a series of deletion mutants which produce altered forms of YB-1 protein lacking some parts of the sequence, domains of the protein necessary for RNA binding were identified. Escherichia coli cells expressing normal or truncated forms of YB-1 protein with the binding capacity acquire resistance against paraquat, a drug that induces oxidative stress in cells, whereas cells with truncated proteins lacking such an activity do not. YB-1 protein may disturb the bacterial system in recognizing oxidatively damaged RNA, thus exerting a dominant negative effect on cell growth. We propose that YB-1 protein may discriminate the oxidized RNA molecule from normal ones, thus contributing to the high fidelity of translation in cells.     

Neighboring base damage induced by permanganate oxidation of 8-oxoguanine in DNA.

We found that single-stranded DNA oligomers containing a 7, 8-dihydro-8-oxoguanine (8-oxo-G) residue have high reactivity toward KMnO4; the oxidation of 8-oxo-G induces damage to the neighboring nucleotide residues. This paper describes the novel reaction in detail, including experiments that demonstrate the mechanism involved in the induction of DNA damage. The results using DNAs of various base compositions indicated that damaged G, T and C (but not A) sites caused strand scissions after hot piperidine treatment and that the damage around the 8-oxo-G occurred at G sites in both single and double strands with high frequency. The latter substrates were less sensitive to damage. Further, kinetic studies of the KMnO4reaction of single-stranded oligomers suggested that thereactivity of the DNA bases at the site 5'-adjacent to the 8-oxo-G was in the order G >A >T, C. This preference correlates with the electron donating abilities of the bases. In addition, we found that the DNA damage at the G site, which was connected with the 8-oxo-G by a long abasic chain, was inhibited in the above order by the addition of dG, dA or dC. On the other hand, the damage reactions proceeded even after the addition of scavengers for active oxygen species. This study suggests the involvement of a redox process in the unique DNA damage initiated by the oxidation of the 8-oxo-G.     

Enhanced mutagenic potential of 8-oxo-7,8-dihydroguanine when present within a clustered DNA damage site

The formation of clustered DNA damage sites is a unique feature of ionizing radiation. Recent studies have shown that the repair of lesions within clusters may be compromised, but little is understood about the mutagenic consequences of such damage sites. Using a plasmid-based method, damaged DNA containing uracil positioned at 1–5 bp separations from 8-oxo-7,8-dihydroguanine on the complementary strand was transfected into wild-type Escherichia coli or into strains lacking the DNA glycosylases Fpg and MutY. Mutation frequencies were found to be significantly higher for clustered damage sites than for single lesions. The loss of MutY gave a large relative increase in mutation frequency and a strain lacking both Fpg and MutY showed even higher mutation frequencies, up to nearly 40% of rescued plasmid. In these strains, the mutation frequency decreases with increasing spacing of the uracil from the 8-oxo-7,8-dihydroguanine site. Sequencing of plasmid DNA carrying clustered damage, following rescue from bacteria, showed that almost all of the mutations are GC→TA transversions. The data suggest that at clustered damage sites, depending on lesion spacing, the action of Fpg is compromised and post-replication processing of lesions by MutY is the most important mechanism for protection against mutagenesis.     

Initiation of 8-oxoguanine base excision repair within trinucleotide tandem repeats

Trinucleotide repeat expansion provides a molecular basis for several devastating neurodegenerative diseases. In particular, expansion of a CAG run in the human HTT gene causes Huntington’s disease. One of the main reasons for triplet repeat expansion in somatic cells is base excision repair (BER), involving damaged base excision and repair DNA synthesis that may be accompanied by expansion of the repaired strand due to formation of noncanonical DNA structures. We have analyzed the kinetics of excision of a ubiquitously found oxidized purine base, 8-oxoguanine (oxoG), by DNA glycosylase OGG1 from the substrates containing a CAG run flanked by AT-rich sequences. The values of k ₂ rate constant for the removal of oxoG from triplets in the middle of the run were higher than for oxoG at the flanks of the run. The value of k ₃ rate constant dropped starting from the third CAG-triplet in the run and remained stable until the 3′-terminal triplet, where it decreased even more. In nuclear extracts, the profile of oxoG removal rate along the run resembled the profile of k ₂ constant, suggesting that the reaction rate in the extracts is limited by base excision. The fully reconstituted BER was efficient with all substrates unless oxoG was near the 3′-flank of the run, interfering with the initiation of the repair. DNA polymerase β was able to perform a strand-displacement DNA synthesis, which may be important for CAG run expansion initiated by BER.     

Repair of clustered DNA lesions. Sequence-specific inhibition of long-patch base excision repair be 8-oxoguanine

Ionizing radiation induces clustered DNA damage where two or more lesions are located proximal to each other on the same or opposite DNA strands. It has been suggested that individual lesions within a cluster are removed sequentially and that the presence of a vicinal lesion(s) may affect the rate and fidelity of DNA repair. In this study, we addressed the question of how 8-oxoguanine located opposite to normal or reduced abasic sites would affect the repair of these sites by the base excision repair system. We have found that an 8-oxoguanine located opposite to an abasic site does not affect either the efficiency or fidelity of repair synthesis by DNA polymerase beta. In contrast, an 8-oxoguanine located one nucleotide 3'-downstream of the abasic site significantly reduces both strand displacement synthesis supported by DNA polymerase beta or delta and cleavage by flap endonuclease of the generated flap, thus inhibiting the long-patch base excision repair pathway.     

The 8-oxoguanine DNA glycosylase 1 (ogg1) decreases the vulnerability of the developing brain to DNA damage

The developing brain is particularly vulnerable to oxidative DNA damage, which may be the cause of most major congenital mental anomalies. The repair enzyme ogg1 initiates the highly conserved base-excision repair pathway. However, its function in the embryonic brain is largely unknown.This study is the first to validate the function of ogg1 during brain development using zebrafish embryos. Ogg1 was found to be highly expressed in the brain throughout early embryonic development, with particularly enrichment observed in the midbrain. The lack of ogg1 causes severe brain defects including changes in brain volume and integrity, destruction of the midbrain–hindbrain boundary, and balance and motor impairment, while overexpression of ogg1 can partially rescue these defects.Multiple cellular and molecular events were involved in the manifestation of brain defects due primarily to the lack of ogg1. These included (1) increased apoptosis; (2) decreased proliferation; and (3) aberrant axon distribution and extension from the inner surface towards the outer layers. The results of a microarray analysis showed that the expression of genes involved in cell cycle checkpoint, apoptosis, and neurogenesis were significantly changed in response to ogg1 knockdown. Cmyb was the key downstream gene that responses to DNA damage caused by ogg1 deficiency. Notably, the recruitment of ogg1 mRNA can alleviate the effects on the brain due to neural DNA damage.In summary, we introduce here that ogg1 is fundamentally required for protecting the developing brain, which may be helpful in understanding the aetiology of congenital brain deficits.     

Mouse MTH2 protein which prevents mutations caused by 8-oxoguanine nucleotides

MutT-related proteins degrade 8-oxo-7,8-dihydrodeoxyguanosine triphosphate (8-oxo-dGTP), a mutagenic substrate for DNA synthesis, in the nucleotide pool, thereby preventing DNA replication errors. During a search of GenBank EST database, we found a new member of MutT-related protein, MTH2, which possesses the 23-amino acid MutT module. The cloned mouse MTH2 (mMTH2) cDNA was expressed in Escherichia coli mutT(-) cells and the protein was purified. mMTH2 protein hydrolyzes 8-oxo-dGTP to 8-oxo-dGMP, with Km of 32 microM. Expression of cDNA for mMTH2 reduced significantly the elevated level of spontaneous mutation frequency of E. coli mutT(-) cells. Thus, MTH2 has a potential to protect the genetic material from the untoward effects of endogenous oxygen radicals. MTH2 could act as an MTH1 redundancy factor.     

Efficient removal of formamidopyrimidines by 8-oxoguanine glycosylases

Under conditions of oxidative stress, the formamidopyrimidine lesions (FapyG and FapyA) are formed in competition with the corresponding 8-oxopurines (OG and OA) from a common intermediate. In order to reveal features of the repair of these lesions, and the potential contribution of repair in mitigating or exacerbating the mutagenic properties of Fapy lesions, their excision by three glycosylases, Fpg, hOGG1 and Ntg1, was examined in various base pair contexts under single-turnover conditions. FapyG was removed at least as efficiently as OG by all three glycosylases. In addition, the rates of removal of FapyG by Fpg and hOGG1 were influenced by their base pair partner, with preference for removal when base paired with the correct Watson-Crick partner C. With the FapyA lesion, Fpg and Ntg1 catalyze its removal more readily than OG opposite all four natural bases. In contrast, the removal of FapyA by hOGG1 was not as robust as FapyG or OG, and was only significant when the lesion was paired with C. The discrimination by the various glycosylases with respect to the opposing base was highly dependent on the identity of the lesion. OG induced the greatest selectivity against its removal when part of a promutagenic base pair. The superb activity of the various OG glycosylases toward removal of FapyG and FapyA in vitro suggests that these enzymes may act upon these oxidized lesions in vivo. The differences in the activity of the various glycosylases for removal of FapyG and FapyA compared to OG in nonmutagenic versus promutagenic base pair contexts may serve to alter the mutagenic profiles of these lesions in vivo.     

Direct visualization of repair of oxidative damage by OGG1 in the nuclei of live cells

Oxidative DNA damage caused by intracellular reactive oxygen species (ROS) is widely considered to be important in the pathology of a range of human diseases including cancer as well as in the aging process. A frequently occurring mutagenic base lesion produced by ROS is 8-oxo deoxyguanine (8-oxo dG) and the major enzyme for repair of 8-oxo dG is 8-oxoguanine-DNA glycosylase 1 (OGG1). There is now substantial evidence from bulk biochemical studies that a common human polymorphic variant of OGG1 (Ser326Cys) is repair deficient, and this has been linked to individual risk of pathologies related to oxidative stress. In the current study, we have used the technique of multiphoton microscopy to induce highly localized oxidative DNA damage in discrete regions of the nucleus of live cells. Cells transfected with GFP-tagged OGG1 proteins demonstrated rapid (<2 min) accumulation of OGG1 at sites of laser-induced damage as indicated by accumulation of GFP-fluorescence. This was followed by repair as evidenced by loss of the localized fluorescence over time. Quantification of the rate of repair confirmed that the Cys326 variant of OGG1 is repair deficient and that the initial repair rate of damage by Cys326 OGG1 was 3 to 4 fold slower than that observed for Ser326 OGG1. These values are in good agreement with kinetic data comparing the Ser326 and Cys326 proteins obtained by biochemical studies.     

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.     

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.