Structural and biochemical studies of a plant formamidopyrimidine-DNA glycosylase reveal why eukaryotic Fpg glycosylases do not excise 8-oxoguanine

Formamidopyrimidine-DNA glycosylase (Fpg; MutM) is a DNA repair enzyme widely distributed in bacteria. Fpg recognizes and excises oxidatively modified purines, 4,6-diamino-5-formamidopyrimidine, 2,6-diamino-4-hydroxy-5-formamidopyrimidine and 8-oxoguanine (8-oxoG), with similar excision kinetics. It exhibits some lesser activity toward 8-oxoadenine. Fpg enzymes are also present in some plant and fungal species. The eukaryotic Fpg homologs exhibit little or no activity on DNA containing 8-oxoG, but they recognize and process its oxidation products, guanidinohydantoin (Gh) and spiroiminohydantoin (Sp). To date, several structures of bacterial Fpg enzymes unliganded or in complex with DNA containing a damaged base have been published but there is no structure of a eukaryotic Fpg. Here we describe the first crystal structure of a plant Fpg, Arabidopsis thaliana (AthFpg), unliganded and bound to DNA containing an abasic site analog, tetrahydrofuran (THF). Although AthFpg shares a common architecture with other Fpg glycosylases, it harbors a zincless finger, previously described in a subset of Nei enzymes, such as human NEIL1 and Mimivirus Nei1. Importantly the "αF-β9/10 loop" capping 8-oxoG in the active site of bacterial Fpg is very short in AthFpg. Deletion of a segment encompassing residues 213-229 in Escherichia coli Fpg (EcoFpg) and corresponding to the "αF-β9/10 loop" does not affect the recognition and removal of oxidatively damaged DNA base lesions, with the exception of 8-oxoG. Although the exact role of the loop remains to be further explored, it is now clear that this protein segment is specific to the processing of 8-oxoG.     

Accumulation of adenine DNA glycosylase-sensitive sites in human mitochondrial DNA

The mitochondrial respiratory chain inevitably produces reactive oxygen species as byproducts of aerobic ATP synthesis. Mitochondrial DNA (mtDNA), which is located close to the respiratory chain, is reported to contain much more 8-oxoguanine (8-oxoG), an oxidatively modified guanine base, than nuclear DNA. Despite such a high amount of 8-oxoG in mtDNA (1-2 8-oxoG/10(4) G), mtDNA is barely cleaved by an 8-oxoG DNA glycosylase or MutM, which specifically excises 8-oxoG from a C:8-oxoG pair. We find here that about half of human mtDNA molecules are cleaved by another 8-oxoG-recognizing enzyme, an adenine DNA glycosylase or MutY, which excises adenine from an A:8-oxoG pair. The cleavage sites are mapped to adenines. The calculated number of MutY-sensitive sites in mtDNA is approximately 1.4/10(4) G. This value roughly corresponds with the electrochemically measured amount of 8-oxoG in mtDNA (2.2/10(4) G), raising the possibility that 8-oxoG mainly accumulates as an A:8-oxoG pair.     

Substrate discrimination by formamidopyrimidine-DNA glycosylase: distinguishing interactions within the active site

Reactive oxygen species are byproducts of normal aerobic respiration and ionizing radiation, and they readily react with DNA to form a number of base lesions, including the mutagenic 8-oxo-7,8-dihydroguanine (8-oxoG), 2,6-diamino-4-hydroxy-5-formamidopyrimidine (FapyG), 4,6-diamino-5-formamidopyrimidine (FapyA), and 8-oxo-7,8-dihydroadenine (8-oxoA). Such oxidative lesions are removed by the base excision repair pathway, which is initiated by DNA glycosylases such as the formamidopyrimidine-DNA glycosylase (Fpg) in Escherichia coli. The 8-oxoG, FapyG, and FapyA lesions are bound and excised by Fpg, while structurally similar 8-oxoA is excised by Fpg very poorly. We carried out molecular modeling and molecular dynamics simulations to interpret substrate discrimination within the active site of E. coli Fpg. Lys-217 and Met-73 were identified as residues playing important roles in the recognition of the oxidized imidazole ring in the substrate bases, and the Watson-Crick edge of the damaged base plays a role in optimally positioning the base within the active site. The recognition and excision of FapyA likely result from the opened imidazole ring, while 8-oxoA's lack of flexibility and closed imidazole ring may contribute to Fpg's inability to excise this base. Different interactions between each base and the enzyme specificity pocket account for differential treatment of the various lesions by this enzyme, and thus elucidate the structure-function relationship involved in an initial step of base excision repair.     

Peroxynitrite-induced oxidation and nitration products of guanine and 8-oxoguanine: structures and mechanisms of product formation.

Peroxynitrite induces DNA base damage predominantly at guanine (G) and 8-oxoguanine (8-oxoG) nucleobases via oxidation reactions. Nitration products are also observed, consistent with the generation of radical intermediates that can recombine with the (.)NO(2) formed during peroxynitrite degradation. The neutral G radical, G(.), reacts with (.)NO(2) to yield 8-nitroguanine (8-nitroG) and 5-nitro-4-guanidinohydantoin (NI), while for 8-oxoG we have proposed a reactive guanidinylidene radical intermediate. The products generated during peroxynitrite-mediated 8-oxoG oxidation depend on oxidant flux, with dehydroguanidinohydantoin (DGh), 2,4,6-trioxo-[1,3,5]triazinane-1-carboxamidine (CAC) and NO(2)-DGh predominating at high fluxes and spiroiminodihydantoin (Sp), guanidinohydantoin (Gh) and 4-hydroxy-2,5-dioxo-imidazolidine-4-carboxylic acid (HICA) predominating at low fluxes. Both product sets are observed at intermediate fluxes. It is therefore important in model systems to ensure that the relative concentrations are well controlled to minimize competing reactions that may not be relevant in vivo. Increasingly sophisticated systems for modeling peroxynitrite production in vivo are being developed and these should help with predicting the products most likely to be formed in vivo. Together with the emerging information on the genotoxic and mutational characteristics of the individual oxidation products, it may be found that the extent of tissue damage, mutational spectra and, hence, cancer risk may change as a function of peroxynitrite fluxes as different product combinations predominate.     

A repair system for 8-oxo-7,8-dihydrodeoxyguanine.

Active oxygen species can damage DNA and may play a role in aging and carcinogenesis. We have tested MutY glycosylase for activity on undamaged mispairs as well as mispairs formed with the oxidatively damaged substrates, 8-oxo-7,8-dihydrodeoxyguanine (GO) or 8-oxo-7,8-dihydrodeoxyadenine (AO). MutY acts as a glycosylase on four of the heteroduplexes tested, A/G, A/GO, A/C, and A/AO, removing the undamaged adenine from each substrate. Genetic data suggest that the primary substrate for MutY glycosylase in vivo is the A/GO mispair. We present biochemical evidence demonstrating that MutY glycosylase is an important part of a repair system that includes the MutM and MutT proteins. The GO repair system is dedicated to the repair of the oxidatively damaged guanine and the mutations it can induce.     

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.     

Biochemical identification of a hydroperoxide derivative of the free 8-oxo-7,8-dihydroguanine base

8-Oxo-7,8-dihydroguanine is one the most abundant base lesions in pro- and eukaryotic DNA. In mammalian cells, it is excised by the 8-oxoguanine DNA glycosylase (OGG1) during DNA base-excision repair, and the generated free 8-oxoG base is one of the DNA-derived biomarkers of oxidative stress in biological samples. The modification of 8-oxoG in the context of nucleoside and DNA has been the subject of many studies; however, the oxidative transformation of the free 8-oxoG base has not been described. By using biochemical and cell biological assays, we show that in the presence of molecular oxygen, the free 8-oxoG base transforms to a highly reactive hydroperoxide (8-oxoG*). Specifically, 8-oxoG* oxidizes Amplex red to resorufin, H(2)DCF to DCF, Fe(2+) to Fe(3+), and GSH to GSSG. This property of 8-oxoG* was diminished by treatment with catalase and glutathione peroxidase, but not superoxide dismutase. 8-OxoG* formation was prevented by reducing agents or nitrogen atmosphere. Its addition to CM-H(2)DCF-DA-loaded cells rapidly increased intracellular DCF fluorescence. There were no such properties observed for 8-oxodeoxyguanosine, 2,6-diamino-4-hydroxy-5-formamidopyrimidine, 2'-deoxyguanosine, guanine, adenine, guanosine, and 8-hydroxyadenine. These data imply that a free 8-oxoG base is more susceptible to oxidation than is its nucleoside form and, consequently, it stands as unique among intact and oxidatively modified purines.     

DNA damage by reactive species: Mechanisms, mutation and repair

DNA is continuously attacked by reactive species that can affect its structure and function severely. Structural modifications to DNA mainly arise from modifications in its bases that primarily occur due to their exposure to different reactive species. Apart from this, DNA strand break, inter- and intra-strand crosslinks and DNA-protein crosslinks can also affect the structure of DNA significantly. These structural modifications are involved in mutation, cancer and many other diseases. As it has the least oxidation potential among all the DNA bases, guanine is frequently attacked by reactive species, producing a plethora of lethal lesions. Fortunately, living cells are evolved with intelligent enzymes that continuously protect DNA from such damages. This review provides an overview of different guanine lesions formed due to reactions of guanine with different reactive species. Involvement of these lesions in inter- and intra-strand crosslinks, DNA-protein crosslinks and mutagenesis are discussed. How certain enzymes recognize and repair different guanine lesions in DNA are also presented.     

Repair activities of 8-oxoguanine DNA glycosylase from Archaeoglobus fulgidus, a hyperthermophilic archaeon.

Oxidative DNA damage is caused by reactive oxygen species formed in cells as by products of aerobic metabolism or of oxidative stress. The 8-oxoguanine (8-oxoG) DNA glycosylase from Archaeoglobus fulgidus (Afogg), which excises an oxidatively-damaged form of guanine, was overproduced in Escherichia coli, purified and characterized. A. fulgidus is a sulfate-reducing archaeon, which grows at between 60 and 95 degrees C, with an optimum growth at 83 degrees C. The Afogg enzyme has both DNA glycosylase and apurinic/apyrimidinic (AP) lyase activities, with the latter proceeding through a Schiff base intermediate. As expected for a protein from a hyperthermophilic organism, the enzyme activity is optimal near pH 8.5 and 60 degrees C, denaturing at 80 degrees C, and is thermally stable at high levels of salt (500mM). The Afogg protein efficiently cleaves oligomers containing 8-oxoG:C and 8-oxoG:G base pairs, and is less effective on oligomers containing 8-oxoG:T and 8-oxoG:A mispairs. While the catalytic action mechanism of Afogg protein is likely similar to the human Ogg1 (hOgg1), the DNA recognition mechanism and the basis for 8-oxoG substrate specificity of Afogg differ from that of hOgg.     

Formation of 8-nitroguanosine in cellular RNA as a biomarker of exposure to reactive nitrogen species

Reactive nitrogen species, such as peroxynitrite, nitrogen oxides and nitryl chloride, have been implicated as a cause of diverse pathophysiological conditions, including inflammation, neurodegenerative and cardiovascular diseases and cancer. We previously reported that 8-nitroguanine is formed by reactions of guanine or calf-thymus DNA with peroxynitrite in vitro. In the present study, we have studied the formation of 8-nitroguanosine and 8-oxo-7,8-dihydroguanosine in reactions of calf-liver RNA with various reactive nitrogen species. 8-Nitroguanosine in RNA was found to be much more stable than 8-nitro-2' -deoxyguanosine in DNA, which rapidly depurinates to release 8-nitroguanine. Both 8-nitroguanosine and 8-oxo-7,8-dihydroguanosine were formed in calf-liver RNA following exposure to various reactive nitrogen species, such as synthetic peroxynitrite. They were also formed in RNA by reactive species formed from nitric oxide and superoxide anion generated concomitantly from 3-morpholino-sydnonimine (SIN-1) and those formed with myeloperoxidase or horseradish peroxidase in the presence of nitrite and hydrogen peroxide. 8-Nitroguanosine was detected by HPLC with an electrochemical detector in enzymatic hydrolyzates of RNA isolated from human lung carcinoma cells incubated with synthetic peroxynitrite. Our results indicate that 8-nitroguanosine in cellular RNA could be measured as a marker of damage caused by endogenous reactive nitrogen species in tissues and cells.     

Chemical and biological consequences of oxidatively damaged guanine in DNA

Of the four native nucleosides, 2'-deoxyguanosine (dGuo) is most easily oxidized. Two lesions derived from dGuo are 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodGuo) and 2,6-diamino-4-hydroxy-5-formamidopyrimidine (Fapy)?dGuo. Furthermore, while steady-state levels of 8-oxodGuo can be detected in genomic DNA, it is also known that 8-oxodGuo is more easily oxidized than dGuo. Thus, 8-oxodGuo is susceptible to further oxidation to form several hyperoxidized dGuo products. This review addresses the structural impact, the mutagenic and genotoxic potential, and biological implications of oxidatively damaged DNA, in particular 8-oxodGuo, Fapy?dGuo, and the hyperoxidized dGuo products.     

Chemical and biological consequences of oxidatively damaged guanine in DNA

Abstract

Of the four native nucleosides, 2′-deoxyguanosine (dGuo) is most easily oxidized. Two lesions derived from dGuo are 8-oxo-7,8-dihydro-2′-deoxyguanosine (8-oxodGuo) and 2,6-diamino-4-hydroxy-5-formamidopyrimidine (Fapy)?dGuo. Furthermore, while steady-state levels of 8-oxodGuo can be detected in genomic DNA, it is also known that 8-oxodGuo is more easily oxidized than dGuo. Thus, 8-oxodGuo is susceptible to further oxidation to form several hyperoxidized dGuo products. This review addresses the structural impact, the mutagenic and genotoxic potential, and biological implications of oxidatively damaged DNA, in particular 8-oxodGuo, Fapy?dGuo, and the hyperoxidized dGuo products.     

Peroxynitrite Mediated Oxidation of Purine Bases of Nucleosides and Isolated DNA

Reaction of nitric oxide with superoxide anion produces the highly reactive species peroxynitrite (ONOO^?). This compound has been shown to be a strong oxidant of lipids and proteins. However, no data are available on its effect on DNA, with the exception of the induction of strand breaks. We report the result of studies on the reactions of peroxynitrite with the adenine and guanine moieties of nucleosides and isolated DNA. The samples were analyzed for 8-oxo-7,8-dihydro-2′-deoxyguano-sine (8-oxo-dGuo), 2,2-diamino-4–[(2-deoxy-β-D-erythro-pentofuranosyl)amino]-5–(2H)-oxazolone (oxazolone) and 8-oxo-7,8-dihydro-2′-deoxyadenosine (8-oxo-dAdo). The effects of peroxynitrite treatment were compared with those of ionizing radiation in aerated aqueous solution, chosen as a source of hydroxyl radicals. At the nucleoside level, both oxidizing conditions led to the formation of oxazolone and 8-oxo-dAdo. In addition, evidence was provided for the formation of the 4R* and 4S* diastereoisomers of 4-hydroxy-8-oxo-4,8-dihydro-2′-deoxyguanosine. The latter dGuo oxidation products were chosen as markers of the release of singlet oxygen (¹O₂) upon reaction of peroxynitrous acid with hydrogen peroxide. Oxidation of purine bases was then studied within isolated DNA. A significant increase in the level of 8-oxp-dGuo, oxazolone and 8-oxo-dAdo was observed within double stranded DNA upon exposure to γ-radiation. Oxazolone and 8-oxo-dAdo were formed upon peroxynitrite treatment but no significant increase in the amount of 8-oxo-dGuo was detected. These results showed that peroxynitrite exhibits oxidizing properties toward purine moieties both in nucleosides and isolated DNA. However, the significant differences in the oxidative damage distribution within DNA observed after exposure to γ radiation by comparison with peroxynitrite treatment questions the involvement of hydroxyl radicals as the main oxidizing species released by decomposition of peroxynitrous acid.     

Enzymatic properties of Escherichia coli and human 7,8-dihydro-8-oxoguanine DNA glycosylases.

Oxidative damage to DNA generates aberrant guanine bases such as 2,6-diamino-4-hydroxy-formamido-pyrimidine (Fapy) and 7,8-dihydro-8-oxoguanine (8-oxoG). Although synthetic oligonucleotides containing a single 8-oxoG have been widely used to study enzymatic processing of this lesion, the synthesis of oligonucleotides containing Fapy as a unique lesion has not been achieved to date. In this study, an oligonucleotide containing a single 2,6-diamino-4-hydroxy-5-(N-methyl)formamido-pyrimidine (me-Fapy, a methylated derivative of Fapy) was prepared by a DNA polymerase reaction and the subsequent alkali treatment. The repair activity of Fpg and hOGG1 proteins were compared using oligonucleotide substrates containing me-Fapy and 8-oxoG.     

Mutagenesis and carcinogenesis caused by the oxidation of nucleic acids

Genomes and their precursor nucleotides are highly exposed to reactive oxygen species, which are generated both as byproducts of oxygen respiration or molecular executors in the host defense, and by environmental exposure to ionizing radiation and chemicals. To counteract such oxidative damage in nucleic acids, mammalian cells are equipped with three distinct enzymes. MTH1 protein hydrolyzes oxidized purine nucleoside triphosphates, such as 8-oxo-2'-deoxyguanosine triphosphate and 2-hydroxy-2'-deoxyadenosine triphosphate (2-OH-dATP), to the corresponding monophosphates. We observed increased susceptibility to spontaneous carcinogenesis in MTH1-null mice, which exhibit an increased occurrence of A:T-->C:G and G:C-->T:A transversion mutations. 8-Oxoguanine (8-oxoG) DNA glycosylase, encoded by the OGG1 gene, and adenine DNA glycosylase, encoded by the MUTYH gene, are responsible for the suppression of G:C to T:A transversions caused by the accumulation of 8-oxoG in the genome. Deficiency of these enzymes leads to increased tumorigenesis in the lung and intestinal tract in mice, respectively. MUTYH deficiency may also increase G:C to T:A transversions through the misincorporation of 2-OH-dATP, especially in the intestinal tract, since MUTYH can excise 2-hydroxyadenine opposite guanine in genomic DNA and the repair activity is selectively impaired by a mutation found in patients with autosomal recessive colorectal adenomatous polyposis.     

A functional analysis of the DNA glycosylase activity of mouse MUTYH protein excising 2-hydroxyadenine opposite guanine in DNA

2-Hydroxy-2-deoxyadenosine triphosphate (2-OH-dATP), generated by the oxidation of dATP, can be misincorporated by DNA polymerases opposite guanine in template DNA during DNA replication, thus causing spontaneous mutagenesis. We demonstrated that mouse MUTYH (mMUTYH) has a DNA glycosylase activity excising not only adenine opposite 8-oxoguanine (8-oxoG) but also 2-hydroxyadenine (2-OH-A) opposite guanine, using purified recombinant thioredoxin-mMUTYH fusion protein. mMUTYH formed a stable complex with duplex oligonucleotides containing an adenine:8-oxoG pair, but the binding of mMUTYH to oligonucleotides containing a 2-OH-A:guanine pair was barely detectable, thus suggesting that mMUTYH recognizes and interacts with these two substrates in a different manner which may reflect the difference in the base excision repair process for each substrate. Mutant mMUTYH with G365D amino acid substitution, corresponding to a G382D germline mutation of human MUTYH found in familial adenomatous polyposis patients, almost completely retained its DNA glycosylase activity excising adenine opposite 8-oxoG; however, it possessed 1.5% of the wild-type activity excising 2-OH-A opposite guanine. Our results imply that the reduced repair capacity of the mutant hMUTYH(G382D), which inefficiently excises 2-OH-A opposite guanine, results in an increased occurrence of somatic G:C to T:A transversion mutations in the APC gene as well as tumorigenesis in the colon.     

Oxidative DNA damage associated with combination of guanine and superoxide radicals and repair mechanisms via radical trapping

In living tissues under inflammatory conditions, superoxide radicals (O(2)*)) are generated and are known to cause oxidative DNA damage. However, the mechanisms of action are poorly understood. It is shown here that the combination of O(2)* with guanine neutral radicals, G(-H)* in single- or double-stranded oligodeoxyribonucleotides (rate constant of 4.7 +/- 1.0 x 10(8) m(-1) s(-1) in both cases), culminates in the formation of oxidatively modified guanine bases (major product, imidazolone; minor product, 8-oxo-7,8-dihydroguanine). The G(-H)* and O(2)* radicals were generated by intense 308 nm excimer laser pulses resulting in the one-electron oxidation and deprotonation of guanine in the 5'-d(CC[2AP]-TCGCTACC) strands and the trapping of the ejected electrons by molecular oxygen (Shafirovich, V., Dourandin, A., Huang, W., Luneva, N. P., and Geacintov, N. E. (2000) Phys. Chem. Chem. Phys. 2, 4399-4408). The addition of Cu,Zn-superoxide dismutase, known to react rapidly with superoxide, dramatically enhances the life-times of guanine radicals from 4 to 7 ms to 0.2-0.6 s in the presence of 5 microm superoxide dismutase. Oxygen-18 isotope labeling experiments reveal two pathways of 8-oxo-7,8-dihydroguanine formation including either addition of O(2)* to the C-8 position of G(-H)* (in the presence of oxygen), or the hydration of G(-H)* (in the absence of oxygen). The formation of the guanine lesions via combination of guanine and superoxide radicals is greatly reduced in the presence of typical antioxidants such as trolox and catechol that rapidly regenerate guanine by the reductive "repair" of G(-H)* radicals. The mechanistic aspects of the radical reactions that either regenerate undamaged guanine in DNA or lead to oxidatively modified guanine bases are discussed.     

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.