Molecular Dynamics Simulation of 7, 8-dihydro-8-Oxoguanine DNA

Abstract

To elucidate the effect of guanine lesion produced by the oxidative damage on DNA, 1 nanosecond molecular dynamics simulations of native and oxidized DNA were performed. The target DNA molecules are dodecamer duplex d(CGCGAATTCGCG)2 and its derivative duplex d(C₁G₂C₃(8-oxoG)₄A₅A₆T₇T₈C₉G₁₀C₁₁G₁₂)·d(C₁₃G₁₄C₁₅G₁₆A₁₇A₁₈T₁₉T₂₀C21_G22C₂₃G₂₄), which has one oxidized guanine, 7, 8-dihydro-8-oxoguanine (8-oxoG), at the fourth position. The local structural change due to the lesion of 8-oxoG and the global dynamic structure of the 8-oxoG DNA were studied. It was found that the 8-oxoG DNA remained structurally stable during the simulation due to newly produced hydrogen bonds around the (8-oxoG)₄ residue. However, there were distinguishable differences in structural parameters and dynamic property in the 8-oxoG DNA. The conformation around the (8- oxoG)₄ residue departed from the usual conformation of native DNA and took an unique conformation of ?-ζ in B_II conformation and χ in high anti orientation at the (8-oxoG)₄ residue, and adopted a very low helical twist angle at the C₃:G₂₂—(8-oxoG)₄:C₂₁ step. Further analysis by principal component analysis indicated that the formation of the hydrogen bonds around the (8-oxoG)₄ residue plays a role as a trigger for the conformational transition of the 8-oxoG DNA in the conformational space.     

Distinct repair activities of human 7,8-dihydro-8-oxoguanine DNA glycosylase and formamidopyrimidine DNA glycosylase for formamidopyrimidine and 7,8-dihydro-8-oxoguanine

7,8-dihydro-8-oxoguanine (8-oxoG) and 2,6-diamino-4-hydroxyformamidopyrimidine (Fapy) are major DNA lesions formed by reactive oxygen species and are involved in mutagenic and/or lethal events in cells. Both lesions are repaired by human 7, 8-dihydro-8-oxoguanine DNA glycosylase (hOGG1) and formamidopyrimidine DNA glycosylase (Fpg) in human and Escherichia coli cells, respectively. In the present study, the repair activities of hOGG1 and Fpg were compared using defined oligonucleotides containing 8-oxoG and a methylated analog of Fapy (me-Fapy) at the same site. The k(cat)/K(m) values of hOGG1 for 8-oxoG and me-Fapy were comparable, and this was also the case for Fpg. However, the k(cat)/K(m) values of hOGG1 for both lesions were approximately 80-fold lower than those of Fpg. Analysis of the Schiff base intermediate by NaBH(4) trapping implied that lower substrate affinity and slower hydrolysis of the intermediate for hOGG1 than Fpg accounted for the difference. hOGG1 and Fpg showed distinct preferences of the base opposite 8-oxoG, with the activity differences being 19.8- (hOGG1) and 12-fold (Fpg) between the most and least preferred bases. Surprisingly, such preferences were almost abolished and less than 2-fold for both enzymes when me-Fapy was a substrate, suggesting that, unlike 8-oxoG, me-Fapy is not subjected to paired base-dependent repair. The repair efficiency of me-Fapy randomly incorporated in M13 DNA varied at the sequence level, but orders of preferred and unpreferred repair sites were quite different for hOGG1 and Fpg. The distinctive activities of hOGG1 and Fpg including enzymatic parameters (k(cat)/K(m)), paired base, and sequence context effects may originate from the differences in the inherent architecture of the DNA binding domain and catalytic mechanism of the enzymes.     

tert-Butoxyl radicals generate mainly 7,8-dihydro-8-oxoguanine in DNA

Like hydroxyl radicals, alkoxyl radicals have been implicated in the generation of cellular oxidative DNA damage under physiological conditions; however, their genotoxic potential has not yet been established. We have analyzed the DNA damage induced by a photochemical source of tert-butoxyl radicals, the water soluble peroxy ester [4-(tert-butyldioxycarbonyl)benzyl]triethylammonium chloride (BCBT), using various repair endonucleases as probes. The irradiation (UV(360)) of BCBT in the presence of bacteriophage PM2 DNA was found to generate a DNA damage profile that consisted mostly of base modifications sensitive to the repair endonuclease Fpg protein. Approximately 90% of the modifications were identified as 7,8-dihydro-8-oxoguanine (8-oxoGua) residues by HPLC/ECD analysis. Oxidative pyrimidine modifications (sensitive to endonuclease III), sites of base loss (AP sites) and single-strand breaks were only minor modifications. Experiments with various scavengers and quenchers indicated that the DNA damage by BCBT+UV(360) was caused by tert-butoxyl radicals as the ultimate reactive species. The mutagenicity associated with the induced damage was analyzed in the gpt gene of plasmid pSV2gpt, which was exposed to BCBT+UV(360) and subsequently transfected into Escherichia coli. The results were in agreement with the specific generation of 8-oxoGua. Nearly all point mutations (20 out of 21) were found to be GC-->TA transversions known to be characteristic for 8-oxoGua. In conclusion, alkoxyl radicals generated from BCBT+UV(360) induce 8-oxoGua in DNA with a higher selectivity than any other reactive oxygen species analyzed so far.     

Gel electrophoretic detection of 7,8-dihydro-8-oxoguanine and 7, 8-dihydro-8-oxoadenine via oxidation by Ir (IV).

Two gel electrophoretic methods are described for detection of 7, 8-dihydro-8-oxoguanine and 7,8-dihydro-8-oxoadenine based on their further oxidation with one-electron oxidants including IrCl62-and IrBr62-. The products of nucleobase oxidation lead to enhanced piperidine-sensitive cleavage and to highly visible stop points in a primer extension assay. 8-oxoG and 8-oxoA lesions may be distinguished by the latter's inability to be oxidized by IrBr62-compared to IrCl62-Comparison is also made to oxidation by MnO4-.     

Induction of reversible complexes between eukaryotic DNA topoisomerase I and DNA-containing oxidative base damages. 7, 8-dihydro-8-oxoguanine and 5-hydroxycytosine

We recently showed that abasic sites, uracil mismatches, nicks, and gaps can trap DNA topoisomerase I (top1) when these lesions are introduced in the vicinity of a top1 cleavage site (Pourquier, P., Ueng, L.-M., Kohlhagen, G., Mazumder, A., Gupta, M., Kohn, K. W., and Pommier, Y. (1997) J. Biol. Chem. 272, 7792-7796; Pourquier, P., Pilon, A. A., Kohlhagen, G., Mazumder, A., Sharma, A., and Pommier, Y. (1997) J. Biol. Chem. 26441-26447). In this study, we investigated the effects on top1 of an abundant base damage generated by various oxidative stresses: 7,8-dihydro-8-oxoguanine (8-oxoG). Using purified eukaryotic top1 and oligonucleotides containing the 8-oxoG modification, we found a 3-7-fold increase in top1-mediated DNA cleavage when 8-oxoG was present at the +1 or +2 position relative to the cleavage site. Another oxidative lesion, 5-hydroxycytosine, also enhanced top1 cleavage by 2-fold when incorporated at the +1 position of the scissile strand. 8-oxoG at the +1 position enhanced noncovalent top1 DNA binding and had no detectable effect on DNA religation or on the incision step. top1 trapping by 8-oxoG was markedly enhanced when asparagine adjacent to the catalytic tyrosine was mutated to histidine, suggesting a direct interaction between this residue and the DNA major groove immediately downstream from the top1 cleavage site. Altogether, these results demonstrate that oxidative base lesions can increase top1 binding to DNA and induce top1 cleavage complexes.     

Photosensitized formation of 8-hydroxyguanine (7,8-dihydro-8-oxoguanine) in DNA by riboflavin.

Potosensitized formation of 8-hydroxyguanine in DNA by riboflavin was observed. A reaction mechanism involving guanine radical cation and hydration reaction was proposed. This hypothesis was confirmed by the incorporation of [18O]-atom within guanine moiety in isotopic experiments using [18O]-H2O. Photosensitized formation of oh8Gua by riboflavin was also observed in cellular DNA.     

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.     

Genetic effects of oxidative DNA damage: comparative mutagenesis of 7,8-dihydro-8-oxoguanine and 7,8-dihydro-8-oxoadenine in Escherichia coli.

A single 7,8-dihydro-8-oxoguanine (G8-OXO; 8-hydroxyguanine) adduct in the lacZ alpha gene of bacteriophage M13 DNA induces a targeted G-->T transversion after replication in Escherichia coli (Biochemistry, 29, 7024-7031 (1990)). This mutation is thought to be due to the facile formation during DNA synthesis of a G8-OXO.base pair, where G8-OXO is in the syn conformation about the deoxyglycosyl bond. A related modified purine, 7,8-dihydro-8-oxoadenine (A8-OXO; 8-hydroxyadenine), is an abundant product found in irradiated and oxidized DNAs. Similar to G8-OXO, as a mononucleoside A8-OXO assumes the syn conformation. This work has assessed the relative mutagenicities of A8-OXO and G8-OXO in the same experimental system. A deoxypentanucleotide containing A8-OXO [d(GCT-A8-OXOG)] was synthesized. After 5'-phosphorylation with [gamma-32P] ATP, the oligomer was ligated into a duplex M13mp19-derived genome at a unique NheI restriction site. Genomes containing either A8-OXO (at position 6275, [+] strand) or G8-OXO (position 6276) were denatured with heat and introduced into E.coli DL7 cells. Analysis of phage DNA from mutant plaques obtained by plating immediately after transformation (infective centers assay) revealed that G8-OXO induced G-->T transversions at an apparent frequency of approximately 0.3%. The frequency and spectrum of mutations observed in DNA sequences derived from 172 mutant plaques arising from the A8-OXO-modified DNA were almost indistiguishable from those generated from transfection of an adenine-containing control genome. We conclude that A8-OXO is at least an order of magnitude less mutagenic than G8-OXO in E.coli cells with normal DNA repair capabilities.     

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.     

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.     

Rat 7,8-dihydro-8-oxoguanine DNA glycosylase: substrate specificity, kinetics and cleavagemechanism at an apurinic site.

Reactive oxygen species produce different lesions in DNA. Among them, 7,8-dihydro-8-oxoguanine (8-oxoG) is one of the major oxidative products implicated in mutagenesis. This lesion is removed from damaged DNA by base excision repair, and genes coding for 8-oxoG-DNA glycosylases have been isolated from bacteria, yeast and human cells. We have isolated and characterized the cDNA encoding the rat 8-oxoG-DNA glycosylase (rOGG1). Expression of the cDNA in the fgp mutY Escherichia coli double mutant allowed the purification of the untagged rOGG1 protein. It excises 8-oxoG from DNA with a strong preference for duplex DNA containing 8-oxoG:C base pairs. rOGG1 also acts on formamidopyrimidine (FaPy) residues, and the K m values on 8-oxoG and FaPy residues are 18.8 and 9.7 nM, respectively. When acting on an oligonucleotide containing an 8-oxoG residue, rOGG1 shows a beta-lyase activity that nicks DNA 3' to the lesion. However, rOGG1 acts on a substrate containing an apurinic site by a beta-delta elimination reaction and proceeds through a Schiff base intermediate. Expression of rOGG1 in E.coli fpg mutY suppresses its spontaneous mutator phenotype.     

Human DNA polymerase lambda is a proficient extender of primer ends paired to 7,8-dihydro-8-oxoguanine

Pol lambda is a DNA repair enzyme with a high affinity for dNTPs, an intrinsic dRP lyase activity, a BRCT domain involved in interactions with NHEJ factors, and also capable to interact with the PCNA processivity factor. Based on this potential, Pol lambda could play a role in BER, V(D)J recombination, NHEJ and TLS. Here we show that human Pol lambda uses a templating 7,8-dihydro-8-oxoguanine (8oxoG) base, a common mutagenic form of oxidative damage, as efficiently as an undamaged dG, but giving rise to the alternative insertion of either dAMP or dCMP. However, Pol lambda strongly discriminated against the extension of the mutagenic 8oxoG:dAMP pair. Conversely, Pol lambda readily extended the non-mutagenic 8oxoG:dCMP pair with an efficiency that was even higher than that displayed on undamaged dG:dCMP pair. A similar capacity for non-mutagenic extension was also shown to occur in the case of O6-methylguanine (m6G), a mutagenic and cytotoxic DNA adduct. A comparison of these novel properties of human Pol lambda with those of other DNA polymerases involved in TLS will be discussed. Interestingly, when double-strand breaks are associated to base damage, modifications as 8oxoG could be eventually part of the synapsis required to join ends, and therefore, the capacity of Pol lambda either to insert opposite 8oxoG or to extend correct base pairs containing such a damage could be beneficial for its role in NHEJ.     

Characterization of DNA with an 8-oxoguanine modification

The oxidation of DNA resulting from reactive oxygen species generated during aerobic respiration is a major cause of genetic damage that, if not repaired, can lead to mutations and potentially an increase in the incidence of cancer and aging. A major oxidation product generated in cells is 8-oxoguanine (oxoG), which is removed from the nucleotide pool by the enzymatic hydrolysis of 8-oxo-2'-deoxyguanosine triphosphate and from genomic DNA by 8-oxoguanine-DNA glycosylase. Finding and repairing oxoG in the midst of a large excess of unmodified DNA requires a combination of rapid scanning of the DNA for the lesion followed by specific excision of the damaged base. The repair of oxoG involves flipping the lesion out of the DNA stack and into the active site of the 8-oxoguanine-DNA glycosylase. This would suggest that thermodynamic stability, in terms of the rate for local denaturation, could play a role in lesion recognition. While prior X-ray crystal and NMR structures show that DNA with oxoG lesions appears virtually identical to the corresponding unmodified duplex, thermodynamic studies indicate that oxoG has a destabilizing influence. Our studies show that oxoG destabilizes DNA (ΔΔG of 2-8 kcal mol(-1) over a 16-116 mM NaCl range) due to a significant reduction in the enthalpy term. The presence of oxoG has a profound effect on the level and nature of DNA hydration indicating that the environment around an oxoG?C is fundamentally different than that found at G?C. The temperature-dependent imino proton NMR spectrum of oxoG modified DNA confirms the destabilization of the oxoG?C pairing and those base pairs that are 5' of the lesion. The instability of the oxoG modification is attributed to changes in the hydrophilicity of the base and its impact on major groove cation binding.     

Mechanism of efficient and accurate nucleotide incorporation opposite 7,8-dihydro-8-oxoguanine by Saccharomyces cerevisiae DNA polymerase eta

Most DNA polymerases incorporate nucleotides opposite template 7,8-dihydro-8-oxoguanine (8-oxoG) lesions with reduced efficiency and accuracy. DNA polymerase (Pol) eta, which catalyzes the error-free replication of template thymine-thymine (TT) dimers, has the unique ability to accurately and efficiently incorporate nucleotides opposite 8-oxoG templates. Here we have used pre-steady-state kinetics to examine the mechanisms of correct and incorrect nucleotide incorporation opposite G and 8-oxoG by Saccharomyces cerevisiae Pol eta. We found that Pol eta binds the incoming correct dCTP opposite both G and 8-oxoG with similar affinities, and it incorporates the correct nucleotide bound opposite both G and 8-oxoG with similar rates. While Pol eta incorporates an incorrect A opposite 8-oxoG with lower efficiency than it incorporates a correct C, it does incorporate A more efficiently opposite 8-oxoG than opposite G. This is mainly due to greater binding affinity for the incorrect incoming dATP opposite 8-oxoG. Overall, these results show that Pol eta replicates through 8-oxoG without any barriers introduced by the presence of the lesion.     

Chemiluminescence enzyme immunoassay of 8-oxoguanine in DNA.

A test system has been developed to determine 8-oxoguanine in DNA, the most important biomarker of damage to DNA bases by reactive oxygen species. The system is based on a chemiluminescence enzyme immunoassay with the use of monoclonal antibodies (mcAB) against 8-oxoguanine. The test involves several stages: 1) immobilization of DNA on nitrocellulose membrane filters using an efficient technique with preliminary formation of a complex with protamine sulfate; 2) formation of antigen--antibody complexes (mcAB with 8-oxoguanine in DNA) with secondary antibodies and with a peroxidase--antiperoxidase complex (PAP method); 3) detection of increased chemiluminescence in a solution of hydrogen peroxide, luminol, and p-iodophenol. The increased chemiluminescence is determined with a conventional liquid scintillation counter for measuring beta-radioactivity. The system was tested by determining 8-oxoguanine formation in DNA upon gamma-irradiation and upon photosensitized oxidation of guanine under visible light in the presence of methylene blue. A linear dose dependence of 8-oxoguanine formation in DNA was shown for gamma-irradiation. The radiation-chemical yield of 8-oxoguanine (G = 0.57 molecule per 100 eV) is convenient to use for calibration of the amount of 8-oxoguanine formed under other conditions. The sensitivity of the method permits the detection of several femtomoles of 8-oxoguanine in a 40 microg sample of DNA.     

Insertion of dGMP and dAMP during in vitro DNA synthesis opposite an oxidized form of 7,8-dihydro-8-oxoguanine.

Oxidative damage to DNA bases commonly resultsin the formation of oxidized purines, particularly 7,8-dihydro-8-oxoguanine (8-oxoG) and 7,8-dihydro-8-oxoadenine (8-oxoA), the former being a well-known mutagenic lesion. Since 8-oxoG is readily subject to further oxidation compared with normal bases, the insertion of a base during DNA synthesis opposite an oxidized form of 8-oxoG was investigated in vitro. A synthetic template containing a single 8-oxoG lesion was first treated with different one-electron oxidants or under singlet oxygen conditions and then subjected to primer extension catalyzed by Klenow fragment exo- (Kf exo-), calf thymus DNA polymerase alpha (pol alpha) or human DNA polymerase beta (pol beta). Consistent with previous reports, dAMP and dCMP are inserted selectively opposite 8-oxoG with all three DNA polymerases. Interestingly, oxidation of 8-oxoG was found to induce dAMP and dGMP insertion opposite the lesion by Kf exo- with transient inhibition of primer extension occurring at the site of the modified base. Furthermore, the lesion constitutes a block during DNA synthesis by pol alpha and pol beta. Experiments with an 8-oxoA-modified template oligonucleotide show that both 8-oxoA and an oxidized form of 8-oxoA direct insertion of dTMP by Kf exo-. Mass spectrometric analysis of 8-oxoG-containing oligonucleotides before and after oxidation with IrCl62-are consistent with oxidation of primarily the 8-oxoG site, resulting in formation of a guanidinohydantoin moiety as the major product. No evidence for formation of abasic sites was obtained. These results demonstrate that an oxidized form of 8-oxoG, possibly guanidinohydantoin, may direct misreading and misinsertion of dNTPs during DNA synthesis. If such a process occurred in vivo, it would represent a point mutagenic lesion leading to G-->T and G-->C transversions. However, the corresponding oxidized form of 8-oxoA primarily shows correct insertion of T during DNA synthesis with Kf exo-.     

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.     

Escherichia coli Nth and human hNTH1 DNA glycosylases are involved in removal of 8-oxoguanine from 8-oxoguanine/guanine mispairs in DNA

The spectrum of DNA damage caused by reactive oxygen species includes a wide variety of modifications of purine and pyrimidine bases. Among these modified bases, 7,8-dihydro-8-oxoguanine (8-oxoG) is an important mutagenic lesion. Base excision repair is a critical mechanism for preventing mutations by removing the oxidative lesion from the DNA. That the spontaneous mutation frequency of the Escherichia coli mutT mutant is much higher than that of the mutM or mutY mutant indicates a significant potential for mutation due to 8-oxoG incorporation opposite A and G during DNA replication. In fact, the removal of A and G in such a situation by MutY protein would fix rather than prevent mutation. This suggests the need for differential removal of 8-oxoG when incorporated into DNA, versus being generated in situ. In this study we demonstrate that E.coli Nth protein (endonuclease III) has an 8-oxoG DNA glycosylase/AP lyase activity which removes 8-oxoG preferentially from 8-oxoG/G mispairs. The MutM and Nei proteins are also capable of removing 8-oxoG from mispairs. The frequency of spontaneous G:C→C:G transversions was significantly increased in E.coli CC103mutMnthnei mutants compared with wild-type, mutM, nth, nei, mutMnei, mutMnth and nthnei strains. From these results it is concluded that Nth protein, together with the MutM and Nei proteins, is involved in the repair of 8-oxoG when it is incorporated opposite G. Furthermore, we found that human hNTH1 protein, a homolog of E.coli Nth protein, has similar DNA glycosylase/AP lyase activity that removes 8-oxoG from 8-oxoG/G mispairs.     

Opposite base-dependent reactions of a human base excision repair enzyme on DNA containing 7,8-dihydro-8-oxoguanine and abasic sites.

The guanine modification 7,8-dihydro-8-oxoguanine (8-oxoG) is a potent premutagenic lesion formed spontaneously at high frequencies in the genomes of aerobic organisms. We have characterized a human DNA repair glycosylase for 8-oxoG removal, hOGH1 (human yeast OGG1 homologue), by molecular cloning and functional analysis. Expression of the human cDNA in a repair deficient mutator strain of Escherichia coli (fpg mutY) suppressed the spontaneous mutation frequency to almost normal levels. The hOGH1 enzyme was localized to the nucleus in cells transfected by constructs of hOGH1 fused to green fluorescent protein. Enzyme purification yielded a protein of 38 kDa removing both formamidopyrimidines and 8-oxoG from DNA. The enzymatic activities of hOGH1 was analysed on DNA containing single residues of 8-oxoG or abasic sites opposite each of the four normal bases in DNA. Excision of 8-oxoG opposite C was the most efficient and was followed by strand cleavage via beta-elimination. However, significant removal of 8-oxoG from mispairs (8-oxoG: T >G >A) was also demonstrated, but essentially without an associated strand cleavage reaction. Assays with abasic site DNA showed that strand cleavage was indeed dependent on the presence of C in the opposite strand, irrespective of the prior removal of an 8-oxoG residue. It thus appears that strand incisions are made only if repair completion results in correct base insertion, whereas excision from mispairs preserves strand continuity and hence allows for error-free correction by a postreplicational repair mechanism.     

Backbone dynamics of DNA containing 8-oxoguanine: importance for substrate recognition by base excision repair glycosylases.

Except for the functional groups sited within the major or minor grooves, the bases of B-DNA are quite protected from the external environment. Enzymes that modify the bases often "flip out" the target into an extrahelical position before the chemistry step is carried out. Examples of this mechanism are the base excision repair glycosylases and the restriction enzyme methylases. The question arises about the mechanism of substrate recognition for these enzymes and how closely it is linked to the base flipping step. Molecular dynamics simulations (AMBER, PME electrostatics) of fully solvated, cation neutralized, DNA sequences containing 8-oxoguanine (8OG) and of appropriate normal (control) DNAs have been carried out. The dynamics trajectories were analyzed to identify those properties of the DNA structure in the vicinity of the altered base, or its dynamics, that could contribute to molecular discrimination between substrate and non-substrate DNA sites. The results predict that the FPG enzyme should flip out the cytosine base paired with the scissile 8OG, not the target base itself.