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.     

Light-induced oxidation of the telomeric G4 DNA in complex with Zn(II) tetracarboxymethyl porphyrin

Structure-specific ligands are convenient tools for the recognition, targeting or probing of non-canonical DNA structures. Porphyrin derivatives exhibit a preference for interaction with G-quadruplex (G4) structures over canonical duplex DNA and are able to cause photoinducible damage to nucleic acids. Here, we show that Zn(II) 5,10,15,20-tetrakis(N-carboxymethyl-4-pyridinium)porphyrin (ZnP1) interacts with different conformations of the telomeric sequence d(TAGGG(TTAGGG)₃) at submicromolar concentrations without any detectible disturbance of the particular fold. Among different folds, potassium (3+1) hybrid G4-structure. reveal the highest affinity to ZnP1. The pattern of guanine oxidation is specific for each telomeric DNA conformation and may serve as an additional tool for probing the G4 topology. The potassium (3+1) and parallel G4 conformations are more susceptible to light-induced oxidation than the sodium G4 conformation or double helix of the telomeric DNA. The major products of the guanine modifications are spiroiminodihydantoin (Sp) and 8-oxoguanine (8-oxoG). ZnP1-induced oxidation of guanines results in the structural rearrangement of parallel and (3+1) G4 conformations yielding an antiparallel-like G4 conformation. The mechanism of the observed light-induced conformational changes is discussed.     

Role of lysine-57 in the catalytic activities of Escherichia coli formamidopyrimidine-DNA glycosylase (Fpg protein).

The Escherichia coli Fpg protein is involved in the repair of oxidized residues. We examined, by targeted mutagenesis, the effect of the conserved lysine residue at position 57 upon the various catalytic activities of the Fpg protein. Mutant Fpg protein with Lys-57-->Gly (K57G) had dramatically reduced DNA glycosylase activity for the excision of 7,8-dihydro-8-oxo-guanine (8-oxoG). While wild type Fpg protein cleaved 8-oxoG/C DNA with a specificity constant ( k cat/ K M) of 0.11/(nM@min), K57G cleaved the same DNA 55-fold less efficiently. FpgK57G was poorly effective in the formation of Schiff base complex with 8-oxoG/C DNA. The efficiency in the binding of 8-oxoG/C DNA duplex for K57G mutant was decreased 16-fold. The substitution of Lys-57 for another basic amino acid Arg (K57R) had a slight effect on the 8-oxoG-DNA glycosylase activity and Schiff base formation. The DNA glycosylase activities of FpgK57G and FpgK57R using 2,6-diamino-4-hydroxy-5N-methylformamidopyrimidine residues as substrate were comparable to that of wild type Fpg. In vivo, the mutant K57G, in contrast to the mutant K57R and wild type Fpg, only partially restored the ability to prevent spontaneously induced transitions G/C-->T/A in E.coli BH990 ( fpg mutY ) cells. These results suggest an important role for Lys-57 in the 8-oxoG-DNA glycosylase activity of the Fpg protein in vitro and in vivo.     

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

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

The C-terminal Lysine of Ogg2 DNA Glycosylases is a Major Molecular Determinant for Guanine/8-Oxoguanine Distinction

7,8-Dihydro-8-oxoguanine (8-oxoG) is a major oxidative lesion found in DNA. The 8-oxoguanine DNA glycosylases (Ogg) responsible for the removal of 8-oxoG are divided into three families Ogg1, Ogg2 and AGOG. The Ogg2 members are devoid of the recognition loop used by Ogg1 to discriminate between 8-oxoG and guanine and it was unclear until recently how Ogg2 enzymes recognize the oxidized base. We present here the first crystallographic structure of an Ogg2 member, Methanocaldococcus janischii Ogg, in complex with a DNA duplex containing the 8-oxoG lesion. This structure highlights the crucial role of the C-terminal lysine, strictly conserved in Ogg2, in the recognition of 8-oxoG. The structure also reveals that Ogg2 undergoes a conformational change upon DNA binding similar to that observed in Ogg1 glycosylases. Furthermore, this work provides a structural rationale for the lack of opposite base specificity in this family of enzymes.     

Efficient and high fidelity incorporation of dCTP opposite 7,8-dihydro-8-oxodeoxyguanosine by Sulfolobus solfataricus DNA polymerase Dpo4

DNA polymerases insert dATP opposite the oxidative damage product 7,8-dihydro-8-oxodeoxyguanosine (8-oxoG) instead of dCTP, to the extent of >90% with some polymerases. Steady-state kinetics with the Y-family Sulfolobus solfataricus DNA polymerase IV (Dpo4) showed 90-fold higher incorporation efficiency of dCTP > dATP opposite 8-oxoG and 4-fold higher efficiency of extension beyond an 8-oxoG:C pair than an 8-oxoG:A pair. The catalytic efficiency for these events (with dCTP or C) was similar for G and 8-oxoG templates. Mass spectral analysis of extended DNA primers showed >/=95% incorporation of dCTP > dATP opposite 8-oxoG. Pre-steady-state kinetics showed faster rates of dCTP incorporation opposite 8-oxoG than G. The measured K(d)(,dCTP) was 15-fold lower for an oligonucleotide containing 8-oxoG than with G. Extension beyond an 8-oxoG:C pair was similar to G:C and faster than for an 8-oxoG:A pair, in contrast to other polymerases. The E(a) for dCTP insertion opposite 8-oxoG was lower than for opposite G. Crystal structures of Dpo4 complexes with oligonucleotides were solved with C, A, and G nucleoside triphosphates placed opposite 8-oxoG. With ddCTP, dCTP, and dATP the phosphodiester bonds were formed even in the presence of Ca(2+). The 8-oxoG:C pair showed classic Watson-Crick geometry; the 8-oxoG:A pair was in the syn:anti configuration, with the A hybridized in a Hoogsteen pair with 8-oxoG. With dGTP placed opposite 8-oxoG, pairing was not to the 8-oxoG but to the 5' C (and in classic Watson-Crick geometry), consistent with the low frequency of this frameshift event observed in the catalytic assays.     

Role of the N-terminal proline residue in the catalytic activities of the Escherichia coli Fpg protein

The Escherichia coli Fpg protein is a DNA glycosylase/AP lyase. It removes, in DNA, oxidized purine residues, including the highly mutagenic C8-oxo-guanine (8-oxoG). The catalytic mechanism is believed to involve the formation of a transient Schiff base intermediate formed between DNA containing an oxidized residue and the N-terminal proline of the Fpg protein. The importance and the role of this proline upon the various catalytic activities of the Fpg protein was examined by targeted mutagenesis, resulting in the construction of three mutant Fpg proteins: Pro-2 --> Gly (FpgP2G), Pro-2 --> Thr (FpgP2T), and Pro-2 --> Glu (FpgP2E). The formamidopyrimidine DNA glycosylase activities of FpgP2G and FpgP2T were comparable and accounted for 10% of the wild-type activity. FpgP2G and FpgP2T had barely detectable 8-oxoG-DNA glycosylase activity and produced minute Schiff base complex with 8-oxoG/C DNA. FpgP2G and FpgP2T mutants did not cleave a DNA containing preformed AP site but readily produced Schiff base complex with this substrate. FpgP2E was completely inactive in all the assays. The binding constants of the different mutants when challenged with a duplex DNA containing a tetrahydrofuran residue were comparable. The mutant Fpg proteins barely or did not complement in vivo the spontaneous transitions G/C --> T/A in E. coli BH990 (fpg mutY) cells. These results show the mandatory role of N-terminal proline in the 8-oxoG-DNA glycosylase activity of the Fpg protein in vitro and in vivo as well as in its AP lyase activity upon preformed AP site but less in the 2,6-diamino-4-hydroxy-5-N-methylformamidopyrimidine-DNA glycosylase activity.     

NMR evidence of the stabilisation by the carcinogen N-2-acetylaminofluorene of a frameshift mutagenesis intermediate.

Two heteroduplexes d(C1A2C3T4C5G6C7A8C9A10C11)-d (G12T13G14T15G16G17A18G19T20G21) containing a bulged guanine either unmodified or modified with the carcinogen N-2-acetylaminofluorene (AAF) have been studied by nuclear magnetic resonance (NMR) as models of slipped mutagenic intermediates (SMI). Conformational equilibria are observed in both the unmodified and the AAF-modified heteroduplexes. The major conformation of the unmodified duplex is one where the extra guanine is stacked in the helix and the major conformation of the AAF-modified heteroduplex is one where the AAF is external to the helix. Unusual sugar proton chemical shifts of C5- and G6-AAF indicate that the AAF ring is pointing out in the 5' direction. A strong increase in the modified heteroduplex melting temperature (+15 degrees C) is observed. Moreover, in contrast to the unmodified heteroduplex, which shows extensive melting in the vicinity of the bulged guanine, the base pairs around the bulge in the AAF-modified heteroduplex remain paired at temperatures up to 30 degrees C. This exceptional stability of the site around the bulged modified guanine is suggested to be responsible for the high rate of -1 mutation induced by AAF at repetitive sequences.     

Impact of the oxidized guanine lesion spiroiminodihydantoin on the conformation and thermodynamic stability of a 15-mer DNA duplex

Spiroiminodihydantoin (Sp) is a hyperoxidized guanine base produced from oxidation of the mutagenic DNA lesion 7,8-dihydro-8-oxo-2'-deoxguanosine (8-oxoG) by a variety of species including peroxynitrite, singlet oxygen, and the high-valent metals Ir(IV) and Cr(V). In this study, the conformation and thermodynamic stability of a 15-mer DNA duplex containing an Sp lesion are examined using spectroscopic techniques and differential scanning calorimetry (DSC). The Sp lesion does not alter the global B-form conformation of the DNA duplex as determined by circular dichroism spectroscopy. Thermal denaturation experiments find that Sp significantly lowers the thermal stability of the duplex by approximately 20 degrees C. The enthalpies, entropies, and free energies of duplex formation for 15-mers containing guanine, 8-oxoG, and Sp were determined by performing DSC experiments as well as van't Hoff analysis of UV melting spectroscopic data. The thermodynamic stability of the Sp duplex is significantly reduced compared to that of both the 8-oxoG and parent G duplexes, with the thermodynamic destabilization being enthalpic in origin. The thermodynamic impact of the Sp lesion is compared to what is found for other types of DNA base damage and discussed in relation to how the presence of this lesion could affect cellular processes, in particular the recognition and repair of these adducts by the base excision repair enzymes.     

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.     

NMR evidence for syn-anti interconversion of a trans opened (10R)-dA adduct of benzo[a]pyrene (7S,8R)-diol (9R,10S)-epoxide in a DNA duplex

2D NMR has been used to examine the structure and dynamics of a 12-mer DNA duplex, d(T(1)A(2)G(3)T(4)C(5)A(6)A(7)G(8)G(9)G(10)C(11)A(12))-d(T(13)G(14)C( 15)C(16)C(17)T(18)T(19)G(20)A(21)C(22)T(23)A(24)), containing a 10R adduct at dA(7) that corresponds to trans addition of the N(6)-amino group of dA(7) to (-)-(7S,8R,9R,10S)-7,8-dihydroxy-9, 10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene [(-)-(S,R,R,S)-BP DE-2]. This DNA duplex contains the base sequence for the major dA mutational hot spot in the HPRT gene when Chinese hamster V79 cells are given low doses of the highly carcinogenic (+)-(R,S,S,R)-BP DE-2 enantiomer. NOE data indicate that the hydrocarbon is intercalated on the 5'-side of the modified base as has been seen previously for other oligonucleotides containing BP DE-2 (10R)-dA adducts. 2D chemical exchange-only experiments indicate dynamic behavior near the intercalation site especially at the 10R adducted dA, such that this base interconverts between the normal anti conformation and a less populated syn conformation. Ab initio molecular orbital chemical shift calculations of nucleotide and dinucleotide fragments in the syn and anti conformations support these conclusions. Although this DNA duplex containing a 10R dA adduct exhibits conformational flexibility as described, it is nevertheless more conformationally stable than the corresponding 10S adducted duplex corresponding to trans opening of the carcinogenic isomer (+)-(R,S,S, R)-BP DE-2, which was too dynamic to permit NMR structure determination. UV and imino proton NMR spectral observations indicated pronounced differences between these two diastereomeric 12-mer duplexes, consistent with conformational disorder at the adduct site and/or an equilibrium with a nonintercalated orientation of the hydrocarbon in the duplex containing the 10S adduct. The existence of conformational flexibility around adducts may be related to the occurrence of multiple mutagenic outcomes resulting from a single DE adduct.     

Comparative studies on the 7-iodo and 7-fluoro derivatives of N-acetoxy-N-2-acetylaminofluorene: binding sites on DNA and conformational change of modified deoxytrinucleotides.

GMP and native DNA were reacted with 7-iodo and 7-fluoro derivatives of N-acetoxy-N-2-acetylaminofluorene. It was shown that the 7-halogeno derivatives react on C-8 of guanine. Furthermore the respective amount of arylamidation (covalent linkage on the C-8 of guanine) and arylation (covalent linkage on 2-NH2 groups of C3 of guanine) addition products was determined in both native and denatured DNA-[14C]AAIF. Two G containing deoxytrinucleotides modified by either AAFF or AAIF were studied comparatively by means of circular dichroism, and as a function of several parameters known to affect the conformation of the deoxytrinucleotides. The induced optical activity in fluorofluorene ring seemed to be very sensible to the conformational changes of the deoxytrinucleotides. On the other hand, the AAIF residue exhibit a lower induced optical activity which remained unchanged when the deoxytrinucleotides conformation was affected. The results presented in this paper led us to conclude that the AAFF and AAIF modified deoxytrinucleotides adopt a conformation which nicely fits with the insertion-denaturation and outside-binding model, respectively.     

NMR studies of a DNA containing 8-hydroxydeoxyguanosine.

The effects of hydroxylation at the C8 of a deoxyguanosine residue in DNA were studied by NMR analysis of a self-complementary dodecanucleotide, d(C1-G2-C3-oh8G4-A5-A6-T7-T8-C9-G10-C11-G12), which has an 8-hydroxy-2'-deoxyguanosine (oh8dG) residue at the 4th position. NMR data indicate that the 8-hydroxyguanine (oh8G) base takes a 6,8-diketo tautomeric form and is base-paired to C with Watson-Crick type hydrogen bonds in a B-form structure. The thermal stability of the duplex is reduced, but the overall structure is much the same as that of the unmodified d(CGCGAATTCGCG) duplex. The structural changes caused by 8-hydroxylation of the deoxyguanosine, if any, are localized near the modification site.     

UV resonance Raman and circular dichroism studies of a DNA duplex containing an A(3)T(3) tract: evidence for a premelting transition and three-centered H-bonds.

The presence of A(n) and A(n)T(n) tracts in double-helical sequences perturbs the structural properties of DNA molecules, resulting in the formation of an alternate conformation to standard B-DNA known as B'-DNA. Evidence for a transition occurring prior to duplex melting in molecules containing A(n) tracts was previously detected by circular dichroism (CD) and calorimetric studies. This premelting transition was attributed to a conformational change from B'- to B-DNA. Structural features of A(n) and A(n)T(n) tracts revealed by X-ray crystallography include a large degree of propeller twisting of adenine bases, narrowed minor grooves, and the formation of three-centered H-bonds between dA and dT bases. We report UV resonance Raman (UVRR) and CD spectroscopic studies of two related DNA dodecamer duplexes, d(CGCAAATTTGCG)(2) (A(3)T(3)) and d(CGCATATATGCG)(2) [(AT)(3)]. These studies address the presence of three-centered H-bonds in the B' conformation and gauge the impact of these putative H-bonds on the structural and thermodynamic properties of the A(3)T(3) duplex. UVRR and CD spectra reveal that the premelting transition is only observed for the A(3)T(3) duplex, is primarily localized to the dA and dT bases, and is associated with base stacking interactions. Spectroscopic changes associated with the premelting transition are not readily detectable for the sugar-phosphate backbone or the cytosine and guanosine bases. The temperature-dependent concerted frequency shifts of dA exocyclic NH(2) and dT C4=O vibrational modes suggest that the A(3)T(3) duplex forms three-centered hydrogen bonds at low temperatures, while the (AT)(3) duplex does not. The enthalpy of this H-bond, estimated from the thermally induced frequency shift of the dT C4=O vibrational mode, is approximately 1.9 kJ/mol or 0.46 kcal/mol.     

Escherichia coli MutY protein has a guanine-DNA glycosylase that acts on 7,8-dihydro-8-oxoguanine:guanine mispair to prevent spontaneous G:C-->C:G transversions.

Low rates of spontaneous G:C-->C:G transversions would be achieved not only by the correction of base mismatches during DNA replication but also by the prevention and removal of oxidative base damage in DNA. Escherichia coli must have several pathways to repair such mismatches and DNA modifications. In this study, we attempted to identify mutator loci leading to G:C-->C:G transversions in E.coli. The strain CC103 carrying a specific mutation in lacZ was mutagenized by random miniTn 10 insertion mutagenesis. In this strain, only the G:C-->C:G change can revert the glutamic acid at codon 461, which is essential for sufficient beta-galactosidase activity to allow growth on lactose. Mutator strains were detected as colonies with significantly increased rates of papillae formation on glucose minimal plates containing P-Gal and X-Gal. We screened approximately 40 000 colonies and selected several mutator strains. The strain GC39 showed the highest mutation rate to Lac+. The gene responsible for the mutator phenotypes, mut39 , was mapped at around 67 min on the E.coli chromosome. The sequencing of the miniTn 10 -flanking DNA region revealed that the mut39 was identical to the mutY gene of E.coli. The plasmid carrying the mutY + gene reduced spontaneous G:C-->T:A and G:C-->C:G mutations in both mutY and mut39 strains. Purified MutY protein bound to the oligonucleotides containing 7,8-dihydro-8-oxo-guanine (8-oxoG):G and 8-oxoG:A. Furthermore, we found that the MutY protein had a DNA glycosylase activity which removes unmodified guanine from the 8-oxoG:G mispair. These results demonstrate that the MutY protein prevents the generation of G:C-->C:G transversions by removing guanine from the 8-oxoG:G mispair in E.coli.     

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.     

Effect ofmutY andmutM/fpg-1 mutations on starvation-associated mutation inEscherichia coli: implications for the role of 7,8-dihydro-8-oxoguanine

MutY specifies a DNA glycosylase that removes adenines unnaturally paired with various bases including oxidized derivatives of guanine, such as 7,8-dihydro-8-oxoguanine (8-oxoG). The rate of mutation in starvedEscherichia coli cells is markedly raised inmutY mutants defective in this glycosylase. As predicted, the mutations produced include G to T transversions. Bacteria carryingmutM orfpg-1 mutations (defective in Fapy glycosylase, which removes oxidized guanine residues such as 8-oxoG) show little or no enhancement of mutation under starvation conditions. When present together withmutY, however,mutM clearly further enhances the rate of mutation in starved cells. Plasmids resulting in overproduction of MutY or Fapy glycosylases reduce the rate of mutation in starved cells. We conclude that, in non-growing bacteria, oxidized guanine residues, including 8-oxoG, constitute an important component of spontaneous mutation. Addition of catalase to the plates did not reduce the mutant yield, indicating that extracellular hydrogen peroxide is not involved in the production of the premutational damage. Singlet oxygen, known to give rise to 8-oxoG, may be the ultimate oxidative species.     

Effect ofmutY andmutM/fpg-1 mutations on starvation-associated mutation inEscherichia coli: implications for the role of 7,8-dihydro-8-oxoguanine

MutY specifies a DNA glycosylase that removes adenines unnaturally paired with various bases including oxidized derivatives of guanine, such as 7,8-dihydro-8-oxoguanine (8-oxoG). The rate of mutation in starvedEscherichia coli cells is markedly raised inmutY mutants defective in this glycosylase. As predicted, the mutations produced include G to T transversions. Bacteria carryingmutM orfpg-1 mutations (defective in Fapy glycosylase, which removes oxidized guanine residues such as 8-oxoG) show little or no enhancement of mutation under starvation conditions. When present together withmutY, however,mutM clearly further enhances the rate of mutation in starved cells. Plasmids resulting in overproduction of MutY or Fapy glycosylases reduce the rate of mutation in starved cells. We conclude that, in non-growing bacteria, oxidized guanine residues, including 8-oxoG, constitute an important component of spontaneous mutation. Addition of catalase to the plates did not reduce the mutant yield, indicating that extracellular hydrogen peroxide is not involved in the production of the premutational damage. Singlet oxygen, known to give rise to 8-oxoG, may be the ultimate oxidative species.     

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.