Mechanisms of oxidation of guanine in DNA by carbonate radical anion, a decomposition product of nitrosoperoxycarbonate

Peroxynitrite is produced during inflammation and combines rapidly with carbon dioxide to yield the unstable nitrosoperoxycarbonate, which decomposes (in part) to CO(3) (.-) and (.)NO(2) radicals. The CO(3) (.-) radicals oxidize guanine bases in DNA through a one-electron transfer reaction process that ultimately results in the formation of stable guanine oxidation products. Here we have explored these mechanisms, starting with a spectroscopic study of the kinetics of electron transfer from 20-22mer double-stranded oligonucleotides to CO(3) (.-) radicals, together with the effects of base sequence on the formation of the end-products in runs of one, two, or three contiguous guanines. The distributions of these alkali-labile lesions were determined by gel electrophoresis methods. The cascade of events was initiated through the use of 308 nm XeCl excimer laser pulses to generate CO(3) (.-) radicals by an established method based on the photodissociation of persulfate to sulfate radicals and the oxidation of bicarbonate. Although the Saito model (Saito et al., J. Am. Chem. Soc. 1995, 117, 6406-6407) predicts relative ease of one-electron oxidations in DNA, following the trend 5'-GGG > 5'-GG > 5'-G, we found that the rate constants for CO(3) (.-)-mediated oxidation of guanines in these sequence contexts (k(5)) showed only small variation within a narrow range [(1.5-3.0)x10(7) M(-1) s(-1)]. In contrast, the distributions of the end-products are dependent on the base sequence context and are higher at the 5'-G in 5'-GG sequences and at the first two 5'-guanines in the 5'-GGG sequences. These effects are attributed to a combination of initial hole distributions among the contiguous guanines and the subsequent differences in chemical reaction yields at each guanine. The lack of dependence of k(5) on sequence context indicates that the one-electron oxidation of guanine in DNA by CO(3) (.-) radicals occurs by an inner-sphere mechanism.     

Oxidative damage by ruthenium complexes containing the dipyridophenazine ligand or its derivatives: a focus on intercalation

Interactions with DNA by a family of ruthenium(II) complexes bearing the dppz (dppz = dipyridophenazine) ligand or its derivatives have been examined. The complexes include Ru(bpy)(2)(dppx)(2+) (dppx = 7,8-dimethyldipyridophenazine), Ru(bpy)(2)(dpq)(2+) (dpq = dipyridoquinoxaline), and Ru(bpy)(2)(dpqC)(2+) (dpqC = dipyrido-6,7,8,9-tetrahydrophenazine). Their ground and excited state oxidation/reduction potentials have been determined using cyclic voltammetry and fluorescence spectroscopy. An intercalative binding mode has been established on the basis of luminescence enhancements in the presence of DNA, excited state quenching, fluorescence polarization values, and enantioselectivity. Oxidative damage to DNA by these complexes using the flash/quench method has been examined. A direct correlation between the amount of guanine oxidation obtained via DNA charge transport and the strength of intercalative binding was observed. Oxidative damage to DNA through DNA-mediated charge transport was also compared directly for two DNA-tethered ruthenium complexes. One contains the dppz ligand that binds avidly by intercalation, and the other contains only bpy ligands, that, while bound covalently, can only associate with the base pairs through groove binding. Long range oxidative damage was observed only with the tethered, intercalating complex. These results, taken together, all support the importance of close association and intercalation for DNA-mediated charge transport. Electronic access to the DNA base pairs, provided by intercalation of the oxidant, is a prerequisite for efficient charge transport through the DNA pi-stack.     

One-electron photooxidation and site-selective strand cleavage at 5-methylcytosine in DNA by sensitization with 2-methyl-1,4-naphthoquinone-tethered oligonucleotides

Photosensitized one-electron oxidation was applied to discriminate a specific base site of 5-methylcytosine (mC) generated in DNA possessing a partial sequence of naturally occurring p53 gene, using a sensitizing 2-methyl-1,4-naphthoquinone (NQ) chromophore tethered to an interior of oligodeoxynucleotide (ODN) strands. Photoirradiation and subsequent hot piperidine treatment of the duplex consisting of mC-containing DNA and NQ-tethered complementary ODN led to oxidative strand cleavage selectively at the mC site, when the NQ chromophore was arranged so as to be in close contact with the target mC. The target mC is most likely to be one-electron oxidized into the radical cation intermediate by the sensitization of NQ. The resulting mC radical cation may undergo rapid deprotonation and subsequent addition of molecular oxygen, thereby leading to its degradation followed by strand cleavage at the target mC site. In contrast to mC-containing ODN, ODN analogs with replacement of normal cytosine, thymine, adenine, or guanine at the mC site underwent less amount of such an oxidative strand cleavage at the target base site, presumably due to occurrence of charge transfer and charge recombination processes between the base radical cation and the NQ radical anion. Furthermore, well designed incorporation of the NQ chromophore into an interior of ODN could suppress a competitive strand cleavage at consecutive guanines, which occurred as a result of positive charge transfer. Thus, photosensitization by an NQ-tethered ODN led to one-electron oxidative strand cleavage exclusively at the target mC site, providing a convenient method of discriminating mC in naturally occurring DNA such as human p53 gene as a positive band on a sequencing gel.     

Mapping the sites for selective oxidation of guanines in DNA

The effective energy of a positive charge when it is localized at a specific guanine nucleobase in DNA was calculated using density functional theory. The results demonstrate that the efficiency of a guanine to act as a hole-trap in DNA strongly depends on the nature of the flanking nucleobases. The presence of a pyrimidine base at the 3' position adjacent to a guanine significantly increases the localization energy of the positive charge. The calculated distributions of a positive charge in sequences of two or three adjacent guanines, flanked by other nucleobases, provide an explanation for experimental literature data on the site-selective oxidation of DNA.     

Oxidation of guanine in G, GG, and GGG sequence contexts by aromatic pyrenyl radical cations and carbonate radical anions: relationship between kinetics and distribution of alkali-labile lesions

Oxidatively generated DNA damage induced by the aromatic radical cation of the pyrene derivative 7,8,9,10-tetrahydroxytetrahydrobenzo[a]pyrene (BPT), and by carbonate radicals anions, was monitored from the initial one-electron transfer, or hole injection step, to the formation of hot alkali-labile chemical end-products monitored by gel electrophoresis. The fractions of BPT molecules bound to double-stranded 20-35-mer oligonucleotides with noncontiguous guanines G and grouped as contiguous GG and GGG sequences were determined by a fluorescence quenching method. Utilizing intense nanosecond 355 nm Nd:YAG laser pulses, the DNA-bound BPT molecules were photoionized to BPT*+ radicals by a consecutive two-photon ionization mechanism. The BPT*+ radicals thus generated within the duplexes selectively oxidize guanine by intraduplex electron-transfer reactions, and the rate constants of these reactions follow the trend 5'-..GGG.. > 5'-..GG.. > 5'-..G... In the case of CO3*- radicals, the oxidation of guanine occurs by intermolecular collision pathways, and the bimolecular rate constants are independent of base sequence context. However, the distributions of the end-products generated by CO3*- radicals, as well as by BPT*+, are base sequence context-dependent and are greater than those in isolated guanines at the 5'-G in 5'-...GG... sequences, and the first two 5'- guanines in the 5'-..GGG sequences. These results help to clarify the conditions that lead to a similar or different base sequence dependence of the initial hole injection step and the final distribution of oxidized, alkali-labile guanine products. In the case of the intermolecular one-electron oxidant CO3*-, the rate constant of hole injection is similar for contiguous and isolated guanines, but the subsequent equilibration of holes by hopping favors trapping and product formation at contiguous guanines, and the sequence dependence of these two phenomena are not correlated. In contrast, in the case of the DNA-bound oxidant BPT*+, the hole injection rate constants, as well as hole equilibration, exhibit a similar dependence on base sequence context, and are thus correlated to one another.     

Influence of intervening mismatches on long-range guanine oxidation in DNA duplexes

A systematic investigation of the efficiency of oxidative damage at guanine residues through long-range charge transport was carried out as a function of intervening base mismatches. A series of DNA oligonucleotides were synthesized that incorporate a ruthenium intercalator linked covalently to the 5' terminus of one strand and containing two 5'-GG-3' sites in the complementary strand. Single base mismatches were introduced between the two guanine doublet steps, and the efficiency of transport through the mismatches was determined through measurements of the ratio of oxidative damage at the guanine doublets distal versus proximal to the intercalated ruthenium oxidant. Differing relative extents of guanine oxidation were observed for the different mismatches. The damage ratio of oxidation at the distal versus proximal site for the duplexes containing different mismatches varies in the order GC approximately GG approximately GT approximately GA > AA > CC approximately TT approximately CA approximately CT. For all assemblies, damage found with the Delta-Ru diastereomer was found to be greater than with the Lambda-diastereomer. The extent of distal/proximal guanine oxidation in different mismatch-containing duplexes was compared with the helical stability of the duplexes, electrochemical data for intercalator reduction on different mismatch-containing DNA films, and base-pair lifetimes for oligomers containing the different mismatches derived from 1H NMR measurements of the imino proton exchange rates. While a clear correlation is evident both with helix stability and electrochemical data monitoring reduction of an intercalator through DNA films, damage ratios correlate most closely with base-pair lifetimes. Competitive hole trapping at the mismatch site does not appear to be a key factor governing the efficiency of transport through the mismatch. These results underscore the importance of base dynamics in modulating long-range charge transport through the DNA base-pair stack.     

Oxidation of DNA Hairpins by Oxoruthenium(IV): Effects of Sterics and Secondary Structure

The effects of steric hindrance on the oxidation of DNA by polypyridyl oxoruthenium(IV) complexes have been investigated. The complexes oxidize DNA by activation either of the 1' ribose C-H bond or by oxo transfer to the guanine nucleobase. A method is presented for determining the relative rates of activation of individual sites from the dependence of the extent of cleavage on the oxidant concentration. This analysis shows that hybridization of the labeled strand to its complement attenuates the rate of oxidation of guanine more effectively than the rate of sugar oxidation. Accordingly, higher ratios of guanine/sugar oxidation are observed in single strands. Among the individual guanine residues, however, the relative reactivities are not altered by hybridization; a similar result is obtained for sugar oxidation. This result implies that sequence-dependent chemical reactivity is partly responsible for the different extents of cleavage observed within the sequence. The ability of hybridization to protect guanine from oxidation is also apparent in hairpin studies, where the stem guanines are much less reactive than the loop guanines, and altered sugar conformations in the loop lead to modulated reactivity. Finally, a set of sterically differentiated complexes shows greater steric effects for oxidation of guanine compared to oxidation of sugar, as expected from the relative rates of the single strand and duplexes.     

The effect of varied ion distributions on long-range DNA charge transport

Long-range oxidative damage to DNA was utilized as a probe to delineate the effects of different ion distributions on DNA charge transport. DNA assemblies were constructed, containing a tethered rhodium intercalating photooxidant, spatially separated from two 5'-GG-3' sites of oxidative damage, with either an A6-tract or a mixed DNA sequence intervening between the guanine doublets; the extent of charge transport was assessed through measurements of the ratio of yields of damage at the guanine doublet distal versus that proximal to the metal binding site. The distal/proximal damage ratios were compared after photooxidation of otherwise identical Rh-tethered assemblies, except for 32P-labeling either at the 5'- or 3'-end; this labeling difference corresponds, in the absence of charge neutralization by condensed counterions, to a shift in negative charge from one end of the duplex to the other. Both with assemblies containing the mixed sequence and the A6-tract, we observed that moving the negative charges to the proximal end of the duplex significantly decreased hole transport to the distal end. We propose that these results reflect variations in the thermodynamic potential at the proximal and distal guanine sites because of the change in charges at the termini of the oligomer. High values for the internal dielectric constant of the stacked base pairs are suggested by these data. Hence, the longitudinal polarizability of DNA may be important to consider in mechanisms for long-range DNA charge transport.     

Rapid radical formation by DNA charge transport through sequences lacking intervening guanines

Using the flash-quench technique to probe DNA charge transport in assemblies containing a tethered ruthenium intercalator, the kinetics and yield of methylindole radical formation as a function of DNA sequence were studied by laser spectroscopy and biochemical methods. In these assemblies, the methylindole moiety serves as an artificial base of low oxidation potential. Hole injection and subsequent formation of the methylindole radical cation were observed at a distance of over 30 A at rates >/=107 s-1 in assemblies containing no guanine bases intervening the ruthenium intercalator and GMG oxidation site. Radical yield was, however, strikingly sensitive to an intervening base mismatch; no significant methylindole radical formation was evident with an intervening AA mismatch. Also critical is the sequence at the injection site; this sequence determines initial hole localization and hence the probability of hole propagation. With guanine rather than inosine near the site of hole injection, decreased yields of radicals and long-range oxidative damage are observed. The presence of the low-energy guanine site in this case serves to localize the hole and therefore diminish charge transport through the base pair stack.     

Emergent functionality of nucleobase radical cations in duplex DNA: prediction of reactivity using qualitative potential energy landscapes

The one-electron oxidation of a series of DNA oligonucleotides was examined. Each oligomer contains a covalently linked anthraquinone (AQ) group. Irradiation of the AQ group with near-UV light results in a one-electron oxidation of the DNA that generates a radical cation (electron "hole"). The radical cation migrates through the DNA by a hopping mechanism and is trapped by reaction with water or molecular oxygen, which results in chemical reaction at particular nucleobases. This reaction is revealed as strand cleavage when the irradiated oligonucleotide is treated with piperidine. The specific oligomers examined reveal the existence of three categories of nucleobase sequences: charge shuttles, charge traps, and barriers to charge migration. The characterization of a sequence is not independent of the identity of other sequences in the oligonucleotide, and for this reason, the function of a particular sequence emerges from an analysis of the entire structure. Qualitative potential energy landscapes are introduced as a tool to assist in the rationalization and prediction of the reactions of nucleobases in oxidized DNA.     

Regulation of one-electron oxidation rate of guanine by base pairing with cytosine derivatives

Effects of base pairing on the one-electron oxidation rate of guanine derivatives, guanine, 8-bromoguanine, and 8-oxo-7,8-dihydroguanine have been studied. The one-electron oxidation rate of guanine derivatives was determined by triplet-quenching experiments, using N,N'-dibutylnaphthaldiimide (NDI) in the triplet excited state (3NDI*) and fullerene (C(60)) in the triplet excited state ((3)C(60*)) as oxidants. In all three guanine derivatives studied here, acceleration of the one-electron oxidation was observed upon hydrogen bonding with cytosine, which demonstrates lowering of the oxidation potential of guanine derivatives by base pairing with cytosine. When a methyl or bromo group was introduced to the C5 position of cytosine, acceleration or suppression of the one-electron oxidation relative to the guanine:cytosine base pair was observed, respectively. The results demonstrate that the one-electron oxidation rate of guanine in DNA can be regulated by introducing a substituent on base pairing cytosine.     

Oxidative charge transport through DNA in nucleosome core particles

Eukaryotic DNA is packaged into nucleosomes, made up of 146 bp of DNA wrapped around a core of histone proteins. We used photoexcited rhodium intercalators to explore DNA charge transport within these assemblies. Although histone proteins inhibit intercalation of the rhodium complex within the core particle, they do not prevent 5'-GG-3' oxidation, the signature of oxidative charge transport through DNA. Moreover, using rhodium intercalators tethered to the 5' terminus of the DNA, we found that guanine bases within the nucleosome can be oxidized from a distance of 24 bp. Histone binding did not affect the pattern and extent of this oxidation. Therefore, although the structure of the nucleosome core particle generally protects DNA from damage by solution-borne molecules, packaging within the nucleosome does not protect DNA from charge transfer damage through the base pair stack.     

Effect of hydrogen bonding on the photo-oxidation of DNA

The one-electron oxidation of DNA has been extensively studied as it leads to the formation of oxidative lesions that cause carcinogenesis and aging. In this paper, experimental results specifically addressing the effect of hydrogen bonding on the one-electron oxidation rate of nucleosides are presented. To separate the hydrogen bonding from the π-stacking effect, experiments were performed in dichloromethane, in which base-pair formation is possible at the monomer level. The effect of base pairing of guanine with cytosine on the rate constant of the electron transfer from guanine to electron acceptor molecules in the triplet excited state was investigated, and a selective enhancement of the electron transfer was observed for the guanine:cytosine base pair. By introducing a methyl or bromo group to the C5 position of cytosine, acceleration or suppression, respectively, of the one-electron oxidation relative to the guanine:cytosine base pair was observed. The results demonstrate that the one-electron oxidation rate of guanine in DNA can be regulated by introducing a substituent on the base-pairing cytosine.     

On the Distance‐Independent Hole Transfer over Long (A⋅T)n‐Sequences in DNA

Electron holes can travel through DNA double strands over long distances in a multistep ‘hopping’ process. But the influence of the DNA sequence on this process is still not understood in all details. We have carried out new experiments to understand the recent observation that the efficiency of the hole transport between guanines (G), which are separated from each other by long adenine?thymine (A?T) sequences, is nearly independent of the length of the (A?T)_n sequence for n ≥ 4. For this purpose, a new synthesis of the modified adenosine 16 and its incorporation into a DNA double strand was worked out. Subsequent experiments demonstrated that the hole transport between GGG units and the H₂O trapping of the guanine radical cation display similar rates. We conclude that the charge must be already partially equilibrated before being trapped by H₂O. Thus, the weak distance effect is caused not only by the rate of the hole transport, but also by its equilibration over the (A ? T)_n sequence.     

Reactivity of guanine at m5CpG steps in DNA: evidence for electronic effects transmitted through the base pairs.

BACKGROUND: Mitomycin C (MC), a DNA cross-linking and alkylating agent, targets guanines in the m5CpG sequence with 2-3-fold preference over guanines in unmethylated CpG. Benzo[a]pyrenediolepoxide (BPDE) and several other aromatic carcinogens form guanine adducts with an identical selectivity for m5CpG, and in certain cancers G to T transversion mutation 'hotspots' in the p53 tumor suppressor gene are more frequent at this sequence than at guanines in other sequences. MC appears suitable to probe the general mechanism of this selectivity. RESULTS: A 162-bp DNA fragment containing C, m5C or f5C (5-fluoro cytosine) at all cytosine positions was cross-linked by MC at guanines in CpG steps. The extent of cross-linking increased in the order f5C < C < m5C. Monoalkylation or cross-linking of duplex 12-mer oligonucleotides containing a single CpG, f5CpG or m5CpG step gave yields of adducts that increased in the same order. The rates showed a correlation with the Hammett sigma constant of the methyl and fluoro substituents of the cytosine. Only the base-pair cytosine substituent influenced reactivity of guanine. CONCLUSIONS: The 2-amino group of guanine in the m5CpG sequence of DNA has a greater nucleophilic reactivity with mitomycin than CpG. Evidence is presented for a novel mechanism: transmission of the electron-donating effect of the 5-methyl substituent of the cytosine to guanine through H-bonding of the m5C.G base pair. The results explain the enhanced reaction of BPDE at m5CpG in DNA and the origin of G-T mutational hotspots in the p53 gene in cancer.     

Guanine of the third strand of C.G*G triplex serves as an effective hole trap

We have examined the structural and electronic effects of the one-electron oxidation of the C.GG triplex, where G is located in a quite different environment from the G of duplex DNA. Upon photoirradiation of an external photosensitizer (riboflavin) with the C.GG triplex, oxidative DNA cleavage occurred exclusively at guanine repeat sequences in the third strand of triple helix DNA. Hole transport through the C.GG triplex also occurred, resulting in selective cleavage at G in the third strand. Thus, the hole generated in the duplex can migrate to GGG in the third strand and is trapped exclusively at Gs in the third strand. These experimental results, together with molecular orbital calculations, suggest that the origin of the selective strand cleavage can be explained as follows: (i) guanine repeat sequences in the third strand are more easily oxidized than in duplex DNA and (ii) in their radical cation states, G of the third strand rapidly deprotonates and reacts with oxygen and/or water, leading to strand cleavage. These results indicate that the oxidative damage preferentially occurred at Gs of the third strand owing to thermodynamic and kinetic features of the one-electron oxidation of the C.GG triplex.     

Long-distance radical cation transport in DNA: Horizontal charge hopping in a dimeric quadruplex

A series of DNA hairpins were synthesized and shown to associate to form quadruplexes formed by stacking five G-quartets in an antiparallel orientation. One of the hairpins in the quadruplex was linked covalently at the 5'-end to an anthraquinone (AQ) group and a 32P label was incorporated either at the 3'-terminus of the AQ-containing hairpin or on its partner hairpin in the quadruplex. Irradiation of the AQ group with UV light leads to the one-electron oxidation of the DNA and concomitant introduction of a radical cation into the DNA. Analysis by PAGE and autoradiography shows that the radical cation reacts at guanines both on the AQ-containing strand and with its partner hairpin in the quadruplex. This observation demonstrates that charge migration in DNA occurs vertically along a DNA chain and horizontally within a G-quartet.     

Long-range oxidative damage to DNA: effects of distance and sequence

INTRODUCTION: Oxidative damage to DNA in vivo can lead to mutations and cancer. DNA damage and repair studies have not yet revealed whether permanent oxidative lesions are generated by charges migrating over long distances. Both photoexcited *Rh(III) and ground-state Ru(III) intercalators were previously shown to oxidize guanine bases from a remote site in oligonucleotide duplexes by DNA-mediated electron transfer. Here we examine much longer charge-transport distances and explore the sensitivity of the reaction to intervening sequences. RESULTS: Oxidative damage was examined in a series of DNA duplexes containing a pendant intercalating photooxidant. These studies revealed a shallow dependence on distance and no dependence on the phasing orientation of the oxidant relative to the site of damage, 5'-GG-3'. The intervening DNA sequence has a significant effect on the yield of guanine oxidation, however. Oxidation through multiple 5'-TA-3' steps is substantially diminished compared to through other base steps. We observed intraduplex guanine oxidation by tethered *Rh(III) and Ru(III) over a distance of 200 A. The distribution of oxidized guanine varied as a function of temperature between 5 and 35 degrees C, with an increase in the proportion of long-range damage (> 100 A) occurring at higher temperatures. CONCLUSIONS: Guanines are oxidized as a result of DNA-mediated charge transport over significant distances (e.g. 200 A). Although long-range charge transfer is dependent on distance, it appears to be modulated by intervening sequence and sequence-dependent dynamics. These discoveries hold important implications with respect to DNA damage in vivo.     

DNA mediated charge transport: characterization of a DNA radical localized at an artificial nucleic acid base

DNA assemblies containing 4-methylindole incorporated as an artificial base provide a chemically well-defined system in which to explore the oxidative charge transport process in DNA. Using this artificial base, we have combined transient absorption and EPR spectroscopies as well as biochemical methods to test experimentally current mechanisms for DNA charge transport. The 4-methylindole radical cation intermediate has been identified using both EPR and transient absorption spectroscopies in oxidative flash-quench studies using a dipyridophenazine complex of ruthenium as the intercalating oxidant. The 4-methylindole radical cation intermediate is particularly amenable to study given its strong absorptivity at 600 nm and EPR signal measured at 77 K with g = 2.0065. Both transient absorption and EPR spectroscopies show that the 4-methylindole is well incorporated in the duplex; the data also indicate no evidence of guanine radicals, given the low oxidation potential of 4-methylindole relative to the nucleic acid bases. Biochemical studies further support the irreversible oxidation of the indole moiety and allow the determination of yields of irreversible product formation. The construction of these assemblies containing 4-methylindole as an artificial base is also applied in examining long-range charge transport mediated by the DNA base pair stack as a function of intervening distance and sequence. The rate of formation of the indole radical cation is >/=10(7) s(-)(1) for different assemblies with the ruthenium positioned 17-37 A away from the methylindole and with intervening A-T base pairs primarily composing the bridge. In these assemblies, methylindole radical formation at a distance is essentially coincident with quenching of the ruthenium excited state to form the Ru(III) oxidant; charge transport is not rate limiting over this distance regime. The measurements here of rates of radical cation formation establish that a model of G-hopping and AT-tunneling is not sufficient to account for DNA charge transport. Instead, these data are viewed mechanistically as charge transport through the DNA duplex primarily through hopping among well stacked domains of the helix defined by DNA sequence and dynamics.     

N(2)-phenyldeoxyguanosine: modulation of the chemical properties of deoxyguanosine toward one-electron oxidation in DNA

We have shown here that (1) substitution of an exocyclic amino group of dG is effective in modulating the chemical properties of dG toward one-electron oxidation and (2) decomposition of the guanine radical cation was effectively suppressed near dPhG. These results indicate that dPhG is a prototype of nucleosides functioning as an intrinsic antioxidant of duplex DNA toward one-electron oxidation.