Possible cause of G-C-->C-G transversion mutation by guanine oxidation product, imidazolone

BACKGROUND: The genome is constantly assaulted by oxidation reactions which are likely to be associated with oxygen metabolism, and oxidative lesions are generated by many types of oxidants. Such genotoxin-induced alterations in the genomic message have been implicated in aging and in several pathophysiological processes, particularly those associated with cancer. The guanine base (G) in genomic DNA is highly susceptible to oxidative stress due to having the lowest oxidation potential. Therefore, G-C-->T-A and G-C-->C-G transversion mutations frequently occur under oxidative conditions. One typical lesion of G is 8-oxo-7,8-dihydro-guanine (8-oxoG), which can pair with A. This pairing may cause G-C-->T-A transversion mutations. Although the number of G-C-->C-G transversions is rather high under specific oxidation conditions such as riboflavin photosensitization, the molecular basis of G-C-->C-G transversions is not known. RESULTS: To determine which oxidative products are responsible for G-C-->C-G transversion mutations, we photooxidized 5'-d(AAAAAAGGAAAAAA)/5'-d(TTTTTTCCTTTTTT) using either riboflavin or anthraquinone (AQ) carboxylate under UV irradiation. Prolonged low-temperature (4 degrees C) enzymatic digestion of photoirradiated sample indicated that under both conditions the amount of 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG) initially increased with decreasing amounts of 2'-deoxyguanosine (dG), then decreased with the formation of 2-amino-5-[(2-deoxy-beta-D-erythro-pentofuranosyl)amino]-4H-imidazol-4-one (dIz), suggesting that nascent 8-oxoG was further oxidized to 2,5-diamino-4H-imidazol-4-one (Iz) in duplex DNA. Photoirradiation of an AQ-linked oligomer with a complementary strand containing 8-oxoG indicated that 8-oxoG residues were oxidized to Iz. These results indicate that Iz is formed from 8-oxoG through long-range hole migration. Primer extension experiments using a template containing Iz demonstrated that only dGTP is specifically incorporated opposite Iz suggesting that specific Iz-G base pairs are formed. The 'reverse' approach consisting of DNA polymerization using dIzTP showed that dIzTP is incorporated opposite G, further confirming the formation of a Iz-G base pair. CONCLUSIONS: HPLC product analysis demonstrated that Iz is a key oxidation product of G through 8-oxoG in DNA photosensitized with riboflavin or anthraquinone. Photoreaction of AQ-linked oligomer confirmed that Iz is formed from 8-oxoG through long-range hole migration. Two sets of primer extension experiments demonstrated that Iz can specifically pair with G in vitro. Specific Iz-G base pair formation can explain the G-C-->C-G transversion mutations that appear under oxidative conditions.     

Combination of nitrogen dioxide radicals with 8-oxo-7,8-dihydroguanine and guanine radicals in DNA: oxidation and nitration end-products

The oxidation and nitration reactions in DNA associated with the combination of nitrogen dioxide radicals with 8-oxo-7,8-dihydroguanine (8-oxoGua) and guanine radicals were explored by kinetic laser spectroscopy and mass spectrometry methods. The oxidation/nitration processes were triggered by photoexcitation of 2-aminopurine (2AP) residues site-specifically positioned in the 2'-deoxyribooligonucleotide 5'-d(CC[2AP]TC[X]CTACC) sequences (X = 8-oxoGua or G), by intense 308 nm excimer laser pulses. The photoionization products, 2AP radicals, rapidly oxidize either 8-oxoGua or G residues positioned within the same oligonucleotide but separated by a TC dinucleotide step on the 3'-side of 2AP. The two-photon ionization of the 2AP residue also generates hydrated electrons that are trapped by nitrate anions thus forming nitrogen dioxide radicals. The combination of nitrogen dioxide radicals with the 8-oxoGua and G radicals occurs with similar rate constants (approximately 4.3 x 10(8) M(-1) s(-1)) in both single- and double-stranded DNA. In the case of 8-oxoGua, the major end-products of this bimolecular radical-radical addition are spiroiminodihydantoin lesions, the products of 8-oxoGua oxidation. Oxygen-18 isotope labeling experiments reveal that the O-atom in the spiroiminodihydantoin lesion originates from water molecules, not from nitrogen dioxide radicals. In contrast, combination of nitrogen dioxide and guanine neutral radicals generated under the same conditions results in the formation of the nitro products, 5-guanidino-4-nitroimidazole and 8-nitroguanine adducts. The mechanistic aspects of the oxidation/nitration processes and their biological implications are discussed.     

Oxidation Chemistry of DNA and p53 Tumor Suppressor Gene

The chemistry of DNA and its repair selectivity control the influence of genomic oxidative stress on the development of serious disorders such as cancer and heart diseases. DNA is oxidized by endogenous reactive oxygen species (ROS) in vivo or in vitro as a result of high energy radiation, non-radiative metabolic processes, and other consequences of oxidative stress. Some oxidations of DNA and tumor suppressor gene p53 are thought to be mutagenic when not repaired. For example, site-specific oxidations of p53 tumor suppressor gene may lead to cancer-related mutations at the oxidation site codon. This review summarizes the research on the primary products of the most easily oxidized nucleobase guanine (G) when different oxidation methods are used. Guanine is by far the most oxidized DNA base. The primary initial oxidation product of guanine for most, but not all, pathways is 8-oxoguanine (8-oxoG). With an oxidation potential much lower than G, 8-oxoG is readily susceptible to further oxidation, and the products often depend on the oxidants. Specific products may control the types of subsequent mutations, but mediated by gene repair success. Site-specific oxidations of p53 tumor suppressor gene have been reported at known mutation hot spots, and the codon sites also depend on the type of oxidants. Modern methodologies using LC–MS/MS for codon specific detection and identification of oxidation sites are summarized. Future work aimed at understanding DNA oxidation in nucleosomes and interactions between DNA damage and repair is needed to provide a better picture of how cancer-related mutations arise.     

Photosensitized oxidative DNA damage: from hole injection to chemical product formation and strand cleavage

Oxidatively generated damage to DNA induced by a pyrenyl photosensitizer residue (Py) covalently attached to a guanine base in the DNA sequence context 5'-d(CAT[G1Py]CG2TCCTAC) in aerated solutions was monitored from the initial one-electron transfer, or hole injection step, to the formation of chemical end-products monitored by HPLC, mass spectrometry, and high-resolution gel electrophoresis. Hole injection into the DNA was initiated by two-photon excitation of the Py residue with 355 nm laser pulses, thus producing the radical cation Py*+ and hydrated electrons; the latter are trapped by O2, thus forming the superoxide anion O2*-. The decay of the Py*+ radical is correlated with the appearance of the G*+/G(-H)* radical on microsecond time scales, and O2*- combines with guanine radicals at G1 to form alkali-labile 2,5-diamino-4H-imidazolone lesions (Iz1Py). Product formation in the modified strand is smaller by a factor of 2.4 in double-stranded than in single-stranded DNA. In double-stranded DNA, hot piperidine-mediated cleavage at G2 occurs only after G1Py, an efficient hole trap, is oxidized thus generating tandem lesions. An upper limit of hole hopping rates, khh < 5 x 103 s-1 from G1*+-Py to G2 can be estimated from the known rates of the combination reaction of the G(-H)* and O2*- radicals. The formation of Iz products in the unmodified complementary strand compared to the modified strand in the duplex is approximately 10 times smaller. The formation of tandem lesions is observed even at low levels of irradiation corresponding to "single-hit" conditions when less than approximately 10% of the oligonucleotide strands are damaged. A plausible mechanism for this observation is discussed.     

Oxidation of guanosine derivatives by a platinum(IV) complex: internal electron transfer through cyclization

Many transition-metal complexes mediate DNA oxidation in the presence of oxidizing radiation, photosensitizers, or oxidants. The DNA oxidation products depend on the nature of the metal complex and the structure of the DNA. Earlier we reported trans-d,l-1,2-diaminocyclohexanetetrachloroplatinum (trans-Pt(d,l)(1,2-(NH(2))(2)C(6)H(10))Cl(4), [Pt(IV)Cl(4)(dach)]; dach = diaminocyclohexane) oxidizes 2'-deoxyguanosine 5'-monophosphate (5'-dGMP) to 7,8-dihydro-8-oxo-2'-deoxyguanosine 5'-monophosphate (8-oxo-5'-dGMP) stoichiometrically. In this paper we report that [Pt(IV)Cl(4)(dach)] also oxidizes 2'-deoxyguanosine 3'-monophosphate (3'-dGMP) stoichiometrically. The final oxidation product is not 8-oxo-3'-dGMP, but cyclic (5'-O-C8)-3'-dGMP. The reaction was studied by high-performance liquid chromatography, (1)H and (31)P nuclear magnetic resonance, and matrix-assisted laser desorption ionization time-of-flight mass spectrometry. The proposed mechanism involves Pt(IV) binding to N7 of 3'-dGMP followed by nucleophilic attack of a 5'-hydroxyl oxygen to C8 of G and an inner-sphere, 2e(-) transfer to produce cyclic (5'-O-C8)-3'-dGMP and [Pt(II)Cl(2)(dach)]. The same mechanism applies to 5'-d[GTTTT]-3', where the 5'-dG is oxidized to cyclic (5'-O-C8)-dG. The Pt(IV) complex binds to N7 of guanine in cGMP, 9-Mxan, 5'-d[TTGTT]-3', and 5'-d[TTTTG]-3', but no subsequent transfer of electrons occurs in these. The results indicate that a good nucleophilic group at the 5' position is required for the redox reaction between guanosine and the Pt(IV) complex.     

The guanine cation radical: investigation of deprotonation states by ESR and DFT

This work reports ESR studies that identify the favored site of deprotonation of the guanine cation radical (G*+) in an aqueous medium at 77 K. Using ESR and UV-visible spectroscopy, one-electron oxidized guanine is investigated in frozen aqueous D2O solutions of 2'-deoxyguanosine (dGuo) at low temperatures at various pHs at which the guanine cation radical, G*+ (pH 3-5), singly deprotonated species, G(-H)* (pH 7-9), and doubly deprotonated species, G(-2H)*- (pH > 11), are found. C-8-deuteration of dGuo to give 8-D-dGuo removes the major proton hyperfine coupling at C-8. This isolates the anisotropic nitrogen couplings for each of the three species and aids our analyses. These anisotropic nitrogen couplings were assigned to specific nitrogen sites by use of 15N-substituted derivatives at N1, N2, and N3 atoms in dGuo. Both ESR and UV-visible spectra are reported for each of the species: G*+, G(-H)*, and G(-2H)*-. The experimental anisotropic ESR hyperfine couplings are compared to those obtained from DFT calculations for the various tautomers of G(-H)*. Using the B3LYP/6-31G(d) method, the geometries and energies of G*+ and its singly deprotonated state in its two tautomeric forms, G(N1-H)* and G(N2-H)*, were investigated. In a nonhydrated state, G(N2-H)* is found to be more stable than G(N1-H)*, but on hydration with seven water molecules G(N1-H)* is found to be more stable than G(N2-H)*. The theoretically calculated hyperfine coupling constants (HFCCs) of G*+, G(N1-H)*, and G(-2H)*- match the experimentally observed HFCCs best on hydration with seven or more waters. For G(-2H)*-, the hyperfine coupling constant (HFCC) at the exocyclic nitrogen atom (N2) is especially sensitive to the number of hydrating water molecules; good agreement with experiment is not obtained until nine or 10 waters of hydration are included.     

Synthesis and characterization of the oxidized dGTP lesions spiroiminodihydantoin-2'-deoxynucleoside-5'- triphosphate and guanidinohydantoin-2'-deoxynucleoside-5'- triphosphate

Two convenient synthetic routes to the oxidized guanosine triphosphate lesions spiroiminodihydantoin-2'-deoxynucleoside-5'-triphosphate (dSpTP) and guanidinohydantoin-2'-deoxynucleoside-5'-triphosphate (dGhTP) are reported. Both two-electron oxidation of 2'-deoxy-7,8-dihydro-8-oxoguanosine-5'-triphosphate (dOGTP) using SO4*- generated photolytically from K2S2O8 or four-electron oxidation of 2'-deoxyguanosine-5'-triphosphate (dGTP) from singlet oxygen provide either dSpTP or dGhTP at pH 8.0 or 4.4, respectively. Highly purified triphosphates are obtained by ion pair reversed-phase HPLC.     

Highly oxidizing excited states of one-electron-oxidized guanine in DNA: wavelength and pH dependence

Excited states of one-electron-oxidized guanine in DNA are known to induce hole transfer to the sugar moiety and on deprotonation result in neutral sugar radicals that are precursors of DNA strand breaks. This work carried out in a homogeneous aqueous glass (7.5 M LiCl) at low temperatures (77-175 K) shows the extent of photoconversion of one-electron-oxidized guanine and the associated yields of individual sugar radicals are crucially controlled by the photon energy, protonation state, and strandedness of the oligomer. In addition to sugar radical formation, highly oxidizing excited states of one-electron-oxidized guanine are produced with 405 nm light at pH 5 and below that are able to oxidize chloride ion in the surrounding solution to form Cl(2)(?-) via an excited-state hole transfer process. Among the various DNA model systems studied in this work, the maximum amount of Cl(2)(?-) is produced with ds (double-stranded) DNA, where the one-electron-oxidized guanine exists in its cation radical form (G(?+):C). Thus, via excited-state hole transfer, the dsDNA is apparently able to protect itself from cation radical excited states by transfer of damage to the surrounding environment.     

Proton-coupled hole hopping in nucleosomal and free DNA initiated by site-specific hole injection

Nucleosomes were reconstituted from recombinant histones and a 147-mer DNA sequence containing the damage reporter sequence 5'-…d([2AP]T[GGG](1)TT[GGG](2)TTT[GGG](3)TAT)… with 2-aminopurine (2AP) at position 27 from the dyad axis. Footprinting studies with ˙OH radicals reflect the usual effects of "in" and "out" rotational settings, while, interestingly, the guanine oxidizing one-electron oxidant CO(3)(˙-) radical does not. Site-specific hole injection was achieved by 308 nm excimer laser pulses to produce 2AP(˙+) cations, and superoxide via the trapping of hydrated electrons. Rapid deprotonation (~100 ns) and proton coupled electron transfer generates neutral guanine radicals, G(-H)˙ and hole hopping between the three groups of [GGG] on micro- to millisecond time scales. Hole transfer competes with hole trapping that involves the combination of O(2)(˙-) with G(-H)˙ radicals to yield predominantly 2,5-diamino-4H-imidazolone (Iz) and minor 8-oxo-7,8-dihydroguanine (8-oxoG) end-products in free DNA (Misiaszek et al., J. Biol. Chem. 2004, 279, 32106). Hole migration is less efficient in nucleosomal than in the identical protein-free DNA by a factor of 1.2-1.5. The Fpg/piperidine strand cleavage ratio is ~1.0 in free DNA at all three GGG sequences and at the "in" rotational settings [GGG](1,3) facing the histone core, and ~2.3 at the "out" setting at [GGG](2) facing away from the histone core. These results are interpreted in terms of competitive reaction pathways of O(2)(˙-) with G(-H)˙ radicals at the C5 (yielding Iz) and C8 (yielding 8-oxoG) positions. These differences in product distributions are attributed to variations in the local nucleosomal B-DNA base pair structural parameters that are a function of surrounding sequence context and rotational setting.     

An Exploration of the Transformation of the 8-Oxo-7,8-Dihydroguanine Radical Cation to Protonated 2-Amino-5-Hydroxy-7,9-Dihydropurine-6,8-Dione in a Base Pair

A theoretical investigation was performed to disclose the transformation mechanism of 8-oxo-7,8-dihydroguanine radical cation (8-oxoG⋅⁺) to protonated 2-amino-5-hydroxy-7,9-dihydropurine-6,8-dione (5-OH-8-oxoG) in base pair. The energy profiles for three possible pathways of the events were mapped. It is shown that direct loss of H7 from base paired 8-oxoG⋅⁺ is the only energetically favorable pathway to generate neutral radical, 8-oxoG(-H7)⋅. Further oxidation of 8-oxoG(-H7)⋅ : C to 8-oxoG(-H7)⁺ : C is exothermic. However, the 8-oxoG(-H7)⁺ : C deprotonation from all possible active sites is infeasible, indicating the inaccessible second proton loss and the lack of essential intermediate 2-amino-7,9-dihydropurine-6,8-dione (8-oxoG^OX). This makes 8-oxoG(-H7)⁺ act as the precursor of hydration leading to the generation of protonated 5-HO-8-oxoG by stepwise fashion in base pair, which would initiate the step down guanidinohydantoin (Gh) pathway. These results clearly specify the structure-dependent transformation for 8-oxoG⋅⁺ and verify the emergence of protonated 5-HO-8-oxoG in base pair.     

Photosensitized xanthone-based oxidation of guanine and its repair: a laser flash photolysis study

The photosensitized oxidation of guanine (G) by the triplet state of xanthone (XT) and the repair for photo-damaged G(-H)(·) by ferulic acid (FCA) were investigated using the laser flash photolysis technique. The rate constants of the reaction of triplet state of XT with G and with FCA were determined as 4.5×10(9) and 8.0×10(9) L mol(-1) s(-1), respectively. Laser exposure was performed on the N(2)-saturated acetonitrile/water (v/v, 1:1) solution containing G, XT and FCA. The transient absorption spectra indicated that the triplet state of XT first reacted with G predominantly to form the oxidized radical G(-H)(·). The radical G(-H)(·) was rapidly repaired by FCA, and the rate constant for the repair reaction was determined as 1.1×10(9) L mol(-1) s(-1). These results demonstrated that non-enzymatic repair is a feasible method for repairing photosensitized DNA bases oxidation.     

Direct observation of guanine radical cation deprotonation in duplex DNA using pulse radiolysis

The dynamics of one-electron oxidation of guanine (G) base mononucleotide and that in DNA have been investigated by pulse radiolysis. The radical cation (G+*) of deoxyguanosine (dG), produced by oxidation with SO(4)-*, rapidly deprotonates to form the neutral G radical (G(-H)*) with a rate constant of 1.8 x 10(7) s(-1) at pH 7.0, as judged from transient spectroscopy. With experiments using different double-stranded oligonucleotides containing G, GG, and GGG sequences, the absorbance increases at 625 nm, characteristic of formation of the G(-H)*, were found to consist of two phases. The rate constants of the faster ( approximately 1.3 x 10(7) s(-1)) and slower phases ( approximately 3.0 x 10(6) s(-1)) were similar for the different oligonucleotides. On the other hand, in the oligonucleotide containing G located at the 5'- and 3'-terminal positions, only the faster phase was seen. These results suggest that the lifetime of the radical cation of the G:C base pair (GC+*), depending on its location in the DNA chain, is longer than that of free dG. In addition, the absorption spectral intermediates showed that hole transport to a specific G site within a 12-13mer double-stranded oligonucleotide is complete within 50 ns; that is, the rate of hole transport over 20 A is >10(7) s(-1).     

Dehydrogenation versus oxygenation in two-electron and four-electron reduction of dioxygen by 9-alkyl-10-methyl-9,10-dihydroacridines catalyzed by monomeric cobalt porphyrins and cofacial dicobalt porphyrins in the presence of perchloric acid

Dehydrogenation of 10-methyl-9,10-dihydroacridine (AcrH(2)) by dioxygen (O(2)) proceeds efficiently, accompanied by the two-electron and four-electron reduction of O(2) to produce H(2)O(2) and H(2)O, which are effectively catalyzed by monomeric cobalt porphyrins and cofacial dicobalt porphyrins in the presence of perchloric acid (HClO(4)) in acetonitrile (MeCN) and benzonitrile (PhCN), respectively. The cobalt porphyrin catalyzed two-electron reduction of O(2) also occurs efficiently by 9-alkyl-10-methyl-9,10-dihydroacridines (AcrHR; R = Me, Et, and CH(2)COOEt) to yield 9-alkyl-10-methylacridinium ion (AcrR+) and H(2)O(2). In the case of R = Bu(t) and CMe(2)COOMe, however, the catalytic two-electron and four-electron reduction of O(2) by AcrHR results in oxygenation of the alkyl group of AcrHR rather than dehydrogenation to yield 10-methylacridinium ion (AcrH+) and the oxygenated products of the alkyl groups, i.e., the corresponding hydroperoxides (ROOH) and the alcohol (ROH), respectively. The catalytic mechanisms of the dehydrogenation vs the oxygenation of AcrHR in the two-electron and four-electron reduction of O(2), catalyzed by monomeric cobalt porphyrins and cofacial dicobalt porphyrins, respectively, are discussed in relation to the C(9)-H or C(9)-C bond cleavage of AcrHR radical cations produced in the electron-transfer oxidation of AcrHR.     

Formation of sugar radicals in RNA model systems and oligomers via excitation of guanine cation radical

In previous work, we have shown that photoexcitation of guanine cation radical (G*+) in frozen aqueous solutions of DNA and its model compounds at 143 K results in the formation of neutral sugar radicals with substantial yield. In this report, we present electron spin resonance (ESR) and theoretical (DFT) evidence regarding the formation of sugar radicals after photoexcitation of guanine cation radical (G*+) in frozen aqueous solutions of one-electron-oxidized RNA model compounds (nucleosides, nucleotides and oligomers) at 143 K. Specific sugar radicals C5'*, C3'* and C1'* were identified employing derivatives of Guo deuterated at specific sites in the sugar moiety, namely, C1'-, C2'-, C3'- and C5'-. These results suggest C2'* is not formed upon photoexcitation of G*+ in one-electron-oxidized Guo and deuterated Guo derivatives. Phosphate substitution at C5'- (i.e., in 5-GMP) hinders formation of C5'* via photoexcitation at 143 K but not at 77 K. For the RNA-oligomers studied, we observe on photoexcitation of oligomer-G*+ the formation of mainly C1'* and an unidentified radical with a ca. 28 G doublet. The hyperfine coupling constants of each of the possible sugar radicals were calculated employing the DFT B3LYP/6-31G* approach for comparison to experiment. This work shows that formation of specific neutral sugar radicals occurs via photoexcitation of guanine cation radical (G*+) in RNA systems but not by photoexcitation of its N1 deprotonated species (G(-H)*). Thus, our mechanism regarding neutral sugar formation via photoexcitation of base cation radicals in DNA appears to be valid for RNA systems as well.     

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.     

Site-specific probing of oxidative reactivity and telomerase function using 7,8-dihydro-8-oxoguanine in telomeric DNA

Telomeres at the ends of human chromosomes contain the repeating sequence 5'-d[(TTAGGG)(n)]-3'. Oxidative damage of guanine in DNAs that contain telomeric and nontelomeric sequence generates 7,8-dihydro-8-oxoguanine (8OG) preferentially in the telomeric segment, because GGG sequences are more reactive in duplex DNA. We have developed a general strategy for probing site-specific oxidation reactivity in diverse biological structures through substitution of minimally modified building blocks that are more reactive than the parent residue, but preserve the parent structure. In this study, 8OG was substituted for guanine at G(8), G(9), G(14), or G(15) in the human telomeric oligonucleotide 5'-d[AGGGTTAG(8)G(9)GTT AG(14)G(15)GTTAGGGTGT]-3'. Replacement of G by 8OG in telomeric DNA can affect the formation of intramolecular G quadruplexes, depending on the position of substitution. When 8OG was incorporated in the 5'-position of a GGG triplet, G quadruplex formation was observed; however, substitution of 8OG in the middle of a GGG triplet produced multiple structures. A clear correspondence between structure and reactivity was observed when oligonucleotides containing 8OG in the 5'-position of a GGG triplet were prepared in the quadruplex or duplex forms and interrogated by mediated electrocatalytic oxidation with Os(bpy)(3)(2+) (bpy = 2,2'-bipyridine). The rate constant for one-electron oxidation of a single 8OG in the 5'-position of a GGG triplet was (6.2 +/- 1.7) x 10(4) M(-1) s(-1) in the G quadruplex form. The rate constant was 2-fold lower for the same telomeric sequence in the duplex form ((3.0 +/- 1.3) x 10(4) M(-1) s(-1)). The position of 8OG in the GGG triplet affects telomerase activity and synthesis of telomeric repeat products. Telomerase activity was decreased significantly when 8OG was substituted in the 5'-position of the GGG triplet, but not when 8OG was substituted in the middle of the triplet. Thus, biological oxidation of G to 8OG in telomeres has the potential to modulate telomerase activity. Further, small molecules that inhibit telomerase by stabilizing telomeric G quadruplexes may not be as effective under oxidative stress.     

Structural characterization of carcinogen-modified oligodeoxynucleotide adducts using matrix-assisted laser desorption/ionization mass spectrometry

The aim of this study was to determine the chemical structure of in vitro 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP)-modified oligodeoxynucleotides (ODNs) by exonuclease digestion and matrix-assisted laser desorption/ionization mass spectrometry. A single-stranded 11-mer ODN, 5'-d(CCATCGCTACC), was reacted with N-acetoxy-PhIP, resulting in the formation of one major and eight minor PhIP-ODN adducts. A 10 min treatment of the major and one minor PhIP-ODN adduct with a 3'-exonuclease, bovine intestinal mucosa phosphodiesterase (BIMP), and a 5'-exonuclease, bovine spleen phosphodiesterase, results in inhibition of the primary exonuclease activity at deoxyguanosine (dG) producing 5'-d(CCATCG(PhIP)) and 5'-d(G(PhIP)CTACC) product ions, respectively. Post-source decay (PSD) of these enzymatic end products identifies dG as the sole modification site in two 11-mer ODN-PhIP adducts. PSD of the minor PhIP-ODN adduct digestion end product, 5'-d(CCATCG(PhIP)), also reveals that the PhIP adducted guanine moiety is in an oxidized form. Prolonged treatment of the PhIP-ODN adducts at 37 degrees C with BIMP induces a non-specific, or endonuclease, enzymatic activity culminating in the formation of deoxyguanosine 5'-monophosphate-PhIP (5'-dGMP-PhIP). The PSD fragmentation pattern of the 5'-dGMP-PhIP [M + H](+) ion of the major adduct confirms PhIP binds to the C-8 position of dG. For the minor adduct, PSD results suggest that PhIP binds to the C-8 position of an oxidized guanine, supporting the hypothesis that this adduct arises from oxidative degradation, resulting in a spirobisguanidino structure.     

Electron-transfer and acid-base properties of a two-electron oxidized form of quaterpyrrole that acts as both an electron donor and an acceptor

Electron-transfer interconversion between the four-electron oxidized form of a quaterpyrrole (abbreviated as P4 for four pyrroles) and the two-electron oxidized form (P4H2) as well as between P4H2 and its fully reduced form (P4H4) bearing analogous substituents in the alpha- and beta-pyrrolic positions was studied by means of cyclic voltammetry and UV-visible spectroelectrochemistry combined with ESR and laser flash photolysis measurements. The two-electron oxidized form, P4H2, acts as both an electron donor and an electron acceptor. The radical cation (P4H2*+) and radical anion (P4H2*-) are both produced by photoinduced electron transfer from dimeric 1-benzyl-1,4-dihydronicotinamide to P4H2, whereas the cation radical form of the compound is also produced by electron-transfer oxidation of P4H2 with [Ru(bpy)3]3+. The ESR spectra of P4H2*+ and P4H2*- were recorded at low temperature and exhibit spin delocalization over all four pyrrole units. Thus, the two-electron oxidized form of the quaterpyrrole (P4H2) displays redox and electronic features analogous to those seen in the case of porphyrins and may be considered as a simple, open-chain model of this well-studied tetrapyrrolic macrocycle. The dynamics of deprotonation from P4H2*+ and disproportionation of P4H2 were examined by laser flash photolysis measurements of photoinduced electron-transfer oxidation and reduction of P4H2, respectively.     

Characterizing DNA photo-oxidation reactions by high-resolution mass measurements with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry

High-resolution mass measurements by matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry were employed to characterize laser-induced oxidation of guanine in a small synthetic deoxyoligonucleotide. The oligonucleotide was exposed to high-intensity UV radiation at 266 nm to produce modifications on the guanine base. The primary product showed a +16 Da mass shift relative to the original strand, whereas secondary products showed mass shifts of +32 and +34 Da. The mass shift of the primary product is consistent with an 8-oxoguanine modification. However, the reactivity of the primary product with hot piperidine and other secondary oxidizing agents was different from that of a synthetic oligonucleotide containing 7,8-dihydro-8-oxo-2'-deoxyguanine (8-oxoG). Based upon the results, a new reaction scheme involving the formation of an epoxide ring across the C-4 and C-5 positions by UV laser-induced oxidation is suggested. The results also illustrate the ability of MALDI to characterize chemical reactivity rapidly at the a low picomolar level.     

Targeted guanine oxidation by a dinuclear copper(II) complex at single stranded/double stranded DNA junctions

A dinuclear copper(II) complex [Cu(II)2(PD'O-)(H2O)2](ClO4)3 (5) with terminal Cu(II)-H(2)O moieties and a Cu...Cu distance of 4.13 A (X-ray structure) has been synthesized and characterized by EPR spectroscopy (ferromagnetic coupling observed) and cyclic voltammetry. Dizinc(II) and mononuclear copper(II) analogues [Zn(II)2(PD'O-)(H2O)2]3+ (7) and [Cu(II)(mPD'OH)(H2O)]2+ (6), respectively, have also been synthesized and structurally characterized. Reacting 5/MPA/O(2) (MPA = 3-mercaptopropionic acid) with DNA leads to a highly specific oxidation of guanine (G) at a junction between single- and double-stranded DNA. Mass spectrometric analysis of the major products indicates a gain of +18 and +34 amu relative to initial DNA strands. The most efficient reaction requires G at the first and second unpaired positions of each strand extending from the junction. Less reaction is observed for analogous targets in which the G cluster is farther from the junction or contains less than four Gs. Consistent with our previous systems, the multinuclear copper center is required for selective reaction; mononuclear complex 6 is not effective. Hydrogen peroxide as a substitute for MPA/O2 also does not lead to activity. Structural analysis of a [Cu(II)2(PD'O-)(G)]3+ complex (8) and dizinc analogue [Zn(II)(2)(PD'O-)(G)](ClO4)3 (9) (G = guanosine) reveals coordination of the G O6 and N7 atoms with the two copper (or zinc) centers and suggests that copper-G coordination likely plays a role in recognition of the DNA target. The Cu2-O2 intermediate responsible for guanine oxidation appears to be different from that responsible for direct-strand scission induced by other multinuclear copper complexes; the likely course of reaction is discussed.