Lifetimes and Reaction Pathways of Guanine Radical Cations and Neutral Guanine Radicals in an Oligonucleotide in Aqueous Solutions

The exposure of guanine in the oligonucleotide 5′-d­(TCGCT) to one-electron oxidants leads initially to the formation of the guanine radical cation G^•+, its deptotonation product G­(-H)^•, and, ultimately, various two- and four-electron oxidation products via pathways that depend on the oxidants and reaction conditions. We utilized single or successive multiple laser pulses (308 nm, 1 Hz rate) to generate the oxidants CO₃^•– and SO₄^•– (via the photolysis of S₂O₈^2– in aqueous solutions in the presence and absence of bicarbonate, respectively) at concentrations/pulse that were ∼20-fold lower than the concentration of 5′-d­(TCGCT). Time-resolved absorption spectroscopy measurements following single-pulse excitation show that the G^•+ radical (p<i>K</i>_a = 3.9) can be observed only at low pH and is hydrated within 3 ms at pH 2.5, thus forming the two-electron oxidation product 8-oxo-7,8-dihydroguanosine (8-oxoG). At neutral pH, and single pulse excitation, the principal reactive intermediate is G­(-H)^•, which, at best, reacts only slowly with H₂O and lives for ∼70 ms in the absence of oxidants/other radicals to form base sequence-dependent intrastrand cross-links via the nucleophilic addition of N3-thymidine to C8-guanine (5′-G*CT* and 5′-T*CG*). Alternatively, G­(-H)^• can be oxidized further by reaction with CO₃^•–, generating the two-electron oxidation products 8-oxoG (C8 addition) and 5-carboxamido-5-formamido-2-iminohydantoin (2Ih, by C5 addition). The four-electron oxidation products, guanidinohydantoin (Gh) and spiroiminodihydantoin (Sp), appear only after a second (or more) laser pulse. The levels of all products, except 8-oxoG, which remains at a low constant value, increase with the number of laser pulses

Water Oxidation Catalyzed by Cobalt(II) Adsorbed on Silica Nanoparticles

A novel, highly efficient, and stable water oxidation catalyst was prepared by a pH-controlled adsorption of Co­(II) on ∼10 nm diameter silica nanoparticles. A <i>lower limit</i> of ∼300 s^–1 per cobalt atom for the catalyst turnover frequency in oxygen evolution was estimated, which attests to a very high catalytic activity. Electron microscopy revealed that cobalt is adsorbed on the SiO₂ nanoparticle surfaces as small (1–2 nm) clusters of Co­(OH)₂. This catalyst is optically transparent over the entire UV–vis range and is thus suitable for mechanistic investigations by time-resolved spectroscopic techniques

Mechanistic Aspects of Hydration of Guanine Radical Cations in DNA

The mechanistic aspects of hydration of guanine radical cations, G^•+ in double- and single-stranded oligonucleotides were investigated by direct time-resolved spectroscopic monitoring methods. The G^•+ radical one-electron oxidation products were generated by SO₄^•– radical anions derived from the photolysis of S₂O₈^2– anions by 308 nm laser pulses. In neutral aqueous solutions (pH 7.0), after the complete decay of SO₄^•– radicals (∼5 μs after the actinic laser flash) the transient absorbance of neutral guanine radicals, G­(-H)^• with maximum at 312 nm, is dominant. The kinetics of decay of G­(-H)^• radicals depend strongly on the DNA secondary structure. In double-stranded DNA, the G­(-H)^• decay is biphasic with one component decaying with a lifetime of ∼2.2 ms and the other with a lifetime of ∼0.18 s. By contrast, in single-stranded DNA the G­(-H)^• radicals decay monophasically with a ∼ 0.28 s lifetime. The ms decay component in double-stranded DNA is correlated with the enhancement of 8-oxo-7,8-dihydroguanine (8-oxoG) yields which are ∼7 greater than in single-stranded DNA. In double-stranded DNA, it is proposed that the G­(-H)^• radicals retain radical cation character by sharing the N1-proton with the N3-site of C in the [G^•+:C] base pair. This [G­(-H)^•:H⁺C ⇆ G^•+:C] equilibrium allows for the hydration of G^•+ followed by formation of 8-oxoG. By contrast, in single-stranded DNA, deprotonation of G^•+ and the irreversible escape of the proton into the aqueous phase competes more effectively with the hydration mechanism, thus diminishing the yield of 8-oxoG, as observed experimentally

Thermodynamic Profiles and Nuclear Magnetic Resonance Studies of Oligonucleotide Duplexes Containing Single Diastereomeric Spiroiminodihydantoin Lesions

The spiroiminodihydantoins (Sp) are highly mutagenic oxidation products of guanine and 8-oxo-7,8-dihydroguanine in DNA. The Sp lesions have recently been detected in the liver and colon of mice infected with <i>Helicobacter hepaticus</i> that induces inflammation and the development of liver and colon cancers in murine model systems [Mangerich, A., et al. (2012) <i>Proc. Natl. Acad. Sci. U.S.A. 109</i>, E1820–E1829]. The impact of Sp lesions on the thermodynamic characteristics and the effects of the diastereomeric Sp-<i>R</i> and Sp-<i>S</i> lesions on the conformational features of double-stranded 11-mer oligonucleotide duplexes have been studied by a combination of microcalorimetric methods, analysis of DNA melting curves, and two-dimensional nuclear magnetic resonance methods. The nonplanar, propeller-like shapes of the Sp residues strongly diminish the extent of local base stacking interactions that destabilize the DNA duplexes characterized by unfavorable enthalpy contributions. Relative to that of an unmodified duplex, the thermally induced unfolding of the duplexes with centrally positioned Sp-<i>R</i> and Sp-<i>S</i> lesions into single strands is accompanied by a smaller release of cationic counterions (Δ<i>n</i>_Na⁺ = 0.6 mol of Na⁺/mol of duplex) and water molecules (Δ<i>n</i>_w = 17 mol of H₂O/mol of duplex). The unfolding parameters are similar for the Sp-<i>R</i> and Sp-<i>S</i> lesions, although their orientations in the duplexes are different. The structural disturbances radiate one base pair beyond the flanking C:G pair, although Watson–Crick hydrogen bonding is maintained at all flanking base pairs. The observed relatively strong destabilization of B-form DNA by the physically small Sp lesions is expected to have a significant impact on the processing of these lesions in biological environments

The Nonbulky DNA Lesions Spiroiminodihydantoin and 5‑Guanidinohydantoin Significantly Block Human RNA Polymerase II Elongation <i>in Vitro</i>

The most common, oxidatively generated lesion in cellular DNA is 8-oxo-7,8-dihydroguanine, which can be oxidized further to yield highly mutagenic spiroiminodihydantoin (Sp) and 5-guanidinohydantoin (Gh) in DNA. In human cell-free extracts, both lesions can be excised by base excision repair and global genomic nucleotide excision repair. However, it is not known if these lesions can be removed by transcription-coupled DNA repair (TCR), a pathway that clears lesions from DNA that impede RNA synthesis. To determine if Sp or Gh impedes transcription, which could make each a viable substrate for TCR, either an Sp or a Gh lesion was positioned on the transcribed strand of DNA under the control of a promoter that supports transcription by human RNA polymerase II. These constructs were incubated in HeLa nuclear extracts that contained active RNA polymerase II, and the resulting transcripts were resolved by denaturing polyacrylamide gel electrophoresis. The structurally rigid Sp strongly blocks transcription elongation, permitting 1.6 ± 0.5% nominal lesion bypass. In contrast, the conformationally flexible Gh poses less of a block to human RNAPII, allowing 9 ± 2% bypass. Furthermore, fractional lesion bypass for Sp and Gh is minimally affected by glycosylase activity found in the HeLa nuclear extract. These data specifically suggest that both Sp and Gh may well be susceptible to TCR because each poses a significant block to human RNA polymerase II progression. A more general principle is also proposed: Conformational flexibility may be an important structural feature of DNA lesions that enhances their transcriptional bypass

Differences in the Access of Lesions to the Nucleotide Excision Repair Machinery in Nucleosomes

In nucleosomes, the access of DNA lesions to nucleotide excision repair is hindered by histone proteins. However, evidence that the nature of the DNA lesions may play a role in facilitating access is emerging, but these phenomena are not well-understood. We have used molecular dynamics simulations to elucidate the structural, dynamic, and energetic properties of the <i>R</i> and <i>S</i> 5′-8-cyclo-2′-dG and the (+)-<i>cis-anti</i>-B­[<i>a</i>]­P-dG lesions in a nucleosome. Our results show that the (+)-<i>cis-anti</i>-B­[<i>a</i>]­P-dG adduct is more dynamic and more destabilizing than the smaller and more constrained 5′,8-cyclo-2′-dG lesions, suggesting more facile access to the more bulky (+)-<i>cis-anti</i>-B­[<i>a</i>]­P-dG lesion

Generation of Guanine–Thymine Cross-Links in Human Cells by One-Electron Oxidation Mechanisms

The one-electron oxidation of cellular DNA in cultured human HeLa cells initiated by intense nanosecond 266 nm laser pulse irradiation produces cross-links between guanine and thymine bases (G*-T*), characterized by a covalent bond between C8 guanine (G*) and N3 thymine (T*) atoms. The DNA lesions were quantified by isotope dilution LC-MS/MS methods in the multiple reaction-monitoring mode using isotopically labeled [¹⁵N, ¹³C]-nucleotides as internal standards. Among several known pyrimidine and 8-oxo-7,8-dihydroguanine lesions, the G*-T* cross-linked lesions were detected at levels of ∼0.21 and 1.19 d­(G*-T*) lesions per 10⁶ DNA bases at laser intensities of 50 and 280 mJ/cm²/pulse, respectively

Differences in the Access of Lesions to the Nucleotide Excision Repair Machinery in Nucleosomes

In nucleosomes, the access of DNA lesions to nucleotide excision repair is hindered by histone proteins. However, evidence that the nature of the DNA lesions may play a role in facilitating access is emerging, but these phenomena are not well-understood. We have used molecular dynamics simulations to elucidate the structural, dynamic, and energetic properties of the <i>R</i> and <i>S</i> 5′-8-cyclo-2′-dG and the (+)-<i>cis-anti</i>-B­[<i>a</i>]­P-dG lesions in a nucleosome. Our results show that the (+)-<i>cis-anti</i>-B­[<i>a</i>]­P-dG adduct is more dynamic and more destabilizing than the smaller and more constrained 5′,8-cyclo-2′-dG lesions, suggesting more facile access to the more bulky (+)-<i>cis-anti</i>-B­[<i>a</i>]­P-dG lesion

Sequence-Dependent Variation in the Reactivity of 8-Oxo-7,8-dihydro-2′-deoxyguanosine toward Oxidation

The goal of this study was to define the effect of DNA sequence on the reactivity of 8-oxo-7,8-dihydro-2′-deoxyguanosine (8-oxodG) toward oxidation. To this end, we developed a quadrupole/time-of-flight (QTOF) mass spectrometric method to quantify the reactivity of site specifically modified oligodeoxyribonucleotides with two model oxidants: nitrosoperoxycarbonate (ONOOCO₂^–), a chemical mediator of inflammation, and photoactivated riboflavin, a classical one-electron oxidant widely studied in mutagenesis and charge transport in DNA. In contrast to previous observations with guanine [Margolin, Y., (2006) Nat. Chem. Biol. 2, 365], sequence context did not affect the reactivity of ONOOCO₂^– with 8-oxodG, but photosensitized riboflavin showed a strong sequence preference in its reactivity with the following order (8-oxodG = O): COA ≈ AOG > GOG ≥ COT > TOC > AOC. That the COA context was the most reactive was unexpected and suggests a new sequence context where mutation hotspots might occur. These results point to both sequence- and agent-specific effects on 8-oxodG oxidation