Conor Crean

Oxidation of single-stranded oligonucleotides by carbonate radical anions: generating intrastrand cross-links between guanine and thymine bases separated by cytosines

The carbonate radical anion is a biologically important one-electron oxidant that can directly abstract an electron from guanine, the most easily oxidizable DNA base. Oxidation of the 5′-d(CCTACGCTACC) sequence by photochemically generated CO3·− radicals in low steady-state concentrations relevant to biological processes results in the formation of spiroiminodihydantoin diastereomers and a previously unknown lesion. The latter was excised from the oxidized oligonucleotides by enzymatic digestion with nuclease P1 and alkaline phosphatase and identified by LC-MS/MS as an unusual intrastrand cross-link between guanine and thymine. In order to further characterize the structure of this lesion, 5′-d(GpCpT) was exposed to CO3·− radicals, and the cyclic nature of the 5′-d(G*pCpT*) cross-link in which the guanine C8-atom is bound to the thymine N3-atom was confirmed by LC-MS/MS, 1D and 2D NMR studies. The effect of bridging C bases on the cross-link formation was studied in the series of 5′-d(GpCnpT) and 5′-d(TpCnpG) sequences with n = 0, 1, 2 and 3. Formation of the G*-T* cross-links is most efficient in the case of 5′-d(GpCpT). Cross-link formation (n = 0) was also observed in double-stranded DNA molecules derived from the self-complementary 5′-d(TTACGTACGTAA) sequence following exposure to CO3·− radicals and enzymatic excision of the 5′-d(G*pT*) product.

Absolute configurations of spiroiminodihydantoin and allantoin stereoisomers: comparison of computed and measured electronic circular dichroism spectra.

The assignment of absolute configurations is of critical importance for understanding the biochemical processing of DNA lesions. The diastereomeric spiroiminodihydantoin (Sp) lesions are oxidation products of guanine and 8-oxo-7,8-dihydroguanine (8-oxoG), and the absolute configurations of the two diastereomers, Sp1 and Sp2, have been evaluated by experimental and computational optical rotatory dispersion (ORD) methods. In order to support our previous assignments by the ORD method, we calculated the electronic circular dichroism spectra (ECD) of the Sp stereoisomers. Comparison of the experimentally measured and computed ECD spectra indicates that Sp1 has (-)-S absolute configuration, while Sp2 has (+)-R absolute configuration. Thus, the S and R assignments, based on the ECD spectra of Sp1 and Sp2, are consistent with our previous assignments of absolute configurations. To further test the validity of this approach, we performed a proof-of-principle computation of the ECD and ORD of the R and S enantiomers of allantoin (similar in chemical composition to Sp) of known absolute configurations. The calculations provide the correct assignment of the absolute configurations of the allantoin enantiomers, indicating that the computational TDDFT approach is robust for identifying the absolute configurations of allantoins and probably the Sp stereoisomers, as has been shown previously for other organic molecules.

Combination Reactions of Superoxide with 8-Oxo-7,8-dihydroguanine Radicals in DNA KINETICS AND END PRODUCTS

One of the major biomarkers of oxidative stress and oxidative damage of cellular DNA is 8-oxo-7,8-dihydroguanine (8-oxoGua), which is more easily oxidized than guanine to diverse oxidative products. In this work, we have investigated further oxidative transformations of 8-oxoGua in single- and double-stranded oligonucleotides to the dehydroguanidinohydantoin, oxaluric acid, and diastereomeric spiroiminodihydantoin lesions. The relative distributions of these end products were explored by a combined kinetic laser spectroscopy and mass spectrometry approach and are shown to depend markedly on the presence of superoxide radical anions. The 8-oxaGua radicals were produced by one-electron oxidation of 8-oxoGua by 2-aminopurine radicals generated by the two-photon ionization of 2-aminopurine residues site specifically positioned in 5′-d(CC[2-aminopurine]TC[8-oxoGua]CTACC). The hydrated electrons also formed in the photoionization process were trapped by dissolved molecular oxygen thus producing superoxide. A combination reaction between the 8-oxoGua and superoxide radicals occurs with the rate constant of (1.3 ± 0.2) × 108 m–1 s–1 and (1.0 ± 0.5) × 108 m–1 s–1 in single- and double-stranded DNA, respectively. The major end products of this reaction are the dehydroguanidinohydantoin lesions that slowly hydrolyze to oxaluric acid residues. In the presence of Cu,Zn-superoxide dismutase, an enzyme that induces the rapid catalytic dismutation of superoxide to the less reactive H2O2 and O2, the yields of the dehydroguanidinohydantion lesions become negligible. Under these conditions, the 8-oxoGua radicals do not exhibit any observable reactivities with oxygen (k < 102 m–1 s–1), decay on the time interval of several seconds, and the major reaction products are the spiroiminodihydantoin lesions. The possible biological implications of the 8-oxoGua oxidation are discussed.

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 Helicobacter hepaticus that induces inflammation and the development of liver and colon cancers in murine model systems [Mangerich, A., et al. (2012) Proc. Natl. Acad. Sci. U.S.A. 109, E1820-E1829]. The impact of Sp lesions on the thermodynamic characteristics and the effects of the diastereomeric Sp-R and Sp-S 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-R and Sp-S lesions into single strands is accompanied by a smaller release of cationic counterions (Δn(Na⁺) = 0.6 mol of Na⁺/mol of duplex) and water molecules (Δn(w) = 17 mol of H₂O/mol of duplex). The unfolding parameters are similar for the Sp-R and Sp-S 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.

Oxidation of Guanine by Carbonate Radicals Derived from Photolysis of Carbonatotetramminecobalt(III) Complexes and the pH Dependence of Intrastrand DNA Cross-links Mediated by Guanine Radical Reactions

The carbonate radical anion CO3.− is a decomposition product of nitrosoperoxycarbonate derived from the combination of carbon dioxide and peroxynitrite, an important biological byproduct of the inflammatory response. The selective oxidation of guanine in DNA by CO3.− radicals is known to yield spiroiminodihydantoin (Sp) and guanidinohydantoin (Gh) products, and also a novel intrastrand cross‐linked product: 5′‐d(CCATCG*CT*ACC), featuring a linkage between guanine C8 (G*) and thymine N3 (T*) atoms in the oligonucleotide (Crean et al., Nucleic Acids Res. 2008, 36, 742–755). Involvement of the T‐N3 (pKa of N3‐H is 9.67) suggests that the formation of 5′‐d(CCATCG*CT*ACC) might be pH‐dependent. This hypothesis was tested by generating CO3.− radicals through the photodissociation of carbonatotetramminecobalt(III) complexes by steady‐state UV irradiation, which allowed for studies of product yields in the pH 5.0–10.0 range. The yield of 5′‐d(CCATCG*CT*ACC) at pH 10.0 is ∼45 times greater than at pH 5.0; this is consistent with the proposed mechanism, which requires N3(H) thymine proton dissociation followed by nucleophilic addition to the C8 guanine radical.

Absolute Configurations of DNA Lesions Determined by Comparisons of Experimental ECD and ORD Spectra with DFT Calculations

The usefulness of modern density functional theory (DFT) methods is considered for establishing the absolute configurations of DNA lesions by comparisons of computed and experimentally measured optical rotatory dispersion (ORD) and electronic circular dichroism (ECD) spectra. Two rigid, structurally different DNA lesions (two spiroiminodihydantoin stereoisomers and four equine estrogen 4-hydoxyequilenin-DNA stereoisomeric adducts) have been investigated. In all cases, the signs and shapes of the computed ORD spectra reproduced the experimentally measured ORD spectra, although the magnitudes of the computed and experimental ORD values do not coincide exactly. The computed ECD spectra also reproduced the shapes of the experimental ECD spectra rather well, but are blue-shifted by 10-20 nm. Since the assignments of the absolute configurations of the DNA lesions studied based on computed and experimental ORD and ECD spectra are fully consistent with one another, the computational DFT method shows significant promise for determining the absolute configurations of DNA lesions. Establishing the stereochemistry of DNA lesions is highly useful for understanding their biological impact, especially when sufficient amounts of material are not available for other methods of structural characterization.