Hydrogen-Rich Cation Radicals of DNA Dinucleotides: Generation and Structure Elucidation by UV-Vis Action Spectroscopy.

Hydrogen-rich DNA dinucleotide cation radicals (dGG + 2H)+•, (dCG + 2H)+•, and (dGC + 2H)+• represent transient species comprising protonated and hydrogen atom adducted nucleobase rings that serve as models for proton and radical migrations in ionized DNA. These DNA cation radicals were generated in the gas phase by electron-transfer dissociation of dinucleotide dication-crown-ether complexes and characterized by UV-vis photodissociation action spectra, ab initio calculations of structures and relative energies, and time-dependent density functional theory calculations of UV-vis absorption spectra. Theoretical calculations indicate that (dGG + 2H)+• cation radicals formed by electron transfer underwent an exothermic conformational collapse that was accompanied by guanine ring stacking and facile internucleobase hydrogen atom transfer, forming 3'-guanine C-8-H radicals. In contrast, exothermic hydrogen transfer from the 5'-cytosine radical onto the guanine ring in (dCG + 2H)+• was kinetically hampered, resulting in the formation of a mixture of 5'-cytosine and 3'-guanine radicals. Conformational folding and nucleobase stacking were energetically unfavorable in (dGC + 2H)+• that retained its structure of a 3'-cytosine radical, as formed by one-electron reduction of the dication. Hydrogen-rich guanine (G + H)• and cytosine (C + H)• radicals were calculated to have vastly different basicities in water, as illustrated by the respective p Ka values of 20.0 and 4.6, which is pertinent to their different abilities to undergo proton-transfer reactions in solution.

Molecular models predict light-induced glutamine tautomerization in BLUF photoreceptors.

The recently discovered photoreceptor proteins containing BLUF (sensor of blue light using FAD) domains mediate physiological responses to blue light in bacteria and euglena. In BLUF domains, blue light activates the flavin chromophore yielding a signaling state characterized by a approximately 10 nm red-shifted absorption. We developed molecular models for the dark and light states of the BLUF domain of the Rhodobacter sphaeroides AppA protein, which are based on the crystal structures and quantum-mechanical simulations. According to these models, photon absorption by the flavin results in a tautomerization and 180 degree rotation of the Gln side chain that interacts with the flavin cofactor. This chemical modification of the Gln residue induces alterations in the hydrogen bond network in the core of the photoreceptor domain, which were observed in numerous spectroscopic experiments. The calculated electronic transition energies and vibrational frequencies of the proposed dark and light states are consistent with the optical and IR spectral changes observed during the photocycle. Light-induced isomerization of an amino acid residue instead of a chromophore represents a feature that has not been described previously in photoreceptors.

A novel approach to DNA damage assessments: measurement of the thymine glycol lesion.

A different approach to the measurement of DNA damage has been developed based on the fact that many lesions can be excised from DNA in the form of modified dinucleoside monophosphates. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) is used in conjunction with isotopically labeled internal standards to quantify the lesion. The method has several advantages, including high sensitivity for the detection of dinucleoside monophosphates. The method was applied to the measurement of the 5,6-dihydroxy-5,6-dihydrothymine (thymine glycol) lesion in the DNA of mouse fibroblast cells exposed in culture to various treatments including ionizing radiation, UVC light and buthionine sulfoximine (BSO), an inhibitor of glutathione synthesis. The application of the method to the measurement of other DNA lesions is discussed.

Singlet oxygen-induced DNA damage.

Singlet oxygen, hydrogen peroxide, hydroxyl radical and hydrogen peroxide are the reactive oxygen species (ROS) considered most responsible for producing oxidative stress in cells and organisms. Singlet oxygen interacts preferentially with guanine to produce 8-oxo-7,8-dihydroguanine and spiroiminodihydantoin. DNA damage due to the latter lesion has not been detected directly in the DNA of cells exposed to singlet oxygen. In this study, the singlet oxygen-induced lesion was isolated from a short synthetic oligomer after exposure to UVA radiation in the presence of methylene blue. The lesion could be enzymatically excised from the oligomer in the form of a modified dinucleoside monophosphate. Using liquid chromatography-tandem mass spectrometry (LC-MS/MS), the singlet oxygen lesion was detected in the form of modified dinucleoside monophosphates in double-stranded DNA and in the DNA of HeLa cells exposed to singlet oxygen. Pentamer containing the singlet oxygen-induced lesion and an isotopic label was synthesized as an internal standard for quantifying the lesion and served as well as for correcting for losses of product during sample preparation.

Phenyl radicals react with dinucleoside phosphates by addition to purine bases and H-atom abstraction from a sugar moiety.

Laser-induced acoustic desorption combined with mass spectrometry has been used to demonstrate that phenyl radicals can attack dinucleoside phosphates at both the sugar and base moieties, that purine bases are more susceptible to the attack than pyrimidine bases, and that the more electrophilic the radical, the more efficient the damage to dinucleoside phosphates.

Isolation and characterization of a novel cross-link lesion in d(CpC) induced by one-electron photooxidation.

Recently we reported the identification and characterization of a novel cross-link lesion formed between two adjacent cytosines in d(CpC), which is the major product formed upon 365 nm photoirradiation of d(CpC) in the presence of 2-methyl-1,4-naphthoquinone. In this study we discuss the isolation and structure characterization of another cross-link lesion formed under the same irradiation condition. Electrospray ionization mass spectroscopy, tandem mass spectrometry and two-dimensional nuclear Overhauser effect spectroscopy results demonstrate that the C6 carbon atom of the 5' cytosine and the N3 nitrogen atom of the 3' cytosine are covalently bonded. In addition, the 5' cytosine moiety is deaminated and the C5 carbon atom in this cytosine is oxidized to a carbonyl group.

Dihydrothymine lesion in X-irradiated DNA: characterization at the molecular level and detection in cells.

PURPOSE: To study the formation of the dihydrothymine lesion produced in DNA by ionizing radiation in an anaerobic environment. MATERIALS AND METHODS: The dihydrothymine lesion, along with other lesions, was isolated from an X-irradiated aqueous solution of the dinucleoside monophosphate d(TpA) and analysed by correlated two-dimensional nuclear magnetic resonance spectroscopy. The dihydrothymine lesion was obtained by enzymatic digestion of irradiated DNA in the form of modified dinucleoside monophosphates, d(T(d)A), where T(d) stands for dihydrothymidine. Liquid chromatography-tandem mass spectrometry was used to detect the lesion in the DNA of X-irradiated mouse fibroblast cells. RESULTS: The modified dinucleoside monophosphate, d(T(d)pA), fragments by two pathways so that altogether the lesion could be detected using two different sets of tandem mass spectrometry (precursor ion mass/daughter ion mass) values. CONCLUSION: The dihydrothymine lesion is a significant lesion in cells exposed to ionizing radiation in an anaerobic environment.

Identification and characterization of a novel cross-link lesion in d(CpC) upon 365-nm irradiation in the presence of 2-methyl-1,4-naphthoquinone.

We report the isolation and characterization for the first time of a cross-link lesion between two adjacent cytosines from the 2-methyl-1,4-naphthoquinone (menadione)-sensitized 365-nm irradiation of d(CpC). Electrospray ionization mass spectrometry (ESI-MS), tandem MS and (1)H NMR results indicate that the cross-link occurs between the C5 carbon atom of one cytosine and the N(4) nitrogen atom of the other cytosine. Furthermore, we synthesized d(CpC) with a (15)N being incorporated on the amino group of either of the two cytosines. We then irradiated the two (15)N-labeled dinucleoside monophosphates, isolated the cross-link products and characterized them by MS and multi-stage tandem MS. The latter results established unambiguously that the N(4) nitrogen atom of the 3'-nucleobase is involved in the covalent bond formation between the two cytosines. This, in combination with two-dimensional nuclear Overhauser effect spectroscopy (NOESY) results, demonstrates that the cross-link arises from the formation of a covalent bond between the C5 carbon atom of the 5' cytosine and the N(4) nitrogen atom of the 3' cytosine. We also show that the solution pH has a significant effect on the formation of the cross-link lesion, which supports that the deprotonation at the exocyclic amino group of cytosine cation radical is essential for the formation of the cross-link lesion.

Mechanisms for the formation of major oxidation products of adenine upon 365-nm irradiation with 2-methyl-1,4-naphthoquinone as a sensitizer.

Recently we reported the isolation and characterization of N6-formyl- and N6-acetyladenine from 365-nm irradiation of dinucleoside monophosphates d(ApA), d(ApC), and d(CpA) in the presence of 2-methyl-1,4-naphthoquinone (menadione) (Wang et al. Biochem. Biophys. Res. Commun. 2002, 291, 1252-7). In this article we investigated the mechanisms for the formation of the two major products by carrying out photoirradiation with isotopically labeled menadione and 2,3-dimethyl-1,4-naphthoquinone. HPLC and electrospray ionization (ESI)-mass spectrometry (MS) and tandem MS studies of the products unambiguously established that the carbonyl group in the products arises from the photosensitizer: The N6-formyl group comes from oxidation of the methyl group and the N6-acetyl group stems from the methyl group and the adjacent ring carbon in menadione. From above results, we proposed mechanisms for the formation of the two products.

Major adenine products from 2-methyl-1,4-naphthoquinone-sensitized photoirradiation at 365 nm.

In this article we report the isolation and characterization of major products of adenine in dinucleoside monophosphates upon 2-methyl-1,4-naphthoquinone (menadione)-sensitized UVA irradiation. Our results show that the major products form via the coupling between the menadione moiety and the exocyclic amino group of adenine. Similar reactions were not observed for cytosine. To our knowledge, this is the first report about the direct reaction between a DNA base and a photosensitizer under 365-nm ultraviolet light irradiation. Our results are consistent with previous observation showing that N(6) radical formed on adenine upon UVA irradiation.

DNA tandem lesions containing 8-oxo-7,8-dihydroguanine and formamido residues arise from intramolecular addition of thymine peroxyl radical to guanine.

Exposure of aerated aqueous solutions of dinucleoside monophosphates bearing both a pyrimidine base and a guanine residue to ionizing radiation leads to the formation of 8-oxo-7,8-dihydroguanine/formamido tandem lesions (8-oxodG/dF). Recent evidence for the formation of the latter damage within isolated DNA emphasized the possible biological relevance of this class of lesions. Therefore, an extensive mechanistic study of the formation of 8-oxodG/dF was carried out with thymine and guanine containing dinucleoside monophosphates (dGpdT and dTpdG). First, the peroxyl radical-induced degradation of guanine within dGpdT and dTpdG was studied in order to assess the possibility of intramolecular electron transfer between guanine and thymine peroxyl radicals. Then, the formation of a series of tandem lesions involving a formamido residue, thymine glycols, 8-oxodG, and oxazolone was monitored within aerated aqueous solutions of dTpdG and dGpdT exposed to gamma-radiation. The absence of formation of tandem lesions other than 8-oxodG/dF in significant yield led to us propose a new mechanism involving addition of the thymine peroxyl radical to the guanine moiety. This received support from (18)O labeling experiments.

Modulation of remote DNA oxidation by hybridization with peptide nucleic acids (PNA).

We have examined the efficiency of DNA photooxidation in DNA/PNA duplex and DNA/(PNA)(2) triplex for the first time. DNA/PNA duplex was cleaved at GG steps by external riboflavin with high efficiency like specific GG cleavage in DNA/DNA duplex. However, the 5'G selectivity of the GG oxidation in DNA/PNA duplex was much lower than that observed in DNA/DNA duplex. Remote DNA oxidation of oxidant-tethered DNA/PNA duplex was considerably suppressed. In contrast, the formation of DNA/(PNA)(2) triplex by hybridization with two PNA strands completely inhibited the remote GG oxidation, indicating that PNA acts as an inhibition for remote oxidative DNA damage.

Individual determination of the yield of the main UV-induced dimeric pyrimidine photoproducts in DNA suggests a high mutagenicity of CC photolesions.

Bipyrimidine photoproducts induced in DNA by UVB radiation include cyclobutane dimers, (6-4) photoproducts, and their related Dewar valence isomers. Even though these lesions have been extensively studied, their rate of formation within DNA is still not known for each possible bipyrimidine site (TT, TC, CT, and CC). Using a method based on the coupling of liquid chromatography to mass spectrometry, we determined the distribution of the 12 possible bipyrimidine photoproducts within isolated and cellular DNA. TT and TC were found to be the most photoreactive sequences, whereas lower amounts of damage were produced at CT and CC sites. In addition to this quantitative aspect, sequence effects were observed on the relative yield of (6-4) adducts with respect to cyclobutane pyrimidine dimers. Another interesting result is the lack of formation of Dewar valence isomers in detectable amounts within the DNA of cells exposed to low doses of UVB radiation. The photoproduct distribution obtained does not fully correlate with the UV mutation spectrum. A major striking observation deals with the low yield of cytosine-cytosine photoproducts which are likely to be associated with the UV-specific CC to TT tandem mutation.

A novel vicinal lesion obtained from the oxidative photosensitization of TpdG: characterization and mechanistic aspects.

A new type of vicinal base lesion was isolated from the photosensitization of TpdG in aerated aqueous solution. One- and two-dimensional NMR measurements were used together with mass spectrometry to accurately characterize the new adduct. Chemical detection of guanidine provided additional structural information on the base moiety at the 3'-OH terminal end. Altogether the experiments results were indicative of the occurrence of a covalent bonding between the pyrimidine ring on the 5'-OH terminal end and the imidazole ring on the 3'-OH terminal end through a methylene bridge. Photosensitization studies of TpdG, thymidine, and 2'-deoxyguanosine in the presence of either benzophenone, menadione, or riboflavin associated with isotopic labeling experiments using enriched oxygen and water provided relevant information on the mechanism of formation of the adduct. The results of these experiments clearly demonstrated that the initial event leading to the formation of the lesion is the abstraction of a hydrogen atom from the methyl group of the thymine base moiety of TpdG. This is followed by the addition of the methyl-centered radical to the C-4 atom of the guanine ring which gives rise to the vicinal lesion after reaction with molecular oxygen and subsequent rearrangement.

Far-UV-induced dimeric photoproducts in short oligonucleotides: sequence effects.

Cyclobutane pyrimidine dimers and pyrimidine(6-4)pyrimidone adducts represent the two major classes of far-UV-induced DNA photoproducts. Because of the lack of appropriate detection methods for each individual photoproduct, little is known about the effect of the sequence on their formation. In the present work, the photoproduct distribution obtained upon exposure of a series of dinucleoside monophosphates to 254 nm light was determined. In the latter model compounds, the presence of a cytosine, located at either the 5'- or the 3'-side of a thymine moiety, led to the preferential formation of (6-4) adducts, whereas the cis-syn cyclobutane dimer was the main thymine-thymine photoproduct. In contrast, the yield of dimeric photoproducts, and particularly of (6-4) photoadducts, was very low upon irradiation of the cytosine-cytosine dinucleoside monophosphate. However, substitution of cytosine by uracil led to an increase in the yield of (6-4) photoproduct. It was also shown that the presence of a phosphate group at the 5'- end of a thymine-thymine dinucleoside monophosphate does not modify the photoproduct distribution. As an extension of the studies on dinucleoside monophosphates, the trinucleotide TpdCpT was used as a more relevant DNA model. The yields of formation of the thymine-cytosine and cytosine-thymine (6-4) photoproducts were in a 5:1 ratio, very close to the value obtained upon photolysis of the related dinucleoside monophosphates. The characterization of the two TpdCpT (6-4) adducts was based on 1H NMR, UV and mass spectroscopy analyses. Additional evidence for the structures was inferred from the analysis of the enzymatic digestion products of the (6-4) adducts of TpdCpT with phosphodiesterases. The latter enzymes were shown to induce the quantitative release of the photoproduct as a modified dinucleoside monophosphate in a highly sequence-specific manner.

Abiogenic synthesis of pyrimidine nucleotides in solid state by vacuum ultraviolet radiation.

The abiogenic synthesis of pyrimidine nucleotides in solid state has been investigated. Our experiment indicates that natural nucleotides are produced in thin films prepared from nucleoside and inorganic phosphate by irradiating with vacuum ultraviolet light (VUV, lambda=100-200 nm). We have investigated the influence of the type of nucleic acids base (thymidine, cytosine, uracil) and the structure of sugar moiety (ribose or deoxyribose) on the course and yield of reaction. We compared the action of vacuum ultraviolet light with action of gamma-radiation, heat and biology significant UV (254 nm) which have been investigated earlier. The occurrence of these reaction in open space is discussed.

Mutation spectra of M13 vectors containing site-specific Cis-Syn, Trans-Syn-I, (6-4), and Dewar pyrimidone photoproducts of thymidylyl-(3'-->5')-thymidine in Escherichia coli under SOS conditions.

The mutations spectra of cis-syn, trans-syn-I, (6-4), and Dewar pyrimidone photoproducts of the TT site of AATTAA and TATTAT in the (-) strand of a heteroduplex M13 vector were obtained in an excision and photoreversal repair deficient Escherichia coli host under SOS conditions. Oligonucleotides containing site-specific photoproducts were annealed to a complementary uracil-containing (+) strand that contained one or more unique pairs of nucleotide mismatches and used to prime (-) strand synthesis with a DNA polymerase and dNTPs. Following DNA synthesis, the reaction mixtures were incubated with T4 DNA ligase and ATP and then used to transfect SOS-induced competent CSRO6F' cells (uvrA6 and phr-1). The transfectants were plated, gridded, and probed by oligonucleotides specific for progeny of the (-) and (+) strands. Individual progeny of the photoproduct-containing (-) strands were plaque purified and sequenced by the dideoxy method. The cis-syn and trans-syn-I dimers were found not to be very mutagenic (<9%), the Dewar product more so (<33%), and the (6-4) product the most mutagenic (<73%). The mutation spectra were similar to those previously reported for the same photoproducts of the TT site of AGTTGG in the (+) strand of an M13 vector [Lawrence, C. W., et al. (1990) Mol. Gen Genet. 222, 166-168; LeClerc, J. E., et al. (1991) Proc. Natl. Acad. Sci. U.S.A. 88, 9685-9689] except that -1 deletion mutations were not observed for the trans-syn-I photoproducts, and a lower frequency of 3'-T-->C mutations was observed for the (6-4) photoproduct. Evidence that a small percentage of (+) strand repair of a double mismatch to the 3'-side of the photoproduct. Evidence that a small percentage of (+) strand repair of a double mismatch to the 3'-side was obtained from transfection experiments in which a second double mismatch was introduced opposite or flanking the photoproduct. Analysis of the minor tandem mutations induced by the (6-4) and Dewar products suggests that the SOS polymerase complex is able to elongate what amounts to double mismatches opposite these photoproducts and is consistent with the action of a highly processive polymerase that lacks proofreading ability.

The solution structure of DNA duplex-decamer containing the (6-4) photoproduct of thymidylyl(3'-->5')thymidine by NMR and relaxation matrix refinement.

The DNA duplex-decamer containing a site-specific (6-4) photoadduct of thymidylyl(3'-->5')thymidine was generated by direct photolytic ultraviolet C irradiation of d(CGCATTACGC). The three-dimensional structure of the duplex (6-4) decamer, d(CGCAT[6-4]TACGC).d(GCGTAATGCG), has been determined by two-dimensional NMR spectroscopy and a relaxation matrix refinement method. NMR data and structural calculations established that the formation of the (6-4) adduct in the B-DNA duplex retains Watson-Crick-type hydrogen bonding throughout the duplex except at the 3'-side of the (6-4) lesion where the T-->C transition mutation is predominantly targeted [LeClerc, J. E., Borden, A. & Lawrence, C. W. (1991) Proc. Natl Acad. Sci. USA 88, 9685-9689], but leads to a 44 degrees bending in the overall DNA helix. Perpendicular base orientation of the (6-4) lesion provides a structural basis where potential hydrogen bonding at the 3' sides of the (6-4) adduct with substituted nucleotide is improbable during replication under stringent conditions.

Free radical-induced double base lesions.

Evidence is presented for the formation of products in irradiated dinucleoside monophosphates in which both bases are damaged. The dinucleoside monophosphates d(GpT), d(GpC), d(TpG) and d(CpG) were X-irradiated in oxygenated aqueous solution. Product identification was by NMR spectroscopy. In products containing double base lesions, guanine is converted to 8-hydroxyguanine and the pyrimidine base is degraded to a formamido remnant.

Dithymine photodimers and photodecomposition products of thymidylyl-thymidine induced by ultraviolet radiation from 150 to 300 nm.

Solid thymidylyl-(3'-->5')-thymidine (dTpdT) was irradiated in a vacuum with monochromatic photons from 150 to 300 nm; the photoproducts were analyzed quantitatively by high-performance liquid chromatography. The results of the experiment were as follows: (1) above 210 nm the major photoproducts were three dithymine photodimers [the cis-syn and trans-syn cyclobutane thymine dimers and the thymine (6-4) photoproduct]; below 210 nm, they were three photodecomposition products (thymine, thymidine 5'-monophosphate and thymidine 3'-monophosphate). This shows that 210 nm is the wavelength at which the major photoproducts change from dithymine photodimers (far-UV type) to photodecomposition products (X-ray type). (2) The yields of the three dithymine photodimers had a similar wavelength dependence with each other: the yields had a peak at 260 nm and gradually decreased toward shorter wavelengths to 150 nm. (3) The yields of the three photodecomposition products also had a very similar wavelength dependence with each other: the yields increased exponentially with a decrease in the wavelength. (4) The average ratios of the yield of the (6-4) photoproduct to that of the cis-syn dimer were 0.30 between 170 and 220 nm, but 0.16 between 240 and 290 nm.