DNA Nucleobase under Ionizing Radiation: Unexpected Proton Transfer by Thymine Cation in Water Nanodroplets.

The effect of ionizing radiation on DNA constituents is a widely studied fundamental process using experimental and computational techniques. In particular, radiation effects on nucleobases are usually tackled by mass spectrometry in which the nucleobase is embedded in a water nanodroplet. Here, we present a multiscale theoretical study revealing the effects and the dynamics of water droplets towards neutral and ionized thymine. In particular, by using both hybrid quantum mechanics/molecular mechanics and full ab initio molecular dynamics, we reveal an unexpected proton transfer from thymine cation to a nearby water molecule. This leads to the formation of a neutral radical thymine and a Zundel structure, while the hydrated proton localizes at the interface between the deprotonated thymine and the water droplet. This observation opens entirely novel perspectives concerning the reactivity and further fragmentation of ionized nucleobases.

NOVEL ANALYTICAL STUDY FOR REACTION INTERMEDIATES IN THE PRIMARY RADIATION INTERACTION OF DNA USING A SYNCHROTRON RADIATION-INDUCED LUMINESCENCE SPECTROSCOPY.

To identify the precise molecular processes to induce DNA lesions, we attempt a novel spectroscopy of X-ray induced luminescence (XIL) using soft X-ray synchrotron radiation, which is a non-destructive analysis of the reaction intermediates in the elementary reaction pathway of damage induction and self-organized restoration. Using a liquid micro-jet technique to introduce aqueous samples in a vacuum chamber, we measure UV-visible luminescence from nucleotide solution as a function of the soft X-ray energy from the nitrogen to oxygen K-edge region. The XIL intensities for the nucleotide solutions are significantly enhanced in the soft X-ray region (410-530 eV) which is ascribed to the K-shell excitation/ionization of nitrogen atoms in the nucleobases. Furthermore, the XIL spectra do not show any signature of X-ray absorption near-edge structure (XANES) of the nucleobases. This is because the luminescence intensities collected from the integral area of the micro-jet only reflect the quantum yield of luminescence of the absorbed X-ray into UV-visible light irrespective of the absorption cross sections, i.e. of XANES. Thus the present result is the first evidence of luminescence as a result of X-ray absorption of aqueous nucleotides.

In vivo mutagenicities of damaged nucleotides produced by nitric oxide and ionizing radiation.

To evaluate the in vivo mutagenicities of damaged DNA precursors (deoxyribonucleoside 5'-triphosphates) produced by exposure to nitric oxide (NO) and ionizing radiation, five damaged deoxyribonucleotides (deoxyxanthosine triphosphate, deoxyoxanosine triphosphate, dITP, dUTP, and 8-hydroxy-dATP) were introduced into competent Escherichia coli cells. Their mutagenic potentials were assayed using the chromosomal rpoB gene as a mutagenesis target. In contrast to 8-hydroxy-dGTP and 2-hydroxy-dATP, which were examined in an earlier study, none of these damaged deoxyribonucleotides significantly increased the rpoB mutant frequency. These results suggest that these five damaged deoxyribonucleotides are weakly mutagenic in vivo if at all. Thus their contributions to mutations induced by NO and ionizing radiation may be small.

Independent generation and reactivity of 2-deoxy-5-methyleneuridin-5-yl, a significant reactive intermediate produced from thymidine as a result of oxidative stress.

2'-Deoxy-5-methyleneuridin-5-yl (1) is produced in a variety of DNA damage processes and is believed to result in the formation of lesions that are mutagenic and refractory to enzymatic repair. 2'-Deoxy-5-methyleneuridin-5-yl (1) was independently generated under anaerobic conditions via Norrish Type I photocleavage during Pyrex filtered photolysis of the benzyl ketone 7. The radical (1) exhibits behavior consistent with that of a resonance-stabilized radical. The KIE for hydrogen atom transfer from t-BuSH was found to be 7.3 +/- 1.7. Competition studies between radical recombination and hydrogen atom donors (2,5-dimethyltetrahydrofuran, kTrap = 46.1 +/- 15.4 M(-1) s(-1); propan-2-ol, kTrap = 13.6 +/- 3.5 M(-1) s(-1)) chosen to mimic the carbohydrate components of 2'-deoxyribonucleotides suggest that 2'-deoxy-5-methyleneuridin-5-yl (1) may be able to transfer damage from the nucleobase to the deoxyribose of an adjacent nucleotide in DNA under hypoxic conditions.

Protonation of nucleobase anions in gamma-irradiated DNA and model systems. Which DNA base is the ultimate sink for the electron?

Electron spin resonance (ESR) spectroscopy has been used to investigate irreversible protonations of the nucleobase anions in gamma-irradiated frozen aqueous solutions of dGMP-dCMP, polyG-polyC, poly[dGdC].poly[dGdC], dAMP-dTMP, poly[dAdT].poly-[dAdT] and DNA itself. Analysis of the ESR spectra at a dose of 22 kGy shows that fractional conversion of total radicals to carbon-protonated species on annealing is in the order: dAMP-dTMP (43%) > pdAdT = DNA (23%) > dGMP.dCMP (15%) > poly-dGdC.polydGdC (6%) > polyG.polyC (3%). Two hydrogen addition radicals make contributions to the polyG.polyC, poly-[dGdC].poly[dGdC] and dGMP.dCMP spectra in H2O on annealing. They are those formed by protonations at C6 of the cytosine anion radical, C(C6)H., and at C8 of the guanine anion radical, G(C8)H.. Computer analysis reveals that anion protonation reaction in dGMP.dCMP results in mainly C(C6)H., whereas protonation reaction in polyG.polyC and poly[dGdC].poly-[dGdC] yields mainly G(C8)H.. In dAMP.dTMP and poly[dAdT].poly[dAdT] as in DNA itself, the only DNA base found to undergo an irreversible protonation at a carbon site is thymine, resulting in T(C6)H.. The conversion of DNA anion to T(C6)H. is found to be dependent on dose. At low doses (5 kGy), about 30% conversion to T(C6)H. is found, whereas at high doses (94 kGy), only 13% conversion is found. The dose dependence is ascribed in part to ion radical recombinations whose probabilities are increased at high doses. A consideration of the rates of protonations of the purine and pyrimidine anion radicals as well as the differences in electron affinities suggest carbon protonation reactions of DNA base anions in irradiated stacked double-strand DNA at 37 degrees C would be predominantly at thymine and perhaps guanine, whereas in single-strand DNA all bases would contribute.

The sizes of cellular deoxynucleoside 5'-triphosphate pools in relation to sensitivity to electron irradiation using sensitive and resistant cell lines.

The deoxynucleoside 5'-triphosphate (dNTP) pool sizes have been determined before and after electron (e-) irradiation in sets of radiation sensitive and resistant cell lines. In the L5178Y mouse lymphoma radiosensitive line (LS), the dTTP pool fell 50% following irradiation, whilst the three other dNTP pools remained unaltered. On the other hand, for the radioresistant line (AII) all four dNTP pools increased by 2-to 3-fold. The dNTP pools of the Chinese hamster radiosensitive (V79) line and radioresistant (V79/79) lines were unaltered by the radiation, but a difference in pool size was present before irradiation, with the pools of the V79 cells being approximately twice those of the V79/79 cells. Two out of the three ataxia telangiectasia cell lines studied show reduced dNTP pools when compared with those of normal human fibroblasts and these pools were also unaltered by the radiation. In the L5178Y and Chinese hamster cells the levels of enzymes involved in the biosynthesis of dNTPs have been determined. In general the higher the level of ribonucleoside diphosphate reductase (RDR) the larger the cellular pools. The observed levels of RDR could, in part, explain the observed results. Increasing the dTTP pool by the addition of deoxythymidine and deoxycytidine to the cell culture with the V79/79 cells reduced their sensitivity to the radiation. These results indicate a relationship between a cell's sensitivity to e- irradiation and the sizes of the cellular dNTP pools. However, the exact nature of any such relationship is unknown.

Gamma-irradiation of homodeoxyoligonucleotides 32P-labelled at one end: computer simulation of the chain length distribution of the radioactive fragments.

Electrophoresis on polyacrylamide gels of the fragments resulting from gamma-irradiation of single-stranded oligodeoxyribonucleotides labelled at their 5'- or 3'-end proved to be a potent tool for the analysis of the radiation-induced chain breakage of DNA. Owing to the fact that the oligonucleotide may be ruptured at more than one site, the counting of the electrophoresis bands must be corrected and it is necessary to assess the influence of the cleavage position on the band intensities. A complicating factor is the inhomogeneity of the system due to the presence of the four bases A, T, C and G. To circumvent this problem, the homooligodeoxyribonucleotides (dA)15, (dC)15, (dT)15 were used as experimental probes. They were gamma-irradiated in solution, heated in alkali and the resulting fragments separated by gel electrophoresis. A computer simulation of the band intensities was compiled based on the general assumption that the chain breakage is homogeneous. The experimental results obtained from the homooligodeoxyribonucleotides labelled at either the 5' or the 3'-end are in excellent agreement with theoretical calculations. Abacus giving the gel band intensities (percentage) against the nucleotide positions and the remaining intensity of the original oligonucleotide have been obtained.

Malondialdehyde precursors in gamma-irradiated DNA, deoxynucleotides and deoxynucleosides.

Gamma-irradiation of DNA, deoxynucleosides, or deoxynucleotides produces material that reacts with thiobarbituric acid to form a chromophore with maximum absorbance at 532 nm. This material is not malondialdehyde. We have identified a new radiation product (thymin-1'-yl)-propenal as the TBA-reactive product of gamma-irradiation of thymidine. Thymine-propenal has been described by other investigators as a product of bleomycin-treatment of DNA. Irradiation of thymidine nucleotides produces phosphorylated precursors to thymine-propenal. Studies of the requirements for formation of TBA-reactivity indicate a mechanism involving reaction of a free radical with the deoxyribose moiety and molecular oxygen. On the basis of these results it is proposed that gamma-irradiation produces TBA-reactive material in DNA by the same reaction sequence in which bleomycin catalyzes the formation of base-propenals in DNA. Bleomycin and gamma-irradiation differ in the extent to which the sequence proceeds to completion with release of free base-propenals.

UV irradiation alters deoxynucleoside triphosphate pools in Escherichia coli.

UV irradiation of exponentially growing Escherichia coli increased intracellular concentration of dATP and dTTP without significantly changing the concentrations of dGTP and dCTP. These selective increases in dATP and dTTP pools are seen in wild-type E. coli K12 and AB1157, as well as in recA and umuC strains, and are proportional to UV dose. The possible significance of these findings with respect to induction of the SOS response and nontargeted mutagenesis are discussed.

UV-induced imbalance of the deoxyribonucleoside triphosphate pool in E. coli.

The effect of UV irradiation on the intracellular DNA precursor pool in E. coli was investigated. UV irradiation of E. coli, followed by post-incubation for 1-1.5 h, altered the relative sizes of the deoxyribonucleoside triphosphate (dNTP) pool. The total amount of dNTPs increased: both dATP and dTTP increased several-fold, dCTP about twofold, while dGTP remained almost unchanged. In recA- and umuC- strains, which are defective in UV-induced mutagenesis, the pattern of nucleotide pool alterations was similar to that of wild-type strains.

Kinetics of UV-induced changes in deoxynucleoside triphosphate pools in Chinese hamster ovary cells and their effect on measurements of DNA synthesis.

Measurements of dNTP pools following exposure of Chinese hamster ovary cells to ultraviolet radiation reveals a rapid accumulation of cellular dTTP and a rapid loss of cellular dCTP. Exposure to 3-, 10- or 20 Jm-2 results in a 3-, 4- or 5.4-fold increase in cellular dTTP, respectively, within the first 10 min after exposure. dTTP levels then decrease noticeably, approaching the control value 3 to 5 hr later. In contrast, dCTP levels decrease rapidly within 10 min after exposure, ultimately to 1/10 that observed in the unirradiated control population. Recovery to normal dCTP levels is slow, taking in excess of 12 hr. No change in dATP is observed for 1-2 hr; subsequently, a moderate decrease in dATP levels occurs which is then followed by recovery, beginning 8 hr after irradiation. These results contrast with changes in dNTP pools observed in Chinese hamster V-79 cells exposed to mutagens. Measurements of rates of DNA synthesis by pulse-labeling cells with [3H]thymidine are also apparently affected by UV-induced transient deviations in the endogenous radiospecific activity of the labeled precursor.