DNA damage induced by the direct effect of He ion particles

We present here evidence showing that the yields of DNA lesions induced by He(2+) ions strongly depend on Linear energy transfer (LET). In this study, hydrated plasmid DNA was irradiated with He(2+) ions with LET values of 19, 63 and 95 keVmicrom(-1). The yields of prompt single-strand breaks (SSBs) are very similar at the varying LET values, whereas the yields of prompt double-strand breaks (DSBs) increase with increasing LET. Further, base lesions were revealed as additional strand breaks by post-irradiation treatment of the DNA with endonuclease III (Nth) and formamidopyrimidine-DNA glycosylase (Fpg). The reduction in the yield of these enzymatically induced SSBs and DSBs becomes significant as the LET increases. These results suggest that the clustering of DNA lesions becomes more probable in regions of high LET.     

Mechanisms of low energy electron damage to condensed biomolecules and DNA

It has recently been shown that 3-20 eV electron impact on vacuum-dry samples of plasmid DNA induced substantial yields of single and double strand breaks (SSBs and DSBs). These results are summarised in the present article along with those obtained from the fragmentation of elementary components (i.e. condensed H2O, bases and sugar analogues) of DNA induced by low energy electron impact under ultra high vacuum conditions. By comparing the results from these experiments, it is possible to determine fundamental mechanisms by which low energy electrons damage DNA. The decay of transient anions formed on the DNA's basic components is found to play a crucial role in producing SSBs and DSBs. Since a large portion of the energy deposited by ionising radiation first leads to the production of low energy secondary electrons, these findings provide basic knowledge necessary to understand the genotoxic effects of high energy radiation and eventually modify these effects at the molecular level.     

Investigation of radiation damage in DNA by using atomic force microscopy

The effect of radiations on supercoiled plasmid DNA has been investigated by using atomic force microscopy (AFM). The DNA molecules were deposited on a substrate and observed by AFM. Alternatively, DNA at different scavenger concentrations was initially exposed to different types of radiations (alpha and X rays) at various doses. After irradiation, fragments (open circular and linearised strands) were observed corresponding to single strand breaks and double strand breaks in DNA. This result indicates the capabilities of AFM for the qualitative detection of strand modifications due to irradiation. The amount of each class of topology enables a quantitative response to be determined for both types of radiation (alpha, X). A value of the radiosensitivity of DNA was obtained as a function of the scavenger concentration. Strong accordance was found between AFM results and those obtained by use of gel electrophoresis. The advantage of AFM in comparison with traditional techniques is the possibility of analysing the radiation effects on one molecule. Indeed, taking the example of alpha particles, it is shown that it is easy to measure the sizes of linear strands by AFM. Such additional or even precise results are difficult to obtain with gel electrophoresis since, in such a case, data are lost through smearing.     

Base excision repair processing of radiation-induced clustered DNA lesions.

Energy from low LET ionising radiation, such as X rays and gamma rays, is deposited in the water surrounding the DNA molecule such that between 2 to 5 radical pairs are generated within a radius of I to 4 nm. As a result, multiple single lesions, including oxidised purine or pyrimidine bases, sites of base loss, and single-strand breaks, can be formed in DNA from the same radiation energy deposition event. The single lesions in these so-called multiply damaged sites or clustered lesions are repaired by base excision repair. Here we show that clustered DNA damages are formed in bacterial cells by ionising radiation and are converted to lethal double-strand breaks during attempted repair. In wild type cells possessing the oxidative DNA glycosylases that recognise and cleave DNA at repairable single damages, double-strand breaks are formed at radiation-induced clusters during post-irradiation incubation and in a dose-dependent fashion. Mutant cells lacking these enzymes do not form double-strand breaks post-irradiation and are substantially more radioresistant than wild type cells. These radioresistant mutant cells can be made radiosensitive by overexpressing one of the oxidative DNA glycosylases. Thus the effect of the oxidative DNA glycosylases in potentiating DNA damage must be considered when estimating radiation risk.     

Nanoscale detection of ionizing radiation damage to DNA by atomic force microscopy

The detection and quantification of ionizing radiation damage to DNA at a single-molecule level by atomic force microscopy (AFM) is reported. The DNA damage-detection technique combining supercoiled plasmid relaxation assay with AFM imaging is a direct and quantitative approach to detect gamma-ray-induced single- and double-strand breaks in DNA, and its accuracy and reliability are validated through a comparison with traditional agarose gel electrophoresis. In addition, the dependence of radiation-induced single-strand breaks on plasmid size and concentration at a single-molecule level in a low-dose (1 Gy) and low-concentration range (0.01 ng microL(-1)-10 ng microL(-1)) is investigated using the AFM-based damage-detection assay. The results clearly show that the number of single-strand breaks per DNA molecule is linearly proportional to the plasmid size and inversely correlated to the DNA concentration. This assay can also efficiently detect DNA damage in highly dilute samples (0.01 ng microL(-1)), which is beyond the capability of traditional techniques. AFM imaging can uniquely supplement traditional techniques for sensitive measurements of damage to DNA by ionizing radiation.     

Yields of strand breaks and base lesions induced by soft X-rays in plasmid DNA

The yields of soft-X-ray-induced DNA damages have been measured by using closed-circular plasmid DNA. Several DNA solutions with three kinds of radical scavenger capacity and also fully hydrated DNA samples were irradiated to compare the contribution by indirect reaction of diffusible water radicals, such as OH*, with those by direct action of secondary electrons. The yields of prompt single- (SSBs) and double-strand breaks (DSBs) decrease with increasing scavenging capacity. The SSB yields for soft X-rays are approximately midway those between gamma-ray and ultrasoft X-ray data previously reported. Heat labile sites are observed only in the low scavenger condition. The yields of the base lesions revealed by post irradiation treatment with base excision repair enzymes showed a similar value for Nth and Fpg protein except in the hydrated sample. These results indicate that the direct effect of soft X-rays induces the damages with different efficiency from those by indirect effect.     

Simulation of light ion induced DNA damage patterns

The biophysical simulation code PARTRAC was extended by a module to handle ions heavier than alpha particles. Cross sections for ion-electron interactions were taken from He(++) ions of the same velocity and scaled by Z(eff(2))/4. Calculated linear energy transfer values, radial dose distributions and secondary electron spectra were found in agreement with experimental results. DNA damage due to irradiation of human fibroblast cells by several light ions from H to S was calculated for various energies complemented by 220 kV(p) X rays as reference radiation. With increasing linear energy transfer, the calculated total yield of double-strand breaks per dose showed saturation behaviour at about twice the value for reference radiation. When data analysis methods for experimental double-strand break yield determination were applied to the simulated DNA damage patterns, the two data sets were found in accord. The calculated patterns of DNA damage clusters were analysed on local and regional scale finding regional clusters in closer correlation to experimental cell inactivation data.     

Calculation of DNA strand breakage by neutralisation effect after 125I decays in a synthetic oligodeoxynucleotide using charge transfer theory

The decay of the radioisotope (125)I into (125)Te is typically followed by the emission of two groups of approximately 10 electrons each via Auger processes. In deoxyribonucleic acid (DNA) with (125)I incorporated, these electrons produce various types of damage to DNA, e.g. single strand breaks (SSBs) and double strand breaks (DSBs) through direct actions of physical tracks, or indirect actions of radicals produced in water. Among the direct actions one should consider not only the excitation and ionisation of DNA by Auger electrons, but also the neutralisation of highly charged (125 m)Te ions with electrons from neighbouring molecules. Comparison between experiment and simulation done recently revealed that without including neutralisation effect the simulated yield of SSBs was 50% less than the measured result. In the present work a calculation of DNA strand breakage by the neutralization effect in a 41-mer synthetic oligodeoxynucleotide (oligoDNA) model was done using the charge transfer theory. Calculation based on transfer rate using the newly evaluated electronic coupling of DNA bases showed that the positive charge (hole) transfer rate is of the order of magnitudes of several 10(13) s(-1), implying that a charge higher than 10 units might not build on a (125 m)Te atom. The potential energy accumulated on the decay base is transferred to bases along the DNA chain nearby and destroys those bases and ionises the sugar-phosphate group, leading a DNA SSB with a frequency of 0.2% per eV in average.     

Use of DNA repair enzymes in electrochemical detection of damage to DNA bases in vitro and in cells

Electrochemical measurements at mercury or solid amalgam electrodes offer a highly sensitive detection of DNA strand breaks. On the other hand, electrochemical detection of damage to DNA bases at any electrode is usually much less sensitive. In this paper, we propose a new voltammetric method for the detection of the DNA base damage based on enzymatic conversion of the damaged DNA bases to single-strand breaks (ssb), single-stranded (ss) DNA regions, or both. Supercoiled DNA exposed to UV light was specifically cleaved by T4 endonuclease V, an enzyme recognizing pyrimidine dimers, the major products of photochemical DNA damage. Apurinic sites (formed in dimethyl sulfate-modified DNA) were determined after treating the DNA with E. coli exonuclease III, an enzyme introducing ssb at the abasic sites and degrading one of the DNA strands. The ssb or ssDNA regions, or both, were detected by adsorptive transfer stripping alternating current voltammetry at the mercury electrode. This technique offers much better sensitivity and selectivity of DNA base damage detection than any other electrochemical method. It is not limited to DNA damage in vitro, but it can detect also DNA base damage induced in living bacterial cells.     

Assessment of genotoxicity of methyl-tert-butyl ether, benzene, toluene, ethylbenzene, and xylene to human lymphocytes using comet assay

Methyl-tert-butyl ether (MTBE) is a gasoline oxygenate and antiknock additive substituting for lead alkyls currently in use worldwide. Benzene, toluene, ethylbenzene, and xylene (BTEX) are volatile monoaromatic hydrocarbons which are commonly found together in crude petroleum and petroleum products such as gasoline. The aim of this study is to evaluate the genotoxic effects of these tested chemicals in human lymphocytes. Using the alkaline comet assay, we showed that all of the tested chemicals induce DNA damage in isolated human lymphocytes. This effect could follow from the induction of DNA strands breaks. The neutral version of the test revealed that MTBE, benzene, and xylenes induce DNA double-strand breaks at 200 microM. Apart from MTBE, the spin traps, 5,5-dimethyl-pyrroline-N-oxide (DMPO) and N-tert-butyl-alpha-phenylnitrone (PBN) can decrease the level of DNA damage in BTEX at 200 microM. This indicated that DNA damage could result from the participation of free radicals in DNA-damaging effect, which was further supported by the fact that post-treatment of formamidopyrimidine-DNA glycosylase (Fpg), enzyme recognizing oxidized DNA purines, gave rise to a significant increase in the extent of DNA damage in cells treated with benzene, and xylene at 200 microM. The results obtained suggested that MTBE and BTEX could induce a variety type of DNA damage such as single-strand breaks (SSBs), double-strand breaks (DSBs), and oxidative base modification.     

Modification of indium tin oxide electrodes with nucleic acids: detection of attomole quantities of immobilized DNA by electrocatalysis

Indium tin oxide electrodes were modified with DNA, and the guanines in the immobilized nucleic acid were used as a substrate for electrocatalytic oxidation by Ru(bpy)3(3+) (bpy = 2,2'-bipyridine). Nucleic acids were deposited onto 12.6-mm2 electrodes from 9:1 DMF/water mixtures buffered with sodium acetate. The DNA appeared to denature in the presence of DMF, leading to adsorption of single-stranded DNA. The nucleic acid was not removed by vigorous washing or heating the electrodes in water, although incubation in phosphate buffer overnight liberated the adsorbed biomolecule. Acquisition of cyclic voltammograms or chronoamperomograms of Ru(bpy)3(2+) at the modified electrodes produced catalytic signals indicative of oxidation of the immobilized guanine by Ru(III). The electrocatalytic current was a linear function of the extent of modification with a slope of 0.5 microA/pmol of adsorbed guanine; integration of the current-time traces gave 2.2+/-0.4 electrons/guanine molecule. Use of long DNA strands therefore gave steep responses in terms of the quantity of adsorbed DNA strand. For example, electrodes modified with a 1497-bp PCR product from the HER-2 gene produced detectable catalytic currents when as little as 550 amol of strand was adsorbed, giving a sensitivity of 44 amol/mm2.     

Simple,fast, label-free,and nanoquencher-free system for operating multivalued DNA logic gates using polythymine templated CuNPs as signal reporters

Boolean logic devices play a key role in both traditional and nontraditional molecular logic circuits.This kind of binary logic,in which each bit is coded by (0,1),has only two output states—on or off (or high/low).Because of the finite computing capacity and variation,it is facing challenges from multivalued logic gates while processing high-density or uncertain/imprecise information.However,a low-cost,simple,and universal system that can perform different multivalued logic computations has not yet been developed,and remains a concept for further study.Herein,taking the ternary OR and INHIBIT logic gates as model devices,we present the fabrication of a novel simple,fast,label-free,and nanoquencher-free system for multivalued DNA logic gates using poly-thymine (T) templated copper nanoparticles (CuNPs) as signal reporters.The mixture of Cu2+ and ascorbic acid (AA) is taken as a universal platform for all ternary logic gates.Different kinds of poly-T strands and delicately designed complementary poly-adenine (A) strands are alternatively applied as ternary inputs to exhibit the ternary output states (low/0,medium/1,high/2).Notably,there are no nanoquenchers in this platform as poly-A strands can function as not only inputs but also efficient inhibitors of poly-T templated CuNPs.Moreover,all DNA are unlabeled single-strand DNA that do not need sophisticated labeling procedures or sequence design.The above design greatly reduces the operating time,costs,and complexity.More importantly,the ternary logic computations can be completed within 20 min because of the fast formation of CuNPs,and all of them share the same threshold values.     

Radiation-induced DNA damage responses

The amazing feature of ionising radiation (IR) as a DNA damaging agent is the range of lesions it induces. Such lesions include base damage, single strand breaks (SSBs), double strand breaks (DSBs) of varying complexity and DNA cross links. A range of DNA damage response mechanisms operate to help maintain genomic stability in the face of such damage. Such mechanisms include pathways of DNA repair and signal transduction mechanisms. Increasing evidence suggests that these pathways operate co-operatively. In addition, the relative impact of one mechanism over another most probably depends upon the cell cycle phase and tissue type. Here, the distinct damage response pathways are reviewed and the current understanding of the interplay between them is considered. Since DNA DSBs are the major lethal lesion induced by IR, the focus lies in the mechanisms responding to direct or indirectly induced DSBs.     

Detection of low dose radiation induced DNA damage using temperature differential fluorescence assay.

A rapid and sensitive fluorescence assay for radiation-induced DNA damage is reported. Changes in temperature-induced strand separation in both calf thymus DNA and plasmid DNA (puc 19 plasmid from Escherichia coli) were measured after exposure to low doses of radiation. Exposures of between 0.004 and 1 Gy were measured with doses as low as 0.008 Gy yielding significant responses. The double-strand, sensitive dye PicoGreen was used as an indicator of DNA denaturation. Calibration plots indicate that fluorescence changes corresponding to amounts as low as 1 ng of double stranded DNA (10(6) copies for plasmid puc 19) are detected by this method.     

Electron photodetachment dissociation of DNA polyanions in a quadrupole ion trap mass spectrometer

We hereby explore the effects of irradiating DNA polyanions stored in a quadrupole ion trap mass spectrometer with an optical parametric oscillator laser between 250 and 285 nm. We studied DNA 6-20-mer single strands and 12-base pair double strands. In all cases, laser irradiation causes electron detachment from the multiply charged DNA anions. Electron photodetachment efficiency directly depends on the number of guanines in the strand, and maximum efficiency is observed between 260 and 275 nm. Subsequent collision-induced dissociation (CID) of the radical anions produced by electron photodetachment results in extensive fragmentation. In addition to neutral losses, a large number of fragments from the w, d, a*, and z* ion series are obtained, contrasting with the w and (a-base) ion series observed in regular CID. The major advantage of this technique, coined electron photodetachment dissociation (EPD) is the absence of internal fragments, combined with good sequence coverage. EPD is therefore a highly promising approach for de novo sequencing of oligonucleotides. EPD of nucleic acids is also expected to give specific radical-induced strand cleavages, with conservation of other fragile bonds, including noncovalent bonds. In effect, preliminary results on a DNA hairpin and on double strands suggest that EPD could also be used to probe intra- and intermolecular interactions in nucleic acids.     

Use of a dual-labelled oligonucleotide as a DNA dosemeter for radiological exposure detection

A reporter molecule consisting of a synthetic oligonucleotide is being characterised for a novel damage detection scenario for its potential use as a field-deployable, personal deoxyribonucleic acid (DNA) dosemeter for radiation detection. This dosemeter is devoid of any biological properties other than being naked DNA and therefore has no DNA repair capabilities. It supports biodosimetry techniques, which require lengthy analysis of cells from irradiated individuals, and improves upon inorganic dosimetry, thereby providing for a more relevant means of measuring the accumulated dose from a potentially mixed-radiation field. Radiation-induced single strand breaks (SSBs) within the DNA result in a quantifiable fluorescent signal. Proof of concept has been achieved over 250 mGy-10 Gy dose range in radiation fields from ??Co, with similar results seen using a linear accelerator X-ray source. Further refinements to both the molecule and the exposure/detection platform are expected to lead to enhanced levels of detection for mixed-field radiological events.     

Viscoelastic modeling of template-directed DNA synthesis

In the present study, we have used the QCM-D technology to study the replication of surface attached oligonucleotide template strands using Escherichia coli DNA polymerase I (Klenow fragment, KF). Changes in resonance frequency (F) and energy dissipation (D) for DNA hybridization and polymerization were recorded at multiple harmonics. Formation of the polymerase/DNA complex led to a significant decrease in energy dissipation, which is consistent with a conformational change induced upon enzyme binding. This interpretation was further strengthened by a data analysis using a Voigt-based viscoelastic model. The analysis revealed a significant increase in shear viscosity and shear modulus during KF binding, whereas the viscoelastic properties of single- and double-stranded templates were almost identical. During the actual DNA synthesis, an initial increase in rigidity (shear viscosity) was followed by a gradual decrease that has two components corresponding to the release of enzyme and to the presence of the catalytically active enzyme/substrate complex. The corresponding decrease in surface concentration was found to underestimate the rate of enzyme release due to viscously coupled water that compensates for the loss in enzyme mass. Furthermore, the modeling elucidates that significant changes in both F and D originate from variations in the viscoelastic properties, which means that changes in F alone should be used with care for estimations of coupled mass and kinetics. Therefore, the modeled temporal variation in effective thickness, being proportional to coupled mass and, thus, independent of structural changes, was used to estimate the catalytic constants of the polymerization reaction. The reported work is the first example providing this type of structural information for the catalytic action of an enzyme, thereby demonstrating the potential of the technique for advanced analysis of complex biological reactions, including proper analysis of enzyme kinetics.     

The production of single strand and double strand breaks in DNA in aqueous solution by vacuum UV photons below 10 eV

The mechanisms of break formation in fully hydrated DNA have been investigated using monochromatic photons below 10 eV. This has been achieved by developing a novel 'wet cell' for irradiating DNA in aqueous solution. Our preliminary data show that 7-10 eV photons readily induce strand breaks even though almost all of the energy is absorbed in the water. Therefore, the mechanism for the induction of single and double strand breaks (SSBs and DSBs) most likely involves indirect damage by OH radicals and is substantiated by data from studies in the presence of the OH radical scavenger Tris, which showed a substantial protective effect. The dose-effect curve for DSB induction is seen to be linear, or near-linear, indicating the involvement of 1-hit mediated induction of DSBs. These data point to single-event induction of DSBs being a significant pathway with all radiation types.     

Fluorescence energy transfer between fluorescein label and DNA intercalators to detect nucleic acids hybridization in homogeneous media

A general approach to detecting nucleic acid sequences in homogeneous media by means of steady-state fluorescence measurements is proposed. The methodology combines the use of a fluorescence-labeled single-strand DNA model probe, the complementary single-strand DNA target, and a DNA intercalator. The probe was fluorescein labeled to a spacer arm at the N4 position of the cytosine amino groups in polyribocytidylic acid (5'), poly(C), which acts as a model DNA probe. The complementary strand was polyriboinosinic acid (5'), poly(I), as a model of the target, and the energy transfer acceptor was an intercalator, either ethidium bromide or ethidium homodimer. In previous papers we have shown that the fluorescence intensity of the fluorescein label decreases when labeled poly(C) hybridizes with poly(I), and this fluorescence quenching can be used to detect DNA hybridization or renaturation in homogeneous media. In this paper we demonstrate that fluorescence resonance energy transfer (FRET) between fluorescein labeled to poly(C) and an intercalator agent takes place when single-stranded poly(C) hybridizes with poly(I), and we show how the fluorescence energy transfer further decreases the steady-state fluorescence intensity of the label, thus increasing the detection limit of the method. The main aim of this work was to develop a truly homogeneous detection system for specific nucleic acid hybridization in solution using steady-state fluorescence and FRET, but with the advantage of only having to label the probe with the energy donor since the energy acceptor is intercalated spontaneously. Moreover, the site label is not critical and can be labeled randomly in the DNA strand. Thus, the method is simpler than those published previously based on FRET. The experiments were carried out in both direct and competitive formats.     

On the biophysical interpretation of lethal DNA lesions induced by ionising radiation

Although DNA damage is widely viewed as a critical target for the induction of cell killing by ionising radiation, the exact nature of DNA damage responsible for these effects is unknown. To address this issue, the probability of forming lethal damage by single proton tracks, derived from published survival data for Chinese hamster V79 cells irradiated by protons with energies from 0.57 to 5.01 MeV, has been compared to estimated yields of clustered DNA lesions and repair outcomes calculated with Monte Carlo models. The reported studies provide new information about the potential relationship between the induction and repair of clustered DNA damage and trends in the expected number of lethal events for protons with increasing linear energy transfer (LET). A good correlation was found between the number of lethal events in V79 cells and the induction of double-strand breaks (DSBs) consisting of three or more elementary DNA lesions. For the yields of other types of DNA damage, as well as point mutations formed through the misrepair of base damage and single-strand breaks, observed trends with increasing LET are not consistent with trends in the yields of lethal events. This observation suggests that the relative biological effectiveness (RBE) of protons of varying quality may be more closely related to the induction of complex DSBs rather than other forms of damage.