Significance of 8-oxoG in the spectrum of DNA damages caused by ionising radiation of different quality

The yields and composition of DNA damages caused by ionising radiation depends on radiation quality. With increasing light energy transfer (LET), the proportion of isolated DNA damages with respect to cluster damaged sites decreases. Non-double strand break complex damages are induced by gamma radiation in mammalian cells at least four times more frequently that prompt DSB. The most important product of oxidative damage to DNA bases is 8-oxo-7,8-dihydroguanine (8-oxoG). The modelling of DNA damage induced by ionising radiation of different qualities was performed to assess frequencies and composition of complex damages containing 8-oxoG. The occurrence of clusters containing 8-oxoG increases from 6 to 11% for LET in the range 0.4-160 keV microm(-1). Distributions of single strand break (SSB) on opposite DNA strand around induced 8-oxoG have similar shape for different ionising radiations, but differ in their occurrence in the whole spectrum of DNA damages. The most probable configuration is a strand break localised at position +/-3 bases from 8-oxoG.     

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.     

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.     

Modification of DNA radiolysis by DNA-binding proteins: structural aspects

Formation of specific complexes between proteins and their cognate DNA modulates the yields and the location of radiation damage on both partners of the complex. The radiolysis of DNA-protein complexes is studied for: (1) the Escherichia coli lactose operator-repressor complex, (2) the complex between DNA bearing an analogue of an abasic site and the repair protein Fpg of Lactococcus lactis. Experimental patterns of DNA damages are presented and compared to predicted damage distribution obtained using an improved version of the stochastic model RADACK. The same method is used for predicting the location of damages on the proteins. At doses lower than a threshold that depends on the system, proteins protect their specific binding site on DNA while at high doses, the studied complexes are disrupted mainly through protein damage. The loss of binding ability is the functional consequence of the amino-acids modification by OH* radicals. Many of the most probably damaged amino acids are essential for the DNA-protein interaction and within a complex are protected by DNA.     

Electronic signature of DNA nucleotides via transverse transport

We report theoretical studies of charge transport in single-stranded DNA in the direction perpendicular to the backbone axis. We find that, if the electrodes which sandwich the DNA have the appropriate spatial width, each nucleotide carries a unique signature due to the different electronic and chemical structure of the four bases. This signature is independent of the nearest-neighbor nucleotides. Furthermore, except for the nucleotides with guanine and cytosine bases, we find that the difference in conductance of the nucleotides is large for most orientations of the bases with respect to the electrodes. By exploiting these differences it may be possible to sequence single-stranded DNA by scanning its length with conducting probes.     

Identifying single bases in a DNA oligomer with electron tunnelling

It has been proposed that single molecules of DNA could be sequenced by measuring the physical properties of the bases as they pass through a nanopore. Theoretical calculations suggest that electron tunnelling can identify bases in single-stranded DNA without enzymatic processing, and it was recently experimentally shown that tunnelling can sense individual nucleotides and nucleosides. Here, we report that tunnelling electrodes functionalized with recognition reagents can identify a single base flanked by other bases in short DNA oligomers. The residence time of a single base in a recognition junction is on the order of a second, but pulling the DNA through the junction with a force of tens of piconewtons would yield reading speeds of tens of bases per second.     

Clustered DNA damages as dosemeters for ionising radiation exposure and biological responses.

Clustered DNA damages--two or more lesions (oxidised bases. abasic sites, or strand breaks) within a few DNA helical turns on opposing strands--are induced in DNA in solution and in vivo in human cells by ionising radiation. They have been postulated to be difficult to repair, and thus of potentially high biological significance. Since the total of clustered damages produced by ionising radiation is at about 3 to 4 times higher levels than double-strand breaks and are apparently absent in unirradiated cells, levels of clustered damages present immediately alter radiation exposure could serve as sensitive dosemeters of radiation exposure. Since some clusters may not be repairable and may accumulate in cells, they might also be useful as integrating dosemeters of biological effects of radiation damage.     

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.     

Nonlinear breathing modes due to a defect in a DNA chain

Recent molecular-dynamics simulations of a DNA duplex containing the 'rogue' base difluorotoluene (F) in place of a thymine (T) base show that breathing events can occur on the nanosecond time-scale, whereas breathing events in a normal DNA duplex take place on the microsecond time-scale. The main aim of this paper is to analyse a nonlinear Klein-Gordon lattice model of the DNA duplex, including both nonlinear interactions between opposing bases and a defect in the interaction at one lattice site, each of which can cause localization of energy. Solutions for a breather mode either side of the defect are derived using multiple-scales asymptotics and are pieced together across the defect to form a solution which includes the effects of the nonlinearity and the defect. We consider defects in the inter-chain interactions and in the along-chain interactions. In most cases we find in-phase breather modes and/or out-of-phase breather modes, with one case displaying a shifted mode.     

Atomistic view of base flipping in DNA

Base flipping is essential for the enzyme-catalysed methylation of DNA. In our previous studies, the flipping of bases out of duplex DNA in DNA alone and when bound to the (cytosine-C5)-methyltransferase from HhaI (M.HhaI) were investigated via potential of mean force calculations. Insights into various experimental observations were obtained. In the present paper we present an overview of previous computational studies of base flipping along with new detailed structural and energetic analysis on atomic events that contribute to the free energy surfaces. The contributions from different intrinsic and environmental effects to the base-flipping process are explored, and experimental data derived from a variety of methods are reconciled. A detailed protein-facilitated base-flipping mechanism is proposed. Ground-state destabilization is achieved via disruption of the target base Watson-Crick interactions by substitution with favourable DNA-protein interactions. In addition, specific DNA-protein interactions and favourable solvation effects further promote target base flipping along the major groove through the protein matrix, and maximal interactions occur between the DNA and the protein upon reaching the fully flipped state. Other DNA binding proteins that involve distortion of DNA's conformation may use a similar mechanism to that by which M.HhaI facilitates base flipping.     

Effects of ionizing treatments on packaging -food simulant combinations

Research relating to ionizing treatment has been carried out on ‘food simulants polyethylene film’ combinations. Pouches have been irradiated by gamma photons or accelerated electrons at a 10kGy dose. Overall, the migration test has given some interesting results, particularly in the case of acid simulant, where a limit of validity has been found for the overall migration method. Many radiolytic products and degradation reactions have been indicated, and it has been established that the high permeability of packaging film to oxygen had influenced the radiolytic product yields. The results show some interactions between packaging film and food simulant under irradiation conditions.     

Quantification of 8-oxodGuo lesions in double-stranded DNA using a photoelectrochemical DNA sensor

Exposure of DNA to oxidative stress conditions results in the generation of 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodGuo). 8-OxodGuo is genotoxic if left unrepaired. We quantified 8-oxodGuo lesions in double-stranded DNA films by using a photoelectrochemical DNA sensor in conjunction with a specific covalent labeling method. A lesion-containing DNA film was assembled on a SnO(2) nanoparticle modified indium tin oxide electrode through layer-by-layer electrostatic adsorption. The lesions were covalently labeled with a biotin conjugated spermine derivative, and ruthenium tris(bipyridine) labeled streptavidin was introduced as the signal reporter molecule. Photocurrent increased with the number of lesions in the strand and decreased as the film was diluted with intact DNA. Quantification of 8-oxodGuo was achieved with an estimated detection limit of ～1 lesion in 650 bases or 1.6 fmol of 8-oxodGuo on the electrode. Incubation of the film with a DNA base excision repair enzyme, E. coli formamidopyrimidine-DNA glycosylase (Fpg), resulted in complete loss of the signal, indicating efficient excision of the isolated lesions in the nucleotide. Oxidatively generated DNA damage to a double-stranded calf thymus DNA film by the Fenton reaction was then assessed. One 8-oxodGuo lesion in 520 bases was detected in DNA exposed to 50 μM Fe(2+)/200 μM H(2)O(2). Treatment with Fpg reduced the photocurrent by 50%, indicating only partial excision of 8-oxodGuo. This suggests that tandem lesions, which are resistant to Fpg excision, are generated by the Fenton reaction. Unlike repair enzyme dependent methods, the sensor recognizes 8-oxodGuo in tandem lesions and can avoid underestimating DNA damage.     

Differential effect of three base modifications on DNA thermostability revealed by high resolution melting

High resolution melting (HRM) can detect and quantify the presence of 5-methylcytosine (5mC) in DNA samples, but the ability of HRM to diagnose other DNA modifications remains unexplored. The DNA bases N6-methyladenine and 5-hydroxymethylcytosine occur across almost all phyla. While their function remains controversial, their presence perturbs DNA structure. Such modifications could affect gene regulation, chromatin condensation and DNA packaging. Here, we reveal that DNA containing N6-methyladenine or 5-hydroxymethylcytosine exhibits reduced thermal stability compared to cytosine-methylated DNA. These thermostability changes are sufficiently divergent to allow detection and quantification by HRM analysis. Thus, we report that HRM distinguishes between sequence-identical DNA differing only in the modification type of one base. This approach is also able to distinguish between two DNA fragments carrying both N6-methyladenine and 5-methylcytosine but differing only in the distance separating the modified bases. This finding provides scope for the development of new methods to characterize DNA chemically and to allow for low cost screening of mutant populations of genes involved in base modification. More fundamentally, contrast between the thermostabilizing effects of 5mC on dsDNA compared with the destabilizing effects of N6-methyladenine (m6A) and 5-hydroxymethylcytosine (5hmC) raises the intriguing possibility of an antagonistic relationship between modification types with functional significance.     

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.     

DNA sequencing by capillary electrophoresis with four-decay fluorescence detection

A scheme for multiplex detection of dye-labeled DNA fragments in DNA sequencing is described in which on-the-fly, frequency-domain fluorescence lifetime detection is used to discriminate among the dye-labeled fragments of the four terminal bases in a single-lane CE separation. Two four-dye systems were evaluated, one excited at 488 nm and the other, at 514 nm. The 488 nm system proved successful for four-decay detection. Base calling was achieved either directly from on-the-fly lifetimes or from lifetime-resolved electropherograms recovered for each base from the electropherogram of the mixture of sequencing reaction products. The latter method was found to be more accurate (99% for two bases and 98.5% for three bases) and could achieve longer read lengths, but it was unsuccessful for sequencing of all four bases. The first method gave a base-calling accuracy of 96% for four-base sequencing over the fragment length range of 41-220 bases.     

Covalent attachment of gold nanoparticles to DNA templates

Functionalized gold nanoparticles have been covalently bound to internal, modified sites on double-stranded DNA. Gold nanoparticles coated with mercaptosuccinic acid or thioctic acid were bound to amino-modified thymine bases on double-stranded DNA. Visible absorption spectra, gel electrophoresis, and atomic force microscopy were used to analyze the products. Thiol groups were added to one end of the gold/nanoparticle product, which was then attached to a gold surface. This method has the potential to allow controlled placement of particles with subnanometer precision and to allow attachment of the product to fixed contacts for nanodevice fabrication.     

Multiscale model of electronic behavior and localization in stretched dry DNA

When the DNA double helix is subjected to external forces it can stretch elastically to elongations reaching 100% of its natural length. These distortions, imposed at the mesoscopic or macroscopic scales, have a dramatic effect on electronic properties at the atomic scale and on electrical transport along DNA. Accordingly, a multiscale approach is necessary to capture the electronic behavior of the stretched DNA helix. To construct such a model, we begin with accurate density-functional-theory calculations for electronic states in DNA bases and base pairs in various relative configurations encountered in the equilibrium and stretched forms. These results are complemented by semi-empirical quantum mechanical calculations for the states of a small size [18 base pair poly(CG)–poly(CG)] dry, neutral DNA sequence, using previously published models for stretched DNA. The calculated electronic states are then used to parametrize an effective tight-binding model that can describe electron hopping in the presence of environmental effects, such as the presence of stray water molecules on the backbone or structural features of the substrate. These effects introduce disorder in the model hamiltonian which leads to electron localization. The localization length is smaller by several orders of magnitude in stretched DNA relative to that in the unstretched structure.     

Microfabricated electrochemical sensor for the detection of radiation-induced DNA damage

An electrochemical biosensor protocol for the detection of radiation-induced DNA damage is described. The procedure employs a dsDNA-coated screen-printed electrode and relies on changes in the guanine-DNA oxidation signal upon exposure to ultraviolet radiation. The decreased signal is ascribed primarily to conformational changes in the DNA and to the photoconversion of the guanine-DNA moiety to a nonelectroactive monomeric base product. Factors influencing the response of these microfabricated DNA sensors, such as irradiation time, wavelength, and distance, are explored, and future prospects are discussed. Similar results are given for the use of bare strip electrodes in connection with irradiated DNA solutions.     

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.     

Single cell DNA damage/repair assay using HaloChip

The molecular level damage to DNA is important due to DNA's susceptibility to free radical attacks and crucial roles in maintaining cell functions. Although a panel of techniques can be used to detect DNA damages, most of them are limited due to low sensitivity, low throughput, incompatibility for automated data analysis, and labor-intensive operations. We have developed a cell array based DNA damage assay in which mammalian cells are attached on an array of microfabricated patterns through electrostatic interactions. After trapping patterned cells inside gels, damaged DNA fragment can diffuse out of the nucleus and form a halo around each cell inside gels. The halo array can be observed fluorescently after labeling DNA with ethidium bromide. DNA damages can be determined sensitively at the single cell level, accurately due to the symmetric shape of the halo, and automatically due to the spatial registry of each cell and the nonoverlapping halos surrounding cells. The HaloChip can be used to detect DNA damages caused by chemicals and ultraviolet and X-ray irradiations with high efficiency. A major advantage of HaloChip is the ability to increase throughout by spatially encoding multiple dosing conditions on the same chip. Most importantly, the method can be used to measure variations in response to DNA damaging agents within the same cell population. Compared with halo assay or comet assay alone, this method allows automated analysis of a million cells without an overlapping issue. Compared with the microwell array based comet assay, this method can selectively capture and analyze cells, and the results can be easily analyzed to provide precise information on DNA damage. This method can be used in a broad range of clinical, epidemiological, and experimental settings.