A novel technique using DNA denaturation to detect multiply induced single-strand breaks in a hydrated plasmid DNA molecule by X-ray and 4He2+ ion irradiation

To detect multiple single-strand breaks (SSBs) produced in plasmid DNA molecules by direct energy deposition from radiation tracks, we have developed a novel technique using DNA denaturation by which irradiated DNA is analysed as single-strand DNA (SS-DNA). The multiple SSBs that arise in both strands of DNA, but do not induce a double-strand break, are quantified as loss of SS-DNA using agarose gel electrophoresis. We have applied this method to X-ray and (4)He(2+) ion-irradiated samples of fully hydrated pUC18 plasmid DNA. The fractions of both SS-DNA and closed circular DNA (CC-DNA) exponentially decrease with the increasing dose of X rays and (4)He(2+) ions. The efficiency of the loss of SS-DNA was half that of CC-DNA for both types of irradiation, indicating that one of two strands in DNA is not broken when one SSB is produced in CC-DNA by irradiation. Contrary to our initial expectation, these results indicate that SSBs are not multiply induced even by high linear energy transfer radiation distributed in both strands.     

Multisphere neutron spectrometric system with thermoluminescence dosemeters: sensitive improvement

When a charged-particle track intercepts the chromatin fibre in DNA of mammalian cells, clustered damage is induced depending on the DNA conformation, local environment and track structure. Intra-track correlated DNA damage may have a higher probability of being mis-repaired or left un-repaired. Fragment size-distributions of DNA double strand breaks (DSBs) induced in primary human fibroblasts by 240 kVp X rays and 238Pu alpha particles (110 keV.micron-1) were resolved using pulsed-field gel electrophoresis (PFGE). By monitoring DSB rejoining kinetics and changes in the fragment size distribution with repair time, the relevance of spatial association of DSBs in determining rejoining kinetics was investigated. Rejoining kinetics appeared bi-phasic and independent of the size of the DNA fragments for both radiation qualities, with high LET radiation-induced DSBs repairing more slowly. Results suggest that local complexity of individual DSBs, rather than spatial association with other breaks is more significant in the determination of rejoining kinetics.     

Induction of DNA double strand breaks in scid cells by carbon ions

The DNA double strand breaks (DSBs) induced by X ray and carbon ion beam irradiation in scid cells were analysed using pulsed-field gel electrophoresis. Scid cells and hybrid cells were ideal to study the DNA DSB repair mechanisms, because their genetic backgrounds were identical except DNA-PK activity. Induction of DNA DSBs was determined after exposure to X rays and carbon beams. DNA DSB repair was by biphasic kinetics with a fast and a slow component. For scid cells only a slow component was observed, whereas the kinetics of DSBs repair was biphasic with a fast and a slow component. It was concluded from the experimental data that the induced DSB rejoining in scid cells was due to the lack of DNA-PK activity.     

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.     

A model for analysis of the yield and the level of clustering of radiation-induced DNA-strand breaks in hydrated plasmids

Although it is widely accepted that the spatial distribution of strand breaks is highly relevant to the biological consequences of radiation, the extent to which strand breaks are clustered is not usually demonstrated directly from the experimental data. To evaluate the spatial distribution of radiation-induced strand breaks, the authors have developed a model for the generation of strand breaks after irradiation. The model assumes that (1) a radiation track has a certain probability of 'hitting' a plasmid, (2) the radiation generates strand break(s) by chance within a relatively small region that could produce double-strand breaks and (3) the number of strand breaks generated within the region follows a Poisson distribution. To find out whether the model is valid, the authors compared the calculated values with the experimental data obtained by a plasmid DNA assay. Taking into account the inherent bias of the plasmid assay, the model described well the experimental results of hydrated plasmids exposed to radiation.     

Radiation damage to DNA-protein specific complexes: estrogen response element-estrogen receptor complex

The exposure of a DNA-protein regulatory complex to ionising radiation induces damage to both partner biomolecules and thus can affect its functioning. Our study focuses on a complex formed by the estrogen response element (ERE) DNA and the recombinant human estrogen receptor alpha (ER), which mediates the signalling of female sex hormones, estrogens. The method of native polyacrylamide retardation gel electrophoresis is used to study the stability of the complex under irradiation by low LET radiation ((60)Co gamma rays) and the ability of the separately irradiated partners to form complexes. The relative probabilities of ERE DNA strand breakage and base damages as well as the probabilities of damages to the ER binding domain are calculated using the Monte Carlo method-based model RADACK.     

Feasibility of a neutron detector-dosemeter based on single-event upsets in dynamic random-access memories

In an attempt to investigate the effect of radiation quality, dose and specific repair pathways on correct and erroneous rejoining of DNA double strand breaks (DSBs), an assay was applied that allows the identification and quantification of incorrectly rejoined DSB ends produced by ionising radiation. While substantial misrejoining occurs in mammalian cells after high acute irradiation doses, decreasing misrejoining frequencies were observed in dose fractionation experiments with X rays. In line with this finding, continuous irradiation with gamma rays at low dose rate leads to no detectable misrejoining. This indicates that the probability for a DSB to be misrejoined decreases drastically when DSBs are separated in time and space. The same dose fractionation approach was applied to determine DSB misrejoining after alpha particle exposure. In contrast to the results with X rays, there was no significant decrease in DSB misrejoining with increasing fractionation. This suggests that DSB misrejoining after alpha irradiation is not significantly affected by a separation of particle tracks. To identify the enzymatic pathways that are involved in DSB misrejoining, cell lines deficient in non-homologous end-joining (NHEJ) were examined. After high X ray doses, DSB misrejoining is considerably reduced in NHEJ mutants. Low dose rate experiments show elevated DSB misrejoining in NHEJ mutants compared with wild-type cells. The authors propose that NHEJ serves as an efficient pathway for rejoining correct break ends in situations of separated breaks but generates genomic rearrangements if DSBs are close in time and space.     

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.     

Chromatin damage induced by fast neutrons or UV laser radiation

Chromatin samples from livers of Wistar rats were subjected to fast neutron irradiation in doses of 10-100 Gy or to a 248 nm excimer laser radiation, in doses of 0.5-3 MJ.m-2. The action of the radiation on chromatin was monitored by chromatin intrinsic fluorescence and fluorescence lifetimes (of bound ethidium bromide to chromatin) and by analysing fluorescence resonance energy transfer between dansyl chloride and acridine orange coupled to chromatin. For the mentioned doses of UV excimer laser radiation, the action on chromatin was more intense than in the case of fast neutrons. The same types of damage are produced by the two radiations: acidic and basic destruction of chromatin protein structure, DNA strand breaking and the increase of the distance between DNA and proteins in chromatin.     

Radiation quality dependence of DNA damage induction

Analysis of DNA fragmentation and repair in relation to radiation quality may give important information about the role of break complexity and correlated double strand breaks (DSBs). DNA fragment analysis was performed by pulsed-field gel electrophoresis after exposure to different radiation qualities. Normal human fibroblasts were irradiated with boron ions (40, 80 and 160 keV.micron-1), nitrogen ions (80, 125, 175 and 225 keV.micron-1) and neon ions (225 and 300 keV.micron-1). The amount of DNA less than 1.1 Mbp decreased with increasing linear energy transfer (LET) for all three ions. When theoretical random distributions were subtracted from the experimental data for 225 keV.micron-1 nitrogen ions in all size intervals (5-5700 kbp), there was a significant non-random distribution of DSBs for sizes up to 1-3 Mbp. This non-random distribution of breaks, probably produced by intra-track correlated DSBs, may constitute a substantial portion of the high-LET induced DSBs.     

Simulation of 125I induced DNA strand breaks in a CAP-DNA complex

The E. coli catabolite gene activator protein (CAP)-DNA complex with 125I located at the position of the H5 atom of the cytosine near the centre was incorporated into the PARTRAC track structure code. DNA strand breaks due to irradiation were calculated by track structure and radical attack simulations; strand breaks due to neutralisation of the highly charged 125Te ion were derived from a semi-empirical distribution. According to the calculations, the neutralisation effect dominates the strand breakage frequency at 2 bases away from the 125I decay site on both strands. The first breakage distribution counted from a 32P labelled end on the strand with 125I agreed well with experimental data, but on the opposite strand, the calculated distribution is more concentrated around the decay site and its yield is about 20% larger than the measured data.     

PX DNA triangle oligomerized using a novel three-domain motif

Structural DNA nanotechnology is directed at building objects, lattices, and arrays from cohesive interactions between DNA molecules. The predominant means of doing this takes advantage of the information inherent in Watson-Crick base pairing in duplex formation and in sticky-ended cohesion. Nevertheless, other forms of nucleic acid cohesion are also known, particularly paranemic edge-sharing interactions (PX). Here we report the formation of a triangular species that has four strands per edge, held together by PX interactions. We demonstrate by nondenaturing gel electrophoresis and by atomic force microscopy (AFM) that we can combine a partial triangle with other strands to form a four-stranded molecule that is robust. By combining them with a new mixed-fusion type of three-domain molecule, we demonstrate by AFM that these triangles can be self-assembled into a linear array.     

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.     

Biological effects induced by K photo-ionisation in and near constituent atoms of DNA

In order to assess the lethal efficiency and other biological effects of inner shell ionisations of constituent atoms of DNA ('K' events), experiments were developed at the LURE synchrotron facility using ultrasoft X rays as a probe of K events. The lethal efficiency of ultrasoft X rays above the carbon K threshold was especially investigated using V79 cells and compared with their efficiency to induce double strand breaks in dry plasmid-DNA. A correlation between the K event efficiencies for these processes is shown. Beams at 340 eV were found to be twice as efficient at killing cells than were beams at 250 eV. In addition, a rough two-fold increase of the relative biological effectiveness for dicentric + ring induction has also been observed between 250 and 340 eV radiations.     

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.     

Biophysical analysis of radiation induced initial DNA fragmentation

Production of double strand breaks (DSBs) and double-stranded DNA fragments after irradiation with high energy nitrogen ions was simulated with stochastic track generation and a high-order chromatin structure (HOCS) model representing a segment of interphase chromosome. The non-random distribution of DNA fragments is predicted for irradiation of HOCS. Agreement with the experimental data is obtained. The influence of HOCS and the role of the background component in DNA fragment distributions are discussed.     

The Influence of Ionizing Radiations Upon Different Forms of Collagen

Collagens received increasing doses of radiations β or γ The structural state of each of them is controlled by differential scanning calorimetry and by electrophoresis on polyacrylamide gel. Each of this method allows the study of only a part of the structure of collagen. The hemostatic activity is analysed by agregation test. The changes in the collagens in relation to irradiation are more important when studied by electrophoresis than by differential scanning calorimetry. Ionizing radiations change hemostatic activity of collagens.     

Induction of DNA DSB and its rejoining in clamped and non-clamped tumours after exposure to carbon ion beams in comparison to X rays

We studied double-strand breaks (DSB) induction and rejoining in clamped and non-clamped transplanted tumours in mice leg after exposure to 80 keV μm(-1) carbon ions and X rays. The yields of DSB in the tumours were analysed by a static-field gel electrophoresis. The OER of DSB after X rays was 1.68±0.31, and this value was not changed after 1 h rejoining time (1.40±0.26). These damages in oxygenated conditions were rejoined 60-70% within 1 h in situ. No difference was found between the exposure to X rays and carbon ions for the induction and rejoining of DSB. Thus, the values of OER and rejoined fraction after exposure to carbon ions were similar to those after X rays, and the calculated relative biological effectivenesses of carbon ion were around 1 under both oxygen conditions. The yields of DSB in vivo depend on exposure doses, oxygen conditions and rejoining time, but not on the types of radiation quality.     

Radiobiological effects of high-let particles: DNA strand breaks

Fundamental studies in radiation biology with high-LET charged particles involve a systematic study of the physical, chemical, molecular and cellular processes. Water molecules and DNA present inside a cell constitute the important targets for energy deposition which eventually lead to either cell death or mutation or transformation. High-LET charged particles are very efficient in causing these types of damages. One of the primary lesions for causing injury to a cell is the production of DNA strand breaks. A good understanding of these breaks is essential before ultimate biological effects of heavy particles can be predicted. Based on known molecular mechanisms of the formation of strand breaks, a theoretical model is presented along with a comparison between the predictions of the model and experimental data. The studies have shown that LET is not a convenient physical parameter to relate the extent of strand breaks and one needs to know the microscopic distribution of energy (track structure). A discussion has also been presented to provide a background on various radiobiological characteristics of high-LET charged particles from the point of view of their uniqueness in damaging cancer cells.     

Point-and-shoot Strategy based on Enzyme-assisted DNA “Paper-Cutting” to Construct Arbitrary Planar DNA Nanostructures

DNA self-assembly provides a “bottom-up” route to fabricating complex shapes on the nanometer scale. However, each structure needs to be designed separately and carried out by professionally trained technicians, which seriously restricts its development and application. Herein, a point-and-shoot strategy based on enzyme-assisted DNA “paper-cutting” to construct planar DNA nanostructures using the same DNA origami as the template is reported. Precisely modeling the shapes with high precision in the strategy based on each staple strand of the desired shape structure hybridizes with its nearest neighbor fragments from the long scaffold strand. As a result, some planar DNA nanostructures by one-pot annealing the long scaffold strand and selected staple strands is constructed. The point-and-shoot strategy of avoiding DNA origami staple strands’ re-designing based on different shapes breaks through the shape complexity limitation of the planar DNA nanostructures and enhances the simplicity of design and operation. Overall, the strategy's simple operability and great generality enable it to act as a candidate tool for manufacturing DNA nanostructures.