The mechanism by which ectoine protects cell components such as proteins and membranes is relatively well understood 5. The beneficial effect is explained by the lower affinity of ectoine, compared to water, to the surface of such biomolecules. The low affinity results in a thermodynamic force that contributes to increased stability of proteins and membranes 9.
The mechanism in protecting living cells against UV radiation, however, is far from being clear. Ectoine reduces the release of certain inflammatory factors and thus it was postulated that ectoine diminishes inflammatory processes caused by UV radiation Likewise, it is hypothesized that ectoine-induced expression of heat shock proteins is the cause for protection against UV radiation In the present study it was investigated whether ectoine is able to protect DNA from damage by ionizing electron radiation in a cell-free setting.
Applying gel electrophoresis and atomic force microscopy AFM it was found that ectoine on its own is a potent protective substance of DNA against ionizing radiation. The primary electrons were generated with the aid of an electron microscope and the experimental setup is described in detail by Hahn et al.
Plasmid DNA has been used for the experiments because its supercoiled isoform is highly sensitive to radiation damage. Assuming one nucleotide unit measures 0. Intact plasmid DNA appears as supercoiled sc structure. Single strand breaks ssb due to mechanical stress or enzymatic activity will lead to an open circular oc isoform, whereas double strand breaks dsb will result in linearized lin DNA. Further degradation of linearized DNA will result in even shorter fragments. Applying gel electrophoresis and intermittent contact AFM enabled identification and quantification of different plasmid isoforms.
After electrophoretic separation, GelRed-stained DNA isoforms were quantified from agarose gels and the amount of undamaged plasmids was plotted against the number of primary electrons and against the dose, respectively Fig.
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Furthermore, binding efficiencies of GelRed for the same amount 75 ng of sc and linear isoform DNA were determined and the correction factor was obtained as 0. The minimally weaker binding to scDNA could therefore be neglected. To estimate how much the sample treatment prior to irradiation has already changed the structure of pUC19, two different control samples were compared. The black triangle shows a control sample without any treatment.
The data denoted by black and red squares are obtained from samples that went through the entire preparation process, which includes the incorporation in the microscope chamber, but without any radiation treatment.
The fraction of undamaged plasmids after irradiation with different electron doses was quantitatively also analyzed by means of intermittent contact AFM, which has developed into a standard technique for imaging susceptible biomolecules 14 with nanometer resolution. In particular, AFM has been used for structural analysis of supercoiled, open circular and linearized plasmid DNA as well as for analysis of contour lengths of linear DNA, which was fragmented by radiation 15 , 16 , 17 , Hence, within the seventeen different irradiated samples we selected four different and characteristic representatives.
The chosen samples comprised pUC19 DNA dissolved in unbuffered water, which was irradiated with a low and a high electron dose of 0. For statistical reasons, three replicates were prepared from each of the four solutions on mica substrate. From each mica surface at least three different sites were picked and multiple images were scanned from these sites by atomic force microscopy.
These results are in good agreement with the data obtained by gel electrophoresis, which revealed the complete loss of scDNA after high-dose irradiation compare Fig. Although linear DNA migrates somewhat faster compared to the open circular isoform, a reliable discrimination of both forms by electrophoresis was often difficult.
Ectoine protects DNA from damage by ionizing radiation | Scientific Reports
Applying a radiation dose of Plasmid pUC19 in water was irradiated with a low electron dose of 0. The present study demonstrates for the first time that ectoine prevents DNA from being damaged by ionizing electron radiation. The argumentation behind the statement above is based on the following approach. Strand breaks become apparent through changes in DNA conformation, which leads to the appearance of open circular and linear DNA isoforms and the decline of supercoiled DNA.
Electrophoresis is a well-established method for structural analysis of plasmids. Atomic force microscopy allows to visualize changes in DNA conformation. The number of single- and double-strand breaks introduced into a single DNA molecule represents the level of damage. Hence, the length distribution of the fragments is of great interest for quantifying the damage caused by radiation.
Ionizing radiation is known to disintegrate DNA. The large number of short fragments is the result of densely localized ionization events in the proximity of the DNA. Hence, it is widely accepted that this is leading to destabilising the secondary structure of DNA 14 , 15 , 16 , 17 , Depending on the dose and the type of radiation, the effect of DNA lesions are ranging from cross-links, base releases and combination of single strand-breaks, double strand-breaks to clustered double-strand breaks 21 , 22 , The damaging effects on DNA dissolved in water have been found to be three magnitudes of order higher compared to radiation experiments with dry DNA 24 , 25 , The mechanisms by which the damage to DNA in water is intensified was explained by the formation of secondary particles such as OH-radicals, H-radicals, ions, prehydrated electrons and low energy electrons.
These particles are generated largely by interactions of ionizing radiation with water molecules The most abundant and damaging species are secondary electrons and radicals. However, after irradiating DNA with Comparing both detection principles it is obvious, that results from gel electrophoresis as an integrating method have their limits concerning the detection of small molecules.
On the contrary, AFM suffers from a limited scan field but benefits from high spatial resolution needed to detect highly damaged species. Such inconsistencies between AFM and electrophoresis were also found by Jiang et al. Some authors have proposed that such differences may result from the chemical fixation on mica during AFM sample preparation, which introduces further strand breaks into already damaged DNA and thereby generating short linear molecules As described by Schmatko and coworkers 30 , the charges on a bare surface without silane can cause damage to DNA and transform supercoiled pUC19 into open circular DNA.
However, such conformational changes of control samples into linearized or even fragmented DNA were not observed by AFM in our experiments. During sample preparation, each mica surface was visually assessed to ensure that the surface was completely silanized. Thus, it was made sure that the observed damage to DNA was caused by radiation and not by sample preparation or substrate adhesion.
In support of our findings, Murakami and coworkers 17 reported that after 60 Co exposure short fragments of DNA can be clearly seen by AFM, whereas those fragments escaped detection by gel electrophoresis. The experiments carried out here, clearly revealed that the compatible solute ectoine protects DNA from damage caused by electron radiation and preserves its native supercoiled conformation, even if inconsistencies between the methods are still worth to be discussed.
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Descriptions of the mechanisms by which ectoine protects DNA against ionizing electron radiation are still under discussion. Ectoine and other compatible solutes protect proteins from denaturation by stressors such as heat or dryness 5 , 9. Stabilizing the native folded state is explained by the preferential exclusion of compatible solutes from the surface of proteins and their first hydration shell. Stabilization by preferential exclusion is explained as the result of the tendency to minimize the surface area 9 , However, this model might not hold true for all molecules. Based on atomistic molecular-dynamics simulations for the compatible solute hydroxyectoine, it was shown that hydroxyectoine can be attracted to surfaces of negatively charged molecules.
Following the preferential exclusion model, the attraction of hydroxyectoine to negative surfaces was coined preferential binding Firstly, the model based on the molecular dynamics simulation confirmed the preferential exclusion of hydroxyectoine from the surface of positively charged proteins and their first hydration shell 33 , 34 , Secondly, the model revealed a preferential binding of hydroxyectoine to negatively charged spheres such as DNA within a distance of 0.
As a consequence, hydroxyectoine will partially replace the first water shell that surrounds DNA in a distance of approximately 0. Assuming that ectoine resembles hydroxyectoine in terms of its binding properties, it is suggested that ectoine as well partially replaces water from the hydration shell of DNA.
Thus, the mechanism by which ectoine stabilizes DNA against ionizing electron radiation can be described as follows: For the interaction of ionizing radiation with water we can assume that a decrease of degradative species in the vicinity of DNA takes place and thus mitigates the damage of DNA. Additional effects such as the scavenging of radicals are already under investigation. In contrast to other studies, the calculation of the radiation doses in this survey is based on a microdosimetric approach Moreover, simulations of electron scattering and of plasmid diffusion suggest that the kinetic energy spectrum of the electrons throughout the water is dominated by low energy electrons.
These data allow to estimate the kinetic energy of electrons in the vicinity of DNA. DNA double-strand breaks are the most critical radiation-induced damage, which eventually will lead to cell death.
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Most ionizing radiation in water ends in an avalanche of low energy electrons, which are together with hydroxyl radicals the key players in damaging DNA. To get closer to the physiological conditions in living cells, a special sample holder had been developed 12 that allowed electron irradiation of DNA in aqueous solution and helped to evaluate the potential of ectoine in protecting DNA.
In water without ectoine, the applied electron radiation caused multiple double-strand breaks in plasmid pUC19 as witnessed by the generation of highly fragmented DNA. Until now, the mechanism on how ectoine protects the stability of plasmid DNA is not fully understood. The replacement of the first hydration shell of DNA by ectoine could possibly lead to this effect, because less secondary electrons will be generated by interaction of ionizing radiation with water in the vicinity of the DNA.
It is, however, also possible that ectoine acts as a radioprotector in terms of its electron as well as radical scavenging properties. Therefore, further studies have to clarify the precise mechanism by which ectoine protects DNA. These studies have to include stability tests for ectoine for electron and UV 43 exposure to find out if ectoine undergoes irreversible chemical changes.
We already know from our recent work that ectoine protects DNA from radiation in a concentration-dependent manner Furthermore, ectoine must be compared in terms of radiation protection to other compatible solutes such as hydroxyectoine, trehalose and glycine betaine. Comparing the different solutes will help to determine whether the specific tetrahydropyrimidine structure of ectoine contribute in stabilizing DNA or whether more common features contribute to the protection, for instance the relatively large hydrophobic surface area of compatible solutes combined with good water solubility.
Moreover, our experiments, which we have carried out in a simple model system have to be expanded to chromosomal DNA and even whole cells. Interestingly, ectoine is not taken up by a number of eukaryotic cells skin cells but it is not clear yet whether this is true for all eukaryotic cells e.
Ectoine protects DNA from damage by ionizing radiation
In conclusion, this work provides strong evidence in support of ectoine as a potent and specific substance in protecting DNA against ionizing radiation. All these operations, however, don't furnish the intrinsic explanation of reality. There are theories, but mystery remains.
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