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Strand breaks and conformational changes of DNA have consequences for the physiological role of DNA. The natural protecting molecule ectoine is beneficial to entire bacterial cells and biomolecules such as proteins by mitigating detrimental effects of environmental stresses. It was postulated that ectoine-like molecules bind to negatively charged spheres that mimic DNA surfaces. We investigated the effect of ectoine on DNA and whether ectoine is able to protect DNA from damages caused by ultraviolet radiation (UV-A). In order to determine different isoforms of DNA, agarose gel electrophoresis and atomic force microscopy experiments were carried out with plasmid pUC19 DNA. Our quantitative results revealed that a prolonged incubation of DNA with ectoine leads to an increase in transitions from supercoiled (undamaged) to open circular (single-strand break) conformation at pH 6.6. The effect is pH dependent and no significant changes were observed at physiological pH of 7.5. After UV-A irradiation in ectoine solution, changes in DNA conformation were even more pronounced and this effect was pH dependent. We hypothesize that ectoine is attracted to the negatively charge surface of DNA at lower pH and therefore fails to act as a stabilizing agent for DNA in our in vitro experiments.
Ectoine, a compatible solute and osmolyte, is known to be an effective protectant of biomolecules and whole cells against heating, freezing and extreme salinity. Protection of cells (human keratinocytes) by ectoine against ultraviolet radiation has also been reported by various authors, although the underlying mechanism is not yet understood. We present the first electron irradiation of DNA in a fully aqueous environment in the presence of ectoine and at high salt concentrations. The results demonstrate effective protection of DNA by ectoine against the induction of single-strand breaks by ionizing radiation. The effect is explained by an increase in low-energy electron scattering at the enhanced free-vibrational density of states of water due to ectoine, as well as the use of ectoine as an ˙OH-radical scavenger. This was demonstrated by Raman spectroscopy and electron paramagnetic resonance (EPR).
Structural changes in plasmid DNA caused by radiation and its protection by Ectoine: an AFM analysis
(2017)
Most ionizing radiation in water ends in an avalanche of low energy electrons which play a dominant role together with OH-radicals in damaging DNA. In the present study we irradiated plasmid DNA with electrons (primary energy 30keV) under physiological conditions, performed with as well as without Ectoine. Ectoine is a compatible solute, synthesized and accumulated in molar concentration within bacteria to withstand osmotic stress or different other stressors.
Plasmid DNA (pUC19, 2686 bp) was studied due to its supercoiled isoform which is highly sensitive to radiation damage. In biochemistry gel electrophoresis is applied for structural analysis of DNA. Although it is a standard technique, a reliable discrimination of short fragments caused by radiation is often difficult. AFM is also commonly used for imaging susceptible biomolecules and, since it is based on a single molecule observation, for analysis of contour lengths of linear DNA as well. Therefore, in our study the structural changes in plasmid DNA after irradiation with different doses were quantitatively analyzed by means of intermittent contact AFM. The figure shows representative AFM images of electron irradiated pUC19 DNA (bar=200nm). For AFM imaging the DNA was chemically fixed on ultra-smooth mica. As can be clearly seen, with increasing radiation dose the number of undamaged DNA declines and fragmented DNA arises (A, B). In aqueous Ectoine solution (1M) the effect of radiation on DNA is dramatically depressed. Ectoine apparently confers protection even against high radiation: the plasmids remain predominantly in the supercoiled isoform (D). Therefore, we strongly believe that Ectoine is a potent protective substance of DNA against ionizing radiation.