The purpose of the present work was to investigate early changes evolved in the DNA macromolecule during its helix to coil phase transition following exposure ...
857 THERMALLY STIMULATED ELECTRIC CHANGES DURING THE HELIX TO COIL TRANSITION OF IRRADIATED DNA Eleftherios G. SIDERIS1, Alexandros G. GEORGAKILAS 1, Costas A. KALFASl and Amalia ANAGNOSTOPOULOU-KONSTA~ 'Institutes of Biology and Nuclear Physics National Centre for Scientific Research "DEMOKRITOS", Agia Paraskevi 153 10 Athens, Greece and 2Department of Physics, National Technical University of Athens, Zografou, 157 73 Athens, Greece. Abstract The purpose of the present work was to investigate early changes evolved in the DNA macromolecule during its helix to coil phase transition following exposure to ionizing radiation. This phase transition measures the thermostability of this macromolecule and it is greatly affected by exposure to ionizing radiations apparently due to induced single strand and double strand breaks on either or the two coils of the double DNA helix. Our results indicate that conductivity changes definitely precede the helix to coil transition. This observation is on line with the suggested precedence of hydrophobic bonds disruption prior to hydrogen bonds disruption which coincides with this type of phase transition that finally leads to the denaturation of the DNA. 1. Introduction
Gradual increase of the temperature of an aqueous DNA solution results onto a gradual reannealing of the DNA double helix form towards a single stranded coil form. The characteristic sigmoidal increase of UV Absorbance at the 260 nm, A260, which accompanies the disruption of the hydrophobic bonds among successive nucleotides and that of the hydrogen bonds between opposite strand nucleotides of the macromolecule. expressed as hyperchromicity, that is AA260 over A260 at arnbient temperature [ I ] , constitutes a sensitive criterion of DNA lesions induced by ionizing radiation [2]. Work from this laboratoiy [3] and other laboratories [2] indicates that exposure of aqueous DNA solutions to ionizing radiation alters not only the hyperchromicity of the DNA-water system but alters also the TM temperature, the DNA melting point temperature, which is defined as the temperature coisesponding to 50% of the maximum hyperchromicity . Changes in the conductivity during the helix to coil transition of the DNA have been reported earlier [4j and more recently have been used for the study of radiation induced breaks in single sh-anded polynucleotides and DNA molecules [mainly 5,6]. In the present work we report changes in the conductivity of aqueous DNA solutions, exposed to ionizing radiation, which precede the changes in the hyperchromicity during the helix to coil transition of this macromolecule.
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2. Experimental
The experimental work was perfoimed with the use of macromolecular native calf thymus DNA, which was repeatedly deproteinized with chlorofoim-butyl alcohol fractionation, precipitated in ethyl alcohol and air dried in acetone [7]. The dried precipitate was solved in deionized water the conductivity and the pH of which were measured to be 1.2 psiemens and 6.8 respectively, at the ambient temperature of 20 O C . Samples of 0.01% wiv aqueous DNA solutions were exposed to 10 and 70 Gy of gamma rays from a Cobalt-60 source of the Atomic Canada Ltd at the rate of 0.7 Gy per min. The same samples were used for the measurements of hyperchrornicity and those of conductivity The theimal transition spectrophotometric measurements were cai-ried out with the recommended ~ S S ~ M O or - ~0.001% wiv DNA, while the conductivity measurements were canied out with 3.5xIO-jM or 0.1% w/v DNA. The A260 changes vs temperature were recorded using a Perkin Elmer UV-VIS double beam spechophotometer with a cuvette holder vested in Peltier units coupled with a digital temperature progi-animable theiinoelechically conholled attachment [8]. Preliminaiy work with t h e n ” hansition spectrophotometiy using 3.5~10-4M or 0.01% wiv DNA solutions, the highest DNA concenhation that can be used for this method, due to instrumentation limitations, resulted in hyperchi-oinicity changes similar to those found with the 3 . 5 ~ 1 0 - 5M or 0.001% wiv DNA solutions. For the conductivity measurements two methods were used alternatively. With the first method the Conductivity of the DNA solutions were measured in glass cylindrical chamber of ,053 cm2 cross section and of 2.3 cin length between the two stainless steal electrodes which were connected to Hewlett Packard function generator and a digital multimeter. The conductivity was measured at the 200 Hz [4]. With the second method conductivity was measured with a Radiometer research conductance meter. In both cases the electrode chambers were vested in a copper mantle peimitting the gradual increase of the temperature through water circulating from a Haacke bath coupled with an exteinal temperature programmer. The actual temperature, in the first case was measured at the heating mantle while in the second case the actual temperature was measured within the cell of the conductivity electrode, during preliminaiy experimental work. In both cases a thennocouple connected to a digital indicator was used. The conductivity of the DNA aqueous solutions from 0.025% io 1% wiv, flanking the 0.1% wiv DNA solution used in our conductivity measurements,was increasing, at the ambient temperature of 20 O C , linearly with the concentration of the DNA. Non-significant differences were found between the conductivity measurements conducted with the two methods. The results given in this repoit wei-e obtained with the first method. The A260 profiles as well as the conductivity profiles were taken within a continu-
859
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The data of the theimal wansition spectrophotometry measurements and those from the conductivity measurernents for non irradiated calf thymus DNA and imdiated with 10 and 70 Gy calf thymus DNA are presented in Figure 1. In this Figure the
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t,igure I . ('ondwrivi~yand A260 changes during lhernial Iransilion of'naiive I ~ ? V ~ I I I . V DNA exposed l o ganinia ionizing radialion. From IO 1oj7 10 bollom: DNA exposed IO 0. I O and 70 Gy.
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when the temperature was gradually raised from 30 to 80 O C . Within this temperature spectrum the conductivity of the used deionized water was linearly raised from 1.2 pSiemens to 6.6 psiemens. As it can be seen in all three cases, that is the non irradiated and the iradiated with 10 and 70 Gy aqueous DNA solutions, the changes recorded from the conductivity ineasurements evolve following a sigmoidal function rathei similar to that deriving from the thei-mal transition spectrophotoinetiy. As it can be seen in Figure I .
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in all three cases, the conductivity changes precede those of hyperchromicity which follow the disruption of the hydrophobic bonds within the stsands and that of the hydrogen bonds between the opposite strands of the native DNA double stranded macromolecule that was used in this work. The first derivatives of the sigmoidal curves were used for the estimation of the mean temperatures where half of the A260 changes were observed.
86 1
that is the TM temperatures and of the mean temperatures, T,, where half of the changes in conductivity were observed. The TM temperatures for DNA samples exposed to 0, I O and 70 Gy were 68, 64 and 61 OC respectively. The Tc temperatures for DNA samples exposed to 0, 10 and 70 Gy were 48, 46 and 44 OC respectively. Thus, conductivity changes in gradually heated aqueous solutions of DNA evolve at distinctly lower temperatures than those where the A260 are observed. In Figure 2 the conductivity changes are plotted against the A260 changes. It is obvious that most of the conductivity changes are taking place prior to the appearance of any significant changes on UV absorbance parameter. This phenomenon might be attributed to the presence of two phases during the helix to coil hansition of native macromolecular DNA. As it was mentioned above, during the first phase, a gradual disruption of hydrophobic bonds between the successive nucleotides within each DNA strand of the DNA molecule is taking place. These changes are followed up by the second phase, that is the disruption of hydrogen bonds between the nucleotides at the opposite strands of the DNA double helix which results on the dramatic increase of the A260 absorbance in the UV region. It is suggested that the known temperature dependency of the mobility of ions in aqueous solutions, in that case the mobility of the Na+ bound to the phosphate moiety of the DNA, facilitates the energy dependent disruption of the hydrophobic bonds and thus, the observed increase of the conductivity, that precedes the increase of ,4260, might coincide with, and be a measure of, the disruption of the hydrophobic bonds ACKNOWLEDGEMENT This work was performed as a part of the P1i.D. thesis of one of the authors (A.G.G.) and was supported by the EEC grants B 16-0224GR and B 17-033C.
REFERENCES A.L. Lerhniger, Biochemistiy (The Molecular Basis of Cell Structure and Function), Worth Publishers Inc., New York. 1972. [2] C. Von Sonntag, The Chemical Basis of Radiation Biology, Taylor & Francis. London, 1987. [?I E.G. Sideris, G. Zan-is, A. Anagnostopoulou-Konsta, A.G. Georgakilas and C A . Kalfas, DNA-water free radicals interaction and the thermostability of the DNA molecule, S.I.F. Conference Proceedings Vol. 43 pp 239-242, 1993. [4] A. Surowiec and S. Somiso, Thennal reversibility of DNA in aqueous solution fi-om dielectric and conductometric measurements. Z. Physikal. Chem. Neue Folge Vol. I3 1 pp 17 1 - 180, 1982. [I]
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151 E. Bothe, G. Ah Qureshi and D. Schulte-Frohlinde, Rate of OH radical induced strand break formation in single stranded DNA under anoxic conditions. An investigation in aqueous soloutions using conductivity methods, 2.Naturforsch. Vol. 3 8 pp ~ 1030-1042, 1983. [ 6 ] E. Bothe and D.Schulte-Ftohlinde, Release of K’ and H+ from Poly U in aqueous solution upon gainma and electron iimdiation, Z. Naturforsch. Vol. 37c pp 1191-1204, 1982. [ 7 ] J.J. M a r ” , A procedure for the isolation of Deoxyribonucleic Acid from microorganisms, J. Mol. Biol Vol. 3 pp 208-218, 1961. [8] E.G. Sideris. C A. Kalfas and N. Katsaros, Binding of Indium, used in Perturbed y-y Conelation studies, on DNA and DNA moieties, Inorg. Chim. Acta Vol 123 pp 1-6, 1986 [ 9 ] F. James and M Roos. Minuit - A system for function minimization and analysis of paraineta m o r s and correlations. Computer Physics Communications, Vol IO, pp. 343-367. 1975.
