Changes in properties of DNA caused by gamma and ultraviolet radiation. Dependence of conformational changes on the chemical nature of the damage

Changes in the pulse-polarographic behaviour and circular dichroism spectra of DNA were investigated after gamma and ultraviolet irradiations and after degradation by DNAase I. It was found that moderate doses of radiation cause local conformational changes in the double helix, which are dependent on the chemical nature of the damage. Only the accumulation of structural changes after high doses of the radiations or after extensive enzymic treatment may cause formation of single-stranded regions in DNA.     

Investigation of conformational changes of DNA molecule caused by gamma-irradiation in water-ethanol solutions

The intrinsic viscosity, optical anisotropy and spectral properties of DNA molecule gamma-irradiated with the doses of 10, 20 and 30 Gy in water-ethanol solutions with ethanol concentrations 0-6 mol/l are investigated in the work. Specific volume of DNA at all doses used shows a complex non-monotone dependence on the ethanol content with a peculiarity at the alcohol concentration corresponding to the destruction of water structure in the mixed solvent (so-called, critical concentration, 3.5 mol/l). Ethanol presence at the concentrations below the critical one protects macromolecule from the radiation action. At the alcohol concentrations larger the critical an inversion of the dose dependence of the DNA specific volume is observed. At that the equilibrium rigidity and secondary structure of macromolecule do not change noticeably. The results obtained indicate a significant role of the solvent structure in radiation damage of DNA molecule.     

An electrochemical analysis of changes in properties of DNA caused by ionizing and ultraviolet radiations

Summary Electrooxidation and electroreduction of- and u.v.-irradiated DNA were studied by means of differential pulse voltammetry at the graphite electrode and differential pulse polarography at the dropping mercury electrode. Two separated voltammetric oxidation peaks G and A were used for monitoring conformational changes in guanine - cytosine (GC) and adenine - thymine (AT) pairs respectively in irradiated double-stranded (ds) DNA. Pulse-polarography reduction peak III was used for detection of denatured DNA in the irradiated samples of ds DNA. It was found that the heights of peaks G and A of ds DNA did not change with the radiation dose after relatively low doses of- and u.v.-radiations (up to ca. 40 krads and 1 × 10⁴ Jm^–2, respectively), when no single-stranded (ss) DNA was detected in the irradiated DNA samples. After higher doses of radiation the occurrence of ss DNA or ss segments in the irradiated samples of ds DNA was accompanied by an increase of peaks G and A; however, peak A grew more rapidly with the increasing dose than peak G. It was concluded that the results obtained support the assumption, according to which regions of ds DNA rich in AT pairs are more susceptible to denaturation caused by- and u.v.-radiations.This dose concerns the DNA solution at a concentration of 600 µg/ml^–1     

Comparison studies on the effects of ultraviolet irradiation on photosynthesis

1. 1. A comparison of chloroplasts from which plastoquinone had been extracted with ultraviolet irradiation supports the conclusion that plastoquinone destruction is not the major cause of ultraviolet inhibition of photosynthesis. No photodestruction of chloroplast lipids, carotenoids or soluble proteins by ultraviolet irradiation was detected.

2. 2. Phenazine methosulfate-mediated cyclic photophosphorylation and variable yield fluorescence were inhibited at the same rate as the Hill reaction. Examination of fluorescence emission spectra of chloroplasts and whole algal cells revealed decreases in both the 685-nm and long-wavelength emission peaks.

3. 3. Digestion of chloroplasts with lipase decreased fluorescence in a manner similar to ultraviolet irradiation. Hill reaction activity was also inhibited by lipase digestion.

4. 4. It is concluded that the inhibition of photosynthesis by ultraviolet irradiation is most likely due to a disruption of the structural integrity of the lamellar membranes which results in the loss of System II activity and associated reactions.

CD studies of conformational changes of DNA upon photosensitized UV-irradiation at 313 nm.

The role of thymine dimerization for previously reported U.V. induced conformational changes has been proved using acetophenone as sensitizer for the specific thymine dimerization upon irradiation at 313 nm. CD results demonstrate that formation of pyrimidine dimers cause typical conformational changes of the DNA B helix as observed on irradiation at 254 nm. Thus the primary role of adenine photoproducts may be excluded.     

Formation of a thymine photoproduct in transforming DNA by near ultraviolet irradiation.

Irradiation at 334 and 365 nm of a highly purified preparation of thymine-labeled transforming DNA from Haemophilus influenzae produced a photo product containing label from thymine but different from the cyclobutane dimer. The photoproduct is soluble in water and in ethanol and Rf values in a number of solvents are presented. The photoproduct has properties similar in a number of respects to those of the spore photoproduct, 5-thyminyl-5,6-dihydrothymine. The near ultraviolet photoproduct is more likely to affect the oxygen independent inactivation of transforming DNA rather than its mutagenesis, as judged by the quantitative relationship between amount of photboproduct and inactivation and mutagenesis.     

An infrared spectroscopy study of the molecular structure changes of DNA after electron and ultraviolet irradiation

Samples of calf thymus DNA and two different leukemic leukocyte DNA's in the solid state have been irradiated (at 300°K) with 800 keV electrons and 4.9 eV (2537Å) ultraviolet rays. The subsequent effects on the DNA's have been studied using infrared spectroscopy as the probe for radiation-produced molecular alterations. The region of the infrared spectrum studied covered the wave number range from 4000 cm⁻¹ to 300 cm⁻¹. Our results indicate that under electron and ultraviolet irradiation, the prominent infrared active absorption peaks of all three DNA's are altered. The infrared results of our ultraviolet irradiation of DNA indicate that similar molecular bonds are broken as for the case of DNA irradiated with 800 keV electrons. The results indicate that up to high doses, calf thymus DNA is more sensitive to electron irradiation than the leukemic leukocyte DNA's. The infrared active absorption peaks of the two leukemic leukocyte DNA's respond similarly to electrons. The ultraviolet results indicate some difference between calf thymus DNA and the two leukocyte DNA's in their response to 4.9 eV light.     

Folding of thermolysin fragments. Correlation between conformational stability and antigenicity of carboxyl-terminal fragments

Circular dichroism (CD) and immunochemical measurements have been used to examine conformational properties of COOH-terminal fragments 121-316, 206-316 and 225(226)-316 of thermolysin, and to compare these properties to those of native thermolysin and thermolysin S, the stable partially active two-fragment complex composed of fragments 5-224(225) and 225(226)-316. In aqueous solution at neutral pH, all the COOH-terminal fragments attain a native-like conformation, as judged both by the content of secondary structure deduced from far-ultraviolet CD spectra and by the recognition of rabbit polyclonal antibodies specific for the COOH-terminal region in native thermolysin. The three fragments showed reversible cooperative unfolding transitions mediated by both heat and guanidine hydrochloride (Gdn X HCl). The phase transition curves were analyzed for Tm (temperature of half-denaturation) and Gibbs free energies (delta GD) of unfolding from native to denatured state. The observed order of thermal stability is 225(226)-316 less than or equal to 206-316 less than 121-316 less than thermolysin S less than thermolysin. The ranking of delta GD values for the three fragments correlates with the size of each fragment. Competitive binding studies by radioimmunoassay using 14C-labeled thermolysin and affinity purified antibodies specific for native antigenic determinants in segment 206-316 of native thermolysin indicate that the COOH-terminal fragments adopt native-like conformations which are in equilibrium with non-native conformations. These equilibria are shifted towards the native state as the fragment size increases from 225(226)-316, to 206-316, to 121-316. Fragment 225(226)-316, when combined with fragment 5-224(225) in the thermolysin S complex, adopts a more stable native-like conformation and becomes much more antigenic. It has been shown that the degree of antigenicity of COOH-terminal fragments towards thermolysin antibodies correlates directly with their conformational stability. The results of this study are discussed in relation to the recently proposed correlation between antigenicity and segmental mobility of globular proteins.     

Fluorescence and conformational changes caused by proton binding to troponin C

The binding of H⁺ to troponin C induces a large conformational change and an enhancement of the tyrosyl fluorescence. Carboxyl groups with abnormal pK' values of 6.0 appear to be controlling these changes.     

Histone-induced conformational changes in DNA as probed by quasi-elastic light scattering.

Quasi-elastic light scattering as measured by intensity fluctuation (self-beat) spectroscopy in the time domain can be profitably used to follow both the translational diffusion D and the dominant internal flexing mode τ_int of DNA and its complexes with various histones in aqueous salt solutions. Without histones, DNA is found to have D = 1.6 × 10^?8 cm²/sec and τ_int ? 5 × 10^?4 sec in 0.8 M NaCl, 2 M urea at 20°C. Total histone as well as fraction F2A induce supercoiling (D = 2.6 × 10^?8 cm²/sec, τ_int ? 2.8 × 10^?4 sec) whereas fraction F1 induces uncoiling (D = 1.0 × 10^?8 cm²/sec, τ_int ? 9.4 × 10^?4 sec). Upon increasing the salt concentration to 1.5 M the DNA–histone complex dissociates (D = 1.8 × 10^?8 cm²/sec). Upon decreasing the salt concentration to far below 0.8 M, the DNA–histone complex eventually precipitates as a chromatin gel.     

Genomic integrity of T1 DNA after gamma-and ultraviolet irradiation.

T1 DNA, gamma-irradiated in the phage particle or irradiated with ultraviolet light was checked for structural integrity by kinetics of melting and reannealing. gamma-Irradiated DNA differed in all thermokinetic properties by a factor of 3-4 from DNA degraded by mechanical or enzymatical treatments. Ultraviolet irradiation caused much smaller effects than gamma-irradiation. Considering the frequency of pyrimidine dimers in relation to the gamma-ray induced lesions, strong evidence can be derived, that in addition to single base damages, local denatured regions are produced by gamma-irradiation. Such regions, formed possibly by direct absorption of radiation energy in DNA, i.e. by primary ionizations, are associated with base lesions and are passed over during reannealing.