Dietrich Schulte-Frohlinde

Radiation-Induced Strand Breaks in DNA: Chemical and Enzymatic Analysis of End Groups and Mechanistic Aspects

Publisher Summary This chapter reviews the results of chemical and enzymatic analyses of radiation-induced strand breaks together that allows proposing a detailed mechanism of DNA strand break formation by ionizing radiation. Chemical analysis enables one to describe radiation-induced reactions, and the final structure of the sugar moiety and enzymatic analysis determines the biochemical reactivity of the end groups on the 3’ or 5’ terminal of the strand break. Enzymatic methods may be more sensitive than chemical analysis, but it has become evident that both approaches are necessary for an understanding of the mechanisms of radiation-induced strand breakage in DNA. Exposure of DNA to ionizing radiation produces an interruption of the nucleotide strand that can be considered as an important lesion responsible for the inactivation of the biological function of DNA. The chapter describes an experimental work that leads to a detailed understanding of the chemical structure of radiation-induced strand breaks and to knowledge of the mechanisms of their formation. The application of different techniques such as pulse radiolysis, ESR spectroscopy, product analysis in one laboratory, and sedimentation and enzymatic analysis in another laboratory lead to comparable conclusions. The chapter describes the frequency of strand breaks under various radiation conditions for DNA in aqueous solution, in dry DNA, and in cells. The presence of intact hydroxyl or phosphate end groups on DNA strand breaks can be demonstrated by a variety of enzymes each of which reacts specifically with a certain end group on the 3’ terminal or on the 5’ terminal. The chapter also describes the details of the specificity of these enzymes and their reactivity with respect to radiation-induced strand breaks.

Yields of Radiation-induced Main Chain Scission of Poly U in Aqueous Solution: Strand Break Formation Via Base Radicals

The G values for single-strand breaks G(ssb) in polyuridylic acid (poly U) have been measured by low-angle laser light scattering in aqueous solutions under various conditions (e.g. in the presence of N2O, Ar and t-butanol). In N2O-saturated solutions at room temperature and pH 5.6, the G(ssb) is 2.3. The efficiency of ssb formation was found to be 41 per cent for OH radicals, 19 per cent for H atoms and congruent to zero for e-aq. On the basis of 20 per cent and less than 5 per cent attack on the sugar moiety by OH radicals and H atoms, respectively, the large G(ssb) values obtained cannot be explained solely as resulting from radicals produced by reaction of OH radicals and H atoms on the sugar moiety. It is therefore proposed that base radicals produced by the reaction of OH radicals or H atoms with the uracil moiety can also lead to chain break formation in poly U via radical transfer to the sugar moiety.

HO2 ELIMINATION FROM α-HYDROXYALKYLPEROXYL RADICALS IN AQUEOUS SOLUTION

Abstract— In aqueous solutions α‐hydroxyalkylperoxyl radicals undergo a spontaneous and a base catalysed HO2 elimination. From kinetic deuterium isotope effects, temperature dependence, and the influence of solvent polarity it was concluded that the spontaneous reaction occurs via an HO2 elimination followed by the dissociation of the latter into H+ and O2‐. The rate constant of the spontaneous HO2 elimination increases with increasing methyl substitution in α‐position (k(CH2(OH)O2) < 10s‐1k(CH3CH(OH)O2) = 52s‐1k((CH3)2C(OH)O2) = 665 s‐1). The OH‐ catalysed reaction is somewhat below diffusion controlled. The mixture of peroxyl radicals derived from polyhydric alcohols eliminate HO2 at two different rates. Possible reasons for this behaviour are discussed. The mixture of the six peroxyl radicals derived from d‐glucose are observed to eliminate HO2 with at least three different rates. The fastest rate is attributed to the HO2 elimination from the peroxyl radical at C‐l (k > 7000s‐1). Because of the HO2 eliminations the peroxyl radicals derived from d‐glucose do not undergo a chain reaction in contrast to peroxyl radicals not containing an α‐OH group. In competition with the first order elimination reactions the α‐hydroxylalkylperoxyl radicals undergo a bimolecular decay. These reactions are briefly discussed.

Laser-induced strand break formation in DNA and polynucleotides.

The main objective of this review is to summarize the current knowledge of the chemical steps leading to laser pulse-induced strand breakage in nucleic acids. One of the motivations behind these photoionization studies is to gain a better understanding of the direct effects of high-energy radiation on DNA which leads to strand breaks (SchulteFrohlinde, 1986a, 1986b, 1989). The radiationinduced double-strand breaks are the most serious damage to DNA in cells which, if unrepaired, lead to cell transformation, mutation and cell death (Ward, 1975, 1990; Schulte-Frohlinde, 1987a, 1987b). Strand break (sb) formation induced by UV light is discussed only in so far as it is not incorporated in recent reviews by Nikogosyan and Letokhov (1983); Peak et al. (1987); HClkne (1987); Cadet and Vigny (1990); Nikogosyan (1990). Physical and spectroscopically observable primary processes have been summarized by Nikogosyan and Letokhov (1983) and Nikogosyan (1990). Photosensitized sb formation has been reviewed by numerous authors, see for example: Amagasa (1981); Cadet et al. (1986); Piette and van de Vorst (1987); Kochevar and Dunn (1990). Also not incorporated in the present work is the content of articles edited by Wang (1976) under the title: “Photochemistry and Photobiology of Nucleic Acids” since strand breakage is virtually not discussed in these reviews. Furthermore, most of the earlier articles which do not explicitly contribute to the elucidation of mechanistic steps are also not included. The present article describes mainly the chemical effects from biphotonic excitation of polynucleotides and DNA leading to singleand double-strand breaks (ssb and dsb). In addition some recent results concerning monophotonic generation of sb are discussed. Methods for the determination of sb are reviewed in Section 2. Of special importance is the time dependence of sb formation since this allows conclusions concerning the mechanism to be drawn. In Section 3 photoionization of nucleic acids as initiating process is outlined and the following Sections (4-12) deal with the chemical mechanisms of ssb and dsb initiation of polynucleotides and DNA under a wide range of experimental conditions. 2 . Methods

Lifetime of peroxyl radicals of poly(U), poly(A) and single-and double-stranded DNA and the rate of their reaction with thiols.

Peroxyl radicals of poly(U), poly(A), and single- and double-stranded DNA have been produced by photolysing H2O2 in oxygenated aqueous solution in presence of the substrates. The peroxyl radicals are formed by the reaction of OH radicals with the polynucleotides followed by addition of oxygen. The lifetime of the peroxyl radicals and the rate constant of their reactions with the thiols cysteamine, glutathione and dithiothreithol have been measured by time-resolved e.s.r. spectroscopy. The unusually long lifetimes range from 0.2 to 3.3 s. The activation energy for the decay for all four substrates is 10.3 +/- 1 kcal/mol (43 kJ mol-1). The reaction rate constants with the thiols range from k = 0.8 X 10(4) to 1.3 X 10(5) dm3 mol-1 s-1. The reactions of the thiols with the peroxyl radical of poly(U) are known to prevent strand break formation. This shows that the peroxyl radicals of poly(U) observed by e.s.r. are intermediates in the pathway leading to strand break formation.

CIS-TRANS PHOTOISOMERIZATION OF 4-NITROSTILBENES

Abstract Results on cis-trans photoisomerization of 4-nitrostilbenes obtained in recent years are summarized and discussed. Mechanisms for the direct trans → cis and cis → trans photoisomerization are presented. The trans → cis isomerization occurs via a triplet state as intermediate whereas in the cis → trans isomerization the main route bypasses the triplet state. The triplet state shows a configurational equilibrium between the planar trans (tr3) and more twisted forms (p3 or c3). The equilibrium is mainly on the trans side. The influence of substitution, temperature, viscosity, solvent, and addition of quenchers on quantum yields of isomerization, fluorescence studies, and spectroscopic as well as kinetic laser flash photolysis results support the above conclusions.

γ-Radiolysis of DNA in Oxygenated Aqueous Solutions: Alterations at the Sugar Moiety

On gamma-irradiation of DNA in N2O/O2-saturated aqueous solutions alterations at the sugar moiety are observed. In the present study three new lesions were recognized: (i) 2-deoxytetrodialdose bound via a phosphoric acid ester linkage to a (broken) DNA strand, (ii) 2-deoxypentos-4-ulose bound to DNA via one (or two?) phosphoric acid ester linkage(s), and (iii) 2-deoxy-D-erythro-pentose bound to DNA via two phosphoric acid ester linkages. Lesion (i) is directly connected with a DNA strand break. Lesion (ii) might be related to a DNA strand break if bound via only one phosphoric acid ester linkage, or has to be considered as an alkali-labile site if bound via two phosphoric acid ester linkages. Lesion (iii) results from base damage, when the damaged base is hydrolysed from the sugar. This lesion is an alkali-labile site which turns into a strand break on alkali treatment. Attempts have been made to quantify these lesions. A lower limit of sugar damage (including lesions observed in preceding studies, but not lesion (iii) of G = 0.25 has been estimated.

Loss of transforming activity of plasmid DNA (pBR322) in E. coli caused by singlet molecular oxygen

Plasmid DNA pBR322 in aqueous solution was exposed to singlet molecular oxygen (1O2) generated by microwave discharge. DNA damage was detected as loss of transforming activity of pBR322 in E.coli (CMK) dependent on the time of exposure. DNA damage was effectively decreased by singlet‐oxygen quenchers such as sodium azide and methionine. Replacement of water in the incubation buffer by D2O led to an increase in DNA damage. 9,10‐Bis(2‐ethylene)anthracene disulfate was used as a chemical trap for 1O2 quantitation by HPLC analysis of the endoperoxide formed.

Hydroxyl Radical-induced Strand Break Formation of Poly(U) in the Presence of Oxygen: Comparison of the Rates as Determined by Conductivity, e.s.r. and Rapid-mix Experiments with a Thiol

The rate of OH radical-induced strand break formation of single-stranded poly(U) in N2O/O2-saturated aqueous solution was studied by measuring the time-dependence of the electrical conductivity following pulse radiolysis. The first half-life of the total conductivity increase depends slightly on pH and the molecular weight and on the dose per pulse. The activation parameters for strand break formation were found to be EA = 52 kJ mol-1 and A = 5 X 10(8) s-1. Similar first half-lives were observed when the decay of peroxyl radicals of poly(U) was measured by e.s.r. under various conditions. This indicates that poly(U)-peroxyl radicals are involved in the rate-determining step of strand break formation. After pulse radiolysis, strand break formation can be inhibited by the addition of dithiothreitol (DTT) in a rapid-mix apparatus. It is postulated that peroxyl radicals of poly(U) react with DTT by formation of hydroperoxides, thereby preventing strand breakage.

Radiation-induced DNA Strand Breaks in Deoxygenated Aqueous Solutions. The Formation of Altered Sugars as End Groups

Gamma irradiation of DNA in deoxygenated, N2O-saturated aqueous solution leads to three bound altered sugars present as end groups in broken DNA strands. These sugars are linked to the DNA by phosphoric acid ester bonds. Two of the end groups have the structures (4) and (5). (Formula: see text) The third end group after dephosphorylation has structure (3). The formation of the bound sugars (4) and (5) is explained by a mechanism postulated earlier for the formation of free altered sugars. Except for the phosphoric acid ester linkage, the free altered sugars have the same chemical structures as the bound altered sugars.