Hydroxyl radical induced cross-linking of cytosine and tyrosine in nucleohistone

Hydroxyl radical induced formation of a DNA-protein cross-link involving cytosine and tyrosine in nucleohistone in buffered aqueous solution is reported. The technique of gas chromatography-mass spectrometry was used for this investigation. A gamma-irradiated aqueous mixture of cytosine and tyrosine was first investigated in order to obtain gas chromatographic-mass spectrometric properties of possible cytosine-tyrosine cross-links. One cross-link was observed, and its structure was identified as the product from the formation of a covalent bond between carbon 6 of cytosine and carbon 3 of tyrosine. With the use of gas chromatography-mass spectrometry with selected-ion monitoring, this cytosine-tyrosine cross-link was identified in acidic hydrolysates of calf thymus nucleohistone gamma-irradiated in N2O-saturated aqueous solution. The yield of this DNA-protein cross-link in nucleohistone was found to be a linear function of the radiation dose in the range of 100-500 Gy (J.kg-1). This yield amounted to 0.05 nmol.J-1. Mechanisms underlying the formation of the cytosine-tyrosine cross-link in nucleohistone were proposed to involve radical-radical and/or radical addition reactions of hydroxyl adduct radicals of cytosine and tyrosine moieties, forming a covalent bond between carbon 6 of cytosine and carbon 3 of tyrosine. When oxygen was present in irradiated solutions, no cytosine-tyrosine cross-links were observed.     

DNA-protein cross-linking between thymine and tyrosine in chromatin of gamma-irradiated or H2O2-treated cultured human cells.

Formation of DNA-protein cross-links between thymine and tyrosine in chromatin of gamma-irradiated or H2O2-treated cultured human cells is reported. Chromatin was isolated from cells, and subsequently hydrolyzed and derivatized. Analysis of derivatized hydrolysates by gas chromatography/mass spectrometry with selected-ion monitoring showed that 3-[(1,3-dihydro-2,4-dioxopyrimidin-5-yl)-methyl]-L-tyrosine (Thy-Tyr cross-link) was formed. The presence of this DNA-protein cross-link in control cells was also observed at a level of approximately 7 molecules per 10(6) DNA nucleotides. Exposure of cells to ionizing radiation at doses between 8.7 and 82 Gy (J.kg-1) increased the amount of the Thy-Tyr cross-link linearly up to approximately fourfold over the background level. At doses higher than 82 Gy, the yield approached a plateau. Treatment of cells with H2O2 (0.5 to 10 mM) also increased the amount of the Thy-Tyr cross-link in a concentration-dependent manner. Addition of dimethyl sulfoxide and o-phenanthroline in the culture medium afforded partial inhibition of cross-link formation. Addition of catalase inhibitor KCN prior to H2O2 treatment increased the yield of cross-linking over the level observed with H2O2 treatment alone. Pretreatment of cells with ascorbic acid for 24 h without H2O2 caused formation of the Thy-Tyr cross-link. This DNA-protein cross-link in chromatin of cells is proposed to be formed by mechanisms involving a radical addition reaction and/or a radical-radical combination involving thymine and tyrosine radicals. Hydroxyl radical mediated by chromatin-bound metal ions is proposed to cause the formation of the Thy-Tyr cross-link in H2O2-treated cells.     

Structure of a hydroxyl radical induced cross-link of thymine and tyrosine

DNA-protein cross-links are formed when living cells or isolated chromatin is exposed to ionizing radiation. Little is known about the actual cross-linked products of DNA and proteins. In this work, a novel hydroxyl radical induced cross-link of thymine and tyrosine has been isolated along with a tyrosine dimer by high-performance liquid chromatography of aqueous mixtures of tyrosine and thymine that had been exposed to hydroxyl radicals generated by ionizing radiation. The isolated compounds have been examined by gas chromatography-mass spectrometry, high-resolution mass spectrometry, and 1H and 13C nuclear magnetic resonance spectroscopy. The structure of the thymine-tyrosine cross-link has been identified as the product from the formation of a covalent bond between the methyl group of the thymine and carbon 3 of the tyrosine ring. In addition, the 3,3' tyrosine dimer was isolated and characterized. The mechanism of the formation of these compounds is discussed. This work presents the first complete chemical characterization of a hydroxyl radical induced DNA base-amino acid cross-link.     

Chemical nature of DNA-protein cross-links produced in mammalian chromatin by hydrogen peroxide in the presence of iron or copper ions

We report on the elucidation of DNA-protein cross-links formed in isolated mammalian chromatin upon treatment with H2O2 in the presence of iron or copper ions. Analysis of chromatin samples by gas chromatography/mass spectrometry after hydrolysis and derivatization showed the presence of 3-[(1,3-dihydro-2,4-dioxopyrimidin-5-yl)methyl]-L-tyrosine (thymine-tyrosine cross-link) on the basis of the gas chromatographic and mass spectrometric characteristics of the trimethylsilylated authentic compound. Other DNA-protein cross-links involving thymine and the aliphatic amino acids and cytosine and tyrosine, which were known to occur in nucleohistone gamma-irradiated under anoxic conditions, were not observed. This was due to inhibition by oxygen as clearly shown by experiments that were carried out using ionizing radiation under both oxic and anoxic conditions instead of using H2O2 and metal ions. However, oxygen did not inhibit formation of the thymine-tyrosine cross-link in gamma-irradiated chromatin or in chromatin treated with H2O2 and metal ions. The yield of the thymine-tyrosine cross-link was higher upon treatment with H2O2/chelated Fe3+ ions than with H2O2/unchelated Fe3+ ions. By contrast, H2O2/unchelated Cu2+ ions produced a higher yield than H2O2/chelated Cu2+ ions. Almost complete inhibition of cross-link formation was provided by the hydroxyl radical scavengers mannitol and dimethyl sulfoxide when H2O2/chelated metal ions were used. On the other hand, scavengers only partially inhibited formation of cross-links when H2O2/unchelated metal ions were used, possibly indicating the site-specific nature of cross-linking. Superoxide dismutase afforded partial inhibition only when chelated ions were used.(ABSTRACT TRUNCATED AT 250 WORDS)     

Generation of 5-(2'-deoxycytidyl)methyl radical and the formation of intrastrand cross-link lesions in oligodeoxyribonucleotides

Hydroxyl radical is one of the major reactive oxygen species (ROS) formed from γ-radiolysis of water or Fenton reaction, and it can abstract one hydrogen atom from the methyl carbon atom of thymine and 5-methylcytosine to give the 5-methyl radical of the pyrimidine bases. The latter radical can also be induced from Type-I photo-oxidation process. Here, we examined the reactivity of the independently generated 5-(2′-deoxycytidyl)methyl radical (I) in single- and double-stranded oligodeoxyribonucleotides (ODNs). It was found that an intrastrand cross-link lesion, in which the methyl carbon atom of 5-methylcytosine and the C8 carbon atom of guanine are covalently bonded, could be formed from the independently generated radical at both GmC and mCG sites, with the yield being much higher at the former site. We also showed by LC-MS/MS that the same cross-link lesions were formed in mC-containing duplex ODNs upon γ irradiation under both aerobic and anaerobic conditions, and the yield was ~10-fold higher under the latter conditions. The independently generated radical allows for the availability of pure, sufficient and well-characterized intrastrand cross-link lesion-bearing ODN substrates for future biochemical and biophysical characterizations. This was also the first demonstration that the coupling of radical I with its 5′ neighboring guanine can occur in the presence of molecular oxygen, suggesting that the formation of this and other types of intrastrand cross-link lesions might have important implications in the cytotoxic effects of ROS.     

Photoreactions of thymine and thymidine with N-acetyltyrosine.

We report here the photoinduced formation of a thymine-N-acetyltyrosine adduct. Irradiation of dilute solutions of thymine in the presence of N-acetyltyrosine (NAT) leads to the formation of N-acetyl-4-hydroxy-3-(6-hydrothymin-5-yl)phenylalanine (I), isolated as a mixture of the 5R and 5S diastereoisomers; the photoreaction occurs when irradiation is done either at lambda = 254 nm or at wavelengths of lambda > 290 nm. Irradiation of thymidine in the presence of NAT and of thymine in the presence of tyrosine leads to analogous photoadducts. The photoreaction of thymine with NAT is completely quenched by oxygen and cannot be sensitized by acetone. The likely mechanism involves initial photoionization of the amino acid and deprotonation to form the phenoxyl radical. Thymine then probably captures the released aqueous electron, leading to protonation at C6 of the resulting radical anion. Combination of the phenoxyl and 5,6-dihydrothymin-5-yl radicals would then lead to formation of the final products. The quantum yield for production of the thymine-NAT adduct at pH 7.8 was estimated to be about 5.5 x 10(-4), while a value of 2.3 x 10(-3) was estimated for production of corresponding thymidine adduct at pH 8.1. The dependence of the quantum yield for adduct formation on pH has been determined for both the thymine and thymidine reactions with NAT; the maxima in the quantum yield profiles occur at pH 8-8.5, while appreciable values were measured at pH 7.5. We have also demonstrated that a similar reaction occurs when tyrosine is located within a peptide.(ABSTRACT TRUNCATED AT 250 WORDS)     

Intramolecular H atom abstraction from the sugar moiety by thymine radicals in oligo- and polydeoxynucleotides

Hydroxyl radical addition to uracil (U) has been suggested to lead to strand breaks in polyuridylic acid, an occurrence attributed in part to H atom abstraction by .U-OH radicals from the ribose moiety [D.G.E. Lemaire et al., Int. J. Radiat. Biol. 45, 351-358 (1984)]. We have investigated this particular reaction by means of the hydroxyl radical-induced products of thymine (T), pT, TpT, TpTpT, polythymidylic acid (poly-T), (T + dR) poly-dA.poly-T, and a mixture of T and 2-deoxyribose (dR). The major monomeric product of .T-OH in TpT, TpTpT, poly-T, and poly-dA.poly-T was found to be 5-hydroxy-6-hydrothymine (H-T-OH), while that in T, pT, and T plus dR was thymine glycol (HO-T-OH). These results indicated that the intramolecular H atom abstraction from a nearby sugar (in this case, deoxyribose) moiety by base radicals, i.e., .T-OH, occurs in oligo- and polydeoxynucleotides of T. In poly-T, the yield of H-T-OH is not much greater than in TpT or TpTpT, indicating that the abstraction of an H atom from the sugar moiety of a nucleotide subunit further than two nucleotides along the chain may not be significant. Additionally, a corresponding decrease in the yield of HO-T-OH with an increase in the yield of H-T-OH suggests that the formations of these two types of thymine products are competitive.     

The deoxyribose method: a simple "test-tube" assay for determination of rate constants for reactions of hydroxyl radicals

Hydroxyl radicals, generated by reaction of an iron-EDTA complex with H2O2 in the presence of ascorbic acid, attack deoxyribose to form products that, upon heating with thiobarbituric acid at low pH, yield a pink chromogen. Added hydroxyl radical "scavengers" compete with deoxyribose for the hydroxyl radicals produced and diminish chromogen formation. A rate constant for reaction of the scavenger with hydroxyl radical can be deduced from the inhibition of color formation. For a wide range of compounds, rate constants obtained in this way are similar to those determined by pulse radiolysis. It is suggested that the deoxyribose assay is a simple and cheap alternative to pulse radiolysis for determination of rate constants for reaction of most biological molecules with hydroxyl radicals. Rate constants for reactions of ATP, ADP, and Good's buffers with hydroxyl radicals have been determined by this method.     

Hydroxyl radical induced cross-linking between phenylalanine and 2-deoxyribose

Hydroxy radicals induce cross-linking between phenylalanine (Phe) and 2-deoxyribose (dR) via formation of corresponding free radical intermediates. The cross-linked products were separated and identified by capillary gas chromatography-mass spectrometry. When phenylalanine and 2-deoxyribose radicals were generated in a 1:1 ratio, the predominant interaction was between Phe and dR radicals while the Phe-Phe and dR-dR cross-links were less abundant. The newly discovered cross-link between 2-deoxyribose and phenylalanine may serve as a model for radiation or free radical induced cross-linking between DNA and proteins and in general between sugar moieties and amino acids.     

Effect of non-volatile scavengers of hydroxyl radicals on thymine radical formation induced by gamma-rays and ultrasound

In order to investigate the mechanism of sonolysis of nucleic acid constituents, the yield of thymine radicals generated by 50 kHz ultrasound in Ar-saturated aqueous solution was compared with that formed by gamma-radiolysis in N2O-saturated solutions in the presence of various non-volatile scavengers, which cannot act in the gas phase of the cavitation bubbles. For comparison of thymine radical yields by sonolysis and gamma radiolysis, the method of spin trapping with 3,5-dibromo-4-nitrosobenzenesulphonate (a water-soluble, non-volatile, aromatic nitroso spin trap) combined with ESR was used. The efficiency of OH radical scavenging is expressed by the reciprocal value of C1/2, the scavenger concentration at which the thymine radical yield is decreased by 50 per cent. In gamma radiolysis the scavenging efficiencies of the solutes depend on their rate constants with OH radicals. For sonolysis the C1/2 values were similar to those obtained for gamma radiolysis except for the hydrophobic 5,5-dimethyl-1-pyrroline-N-oxide. These results suggest that thymine radicals induced by ultrasound are produced in the bulk of the solution as well as in the interfacial region.     

Footprinting protein-DNA complexes using the hydroxyl radical

Hydroxyl radical footprinting has been widely used for studying the structure of DNA and DNA-protein complexes. The high reactivity and lack of base specificity of the hydroxyl radical makes it an excellent probe for high-resolution footprinting of DNA-protein complexes; this technique can provide structural detail that is not achievable using DNase I footprinting. Hydroxyl radical footprinting experiments can be carried out using readily available and inexpensive reagents and lab equipment. This method involves using the hydroxyl radical to cleave a nucleic acid molecule that is bound to a protein, followed by separating the cleavage products on a denaturing electrophoresis gel to identify the protein-binding sites on the nucleic acid molecule. We describe a protocol for hydroxyl radical footprinting of DNA-protein complexes, along with a troubleshooting guide, that allows researchers to obtain efficient cleavage of DNA in the presence and absence of proteins. This protocol can be completed in 2 d.     

Radiation-induced crosslinks between thymine and phenylalanine

OH radicals generated by ionizing radiation in aqueous solutions of thymine (T) and phenylalanine (Phe) induce crosslinking between thymine and phenylalanine. The crosslinked products were isolated and characterized by capillary gas chromatography-mass spectrometry. They are formed via OH radical adducts to thymine and phenylalanine and the reaction between dissimilar radicals is greatly favoured (T-Phe:Phe-Phe:T-T = 0.46:0.14:0.05). The reaction mechanism presented may serve as a model for radiation or any free radical-induced crosslinks between DNA and proteins.     

Kinetics of oxidation of tyrosine by a model alkoxyl radical

Oxidation of tyrosine moieties by radicals involved in lipid peroxidation is of current interest; while a rate constant has been reported for reaction of lipid peroxyl radicals with a tyrosine model, little is known about the reaction between tyrosine and alkoxyl radicals (also intermediates in the lipid peroxidation chain reaction). In this study, the reaction between a model alkoxyl radical, the tert-butoxyl radical and tyrosine was followed using steady-state and pulse radiolysis. Acetone, a product of the β-fragmentation of the tert-butoxyl radical, was measured; the yield was reduced by the presence of tyrosine in a concentration- and pH-dependent manner. From these data, a rate constant for the reaction between tert-butoxyl and tyrosine was estimated as 6 ± 1 × 10(7) M(-1) s(-1) at pH 10. Tyrosine phenoxyl radicals were also monitored directly by kinetic spectrophotometry following generation of tert-butoxyl radicals by pulse radiolysis of solutions containing tyrosine. From the yield of tyrosyl radicals (measured before they decayed) as a function of tyrosine concentration, a rate constant for the reaction between tert-butoxyl and tyrosine was estimated as 7 ± 3 × 10(7) M(-1) s(-1) at pH 10 (the reaction was not observable at pH 7). We conclude that reaction involves oxidation of tyrosine phenolate rather than undissociated phenol; since the pK(a) of phenolic hydroxyl dissociation in tyrosine is ≈ 10.3, this infers a much lower rate constant, about 3 × 10(5) M(-1) s(-1), for the reaction between this alkoxyl radical and tyrosine at pH 7.4.     

Modeling the influence of histone proteins on the sensitivity of DNA to ionizing radiation

The DNA-binding proteins that are present in chromatin significantly affect the sensitivity of cells to ionizing radiation and to the radiation chemistry of DNA damage. The interaction between protein and DNA modifies the radiation chemistry of the latter. To model these processes, we have examined the effects of ionizing radiation on the minichromosome form of SV40 (which contains histone proteins arranged in nucleosomes) and also on plasmid DNA in the presence of lysozyme. Although high concentrations of lysozyme can bring about an extensive radioprotection by condensation of the plasmid, at lower levels it still produces significant radioprotective effects under conditions where this associative phase separation does not take place. The presence of histones or of lysozyme decreases the yield of modified guanines produced by ionizing radiation. Comparison with previous observations made with oligopeptides suggests that the mechanism responsible is electron donation to guanyl radicals in the DNA by tryptophan and tyrosine residues in the proteins. However, there was no evidence for DNA-protein crosslink formation.     

Mechanism of the formation of DNA-protein cross-links during the gamma irradiation of chromatin in aqueous solutions

The method of gel electrophoresis was used to study DNA-protein cross-link formation in fragmentized chromatin gamma-irradiated in water solutions (0.03%). By introducing changes into irradiation conditions (for instance, the use of different gases saturating the solution and the administration of radical acceptors) and by the subsequent electrophoretic analysis (treatment of the exposed chromatin by dissociating mixtures and enzymes) the authors showed a covalent nature of the cross-links in a radiation-induced DNA-protein complex and found the value of G (a cross-link) to be 0.02.     

Identification of tyrosine 204 as the photo-cross-linking site in the DNA-EcoRI DNA methyltransferase complex by electrospray ionization mass spectrometry

We describe a highly sensitive strategy combining laser-induced photo-cross-linking and HPLC-based electrospray ionization mass spectrometry to identify amino acid residues involved in protein-DNA recognition. The photoactivatible cross-linking thymine isostere, 5-iodoracil, was incorporated at a single site within the sequence recognized by EcoRI DNA methyltransferase (GAATTC). UV irradiation of the DNA-protein complex at 313 nm results in a >60% cross-linking yield. SDS-polyacrylamide gel electrophoresis and mass spectrometry were used to analyze the covalent cross-linked complex. The total mass is consistent with covalent bond formation between one strand of DNA and the protein with 1:1 stoichiometry. Protease digestion of the cross-linked complex yields several peptide-DNA adducts that were purified by anion-exchange column chromatography. A combination of mass spectrometric analysis and amino acid sequencing revealed that tyrosine 204 was cross-linked to the DNA. Electrospray mass spectrometric analysis of the peptide-nucleoside adduct confirmed this assignment. Tyrosine 204 resides in a peptide motif previously thought to be involved in AdoMet binding and methyl transfer. Thus, amino acids within loop segments but outside of "DNA binding" motifs can be critical to DNA recognition. Our method provides an accurate characterization of picomole quantities of DNA-protein complexes.     

Oxidation of single-stranded oligonucleotides by carbonate radical anions: generating intrastrand cross-links between guanine and thymine bases separated by cytosines

The carbonate radical anion is a biologically important one-electron oxidant that can directly abstract an electron from guanine, the most easily oxidizable DNA base. Oxidation of the 5′-d(CCTACGCTACC) sequence by photochemically generated CO₃^·− radicals in low steady-state concentrations relevant to biological processes results in the formation of spiroiminodihydantoin diastereomers and a previously unknown lesion. The latter was excised from the oxidized oligonucleotides by enzymatic digestion with nuclease P1 and alkaline phosphatase and identified by LC-MS/MS as an unusual intrastrand cross-link between guanine and thymine. In order to further characterize the structure of this lesion, 5′-d(GpCpT) was exposed to CO₃^·− radicals, and the cyclic nature of the 5′-d(G*pCpT*) cross-link in which the guanine C8-atom is bound to the thymine N3-atom was confirmed by LC-MS/MS, 1D and 2D NMR studies. The effect of bridging C bases on the cross-link formation was studied in the series of 5′-d(GpC_npT) and 5′-d(TpC_npG) sequences with n = 0, 1, 2 and 3. Formation of the G*-T* cross-links is most efficient in the case of 5′-d(GpCpT). Cross-link formation (n = 0) was also observed in double-stranded DNA molecules derived from the self-complementary 5′-d(TTACGTACGTAA) sequence following exposure to CO₃^·− radicals and enzymatic excision of the 5′-d(G^*pT^*) product.     

Chemical and biochemical aspects of superoxide radicals and related species of activated oxygen

The spectrum of biological processes in which oxygen is used by living systems is quite large, and the products include some damaging species of activated oxygen, particularly the superoxide radical (O-.2) and hydrogen peroxide (H2O2). Superoxide radicals and hydrogen peroxide, in turn, can lead to the formation of other damaging species: hydroxyl radicals (.OH) and singlet oxygen (1O2). Hydroxyl radicals react with organic compounds to give secondary free radicals that, in the presence of oxygen, yield peroxy radicals, peroxides, and hydroperoxides. Formation, interconversion, and reactivity of O-.2 and related activated oxygen species, methods available for their detection, and the basis of their biological toxicity are briefly reviewed.     

Oxidative breakdown and conversion of urocanic acid isomers by hydroxyl radical generating systems.

cis-Urocanic acid (cis-UCA), formed from trans-urocanic acid (trans-UCA) by photoisomerization, has been shown to mimic suppressive effects of UV on the immune system. It is our hypothesis that UCA oxidation products in the skin play a role in the process of immunosuppression. Recently, both UCA isomers were found to be good hydroxyl radical scavengers and in this context we investigated the formation of products resulting from the interaction of hydroxyl radicals with UCA. Hydroxyl radicals were generated by (1) UV/H(2)O(2) (photooxidation), (2) ferrous ions/H(2)O(2) (Fenton oxidation) and (3) cupric ions/ascorbic acid. Oxidation products were identified by spectrometric methods and assessed by reversed-phase HPLC analysis. The photooxidation of UCA was induced by UV-B and UV-C, but not by UV-A radiation. Photooxidation and Fenton oxidation of trans-UCA, as well as of cis-UCA yielded comparable chromatographic patterns of UCA oxidation products. Several of the formed products were identified. The formation of three identified imidazoles was shown in UV-B exposed corneal layer samples, derived from human skin.