Radiation chemistry of a dinucleoside monophosphate and its sequence isomer

A combination of high-performance liquid chromatography (HPLC) and nuclear magnetic resonance (NMR) spectroscopy was used to analyze the products of X-irradiated aqueous solutions of the dinucleoside monophosphate thymidylyl(3'-5')-2'-deoxyadenosine, d(TpA), and its sequence isomer 2'-deoxyadenylyl(3'-5')thymidine, d(ApT). The products of d(TpA) include both bases and nucleotides and a variety of thymine modifications of d(TpA) including the two cis and two trans glycol stereoisomers, two cis monohydroxy derivatives, an N-formamide derivative, and the hydroxymethyl derivative. Attention is focused on using NMR spectral features to distinguish among the various stereoisomers. The radiation chemistry of d(ApT) is also explored and differences in product formation compared with d(TpA) are described, particularly the formation of two products involving modification of adenine base. The potential of the HPLC-NMR approach to the study of radiation chemistry in DNA model compounds is discussed.     

Conformations of dinucleoside monophosphates in relation to duplex DNA structures

Mononucleotide conformations are important in understanding the structural aspects of nucleic acids and polynucleotides. In order to study the influence of stacking interactions between adjacent bases in a polynucleotide on the preferred conformations of mononucleotides, conformational energy calculations have been carried out on dinucleoside monophosphate fragments. Four base sequences—d(ApT), d(TpA), d(CpG), and d(GpC)— have been analyzed in the framework of helical structures. Flexibility of the furanose ring has been incorporated in the investigations. Energetically favored conformers of the four compounds correspond to a variety of left- and right-handed uniform helical structures, similar to those of the commonly observed polymorphous forms. Implications of these investigations on the further understanding of double-helical polynucleotide conformations are briefly discussed.     

Invariant and variable base stacking geometries in B-DNA and A-DNA

We calculated the interatomic distances between all couples of non-hydrogen atoms belonging to the neighboring Watson-Crick base pairs in the available crystal structures of DNA. Their standard deviations revealed remarkably large differences in the variability of the base stacking geometries of the particular steps. In line with experimental studies in solution, (CpA)-(TpG) and (TpA).(TpA) were identified as the most variable or flexible steps in the crystal structures of B-DNA. On the other hand, base stacking geometries of the (ApT).(ApT) steps were the most invariant, which was very surprising because all three steps composed only of C and G were much more flexible. This finding suggests that conformational stability of DNA and the rigidity have different origins. Furthermore, the nucleotide sequence dependence of the flexibility was almost reversed in A-DNA because the most flexible steps in B-DNA were the least flexible in A-DNA. The most invariant steps of B-DNA were variable in A-DNA. The (ApT).(ApT) step was a notable exception to this rule because it belonged to the most rigid steps in both B-DNA and A-DNA. The present results are fully consistent with the properties that poly(dA-dT).poly(dA-dT), poly(dA).poly(dT), poly(dAdC).poly(dG-dT) and poly(dA-dG).poly(dC-dT) exhibit in solution.     

N.m.r. and c.d. studies of the DNA fragments d(TATATATA) and d(TATATA) in solution

DNA fragments d(TATATATA) and d(TATATA) were studied in low-salt aqueous solutions and found to coexist in more than one conformer. 1H-n.m.r. demonstrates that single-stranded and double-stranded states are involved in the conformational coexistence. Circular dichroism spectroscopy indicates a global B-DNA stacking of bases in the fragments. 31P-n.m.r. resonances of the TpA and ApT phosphodiester bonds are substantially separated in the spectra of both d(TATATATA) and d(TATATA) duplexes to suggest an alternating architecture of their backbones. In fact, the oligonucleotide duplexes are much more alternating than the corresponding polynucleotide under the same solution conditions. The alternating character of the d(TATATATA) double helix is further enhanced in molar caesium fluoride solutions. The oligonucleotide isomerization into X-DNA is, however, accompanied by gel formation, which makes high resolution n.m.r. measurements impossible.     

Echinomycin binding to the sequence CG(AT)nCG alters the structure of the central AT region.

DNA fragments containing the sequence CG(AT)nCG have been used in footprinting experiments to assess the effect of echinomycin, which binds to CG steps, on the structure of the central AT region. DNAase I normally cuts ApT much better than TpA; in the presence of the drug this preference is retained but cleavage at TpA is enhanced. Changes in cleavage by micrococcal nuclease have also been observed. Echinomycin renders alternate adenines hyperreactive to diethylpyrocarbonate. The results suggest that echinomycin induces structural changes in regions surrounding its binding site and that these can be cooperatively propagated over several turns of the DNA helix.     

The effects of actinomycin on the structure of dAn.dTn and (dA-dT)n regions surrounding its GC binding site. A footprinting study.

The effect of actinomycin on the structure of DNA fragments containing the sequences (AT)5GC(AT)5, (TA)5GC(TA)5, A9GCT9, and T9GCA9, cloned into the SmaI site of pUC19, has been studied by footprinting analysis using a variety of probes known to be sensitive to DNA structure. In each case clear footprints are found around the central GC sites. DNase I cleavage of fragments containing alternating AT shows much greater cutting at ApT than TpA; in the presence of actinomycin, although this preference is retained, there is a large increase in the cutting efficiency at the closest TpA steps. DNase I cleavage in homopolymeric regions of A and T, which is normally very poor, is greatly enhanced by drug binding. With T9GCA9 the enhancements are propagated in both directions, whereas changes are only found to the 5'-side of the GC site in A9GCT9. The results are confirmed by similar experiments with micrococcal nuclease and DNase II. Small increases in sensitivity to diethylpyrocarbonate are found at adenines proximal to GC. Experiments performed at 4 degrees C suggest that conformational changes are a necessary consequence of drug binding.     

Mechanisms of DNA damage and insight into mutations by chromium(VI) in the presence of glutathione

The mechanisms of hexavalent chromium(VI) induced DNA damage were unveiled by detecting products of single- and double-stranded DNA in the presence of glutathione. The absence of a detectable hydroxyl radical in the reactions indicates that DNA damage was exclusively by hypervalent chromium species. Polyacrylamide gel electrophoresis (PAGE) experiments with 32-mer single-stranded oligonucleotide and its complementary duplex revealed cleavages largely at purine bases with significant enhancement of such cleavages in the presence of a base. Quantitative estimations of bases released by HPLC before and after enzymatic digestion with exonucleases unequivocally established the excessive release of purine bases. This release was accompanied by the concomitant formation of phosphoglycolate as characterized by liquid chromatography-mass spectrometry (LC-MS). These data connote that the preponderance DNA damage is due to an oxidation specifically at H4' of the ribose moiety leading to the formation of apurinic sites. In addition to the oxidation at H4', DNA oxidation was also initiated through H5' site as evidenced by the identification of furfural. This pathway appears to be non-selective and more abundant for ssDNA as cleavages were observed at both purine and pyrimidine bases. Finally, the detection of guanidinohydantoin as a minor product points the involvement of an oxygen activated hypervalent chromium species, perhaps a peroxo-chromium species. Both major and minor pathways lead to cleavages at purine sites for ds-DNA and are consistent with the observation that DNA cleavage was enhanced in the presence of a base. In contrast, when hydrogen peroxide was added to the reactions, random DNA cleavages were apparent indicating involvement of multiple species including a hydroxyl radical. These data pinpoint mutation mechanisms induced by chromium(VI) in the presence of glutathione due to transversion either by inserting the wrong bases opposite to the apurinic sites during replication or by purine-purine mismatch.     

Conformation of the oligonucleotide d(AAAAAATTTTTT)2 by two-dimensional nuclear Overhauser effect spectroscopy and its relevance to poly(dA).poly(dT)

Conformational analysis from the pattern and intensities of cross-peaks in the two-dimensional nuclear Overhauser effect proton nmr spectra of the homopolymer, poly(dA) · poly(dT), and the analogous oligomer, d(AAAAAATTTTTT)₂, indicate that they both exist in the B-conformation. The conformation of the ApT/TpA junction in the oligomer is significantly different from the rest of the base pairs.     

Induction of oxidative cell damage by photo-treatment with zinc meta N-methylpyridylporphyrin

We have previously reported that isomeric Zn(II) N-methylpyridylporphyrins (ZnTM-2(3,4)-PyP^4 + ) can act as photosensitizers with efficacy comparable to that of hematoporphyrin derivative (HpD) in preventing cell proliferation and causing cell death in vitro. To better understand the biochemical basis of this activity, the effects of photo-activated ZnTM-3-PyP^4 +  on GSH/GSSG ratio, lipid peroxidation, membrane permeability, oxidative DNA damage, and the activities of SOD, catalase, glutathione reductase, and glutathione peroxidase were evaluated. Light exposure of ZnTM-3-PyP^4 + -treated colon adenocarcinoma cells caused a wide spectrum of oxidative damage including depletion of GSH, inactivation of glutathione reductase and glutathione peroxidase, oxidative DNA damage and peroxidation of membrane lipids. Cell staining with Hoechst-33342 showed morphological changes consistent with both necrotic and apoptotic death sequences, depending upon the presence of oxygen.     

Proton NMR studies of [N-MeCys3,N-MeCys7]TANDEM binding to DNA oligonucleotides: sequence-specific binding at the TpA site.

[N-MeCys3,N-MeCys7]TANDEM, an undermethylated analogue of Triostin A, contains two N-methyl groups on the cysteine residues only. Footprinting results showed that [N-MeCys3,N-MeCys7]TANDEM binds strongly to DNA rich in A.T residues [Low, C. M. L., Fox, K. R., Olsen, R. K., & Waring, M. J. (1986) Nucleic Acids Res. 14, 2015-2033]. However, it was not known whether specific binding of [N-MeCys3,N-MeCys7]TANDEM requires a TpA step or an ApT step. In 1:1 saturated complexes with the octamers [d(GGATATCC)]2 and [d(GGTTAACC)]2, [N-MeCys3,N-MeCys7]TANDEM binds to each octamer as a bis-intercalator bracketing the TpA step. The octadepsipeptide ring binds in the minor groove of the DNA. Analysis of sugar coupling constants from the phase-sensitive COSY data indicates that the sugar of the thymine in the TpA binding site adopts predominantly an N-type sugar conformation, while the remaining sugars on the DNA adopt an S-type conformation, as has been observed in other Triostin A and echinomycin complexes. The drug does not bind to the octamer [d(GGAATTCC)]2 as a bis-intercalator. Only weak nonintercalative binding is observed to this DNA octamer. These results show unambiguously that [N-MeCys3,N-MeCys7]TANDEM binds sequence specifically at TpA sites in DNA. The factors underlying the sequence specificity of [N-MeCys3,N-MeCys7]TANDEM binding to DNA are discussed.     

Effect of oxygen on radiation-induced DNA damage in isolated nuclei.

In intact mammalian cells, ionizing radiation causes substantially less damage to DNA in the absence of oxygen than in the presence of oxygen. In contrast, when DNA is isolated (usually from viruses) and irradiated in solution, the absence of oxygen does not lead to a decrease in damage unless low-molecular-weight thiols are also present. We investigated an intermediate condition: that of DNA irradiated in isolated nuclei. Using an HPLC-based assay of thiols with electrochemical detection, we have determined that the nuclear isolation procedure leads to the elimination of virtually all low-molecular-weight thiols (predominantly glutathione and cysteine). Thus it was our expectation that the thiol-depleted state would concurrently eliminate the OER, and thereby mimic the isolated DNA system, while retaining structural characteristics of chromosomal DNA. We evaluated radiation-induced DNA damage in isolated nuclei by measuring single-strand breaks using alkaline elution and by measuring double-strand breaks using neutral elution and pulsed-field gel electrophoresis. Despite the removal of low-molecular-weight thiol compounds, the oxygen dependence of radiation-induced damage more closely paralleled that of whole cells than that of DNA in solution. Thus damage of DNA irradiated in isolated nuclei is dependent on oxygen.     

Gliotoxin causes oxidative damage to plasmid and cellular DNA

The cytotoxic effects of gliotoxin (Müllbacher, A., and Eichner, R. D. (1984) Proc. Natl. Acad. Sci. U.S.A. 81, 3835-3837), a fungal secondary metabolite, and related epipolythiodioxopiperazines have been investigated using plasmid and eukaryotic DNA. Incubation of the dithiol derivative of these compounds with DNA and Fe3+ is sufficient to cause single- and double-stranded breaks as determined by neutral agarose gel electrophoresis. The disulfide form is inactive except in the presence of a suitable reducing agent, such as reduced glutathione, dithiothreitol, or reduced pyridine coenzymes. The autooxidation of these dithiols produces reducing equivalents as evidenced by (i) the production of H2O2 and (ii) the generation of thiobarbituric acid reactive products when incubated with deoxyribose. The latter process is inhibited by ethanol and desferrioxamine. The DNA damage is abrogated by metal chelators and catalase. We conclude that the antiproliferative action of gliotoxin may be caused by DNA damage effected by reactive oxygen species or other radicals generated through redox cycling.     

Binding of a Hoechst dye to d(CGCGATATCGCG) and its influence on the conformation of the DNA fragment

Hoechst dye 33258 is a planar drug molecule that binds to the minor groove of DNA, especially where there are a number of A.T base pairs. We have solved the structure of the Hoechst dye bound to the DNA dodecamer d(CGCGATATCGCG) at 2.3 A. This structure is compared to that of the same dodecamer with the minor-groove-binding drug netropsin bound to it, as well as to structures that have been solved for this Hoechst dye bound to a DNA dodecamer containing the central four base pairs with the sequence AATT. We find that the position of the Hoechst drug in this dodecamer is quite different from that found in the other dodecamer since it has an opposite orientation compared to the other two structures. The drug covers three of the four A.T base pairs and extends its piperazine ring to the first G.C base pair adjacent to the alternating AT segment. Furthermore, the drug binding has modified the structure of the DNA dodecamer. Other DNA dodecamers with alternating AT sequences show an alternation in the size of the helical twist between the ApT step (small twist) and the TpA step (large twist). In this structure the alternation is reversed with larger twists in the ApT steps than in the TpA step. In addition, there is a rotation of one of the thymine bases in the DNA dodecamer that is associated with hydrogen bonding to the Hoechst drug. This structure illustrates the considerable plasticity found in the DNA molecule when it binds to different planar molecules inserted into the minor groove.     

High-resolution crystal structure of the intramolecular d(TpA) thymine-adenine photoadduct and its mechanistic implications

A high-resolution crystal structure is reported for d(TpA)*, the intramolecular thymine–adenine photoadduct that is produced by direct ultraviolet excitation of the dinucleoside monophosphate d(TpA). It confirms the presence of a central 1,3-diazacyclooctatriene ring linking the remnants of the T and A bases, as previously deduced from heteronuclear NMR measurements by Zhao et al. (The structure of d(TpA)*, the major photoproduct of thymidylyl-(3′-5′)-deoxyadenosine. Nucleic Acids Res., 1996, 24, 1554–1560). Within the crystal, the d(TpA)* molecules exist as zwitterions with a protonated amidine fragment of the eight-membered ring neutralizing the charge of the internucleotide phosphate monoanion. The absolute configuration at the original thymine C5 and C6 atoms is determined as 5S,6R. This is consistent with d(TpA)* arising by valence isomerization of a precursor cyclobutane photoproduct with cis–syn stereochemistry that is generated by [2 + 2] photoaddition of the thymine 5,6-double bond across the C6 and C5 positions of adenine. This mode of photoaddition should be favoured by the stacked conformation of adjacent T and A bases in B-form DNA. It is probable that the primary photoreaction is mechanistically analogous to pyrimidine dimerization despite having a much lower quantum yield.     

Singlet oxygen-induced DNA damage

Singlet oxygen, hydrogen peroxide, hydroxyl radical and hydrogen peroxide are the reactive oxygen species (ROS) considered most responsible for producing oxidative stress in cells and organisms. Singlet oxygen interacts preferentially with guanine to produce 8-oxo-7,8-dihydroguanine and spiroiminodihydantoin. DNA damage due to the latter lesion has not been detected directly in the DNA of cells exposed to singlet oxygen. In this study, the singlet oxygen-induced lesion was isolated from a short synthetic oligomer after exposure to UVA radiation in the presence of methylene blue. The lesion could be enzymatically excised from the oligomer in the form of a modified dinucleoside monophosphate. Using liquid chromatography-tandem mass spectrometry (LC-MS/MS), the singlet oxygen lesion was detected in the form of modified dinucleoside monophosphates in double-stranded DNA and in the DNA of HeLa cells exposed to singlet oxygen. Pentamer containing the singlet oxygen-induced lesion and an isotopic label was synthesized as an internal standard for quantifying the lesion and served as well as for correcting for losses of product during sample preparation.     

Modification of high LET radiation-induced damage and its repair in yeast by hypoxia.

The lethal response of a diploid yeast strain BZ34 to densely ionizing radiations from the reaction 10B(n, alpha)7 Li was studied. The values for relative biological effectiveness (r.b.e.) and oxygen enhancement ratio (o.e.r.) for this radiation compare favourably with the data obtained with charged particles on the same strain of yeast. Recovery from potentially lethal damage was also studied by post-irradiation holding under non-nutrient conditions. In order to understand the role of oxygen in the recovery process, the investigation covered the following treatment regimens: (a) aerobic irradiation and aerobic holding (A-A), (b) aerobic irradiation and hypoxic holding (A-H), (c) hypoxic irradiation and hypoxic holding (H-H) and (d) hypoxic irradiation and aerobic holding (H-A). It has been found that the presence of oxygen is essential for recovery from the damage induced by both gamma rays and high linear energy transfer (LET) radiations. The extent of recovery was larger for gamma-induced damage than for damage induced by high LET radiation (alpha + 7Li) for the A-A condition. In the H-H condition, while only a slight recovery was seen for gamma-induced damage, it was totally absent for high LET damage. For the modality A-H, it was found that there is not recovery from the sparsely ionising gamma radiation-induced damage. The implications of these results for the treatment of malignant tumours by radiotherapy are briefly discussed.     

DNA sequence recognition by under-methylated analogues of triostin A.

Two new analogues of TANDEM (des-N-tetramethyl triostin A) have been synthesised in an effort to elucidate the molecular basis of DNA nucleotide sequence recognition in this series of compounds. Their binding preferences have been investigated by DNAase I footprinting and differential inhibition of restriction nuclease attack. The presence of a single N-methyl group on only one valine residue (in [N-MeVal4] TANDEM) abolishes the ability to recognise DNA, presumably because this antibiotic analogue has suffered an unfavourable conformational change in the depsipeptide ring. A bis-methylated analogue, [N-MeCys3, N-MeCys7]TANDEM, was found to interact quite strongly with DNA and afforded binding sites, rich in AT residues, identical to those of TANDEM. Footprinting with various DNA fragments of known sequence showed that this analogue recognises sequences containing the dinucleotide TpA, although we cannot exclude the possibility that it binds to ApT as well. [N-MeCys3, N-MeCys7]TANDEM inhibits cutting by RsaI, a restriction enzyme that recognises GTAC but not by Sau3AI which recognises GATC. This provides further supportive evidence that the ligand (and, by extension, TANDEM itself) prefers binding to sequences containing the dinucleotide step TpA.     

Chromium(VI)-mediated DNA damage: oxidative pathways resulting in the formation of DNA breaks and abasic sites

Inside cells chromium(VI) is activated to its ultimate carcinogenic form by reducing agents including glutathione (GSH) and ascorbate (AsA). The precise mechanism by which DNA damaging species are formed is unclear. In earlier in vitro work with isolated DNA we have shown that chromium(VI) in combination with GSH or AsA is able to induce similar numbers of single strand breaks and apurinic/apyrimidinic sites (AP-sites). Moreover, the formation of both lesions followed a similar temporal pattern. It is conceivable that the two forms of DNA damage arise from a common precursor lesion (e.g. hydrogen abstraction at C4' of the DNA sugar moiety) with a partitioning along two pathways, one yielding an AP-site, the other a single strand break (SSB) and a base propenal. The present study is intended to test this hypothesis by analysing whether oxidation products of deoxyribose can be formed in the presence of chromium(VI) and GSH or AsA. It was found that mixtures of chromium(VI) and GSH or AsA were able to oxidise 2-deoxyribose to yield malondialdehyde, which was detected by reaction with thiobarbituric acid. The characteristic pink chromogen, which forms upon reaction with thiobarbituric acid, was also observed with calf thymus DNA as the substrate. In both experimental systems the addition of catalase prevented the formation of deoxyribose breakdown products. Hydroxyl radicals did not seem to be important for the generation of DNA damage as the characteristic modified DNA bases could not be detected by using gas chromatography-mass spectrometry. These results lead us to conclude that the formation of SSB during the reductive conversion of chromium(VI) proceeds primarily via hydrogen abstraction from C4'. The observation that Fenton chemistry is not involved in these processes is intriguing and necessitates further research into the ways in which chromium can activate molecular oxygen to form DNA damaging species.     

DNA structure influences sequence specific cleavage by bleomycin.

We have examined the cleavage of several synthetic DNA sequences by iron(II)-bleomycin. We find that, although bleomycin cuts mixed sequence DNAs with a preference for GC = GT > GA >> GG, it efficiently cleaves regions of (AT)n cutting exclusively at ApT, not TpA. Isolated ApT steps show very little cleavage while blocks of three or more contiguous ATs are cut as efficiently as GpT. This cleavage is specific for (AT)n, since sequences of the type (TAA)n.(TTA)n and (ATT)n.(AAT)n are hardly cut at all. No cleavage is observed at ApC or CpA within sequences of the type (AC)n.(GT)n; regions of An.Tn are also not cut. Although the cobalt-bleomycin complex (which binds to but does not cleave DNA) yields good DNase I footprints at GT and GC sites, no footprints are observed within (AT)n, suggesting that although the cleavage reaction is efficient, the binding affinity is relatively weak. We propose a model in which bleomycin cleavage is determined by local DNA structure, while strong binding requires the presence of a guanine residue.     

Diastereomeric dinucleoside-methylphosphonates: determination of configuration with the 2-D NMR ROESY technique.

The determination of configuration at phosphorus in diastereomeric dinucleoside-methylphosphonates having the -O-P(= O)(-CH3)-O- internucleotide linkage with the NOE derived ROESY NMR technique is described for ApT, TpT, ApA, TpA and CpG. For this purpose ROE's from the P-CH3 group to the protons in the nearest neighbourhood were measured. These ROE's are different within diastereomeric pairs of a dimer enabling us to deduce the individual configuration. The validity of the method is proven in comparison with dimers of known configuration (ApT, TpT). Together with a recently published diastereoselective synthesis method a more homogeneous picture between physical properties and the corresponding configuration is provided. There is an improvement in our knowledge about the stereochemistry of these substances which could not be deduced from the data known before.