Photooxidation of d(TpG) by riboflavin and methylene blue. Isolation and characterization of thymidylyl-(3',5')-2-amino-5-[(2-deoxy-beta-D- erythro-pentofuranosyl)amino]-4H-imidazol-4-one and its primary decomposition product thymidylyl-(3',5')-2,2-diamino-4-[(2-deoxy-beta-D- erythro-pentofuranosyl)amino]-5(2H)-oxazolone.

The major initial product of riboflavin- and methylene blue-mediated photosensitization of 2'-deoxyguanosine (dG) in oxygen-saturated aqueous solution has previously been identified as 2-amino-5-[(2-deoxy-beta-D-erythro-pentofuranosyl)amino] 4H-imidazol-4-one (dlz). At room temperature in aqueous solution dlz decomposes quantitatively to 2,2-diamino-4-[(2-deoxy-beta-D-erythro- pentofuranosyl)amino]-5(2H)-oxazolone (dZ). The data presented here show that the same guanine photooxidation products are generated following riboflavin- and methylene blue-mediated photosensitization of thymidylyl-(3',5')-2'-deoxyguanosine [d(TpG)]. As observed for the monomers, the initial product, thymidylyl-(3',5')-2-amino-5-[(2-deoxy- beta-D-erythro-pentofuranosyl)amino]-4H-imidazol-4-one [d(Tplz)], decomposes in aqueous solution at room temperature to thymidylyl-(3',5')-2,2-diamino-4- [(2-deoxy-beta-D-erythro-pentofuranosyl)amino]-5(2H)-oxazolone [d(TpZ)]. Both modified dinucleoside monophosphates have been isolated by HPLC and characterized by proton NMR spectrometry, fast atom bombardment mass spectrometry, chemical analyses and enzymatic digestions. Among the chemical and enzymatic properties of these modified dinucleoside monophosphates are: (i) d(Tplz) and d(TpZ) are alkali-labile; (ii) d(Tplz) reacts with methoxyamine, while d(TpZ) is unreactive; (iii) d(Tplz) is digested by snake venom phosphodiesterase, while d(TpZ) is unaffected; (iv) relative to d(TpG), d(TpZ) and d(Tplz) are slowly digested by spleen phosphodiesterase; (v) d(Tplz) and d(TpZ) can be 5'-phosphorylated by T4 polynucleotide kinase. The first observation suggests that dlz and dZ may be responsible for some of the strand breaks detected following hot piperidine treatment of DNA exposed to photosensitizers.     

Measurement of the main photooxidation products of 2'-deoxyguanosine using chromatographic methods coupled to mass spectrometry

Analytical methods were developed for the measurement of the main photooxidation products of 2'-deoxyguanosine (dGuo), arising from either the type I (electron transfer) or the type II (singlet oxygen) photosensitization mechanism. Oxidation of dGuo by a type I mechanism leads to the predominant formation of 2, 2-diamino-5-[2-deoxy-beta-d-erythro-pentofuranosyl)amino]-5(2H)-oxazo lone. On the other hand, the two 4R and 4S diastereomers of 4-hydroxy-8-oxo-4,8-dihydro-2'-deoxyguanosine are the main singlet oxygen-mediated dGuo oxidation products. The modified nucleosides were measured by either gas chromatography coupled to mass spectrometry after silylation of the sample or by high-performance liquid chromatography associated to tandem mass spectrometry. In order to improve the accuracy of the assays, isotopically labeled internal standards were synthesized for an isotope dilution mass spectrometry quantitation. The methods were successfully applied to the measurement of methylene blue- and riboflavin-mediated 2'-deoxyguanosine photooxidation reactions. The advantages of the two above-mentioned methods are discussed on the basis of comparative sensitivity and accuracy.     

Peroxynitrite Mediated Oxidation of Purine Bases of Nucleosides and Isolated DNA

Reaction of nitric oxide with superoxide anion produces the highly reactive species peroxynitrite (ONOO^?). This compound has been shown to be a strong oxidant of lipids and proteins. However, no data are available on its effect on DNA, with the exception of the induction of strand breaks. We report the result of studies on the reactions of peroxynitrite with the adenine and guanine moieties of nucleosides and isolated DNA. The samples were analyzed for 8-oxo-7,8-dihydro-2′-deoxyguano-sine (8-oxo-dGuo), 2,2-diamino-4–[(2-deoxy-β-D-erythro-pentofuranosyl)amino]-5–(2H)-oxazolone (oxazolone) and 8-oxo-7,8-dihydro-2′-deoxyadenosine (8-oxo-dAdo). The effects of peroxynitrite treatment were compared with those of ionizing radiation in aerated aqueous solution, chosen as a source of hydroxyl radicals. At the nucleoside level, both oxidizing conditions led to the formation of oxazolone and 8-oxo-dAdo. In addition, evidence was provided for the formation of the 4R* and 4S* diastereoisomers of 4-hydroxy-8-oxo-4,8-dihydro-2′-deoxyguanosine. The latter dGuo oxidation products were chosen as markers of the release of singlet oxygen (¹O₂) upon reaction of peroxynitrous acid with hydrogen peroxide. Oxidation of purine bases was then studied within isolated DNA. A significant increase in the level of 8-oxp-dGuo, oxazolone and 8-oxo-dAdo was observed within double stranded DNA upon exposure to γ-radiation. Oxazolone and 8-oxo-dAdo were formed upon peroxynitrite treatment but no significant increase in the amount of 8-oxo-dGuo was detected. These results showed that peroxynitrite exhibits oxidizing properties toward purine moieties both in nucleosides and isolated DNA. However, the significant differences in the oxidative damage distribution within DNA observed after exposure to γ radiation by comparison with peroxynitrite treatment questions the involvement of hydroxyl radicals as the main oxidizing species released by decomposition of peroxynitrous acid.     

Singlet oxygen induces oxidation of cellular DNA

The aim of the present work was to evaluate the potential for (1)O(2) to induce oxidation of cellular DNA. For this purpose cells were incubated in the presence of a water-soluble endoperoxide whose thermal decomposition leads to the formation of singlet oxygen. Thereafter, DNA was extracted and the level of several modified DNA bases was determined by HPLC analysis coupled to a tandem mass spectrometric detection. A significant increase in the level of 8-oxo-7,8-dihydro-2'-deoxyguanosine was observed upon incubation of the cells with the chemical generator of (1)O(2), whereas the level of the other DNA bases measured remained unchanged. To demonstrate that singlet oxygen is directly involved in the formation of 8-oxo-7, 8-dihydro-2'-deoxyguanosine, the corresponding (18)O-labeled endoperoxide was used. Incubation of the cells with such a generator of (18)O-labeled singlet oxygen results in the formation of (18)O-labeled 8-oxo-7,8-dihydro-2'-deoxyguanosine in the nuclear DNA. This result clearly demonstrates that singlet oxygen, when released within cells, is able to directly oxidize cellular DNA.     

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.     

18O]-labeled singlet oxygen as a tool for mechanistic studies of 8-oxo-7,8-dihydroguanine oxidative damage: detection of spiroiminodihydantoin,imidazolone and oxazolone derivatives

A water-soluble [18O]-labeled endoperoxide derived from N,N'-di(2,3-dihydroxypropyl)-1,4-naphthalene-dipropanamide (DHPN18O2) has been shown to act as a clean chemical source of [18O]-labeled molecular singlet oxygen. This allows the assessment of the singlet oxygen (1O2) reactivity toward biological targets such as DNA. The present work focuses on the qualitative identification of the main 1O2-oxidation products of 8-oxo-7,8-dihydro-2'-deoxyguanosine, which was achieved using high performance liquid chromatography coupled to electrospray ionization tandem mass spectrometry (HPLC-ESI-MS/MS). Thus, the [18O]-labeled and unlabeled imidazolone and oxazolone, together with the diastereoisomeric spiroiminodihydantoin nucleosides, were detected as the main degradation products. In addition, a modified nucleoside that exhibits similar features as those of the oxidized guanidinohydantoin molecule was detected. Our data strongly suggest that the imidazolone and oxazolone nucleosides are generated via the rearrangement of an unstable 5-hydroperoxide intermediate. Interestingly, the combined use of appropriate tools, including isotopically labeled singlet oxygen and the high- resolution HPLC-ESI-MS/MS technique, has allowed to shed new light on the 1O2-mediated oxidation reactions of guanine DNA components.     

Singlet oxygen induces oxidation of cellular DNA

The aim of the present work was to evaluate the potential for (1)O(2) to induce oxidation of cellular DNA. For this purpose cells were incubated in the presence of a water-soluble endoperoxide whose thermal decomposition leads to the formation of singlet oxygen. Thereafter, DNA was extracted and the level of several modified DNA bases was determined by HPLC analysis coupled to a tandem mass spectrometric detection. A significant increase in the level of 8-oxo-7,8-dihydro-2'-deoxyguanosine was observed upon incubation of the cells with the chemical generator of (1)O(2), whereas the level of the other DNA bases measured remained unchanged. To demonstrate that singlet oxygen is directly involved in the formation of 8-oxo-7, 8-dihydro-2'-deoxyguanosine, the corresponding (18)O-labeled endoperoxide was used. Incubation of the cells with such a generator of (18)O-labeled singlet oxygen results in the formation of (18)O-labeled 8-oxo-7,8-dihydro-2'-deoxyguanosine in the nuclear DNA. This result clearly demonstrates that singlet oxygen, when released within cells, is able to directly oxidize cellular DNA.     

Photosensitized DNA damage induced by NADH: site specificity and mechanism

Increasing evidence reveals the carcinogenicity of UVA radiation. We demonstrated that UVA-irradiated NADH induced damage to (32)P-labeled DNA fragments obtained from the p53 gene in the presence of Cu(II). Formamidopyrimidine glycosylase (Fpg)-sensitive lesions were formed at guanine residues, whereas piperidine-labile lesions occurred frequently at thymine residues. Formation of 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG), upon UVA exposure in the presence of Cu(II), increased depending on NADH concentration. Catalase and bathocuproine, a Cu(I)-specific chelator, inhibited the DNA damage, suggesting the involvement of reactive species derived from H(2)O(2) and Cu(I). UVA-irradiated riboflavin induced DNA cleavage through electron transfer at 5' guanine of the 5'-GG-3' sequence with both Fpg and piperidine treatments; Fpg induced less cleavage at the guanine residues than piperidine. These results imply that NADH may participate as an endogenous photosensitizer in UVA carcinogenesis via H(2)O(2) generation, producing metal-mediated mutagenic lesions such as 8-oxodG.     

Selective hydrolysis of damaged DNA by nuclease P1

The turnover rates for hydrolysis by nuclease P1 of the 16 unmodified dideoxynucleoside monophosphates were measured. In addition, the turnover rates were measured in a variety of dideoxynucleoside monophosphates containing free radical-induced base modifications. The modified bases included cis-5,6-dihydroxy-5,6-dihydrothymine (thymine glycol), 5,6-dihydrothymine, 5-hydroxymethyuracil, 8-hydroxyguanine, 5-hydroxy-5-methylhydantoin and the formamido remnant which can be derived from either a thymine or a cytosine base. The turnover rate for dinucleoside monophosphates containing 4,8-dihydro-4-hydroxy-8-oxo-guanine modifications, which are induced by singlet oxygen, were also measured. A model was devised for the hydrolysis of DNA by nuclease P1 which uses the observed turnover rates as parameters. The model predicts the abundance of monomers and dimers as hydrolysis proceeds. Whereas the level of monomers increases monotonically, the level of each dimer first increases and then falls off. There are advantages to phosphorylating dimers, as compared with monomers, using polynucleotide kinase. Consequently this model may be of interest in connection with ³²P-postlabeling applied to the measurement of DNA damage in nuclease P1 partial hydrolysates of DNA.     

Mechanism of site-specific DNA damage induced by ozone

Ozone has been shown to induce lung tumors in mice. The reactivity of ozone with DNA in an aqueous solution was investigated by a DNA sequencing technique using 32P-labeled DNA fragments. Ozone induced cleavages in the deoxyribose-phosphate backbone of double-stranded DNA, which were reduced by hydroxyl radical scavengers, suggesting the participation of hydroxyl radicals in the cleavages. The ozone-induced DNA cleavages were enhanced with piperidine treatment, which induces cleavages at sites of base modification, but the inhibitory effect of hydroxyl radical scavengers on the piperidine-induced cleavages was limited. Main piperidine-labile sites were guanine and thymine residues. Cleavages at some guanine and thymine residues after piperidine treatment became more predominant with denatured single-stranded DNA. Exposure of calf thymus DNA to ozone resulted in a dose-dependent increase of the 8-oxo-7,8-dihydro-2'-deoxyguanosine formation, which was partially inhibited by hydroxyl radical scavengers. ESR studies using 5,5-dimethylpyrroline-N-oxide (DMPO) showed that aqueous ozone produced the hydroxyl radical adduct of DMPO. In addition, the fluorescein-dependent chemiluminescence was detected during the decomposition of ozone in a buffer solution and the enhancing effect of D2O was observed, suggesting the formation of singlet oxygen. However, no or little enhancing effect of D2O on the ozone-induced DNA damage was observed. These results suggest that DNA backbone cleavages were caused by ozone via the production of hydroxyl radicals, while DNA base modifications were mainly caused by ozone itself and the participation of hydroxyl radicals and/or singlet oxygen in base modifications is small, if any. A possible link of ozone-induced DNA damage to inflammation-associated carcinogenesis as well as air pollution-related carcinogenesis is discussed.     

Synthesis and enzymatic processing of oligodeoxynucleotides containing tandem base damage.

Several studies have shown that ionizing radiation generates a wide spectrum of lesions to DNA including base modifications, abasic sites, strand breaks, crosslinks and tandem base damage. One example of tandem base damage induced by @OH radical inX-irradiated DNA oligomers is N -(2-deoxy-beta-d- erythro -pentofuranosyl)-formylamine/8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodGuo). In order to investigate the biological significance of such a tandem lesion, both 8-oxo-7,8-dihydroguanine and formylamine were introduced into synthetic oligonucleotides at vicinal positions using the solid phase phosphoramidite method. For this purpose, a new convenient method of synthesis of 8-oxodGuo was developed. The purity and integrity of the modified synthetic DNA fragments were assessed using different complementary techniques including HPLC, polyacrylamide gel electrophoresis, electrospray and MALDI-TOF mass spectrometry. The piperidine test applied to the double modified base-containing oligonucleotides revealed the high alkaline lability of formylamine in DNA. In addition, various enzymatic experiments aimed at determining biochemical features of such multiply damaged sites were carried out using the synthetic substrates. The pro-cessing of the vicinal lesions by nuclease P1, snake venom phosphodiesterase, calf spleen phospho-diesterase and repair enzymes including Escherichia coli endonuclease (endo) III and Fapy-glycosylase was studied and is reported.     

Site-specific targeting of aflatoxin adduction directed by triple helix formation in the major groove of oligodeoxyribonucleotides.

The targeted adduction of aflatoxin B1- exo -8,9-epoxide (AFB1- exo -8,9-epoxide) to a specific guanine within an oligodeoxyribonucleotide containing multiple guanines was achieved using a DNA triplex to control sequence selectivity. The oligodeoxyribonucleotide d(AGAGAAGATTTTCTTCTCTTTTTTTTCTCTT), designated '3G', spontaneously formed a triplex in which nucleotides C27*G2*C18 and C29*G4*C16 formed base triplets, and nucleotides G7*C13formed a Watson-Crick base pair. The oligodeoxyribonucleotide d(AAGAAATTTTTTCTTTTTTTTTTCTT), designated '1G', also formed a triplex in which nucleotides C24*G3*C24 formed a triplet. Reaction of the two oligodeoxyribonucleotides with AFB1-exo-8,9-epoxide revealed that only the 3G sequence formed an adduct, as determined by UV absorbance and piperidine cleavage of the 5'-labeled adduct, followed by denaturing polyacrylamide gel electrophoresis. This site was identified as G7by comparison to the guanine-specific cleavage pattern. The chemistry was extended to a series of nicked bimolecular triple helices, constructed from d(AAAGGGGGAA) and d(CnTTCTTTTTCCCCCTTTATTTTTTC5-n) (n = 1-5). Each oligomer in the series differed only in the placement of the nick. Reaction of the nicked triplexes with AFB1- exo -8,9-epoxide, piperidine cleavage of the 5'-labeled adduct, followed by denaturing polyacrylamide gel electrophoresis, revealed cleavage corresponding to the guanine closest to the pyrimidine strand nick. By using the appropriate pyrimidine sequence the lesion was positioned within the purine strand.     

Identification of the main oxidation products of 8-methoxy-2'-deoxyguanosine by singlet molecular oxygen

It is now well established that oxidation of 2'-deoxyguanosine (dGuo) in DNA by singlet molecular oxygen [O2 (1Delta(g))] produces 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodGuo), whereas the main degradation products of free dGuo in aqueous solution have been identified as the two diastereomers of spiroiminodihydantoin nucleoside. Interestingly, O2 (1Delta(g))-mediated oxidation of free 8-oxodGuo gives rise to a pattern of degradation products that is different from that observed when the nucleoside is inserted into DNA. The reasons for these differences and the mechanisms involved in the oxidation reactions are not yet completely understood for either dGuo or 8-oxodGuo, either free or within DNA. In the present work, we report a study of the reaction of O2 (1Delta(g)) toward a modified nucleoside, 8-methoxy-2'-deoxyguanosine (8-MeOdGuo), either free or incorporated into an oligonucleotide. The reason for the choice of 8-MeOdGuo as a chemical model to study in more detail the oxidation pathways of 8-oxodGuo or, more precisely, of the tautomeric 8-hydroxy-2'-deoxyguanosine was dictated by the fact that only the 7,8-enolic tautomer is present in the molecule. The thermolysis of an endoperoxide of a naphthalene derivative as a clean chemical source of 18O-labeled O2 (1Delta(g)) was used to oxidize 8-MeOdGuo. The main O2 (1Delta(g)) oxidation products that were separated and analyzed by HPLC coupled to tandem mass spectrometry were identified as the 2'-deoxyribonucleoside derivatives of 2,2,4-triamino-5-(2H)oxazolone, 2,5-diamino-4H-imidazol-4-one together with the methyl-substituted derivatives of spiroiminodihydantoin, oxidized iminoallantoin and urea. On the other hand, O2 (1Delta(g)) oxidation of 8-MeOdGuo-containing oligonucleotide generated imidazolone as the predominant degradation product. These results provided new mechanistic insights into the reactions of O2 (1Delta(g)) with purine nucleosides.     

Reaction of acid-activated mitomycin C with calf thymus DNA and model guanines: elucidation of the base-catalyzed degradation of N7-alkylguanine nucleosides

Mitomycin C (MC, 1) forms covalent adducts under acidic activating conditions (pH approximately 4) with deoxyguanosine, d(GpC), and guanine residues of calf thymus DNA. In the case of deoxyguanosine, five adducts arise from a common precursor, N7-(2' beta, 7'-diaminomitosen-1'-yl)-2'-deoxyguanosine (10a; not isolated), which hydrolyzes spontaneously via two pathways: scission of the glycosidic bond to form N7-(2' beta, 7'-diaminomitosen-1' alpha-yl)guanine (5) and its 1' beta-isomer (6) and imidazolium ring opening to generate three 2,6-diamino-4-hydroxy-5-(N-formyl-2' beta, 7'-diaminomitosen-1' beta-yl)pyrimidine (FAPyr) derivatives that are substituted at N6 by isomeric 2'-deoxyribose units [i.e., 1' beta-furanose (7), 1' alpha-furanose (8), and 1' beta-pyranose (9)]. The structures of 5-9 were determined by spectroscopic methods. The same five adducts were obtained from d(GpC), but only the guanine adducts 5 and 6 were formed in DNA. Adducts 7-9 interconvert during high-performance liquid chromatography (HPLC). The unexpected isomerization of the deoxyribose moiety of the initially formed 1' beta-furanose adduct 7 to those of 8 and 9 occurs upon imidazolium ring opening, as discerned by the course of imidazolium cleavage of the simple models N7-ethyl- and N7-methylguanosine and N7-methyl-2'-deoxyguanosine. All ring-opened N7-alkylguanosine derivatives studied here exist as a mixture of distinct N-formyl rotamers, manifested by multiple interconverting peaks on HPLC and in the 1H NMR spectra. In the UV spectra of such derivatives, a new and diagnostic maximum at 218 nm (at pH 7) is observed. Acid-activated MC is found to alkylate preferentially the Gua-N7 position in deoxyguanosine or d(GpC), in contrast to reductively activated MC, which preferentially alkylates the Gua-N2 position. This finding is explained by the different electronic structures of acid- and reduction-activated MC. In DNA, the N7 specificity of acid-activated MC is partially offset by steric factors.     

Boron-containing oligodeoxyribonucleotide 14mer duplexes: enzymatic synthesis and melting studies.

A set of three 14mer oligodeoxyribonucleotides of sequence d(5'-CTATGGCCTCAG*CT-3'/3'-GATACCGGAGTCGA-5') containing G* variants either as 2'-deoxyguanosine phosphate (unmodified), N7-cyanoborane 2'-deoxyguanosine phosphate (base-modified) or 2'-deoxyguanosine boranophosphate (backbone-modified) were synthesized by template-directed primer extension. Both the N7-cyanoborane 2'-deoxyguanosine triphosphate and 2'-deoxyguanosine alpha-boranotriphosphate nucleotides are good substrates for Sequenase. We infer that a single Sp boranophosphate linkage (which has a stereochemistry equivalent to the corresponding Rp thiophosphate analog) is formed in the backbone-modified 14mer. Thermally induced helix-coil transitions were monitored for the hybridized duplexes using UV and circular dichroism (CD) spectroscopy. The CD spectra of the two types of boron-modified hybrids closely resemble the unmodified parent duplex, forming B-type helices in 150 mM NaCl, 1 mM EDTA, 10 mM phosphate, pH 7.4, buffer. UV melting results indicate that both hybrids have stabilities comparable with the parent duplex as measured by Tm or delta G degree 25. These studies indicate that singly modified base- or backbone-boronated DNA are good analogs of normal DNA.     

Photodynamic guanine modification by hematoporphyrin is specific for single-stranded DNA with singlet oxygen as a mediator

Photodynamic modification of DNA by hematoporphyrin (Hp) was characterized by the DNA sequencing technique using 32P-labeled DNA fragments, and the reaction mechanism was investigated by ESR spectroscopy. Mild photodynamic treatment of single-stranded DNA with Hp induced an alteration of guanine residues, and subsequent treatment with piperidine led to chain cleavages at each guanine residue. On the other hand, methylene blue plus light modified the guanine residues in both single-stranded and double-stranded DNA. ESR studies using 2,2,6,6-tetramethylpiperidine and 2,2,6,6-tetramethyl-4-piperidone as singlet oxygen traps demonstrated that Hp plus light produced almost the same amount of singlet oxygen as methylene blue plus light and that the photochemically generated singlet oxygen reacts significantly with guanylate but only slightly with other mononucleotides. An ESR spin destruction method revealed that photoexcited Hp generated porphyrin radical, but guanylate did not react with this radical. These results indicate that photoexcited Hp reacts with oxygen to generate singlet oxygen which oxidizes the guanine residues of single-stranded DNA and that the difference in photoreactivities of DNA with Hp and methylene blue may be explained in terms of the structural difference in their intercalating abilities.     

Analysis of DNA damage at the dinucleoside monophosphate level: application to the formamido lesion.

The dinucleoside monophosphates d(TpG), d(TpC), and d(TpT) were X-irradiated in oxygenated solution. In each case the modification of the dinucleoside in which the thymine base is degraded to a formamido remnant was observed as a principal product. The hydrolysis of the phosphoester bond of formamido-modified dinucleosides is much slower than that of the corresponding unmodified dinucleosides. This effect is also observable in the hydrolysis of irradiated DNA, where hydrolysis by nuclease P1 (plus acid phosphatase) generates the modified dinucleosides d(TFpN), TF being the modified thymidine. The total yield of the formamido lesion in all its forms, d(TFpN), exceeds the yield of any other base modification.     

Formation of a diimino-imidazole nucleoside from 2'-deoxyguanosine by singlet oxygen generated by methylene blue photooxidation

Singlet oxygen ((1)O(2)) is capable of inducing genotoxic, carcinogenic and mutagenic effects. It has previously been reported that the reaction of (1)O(2) with 2'-deoxyguanosine, which is a major target of (1)O(2) among the DNA constituents, leads to formation of various oxidized products including 8-oxo-7,8-dihydro-2'-deoxyguanosine and spiroiminodihydantoin, amino-imidazolone and diamino-oxazolone nucleosides. In addition to these products, we report that a novel diimino-imidazole nucleoside, 2,5-diimino-4-[(2-deoxy-beta-D-erythro-pentofuranosyl)amino]-2H,5H-imidazole (dD), is formed by reaction of 2'-deoxyguanosine with (1)O(2) generated by irradiation with visible light in the presence of methylene blue under aerobic conditions. Its identification is based on identical chromatographic and spectroscopic data with an authentic compound, which we recently isolated and characterised from the reaction mixture of 2'-deoxyguanosine with reagent HOCl and a myeloperoxidase-H(2)O(2)-Cl(-) system. The yield of dD was increased by D(2)O and decreased by azide. dD was not generated from 8-oxo-7,8-dihydro-2'-deoxyguanosine. These results indicate that dD is generated by (1)O(2) directly from 2'-deoxyguanosine, but not via 8-oxo-7,8-dihydro-2'-deoxyguanosine. dD may play a role in the genotoxicity of singlet oxygen in cells.     

Oxidative DNA damage photo-induced by 3-carbethoxypsoralen and other furocoumarins. Mechanisms of photo-oxidation and recognition by repair enzymes

DNA photosensitization by several furocoumarins (including 3-carbethoxypsoralen (3-CPs), 8-methoxypsoralen (8-MOP), 5-methoxypsoralen (5-MOP) and angelicin was investigated by using DNA sequencing methodology. 3-CPs induces photo-oxidation of guanine residues leading to alkali-labile sites in DNA (revealed by hot piperidine), whereas 8-MOP, 5-MOP and angelicin do not. There is a preferential photo-oxidation of G when located on the 5' side of GG doublets, likely to reflect a better accessibility of the G moiety in such a context. Mechanisms operating via both radicals (type I) and singlet oxygen (type II) are involved in the photo-oxidation of G residues by 3-CPs. Photo-oxidized G residues are produced independently of the formation of photoadducts, and scavengers of singlet oxygen or radicals do not inhibit photobinding of 3-CPs to DNA. This leads us to propose that covalent photoadducts arise from the intercalated excited sensitizer molecules, whereas G photo-oxidations are produced either by electron transfer reactions involving bound 3-CPs or by energy transfer to molecular oxygen, thereby producing singlet oxygen that subsequently reacts with guanine bases. Quantification of both types of DNA lesions indicated that in vitro photo-oxidized G residues are produced in DNA by 3-CPs plus ultraviolet light at least to the same extent as photoadducts, under our conditions. A calf thymus redoxyendonuclease, equivalent to the endonuclease III of Escherichia coli, specific for oxidative DNA damages, recognizes and cleaves DNA at sites of photo-oxidized G residues. The extent of the cleavage by this enzyme was close to that observed by hot piperidine and followed the amount of photo-oxidized G residues produced when the lifetime of excited oxygen species is modified. The redoxyendonuclease did not incise DNA treated with 8-MOP, 5-MOP or angelicin plus ultraviolet light. The exonuclease III and endonuclease IV of E. coli also involved in the repair of oxidative DNA damage, convert the replicative form I of 3-CPs-treated DNA to replicative form II. This suggests that the lesions recognized by these enzymes are apurinic-like lesions. In view of the low toxicity and mutagenicity of 3-CPs, DNA photo-oxidation products induced by the photodynamic effect of 3-CPs are likely to be efficiently taken care of by the DNA repair system(s). It is clear that 3-CPs photo-induces several classes of DNA damage, including oxidative damage.(ABSTRACT TRUNCATED AT 400 WORDS)     

Palindromic oligonucleotides containing 7-deaza-2''-deoxyguanosine: solid-phase synthesis of d[(p)GG*AATTCC] octamers and recognition by the endodeoxyribonuclease EcoRI.

Octadeoxynucleotides with the sequence d[(p)GG*AATTCC] have been prepared by solid-phase synthesis employing regular and base-modified phosphoramidites. These oligomers which contain an isosterically altered recognition sequence of the endodeoxyribonuclease Eco RI form duplexes under appropriate salt conditions. Since G* can represent 7-deaza-2'-deoxyguanosine the oligomers were used as probes to study their cleavage by the endodeoxyribonuclease Eco RI. The enzymatic hydrolysis of the modified octamer was strongly decreased compared to the regular DNA-fragment. This shows that guanine N-7 located at the cleavage site is important for the recognition process by the enzyme. The residual enzymatic activity is discussed on the basis of reduced specificity towards the recognition fragment. The fact that this cleavage occurs already under regular conditions indicates that the process described here bases on an intrinsic property of the oligomer and is different from the star activity.