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Free-radical-induced formation of an 8,5'-cyclo-2'-deoxyguanosine moiety in deoxyribonucleic acid.

ABSTRACT: Isolation and identification of a novel .OH-induced product, namely an 8,5'-cyclo-2'-deoxyguanosine moiety, in DNA and 2'-deoxyguanosine are described. .OH radicals were generated in dilute aqueous solutions by gamma-irradiation. Analyses of 2'-deoxyguanosine and enzymic hydrolysates of DNA by gas chromatography-mass spectrometry (g.c.-m.s.) after trimethylsilylation showed the presence of 8,5-cyclo-2'-deoxyguanosine on the basis of its fragment ions. This product was isolated by h.p.l.c. Its u.v. and n.m.r. spectra taken were in agreement with the structure suggested by its mass spectrum. Exact masses of the typical ions from the mass spectrum of the trimethylsilyl derivative of this product were measured by high-resolution m.s. The values found were in excellent agreement with the theoretical mass derived from the suggested fragmentation patterns. Both (5'R)- and (5'S)-epimers of 8,5'-cyclo-2'-deoxyguanosine were observed. These two diastereomers were separated from each other by g.c. as well as by h.p.l.c. The assignment of the epimers was accomplished on the basis of the n.m.r. data. The formation of 8,5'-cyclo-2'-deoxyguanosine was suppressed by the presence of O2 in the solutions. The use of g.c.-m.s. with the selected-ion monitoring technique facilitated the detection of 8,5'-cyclo-2'-deoxyguanosine in DNA at radiation doses as low as 1 Gy. Its mechanism of formation probably involves hydrogen atom abstraction by .OH radicals from the C-5' of the 2'-deoxyguanosine moiety followed by intramolecular cyclization with the formation of a covalent bond between the C-5' and C-8 and subsequent oxidation of the resulting N-7-centred radical.

SUBMITTER: Dizdaroglu M

PROVIDER: S-EPMC1147122 | biostudies-other | 1986 Aug

REPOSITORIES: biostudies-other

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Project description:The mechanistic aspects of the methylation of guanine in DNA initiated by methyl radicals that are derived from the metabolic oxidation of some chemical carcinogens remain poorly understood. In this work, we investigated the kinetics and the formation of methylated guanine products by two methods: (i) the combination of *CH3 radicals and guanine neutral radicals, G(-H)*, and (ii) the direct addition of *CH3 radicals to guanine bases. The simultaneous generation of *CH3 and dG(-H)* radicals was triggered by the competitive one-electron oxidation of dimethyl sulfoxide (DMSO) and 2'-deoxyguanosine (dG) by photochemically generated sulfate radicals in deoxygenated aqueous buffer solutions (pH 7.5). The photolysis of methylcob(III)alamin to form *CH3 radicals was used to investigate the direct addition of these radicals to guanine bases. The major end products of the radical combination reactions are the 8-methyl-dG and N2-methyl-dG products formed in a ratio of 1:0.7. In contrast, the methylation of dG by *CH3 radicals generates mostly the 8-methyl-dG adduct and only minor quantities of N2-methyl-dG (1:0.13 ratio). The methylation of the self-complementary 5'-d(AACGCGAATTCGCGTT) duplexes was achieved by the selective oxidation of the guanines with carbonate radical anions in the presence of DMSO as the precursor of *CH3 radicals. The methyl-G lesions formed were excised by the enzymatic digestion and identified by LC-MS/MS methods using uniformly 15N-labeled 8-methyl-dG and N2-methyl-dG adducts as internal standards. The ratios of 8-methyl-G/N2-methyl-G lesions derived from the combination of methyl radicals with G(-H)* radicals positioned in double-stranded DNA or that with the free nucleoside dG(-H)* radicals were found to be similar. Utilizing the photochemical method and dipropyl or dibutyl sulfoxides as sources of alkyl radicals, the corresponding 8-alkyl-dG and N2-alkyl-dG adducts were also generated in ratios similar to those obtained with DMSO.

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Free-radical-induced formation of an 8,5'-cyclo-2'-deoxyguanosine moiety in deoxyribonucleic acid.

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