Effect of DNA Conformation on the Hydroxyl Radical-induced Formation of 8,5′-cyclopurine 2′-deoxyribonucleoside Residues in DNA

Abstract

Reactions of hydroxyl radicals with DNA form a variety of base and sugar products and 8,5'-cyclopurine 2'-deoxyribonucleoside residues in DNA. Here we report the effect of DNA conformation on the yields of 8,5'-cyclopurine 2'-deoxynucleosides and the ratios of their (5'R)- and (5'S)-diastereomers. Calf thymus DNA in native (double-stranded DNA) or heat-denatured form (single-stranded DNA) was exposed to hydroxyl radicals generated by ionizing radiation in nitrous oxide-saturated phosphate buffer. Doses ranging from 10 to 40 Gy were used to ensure low levels of damage to DNA and thus to preserve its secondary structure in experiments with double-stranded DNA (ds-DNA). After irradiation, DNA was hydrolysed enzymatically to 2'-deoxyribonucleosides. The hydrolysates were dried, trimethylsilylated, and analyzed by capillary gas chromatography-mass spectrometry with selected-ion monitoring. An internal standard was used for quantitative measurements and added to DNA samples prior to enzymatic hydrolysis. The yields of 8,5'-cyclo-2'-deoxyadenosine and 8,5'-cyclo-2'-deoxyguanosine in single-stranded DNA (ss-DNA) were higher than those in ds-DNA. The (5'R)-diastereomers of both compounds were found to predominate over their (5'S)-diastereomers in ss-DNA. In contrast, the yields of the (5'S)-diastereomers in ds-DNA were slightly higher than those of the (5'R)-diastereomers. The G values of 8,5'-cyclo-2'-deoxyadenosine in ss-DNA and ds-DNA were 0.042 and 0.025, respectively. Those of 8,5'-cyclo-2'-deoxyguanosine in ss-DNA and ds-DNA were 0.038 and 0.017, respectively.

Removal of oxygen free-radical-induced 5′,8-purine cyclodeoxynucleosides from DNA by the nucleotide excision-repair pathway in human cells

“…The cyPu lesions appear to be relatively abundant forms of DNA damage after exposure to reactive oxygen species, introduced at 20-30% of the levels of the major lesions 8-hydroxyguanine and thymine glycol, although the relative rates of formation vary with experimental conditions (11,23). Until recently, it has not been feasible to synthesize oligodeoxyribonucleotide substrates containing this unique type of cyclic lesion.…”

“…Discussion cyPu formation in DNA as a consequence of hydroxyl radical damage in vivo and in vitro has been demonstrated by gas chromatography͞mass spectrometry analysis and by HPLC of hydrolysates (8)(9)(10)(11). The cyPu lesions appear to be relatively abundant forms of DNA damage after exposure to reactive oxygen species, introduced at 20-30% of the levels of the major lesions 8-hydroxyguanine and thymine glycol, although the relative rates of formation vary with experimental conditions (11,23).…”

“…1) with generation of the 5ЈR diastereoisomer being predominant to that of the 5ЈS analogue in single-stranded DNA, whereas both diastereoisomers are produced in similar amounts in double-stranded DNA. Cyclodeoxyadenosines are the major lesions, being more abundant than cyclodeoxyguanosines (8)(9)(10)(11). Until recently, the difficult synthesis of oligonucleotides containing cyPu residues had not been achieved.…”

Effect of Oxidative Stress on DNA Repairing Genes

“…A unique reaction of the C5'-centered sugar radical is the addition to the C8-position of the purine ring of the same nucleoside. This intramolecular cyclisation followed by oxidation yields 8, 5'-cyclopurine-2'-deoxynucleosides [Dizdaroglu, 1986, Dirksen et al, 1988. Both 5'R-and 5'S-diastereomers of 8, 5'-cyclo-2'-deoxyguanosine (cyclo-dG) and 8, 5'-cyclo-2'-deoxyadenosine (cyclo-dA) are generated in DNA [Dizdaroglu, 1986, Dirksen et al, 1988.…”

Oxidative Processes and Antioxidative Metaloenzymes

“…Dialuric acid is oxidized in the presence of O2 to alloxan [44]. C5-OH-6-hydroperoxide gives rise to trans-1-carbamoyl-2-oxo-4,5-dihydroxyimidazolidine as a major product of cytosine [34,188] but as a minor product from DNA [43].…”