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, trimethylsilyated, 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.