EPR Spin-trapping studies of radical damage to DNA

Abstract

EPR spectroscopy has been employed, in conjunction with spin-trapping [using 2-methyl-2-nitrosopropane dimer (MNP)] to study the attack of ˙OH (generated by the Fe²⁺–EDTA/H₂O₂ couple) on deoxyribonucleic acid (DNA) and its components. The results confirm that for pyrimidine deoxyribonucleosides and deoxyribonucleotides the alkenic C₅–C₆ double bond of the base moiety is the predominant site of attack (as with the pyrimidine bases, nucleosides and nucleotides) though weak signals from sugar–derived radicals are also observed. Both sugar and base radicals have also been detected for purines (both ribose and deoxyribose derivatives), providing evidence that rapid transfer occurs from the initially formed base radicals to the sugarphosphate backbone.

The spectra of the adducts from double-stranded (ds) and single-stranded (ss) DNA are found to vary with pH and ˙OH flux. With dsDNA at pH 7, an anisotropic spectrum due to a slowly tumbling adduct of a high molecular weight polymer radical-adduct is accompanied by a more isotropic signal due to a mobile, low molecular weight radical-adduct, the relative yield of which increases as the pH is lowered. Close similarities are observed between the spectra of dsDNA at pH 2 and ssDNA at pH 7, with a greater occurrence of strand-breakage under these conditions. Treatment of the partially-immobilised DNA adducts formed at pH 6–7 either with the enzyme DNase or with acid releases more mobile adducts enabling information to be obtained about sites of initial attack (at pyrimidine bases) and mechanisms of transfer of damage.