DNA damage induced via independent generation of the radical resulting from formal hydrogen atom abstraction from the C1′-position of a nucleotide

Background: Deoxyribonucleotide radicals resulting from formal C1'-hydrogen atom abstraction are important reactive intermediates in a variety of DNA-damage processes. The reactivity of these radicals can be affected by the agents that generate them and the environment in which they are produced. As an initial step in determining the factors that control the reactivity of these important radical species, we developed a mild method for their generation at a defined site within a biopolymer. Results: Irradiation of oligonucleotides containing a photolabile nucleotide produced C1'-DNA radicals, In the absence of potential reactants other than O-2, approximately 90% of the damage events involve formation of alkaline-labile lesions, with the remainder resulting in direct strand breaks. The ratio of alkaline-labile lesions to direct strand breaks (similar to 9:1) is independent of whether the radical is generated in single-stranded DNA or double-stranded DNA, Strand damage is almost completely quenched under anaerobic conditions in the presence of low thiol concentrations. Competition studies with O-2 indicate that the trapping rate of C1'-DNA radicals by beta-mercaptoethanol is -1.1 x 10(7) M-1 s(-1). Conclusions: The mild generation of the C1'-DNA radical in the absence of exogenous oxidants makes it possible to examine their intrinsic reactivity. In the absence of other reactants, the formation of direct strand breaks from C1'-radicals is, at most, a minor pathway. Competition studies between beta-mercaptoethanol and O-2 indicate that significantly higher thiol concentrations than those in vivo or some means of increasing the effective thiol concentration near DNA are needed for these reagents to prevent the formation of DNA lesions arising from the C1'-radical under aerobic conditions.