Oxidation of nucleic acids occurs at both the nucleobase and the sugar moieties. In this work, we explore an example of each type of oxidation. The RNA strand scission induced by hydroxyl radical attack has been used as a model to study the effects of γ-radiolysis on DNA, and as a reporter for the solvent accessible surface area in RNA footprinting. While less than 20% of the hydroxyl radicals react with the sugar moiety, the observation of significantly more cleavage indicates that there is an indirect pathway to produce strand scission from a nucleobase radical. We site-specifically incorporated a photo-labile synthetic precursor of the 5,6-dihydrouridin-6-y1 radical into various RNA oligonucleotides and analyzed the photolysis products. The C6-radical abstracted H2' from either its own sugar (intranucleotidyl) or the 5'-flanking sugar (internucleotidyl) in an oxygen-dependent and secondary-structure-dependent manner with yields as high as 40% cleavage after 4 h. The rate of strand scission was measured by competition with thiol. The observed rate of 10-30 s^-1 is consistent with rate-limiting H-atom abstraction. The photolysis products were characterized by PAGE mobility and by MALD1-TOF mass spectrometry. Possible mechanisms for the generation of the observed products are presented.

DNA oxidation has received considerably more attention due to the impact of oxidized DNA lesions on cells. The C4'-oxidized abasic lesion (C4-AP), formed by abstraction of H4' from DNA, is particularly interesting because it forms a three-nucleotide deletion product when bypassed by error prone polymerases. We synthesized an improved phosphoramidite for the C4-AP precursor, and incorporated it into oligonucleotides to observe the lesion's reactivity. Mechanistic explanations are explored for the three-three nucleotide deletion by comparing C4-AP to analogues of the lesion. In vivo and in vitro analyses are reported, but none of the hypothetical mechanisms is supported by the data.

The repair of C4-AP by the base excision repair pathway has been previously proposed. In short-patch base excision repair, after the initial incision by AP endonuclease 1 a lyase is necessary to remove the 5'-phosphorylated lesion from the end of the oligonucleotide. The reaction of C4-AP with the lyase of polymerase β produced an interesting adduct that prevented turnover of the enzyme. We confirmed the inhibition through a variety of steady-state and single-turnover kinetic experiments. We also analyzed the adduct by protease digestion of polymerase β in the presence and absence of C4-AP and characterized the products via mass spectrometry. Although C4-AP was ultimately shown to be a weak substrate for long-patch base excision repair, the lesion is difficult to repair due to its ability to inhibit polymerase β.