Tandem Lesions Arising from 5-(Uracilyl)methyl Peroxyl Radical Addition to Guanine: Product Analysis and Mechanistic Studies.

The reaction of hydroxyl radical (HO•) with thymine in DNA generates 5-(uracilyl)-methyl radicals (T•) and the corresponding methylperoxyl radical (TOO•) in the presence of O2, which in turn propagates damage by reacting with a vicinal nucleobase. This leads to so-called double or tandem lesions. Because methyl oxidation products of thymine are major products, we investigated the reactivity of TOO• using a photolabile precursor: 5-(phenylthiomethyl)uracil (TSPh). The precursor was prepared and incorporated into a DNA trinucleotide: 5'-d(GpTSPhpA)-3' (G-TSPh-A). Upon photolysis, the resulting products were characterized by LC-MS/MS. Thereby, we identified four tandem lesions involving GpT, which include either 2,6-diamino-4-hydroxy-5-formamidopyrimidine (fapyG) or 8-oxo-7,8-dihydroguanine (oxoG) in tandem with either 5-formyluracil (fU) or 5-hydroxymethyluracil (hmU). The formation of these tandem lesions is explained by initial addition of TOO• to the C8 of guanine moiety, giving an N7-guanine cross-linked radical. The latter radical undergoes either reduction to an 7,8-saturated endoperoxide or oxidation to an 7,8-unsaturated endoperoxide, which transform into fapyG-fU-A and oxoG-fU-A, respectively. This is supported by the effect of a reducing (dithiothreitol) and oxidizing agent (Fe3+) on product formation. This study expands the repertoire of tandem lesions that can occur at GpT sequences and underlines the importance of redox environment.