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

Abstract

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.

Ozone-Induced DNA Damage: A Pandora’s Box of Oxidatively Modified DNA Bases

“…Tandem lesions containing N -formamide and 8oxoG have previously been characterized and detected in oligonucleotides and isolated DNA exposed to ionizing radiation and Fenton-like reactions implying the generation of hydroxyl radicals. ,,,, The mechanism of formation of these and other tandem lesions has been proposed to involve initial attack of • OH and incorporation of oxygen to form peroxyl radicals, which subsequently react with the adjacent G moiety to form either 8oxoG or 2,6-diamino-4-hydroxy-5-formamidopyrimidine (FapyG). , A similar mechanism may be responsible for the formation of tandem lesions by ozone. In addition to the reaction pathway involving a bipolar carbonyl intermediate (analogous to I → II ), Pryor proposed O–O bond homolysis of the initial ozonide into a biradical species to explain the effects of temperature, metal chelators, and H 2 O on the formation of radical species during ozone-mediated oxidation of a biological target .…”

Damage Induced to DNA and Its Constituents by 0–3 eV UV Photoelectrons†

“…We can therefore conjecture that in cells, the initial radicals that produced this type of CLs (143) would also be effective in crosslinking DNA with proteins. Moreover, it has been demonstrated that a single radical, such as thymine radical, could produce tandem lesions and protein‐DNA crosslinks (151,152). As shown in Fig.…”

Early Events in Radiobiology: Isolated and Cluster DNA Damage Induced by Initial Cations and Nonionizing Secondary Electrons

“…Tandem base modifications have also been reported; they are induced following the initial formation of peroxyl pyrimidine radicals that subsequently react with vicinal bases . Under UHV, such reactions would not occur, but we cannot rule out the possibility of reaction with oxygen, during the sample analysis procedure under atmospheric conditions. , …”

“…30 Under UHV, such reactions would not occur, but we cannot rule out the possibility of reaction with oxygen, during the sample analysis procedure under atmospheric conditions. 70,71 Much of our present understanding on the production of cluster lesions has revolved around the concept of random multiple hits by the primary particles and radiation-generated species along tracks, causing two or more simple lesions within 20 bp. This concept was introduced and emphasized by Ward, 72,73 Goodhead, 74,75 and others.…”