Aerobic Fate of the C-3‘-Thymidinyl Radical in Single-Stranded DNA

Abstract

Oxidative events that target the sugar-phosphate backbone of DNA can lead to reactive fragments that interfere with DNA repair, transcription and translation by the formation of cross-links and adducts of proteins and nucleobases. Here we report the formation of several such lesions through the aerobic degradation of an independently generated C-3'-thymidinyl radical in 2'-deoxyoligonucleotides. Individual fragments were identified by independent synthesis and comparison of retention times in high-performance liquid chromatography (HPLC) and/or matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-ToF MS) along with gel electrophoresis. The formation of this reactive intermediate in the presence of oxygen was found to produce 3'-phosphoglycolaldehyde (3'-PGA) as well as 3'-ketoenolether (3'-KEE), 3'-phosphoglycolate (3'-PG), and 5'-aldehyde terminated oligonucleotide fragments. Additionally, a significant outcome of C-3'-thymidinyl radical formation in DNA oligomers is a strand break resulting in one 3'- and two 5'-phosphate-terminated oligomers. These results suggest the involvement of several sugar derived reactive species upon C-3'-radical initiated scission of single-stranded DNA under aerobic conditions. The electrophilic nature of several of these products as well as their formation through a single oxidative event can make the presence of a C-3'-DNA radical more detrimental to the cell than products derived from more frequently occurring DNA sugar radicals.

Reactivity of Nucleic Acid Radicals

“…117 The major products were consistent with those detected by Barton. For instance, the majority of 3′-fragments contained 5′-phosphate termini (82%) and the majority of the 5′-fragments (78%) were attributable to the 3′-keto thymidine ( 35 , Scheme 16 ).…”

Mechanism of Rate Acceleration of Radical C–C Bond Formation Reaction by a Radical SAM GTP 3′,8-Cyclase

“…Perturbations in the active site that prevent the completion of RNR catalysis most frequently result in base dissociation [20][21][22] . C-3′ radical-mediated base dissociation was also reported for oxidative damage of DNA 23 . The reactivity of nucleotide C-3′ radical in oligonucleotides has been studied using photoactivatable nucleotides 24, , in which C-3′ radicals caused strand scission and base dissociation 23,25 .…”

Mechanistic Studies on RNA Strand Scission from a C2′-Radical

“…For instance, the peroxyl radical obtained from the O 2 trapping of the C3′-radical in DNA abstracts a hydrogen atom from the C4′-position. 42,43 The facility of the resulting C4′-radical (26) to yield strand scission via β-fragmentation is well established. 17,44,45 Formation of 3′phosphate (8) from 6 under anaerobic conditions is difficult to explain.…”

“…Oxygen trapping of 6 yields 25 , which may ultimately yield 8 via a mechanism for which there is precedent in nucleic acid radical chemistry (Scheme ). For instance, the peroxyl radical obtained from the O 2 trapping of the C3′-radical in DNA abstracts a hydrogen atom from the C4′-position. , The facility of the resulting C4′-radical ( 26 ) to yield strand scission via β-fragmentation is well established. ,, Formation of 3′-phosphate ( 8 ) from 6 under anaerobic conditions is difficult to explain. However, significant quantities of 8 are only formed under anaerobic conditions in the absence of thiol, and one cannot rule out trace amounts of O 2 that trap 6 , which is generated from <50 nM solutions of radical precursor.…”