Generation of Guanine–Thymidine Cross-Links in DNA by Peroxynitrite/Carbon Dioxide

Abstract

Nitrosoperoxycarbonate derived from the combination of carbon dioxide and peroxynitrite, is an important chemical mediator of inflammation. In aqueous solutions, it rapidly decomposes to the reactive species CO3•− and •NO2 radicals that are known to initiate the selective oxidation and nitration of guanine in DNA. We have previously demonstrated that the reactions of carbonate radical anions with guanine in 2′-deoxyoligoribonucleotides generate a previously unknown intrastrand cross-linked guanine-thymine product G*-T* with a covalent bond between the C8 (G*) and thymine N3 (T*) atoms (Crean et al., Nucleic Acids Res., 2008, 36, 742–755). In this work we demonstrate that G*-T* cross-linked products are also formed when peroxynitrite (0.1 mM) reacts with native DNA in aqueous solutions (pH 7.5–7.7) containing 25 mM carbon dioxide/bicarbonate, in addition to the well known nitration/oxidation products of guanine such as 8-nitroguanine (8-nitroG), 5-guanidino-4-nitroimidazole (NIm), 8-oxo-7,8-dehydroguanine (8-oxoG) and spiroiminodihydantoin (Sp). The yields of these products, after enzymatic digestion with P1 nuclease and alkaline phosphatase to the nucleotide level, and reversed phase HPLC separation, were compared with those obtained with the uniformly, isotopically labeled 15N,13C-labeled 2′-deoxy oligoribonucleotides 5′-dGpT and 5′-dGpCpT. The d(G*pT*) and d(G*-T*) cross-linked products derived from the di- and tri-oligonucleotides, respectively, were used as standards for identifying the analogous lesions in calf thymus DNA by isotope dilution LC-MS/MS methods in the selected reaction-monitoring mode. The Nim and 8nitroG are the major products formed (~ 0.05% each), and lesser amounts of 8-oxoG (~ 0.02%), and d(G*pT*) and d(G*-T*) enzymatic digestion products (~ 0.002% each) were found. It is shown that the formation of d(G*pT*) enzyme digestion product can arise only from intrastrand cross-links, whereas d(G*-T*) can arise from both interstrand and intrastrand cross-linked products.

DNA Base Damage by Reactive Oxygen Species, Oxidizing Agents, and UV Radiation

“…However, the formation of tandem lesions was recently supported by labeling experiments showing that a large percentage (50%) of 8-oxoGua and 8-oxoAde are labeled by 18 O 2 rather than H 2 18 O when DNA is irradiated in aqueous solution (Bergeron et al 2010). Another example of tandem lesions via a single radical involves the formation of a guanine-thymine cross-link between C8 of guanine and N3 of thymine upon initial formation of guanine radical cation, which has thus far only been observed in isolated DNA (Yun et al 2011;Ding et al 2012). …”

One-electron oxidation reactions of purine and pyrimidine bases in cellular DNA

“…Intrastrand G*-T* cross-links can be generated by the chemical mediator of inflammation, nitrosoperoxycarbonate (Yun et al 2011). The latter, derived from the combination of carbon dioxide and peroxynitrite, rapidly decomposes to reactive species, CO 3 • − and • NO 2 radicals (Pacher et al 2007) that are known to initiate the selective oxidation and nitration of guanine in DNA (Shafirovich et al 2009).…”

“…The latter, derived from the combination of carbon dioxide and peroxynitrite, rapidly decomposes to reactive species, CO 3 • − and • NO 2 radicals (Pacher et al 2007) that are known to initiate the selective oxidation and nitration of guanine in DNA (Shafirovich et al 2009). The reaction of peroxynitrite with native DNA in aqueous solutions (25 mM carbon dioxide/bicarbonate, pH 7.5 – 7.8) generates G*T* cross-links in addition to the well-known nitration/oxidation products of guanine, including 8-nitroguanine (8-nitroG), 5-guanidino-4-nitroimidazole (NIm), 8-oxo-7,8-dihydroguanine (8-oxoG) and spiroiminodihydantoin (Sp) (Yun et al 2011). The yields of these products, after enzymatic digestion with P1 nuclease and alkaline phosphatase to the nucleotide level, and reversed phase HPLC separation, were compared with those obtained with uniformly, isotopically labeled [ 15 N, 13 C]-labeled 2′-deoxyoligoribonucleotides 5′-dGpT and 5′-dGpCpT.…”

Base and Nucleotide Excision Repair of Oxidatively Generated Guanine Lesions in DNA

“…In the case of guanine oxidation by peroxynitrite, the 5-guanidino-4-nitroimidazole (NIm) lesion (16 -18) is also produced and can serve as a biomarker of inflammation-related oxidation mechanisms (1). The NIm lesions together with the easily depurinated 8-nitroguanine, are typical products of guanine damage in calf thymus DNA induced by reactions with nitrosoperoxycarbonate (16,19) derived from the combination of carbon dioxide and peroxynitrite (20). The structures of these oxidatively generated guanine lesions are shown in Fig.…”