The Carbonate Radical Is a Site-selective Oxidizing Agent of Guanine in Double-stranded Oligonucleotides

Abstract

The carbonate radical anion (CO 3 . ) is believed to be an . anions and the formation of G(؊H) ⅐ radicals are correlated with one another on the millisecond time scale, whereas the neutral guanine radicals decay on time scales of seconds. Alkali-labile guanine lesions are produced and are revealed by treatment of the irradiated oligonucleotides in hot piperidine solution. The DNA fragments thus formed are identified by a standard polyacrylamide gel electrophoresis assay, showing that strand cleavage occurs at the guanine sites only. The biological implications of these oxidative processes are discussed.There is growing evidence that bicarbonate and carbon dioxide, both present in biological systems in significant amounts, can alter the mechanisms and reaction pathways of reactive oxygen (1-4) and nitrogen (5-13) species formed during normal metabolic activity and under conditions of oxidative stress. It has been proposed that the mechanism of generation of carbonate radical anions (CO 3 . ) 1 from bicarbonate (HCO 3 Ϫ ) or CO 2 can involve the one-electron oxidation of HCO 3 Ϫ at the active site of copper-zinc superoxide dismutase (3, 4) and homolysis of the nitrosoperoxycarbonate anion (ONOOCO 2 Ϫ ) formed by the reaction of peroxynitrite with carbon dioxide (14 -18).The carbonate radical anion is a strong one-electron oxidant that oxidizes appropriate electron donors via electron transfer mechanisms (19). Detailed pulse radiolysis studies have shown that carbonate radicals can rapidly abstract electrons from aromatic amino acids (tyrosine and tryptophan). However, reactions of CO 3 . with sulfur-containing methionine and cysteine are less efficient (20 -22). Hydrogen atom abstraction by carbonate radicals is generally very slow (19), and their reactivities with other amino acids are negligible (20 -22). It is well established that carbonate radicals can play an important role in the modification of selective amino acids in proteins in cellular environments under conditions of oxidative stress, aging, and inflammatory processes (1,11,12). The role of HCO 3 Ϫ /CO 2 in potentiating oxidative DNA damage has received relatively little attention. It has been shown that the presence of HCO 3 Ϫ /CO 2 inhibits direct strand cleavage of DNA induced by ONOO Ϫ but enhances the formation of 8-nitroguanine, alkali-labile and formamidopyrimidine glycosylase-labile DNA lesions (23-25). Peroxynitrite causes direct DNA strand cleavage by oxidizing deoxyribose. However, in the presence of HCO 3 Ϫ /CO 2 there is a shift in product distribution from direct strand cleavage to the formation of oxidative modifications of guanines (26), suggesting that the carbonate radical anion could play an important role in this phenomenon (24). Although guanine is indeed the most easily oxidized base in DNA, the reactions of the carbonate radical anions with the different aromatic DNA residues have not yet been characterized.In this work, we explore the electron transfer reactions from guanine electron donor residues embedded in the self-comp...

Oxidation of Guanine in G, GG, and GGG Sequence Contexts by Aromatic Pyrenyl Radical Cations and Carbonate Radical Anions: Relationship between Kinetics and Distribution of Alkali-Labile Lesions

“…It is therefore important to optimize the reaction conditions in order to predominantly observe the reaction kinetics of the electron transfer between CO 3 •− radicals and the oligonucleotide strands. 27 The optimal conditions 27,28 were determined by considering the set of rate constants in Table 2. In order to minimize the contribution of the reactions of SO 4 •− radicals with oligonucleotides (reaction 7 in Table 2), we used high concentrations of HCO 3 − (300 mM) and much lower concentrations of oligonucleotides (≤ 0.1 mM).…”

“…27,28 Typical decay curves depicted in Figure 6 show that in a solution containing the duplex 3d (100 μM), the decay of the CO 3 •− radicals is faster than in the absence of DNA. The rate constants of DNA oxidation by CO 3 •− radicals are extracted from these decay curves by methods that have been previously described in detail.…”

“…The rate constants of DNA oxidation by CO 3 •− radicals are extracted from these decay curves by methods that have been previously described in detail. 27,28 These rate constants, k 9 (defined in Table 2), have been measured using the same set of oligonucleotide duplexes as in Figure 5A, are summarized in Table S2 (Supporting Information). We found that in the duplexes (2 -5) with single G, and contiguous GG and GGG sequences, the values of k 9 are close to one another ( Figure 5B), in contrast to the effects observed in the case of the oxidation of G by BPT •+ radicals ( Figure 5A).…”

“…27,28 Damage to the guanine base residues is revealed in the form of strand cleavage after treatment of the irradiated sequences with hot piperidine, followed by polyacrylamide gel electrophoresis to resolve the cleavage products. 27,30 These methods were used to explore the distributions of DNA cleavage patterns generated by three different one-electron oxidants, BPT •+ , CO 3 •− radicals, and riboflavin. The latter was included for comparison because it is a typical and extensively studied type 1 photosensitizer that initiates the oxidation of DNA by a one-electron abstraction mechanism.…”

“…The rate constants for oxidation of isolated guanines vs. 5′-guanines in a GG sequence context was elucidated by Sistare et al using an electrochemical method. 25 Recently we have explored the oxidation of guanine in different sequence contexts by nitrosoperoxycarbonate 26 and carbonate radical anion [27][28][29][30] arising from the decomposition of this a chemical mediator of inflammation in vivo. 2 The CO 3 •− radicals oxidize guanine in DNA by a one-electron abstraction reaction that, by a series of subsequent oxidation steps and chemical reactions, culminates in the formation of guanine oxidation products (mainly the hot alkali-and Fpg-labile spiroiminodihydantoin lesions).…”