DNA Lesions Derived from the Site Selective Oxidation of Guanine by Carbonate Radical Anions

Abstract

Carbonate radical anions are potentially important oxidants of nucleic acids in physiological environments. However, the mechanisms of action are poorly understood, and the end products of oxidation of DNA by carbonate radicals have not been characterized. These oxidation pathways were explored in this work, starting from the laser pulse-induced generation of the primary radical species to the identification of the stable oxidative modifications (lesions). The cascade of events was initiated by utilizing 308 nm XeCl excimer laser pulses to generate carbonate radical anions on submicrosecond time scales. This laser flash photolysis method involved the photodissociation of persulfate to sulfate radical anions and the one electron oxidation of bicarbonate anions by the sulfate radicals to yield the carbonate radical anions. The latter were monitored by their characteristic transient absorption band at 600 nm. The rate constants of reactions of carbonate radicals with oligonucleotides increase in the ascending order: 5'-d(CCATCCTACC) [(5.7 +/- 0.6) x 10(6) M(-)(1) s(-)(1)] < 5'-d(TATAACGTTATA), self-complementary duplex [(1.4 +/- 0.2) x 10(7) M(-)(1) s(-)(1)] < 5'-d(CCATCGCTACC [(2.4 +/- 0.3) x 10(7) M(-)(1) s(-)(1)] < 5'-d(CCATC[8-oxo-G]CTACC) [(3.2 +/- 0.4) x 10(8) M(-)(1) s(-)(1)], where 8-oxo-G is 8-oxo-7,8-dihydroguanine, the product of a two electron oxidation of guanine. This remarkable enhancement of the rate constants is correlated with the presence of either G or 8-oxo-G bases in the oligonucleotides. The rate constant for the oxidation of G in a single-stranded oligonuclotide is faster by a factor of approximately 2 than in the double-stranded form. The site selective oxidation of G and 8-oxo-G residues by carbonate radicals results in the formation of unique end products, the diastereomeric spiroiminodihydantoin (Sp) lesions, the products of a four electron oxidation of guanine. These lesions, formed in high yields (40-60%), were isolated by reversed phase HPLC and identified by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. These assignments were supported by the characteristic circular dichroism spectra of opposite signs of the two lesions. The oxidation of guanine to Sp diastereomers occurs, at least in part, via the formation of 8-oxo-G lesions as intermediates. The Sp lesions can be considered as the terminal products of the oxidation of G and 8-oxo-G in DNA by carbonate radical anions. The mechanistic aspects and biological implications of these site selective reactions in DNA initiated by carbonate radicals are discussed.

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

“…The rate constants were determined by least squares fits of the appropriate kinetic equations to the experimentally measured transient absorption profiles as described in detail elsewhere. 28,32 The values reported are averages of five independent measurements. The relevant and important steps leading to the oxidation of oligonucleotides by BPT •+ radicals are summarized in Table 1.…”

“…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).…”

“…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).…”