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Chem. Res. Toxicol. 2000, 13, 658-664
Peroxynitrite-Induced Secondary Oxidative Lesions at Guanine Nucleobases: Chemical Stability and Recognition by the Fpg DNA Repair Enzyme Natalia Y. Tretyakova, John S. Wishnok, and Steven R. Tannenbaum* Division of Bioengineering and Environmental Health and Department of Chemistry, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139 Received April 11, 2000
Synthetic oligodeoxynucleotides containing secondary oxidative lesions at guanine nucleobases have been prepared by the site-specific oxidation by ONOO- of oligomers containing 8-oxoguanine (8-oxo-G). The oligomers have been tested for their stability to the standard hot piperidine treatment that is commonly used to uncover oxidized DNA lesions. While DNA containing oxaluric acid and oxazolone was cleaved at the site of modification under hot piperidine conditions, the corresponding cyanuric acid and 8-oxo-G lesions were resistant to piperidine. The recognition of the oxidative lesions by formamidopyrimidine glycosylase (Fpg enzyme) was examined in double-stranded versions of the synthetic oligodeoxynucleotides. Fpg efficiently excised 8-oxo-G and oxaluric acid and to some extent oxazolone, but not cyanuric acid. These data suggest that some DNA lesions formed via ONOO- exposures (cyanuric acid) are not repaired by Fpg and are not uncovered by assays based on piperidine cleavage at the site of lesion. Our results indicate that cryptic secondary and tertiary oxidation products arising from 8-oxo-G may contribute to the overall mutational spectra arising from oxidative stress.
Introduction Peroxynitrite (ONOO-)1 is formed in vivo by a diffusion-limited reaction of nitric oxide with superoxide anion (1). Under physiological conditions ([CO2] ≈ 1 mM), ONOO- is likely to capture a molecule of carbon dioxide to form nitrosoperoxycarbonate ONOOCO2- (2, 3). Carbonate radicals resulting from ONOOCO2-, along with the peroxynitrite anion and peroxynitrous acid, can damage cell constituents, including thiols, lipids, proteins, and DNA (4-7). Oxidative DNA damage resulting from the exposure to endogenous ONOO- has been implicated in mutagenesis, cancer, and a number of pathological conditions associated with aging (8-12). ONOO- and its secondary radical species are powerful oxidizing and/or nitrating agents. The nitration of DNA gives rise to 8-nitroguanine (8-nitro-G), an unstable lesion that undergoes rapid, spontaneous depurination producing abasic sites (13-15). Guanine (G) is also the preferred site of ONOO--mediated oxidation since it has the lowest redox potential among the DNA nucleobases (16). The major oxidation products of G include 8-oxo-7,8-dihydroguanine (8-oxo-G) (17, 18), 2,2-diamino-4-[(2-deoxyβ-D-erythro-pentofuranosyl)amino]-5(2H)-oxazolone (oxazolone), and its imidazolone precursor (Scheme 1; 1922). 8-Oxo-G has been widely used as an indicator of oxidative DNA damage and as a marker of oxidative stress (23, 24). It has been shown, however, that 8-oxo7,8-dihydro-2′-deoxyguanosine is at least 1000-fold more * To whom correspondence should be addressed. 1 Abbreviations: cyanuric acid, 1,3,5-triazine-1(2H)-carboximidamide tetrahydro-2,4,6-trione; LC/ESI--MS, liquid chromatography/negative ion electrospray ionization mass spectrometry; MALDI, matrix-assisted laser desorption ionization; 8-nitro-G, 8-nitroguanine; oxazolone, 2,2diamino-4-[(2-deoxy-β-D-erythro-pentafuranosyl)amino]-5(2H)-oxazolone; ONOO-, peroxynitrite; 8-oxo-G, 8-oxo-7,8-dihydroguanine.
reactive toward ONOO- than normal 2′-deoxyguanosine, and it is rapidly converted to secondary products (25, 26). These products include the 2-deoxy-β-D-erythro-pentofuranosyl derivatives of cyanuric acid, parabanic acid, oxaluric acid, and their intermediates (Scheme 1; 27). 8-Oxo-G-containing DNA undergoes similar transformations to give cyanuric acid and oxaluric acid at the site of 8-oxo-G (28). Given the high reactivity of 8-oxo-G toward reactive oxygen species, it is possible that the yields of secondary oxidative products of guanine in vivo are similar or greater than those of 8-oxo-G itself. Replication of oxidatively damaged DNA may result in mutations due to the mispairing of lesions with incorrect nucleobases. ONOO- treatment of the supF gene induces mainly G to T and G to C transversions when replicated in bacterial or mammalian cells (8, 9). 8-Oxo-G has been shown to pair with either C or A, the latter mispairing resulting in G to T transversions (29, 30). The base-pairing properties of the oxidation products of 8-oxo-G are not well established. Using the Klenow fragment of DNA polymerase R, the insertion of dGMP and dAMP opposite an oxidized form of 8-oxo-G has been reported (31). Cyanuric acid has been shown to pair with either A or G in one nucleotide extension assay with Klenow polymerase, giving rise to G to T and G to C transversions (32). Both G to T and G to C transversions have been observed in peroxy radical-treated DNA, while 8-oxo-G was not detected (33). These results suggest that the secondary DNA lesions resulting from further oxidation of 8-oxo-G may contribute to ONOO- mutagenicity. Our laboratory recently reported that most of the damage “hotspots” for nucleobase reactions with ONOO- coincided with the mutation hotspots in the supF gene following replication in bacterial cells (34). Since these ONOO--induced lesions were cleaved by both hot pip-
10.1021/tx000083x CCC: $19.00 © 2000 American Chemical Society Published on Web 06/24/2000
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Scheme 1. Secondary Oxidation Products Resulting from ONOO- Treatment of 8-Oxo-G-Containing DNA (*R being the rest of DNA)
eridine treatment and incubation with the Fpg repair enzyme, they differ from 8-oxo-G which is resistant to piperidine (22). The exact structure of these modified guanines, however, could not be established by gel electrophoresis. There are numerous reports on the conversion of 8-oxo-G to piperidine-labile products using a variety of oxidizing agents, e.g., Ir(IV) (31, 35, 36), permanganate (31, 35), singlet oxygen (31, 36), peroxynitrite (37), and photoionization (37). However, the chemical identities of the secondary oxidative products of guanine have not been established with certainty. A guanidinohydantoin structure has been proposed for the product of Ir(IV)induced oxidation of 8-oxo-G-containing oligodeoxynucleotides (31). However, the ESI- mass spectral data presented in support of this assignment were obtained using a complex mixture of oxidized oligomers and are difficult to interpret due to the low signal-to-noise ratio and the possibility of metal ion adducts (31). For γ-irradiated DNA, the formation of alkali-labile 2-deoxyribono-1,4-lactone through a 1′-hydrogen abstraction from deoxyribose has been suggested (38). However, no direct evidence for the formation of the latter lesion is available. In summary, there is still a paucity of information about the chemical nature of the oxidized 8-oxo-G lesions that render DNA subject to cleavage by hot piperidine. Similar uncertainty exits about the chemical structure of oxidative lesion(s) excised by the Fpg protein (39). The objective of this investigation was to determine the stability of the secondary oxidative lesions of guanine under standard hot piperidine treatment. In addition, the recognition of these lesions by Fpg repair enzyme was investigated. While oxaluric acid and oxazolone-containing DNA were cleaved under hot piperidine treatment, cyanuric acid and 8-oxo-G were resistant to piperidine. Fpg efficiently excised oxaluric acid and, to some extent, oxazolone, but not cyanuric acid. These results provide a useful tool for interpreting the gel electrophoresis results of oxidatively damaged DNA and suggest that oxaluric acid may be responsible for the strand breaks observed upon treatment of ONOO--exposed DNA with Fpg and piperidine.
Experimental Procedures Materials. 8-Oxo-G-containing oligodeoxyribonucleotide CCACAACXCAAA (X being 8-oxo-G) was obtained from Synthetic Genetics (San Diego, CA) and purified by HPLC as described below. The Fpg enzyme was a gift from A. Grollman (State University of New York, Stony Brook, NY). All other enzymes were from Sigma (St. Louis, MO). Piperidine, imidazole, acetonitrile, triethylamine, and ammonium acetate were from Aldrich. ONOO- Treatment of DNA. ONOO- was prepared by ozonation of sodium azide in alkaline solution (0.1 N NaOH) and stored at -80 °C until it was used (40). ONOO- concentrations were determined by spectrophotometry ( ) 1670 M-1 cm-1, λ ) 302 nm in 0.1 N NaOH). Oligodeoxyribonucleotides containing 8-oxo-G were HPLC-purified prior to ONOO- reaction and carefully lyophilized to remove most of the ammonium acetate. Reactions between ONOO- and DNA were carried out in 150 mM potassium phosphate/25 mM sodium bicarbonate buffer at pH 7.2. A droplet (1-5 µL) of ONOO- stock solution (≈38 mM) was placed on the underside of a lid of an Eppendorf tube containing buffered oligodeoxynucleotide solutions (∼50 µM, total reaction volume of 50 µL). The tube was carefully closed, and then the contents were vortexed for 1 min. Samples were routinely left at room temperature for 1 h prior analysis. HPLC Purification of Oligodeoxynucleotides. The oligodeoxyribonucleotides were purified by HPLC using a 250 mm × 2.1 mm, 5 µm Supelcosil LC-18-DB column with UV detection at 260 nm. The mobile phase was 150 mM aqueous ammonium acetate (A) and 100% acetonitrile (B) with a gradient of 7 to 12% B over the course of 30 min at a flow rate of 0.25 mL/min. HPLC fractions corresponding to each oligodeoxyribonucleotide product were collected, combined, and thoroughly dried under vacuum to remove most of the ammonium acetate. The quantities of the synthetic oligodeoxyribonucleotides were calculated from their HPLC peak areas (UV detection at 260 nm) using 8-oxo-G oligomer as a standard. MALDI-TOF Mass Spectrometry. A PerSeptive Biosystems (Framingham, MA) Voyager Elite DE MALDI-TOF instrument was used for the analyses. The MALDI matrix consisted of a 90:5:5 mixture of 0.36 M 3-hydroxypicolinic acid (Aldrich, Milwaukee, WI), 0.36 M picolinic acid (Aldrich), and 0.22 M ammonium hydrogen citrate (Mallinckrodt), prepared daily in 50% aqueous acetonitrile. 3-Hydroxypicolinic acid was recrystallized from water prior to being used. HPLC-purified oligodeoxynucleotides (1-30 pmol) were dried under vacuum and dissolved in 4 µL of MALDI matrix solution. One microliter of the mixture was spotted onto the sample plate and allowed to
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air-dry prior to instrument insertion. During the loading procedure, adventitious alkali cations were removed with AG 50W-X8 cation-exchange resin (Bio-Rad, Hercules, CA) in the NH4+ form, activated with ammonium acetate (41). Spectra were obtained in the reflector mode with a laser energy of 29003000, an accelerating voltage of 24 000 V, a grid voltage of 90%, a guide wire voltage of 0.08%, and a delay time of 150 ns. Calibration of the instrument was carried out using synthetic oligodeoxyribonucleotides of known molecular weight bracketing the m/z of each analyte. Each spectrum was an average of 128256 laser shots. Liquid Chromatography/Mass Spectrometry. LC/MS analyses were performed using a model 1100 liquid chromatograph (Hewlett-Packard) interfaced with a TSQ 7000 triplequadrupole mass spectrometer (Finnigan). Separation was achieved using a Supelcosil 150 mm × 1 mm, 5 µm column. The solvent system consisted of 25 mM aqueous ammonium acetate (A) and 100% acetonitrile (B) at a gradient of 0 to 30% B over the course of 30 min at a flow rate of 65 µL/min. The mass spectrometer was operated in the negative ion mode. Electrospray ionization was achieved at a spray voltage of 3-4 kV, and the heated capillary was maintained at 270 °C. The sheath gas pressure was 60-65 psi. The sheath liquid (80% isopropyl alcohol in water containing 50 mM imidazole and 50 mM piperidine) was applied at a rate of 5 µL/min. Full scan experiments were conducted by scanning from m/z 600 to 1800 over the course of 5 s. The spectra were obtained by averaging across the LC peaks with background subtraction at both sides from the peak. Piperidine Lability Studies. HPLC-purified oligodeoxynucleotides (0.8-1 µg, ≈0.2 nmol) were dissolved in 50 µL of freshly prepared 1 M piperidine and heated at 90 °C for 30 min (39). The piperidine was evaporated to dryness under vacuum. Twenty microliters of water was added to the pellet, followed by drying under vacuum (repeated twice). The resulting sample was dissolved in 50 µL of distilled water and analyzed by MALDI-TOF or LC/ESI--MS as described above. Phosphodiesterase Digestion. Oligonucleotides (1-2 µg) were incubated with 1.5 × 10-3 unit of snake venom phosphodiesterase in water for 5-60 min at 37 °C. The digests were dried under vacuum, dissolved in 4 µL of MALDI matrix, and analyzed by MALDI-TOF mass spectrometry as described above. For complete digestion to deoxyribonucleosides, the modified oligomers (3-5 µg) were incubated overnight in 30 mM ammonium acetate, 0.1 mM zinc sulfate buffer (pH 5) containing nuclease P1 (6 units), and acid phosphatase (0.6 unit). The digests were analyzed by on-line LC/ESI--MS in the constant neutral loss mode using the facile loss of 116 mass units (deoxyribose) from 2′-deoxyribonucleosides. Fpg Repair Experiments. The HPLC-pure synthetic oligodeoxynucleotides containing the secondary oxidation products of guanine (0.8-1 µg, ≈0.5 µmol in 25 µL of water) were annealed to their complementary strands by heating at 80 °C for 5 min followed by slow cooling to room temperature. The following day, 25 µL of 2× Fpg buffer was added [final concentrations of 50 mM Tris-HCl (pH 7.5), 100 mM NaCl, 1 mM dithiothreitol, and 2 mM EDTA], followed by the Fpg enzyme (0.2 µg/µg of DNA). Samples were incubated at 37 °C for 2 h. The digests were directly analyzed by negative-ion MALDI-TOF MS and HPLC with UV detection as described above.
Results Preparation of the Oligodeoxynucleotides Containing Secondary Oxidation Products of Guanine. Chemical insertion of the secondary oxidation products of guanine in oligodeoxynucleotides is a challenging task due to their chemical lability (22). This lability inevitably leads to DNA degradation under the alkaline conditions used in the deprotection step of solid-phase synthesis. Therefore, an alternative route, involving the specific
Tretyakova et al.
Figure 1. HPLC separation of CCACAACXCAAA (X being 8-oxo-G) after exposure to ONOO-.
Figure 2. MALDI-TOF mass spectra of the oligodeoxyribonucleotide products resulting from exposure of CCACAACXCAAA (X being 8-oxo-G) to ONOO-.
oxidation of 8-oxo-G-containing DNA with ONOO- followed by HPLC purification of the modified oligodeoxynucleotides, was used. Treatment of the synthetic oligodeoxynucleotide CCACAACXCAAA (X being 8-oxo-G) with ONOO- resulted in several products that were separated by HPLC (Figure 1). The identity of each oxidation product was established from MALDI-TOF mass spectral data obtained for the collected fractions (Figure 2). The earliest eluting peak at 13.1 min contained CCACAACYCAAA, where Y is oxazolone (Scheme 1). The second peak at 13.8 min corresponded to the oxaluric acid-containing oligomer, CCACAACZCAAA, where Z is oxaluric acid. The peak at 15 min contained an unstable intermediate that converted to oxazolone upon drying under vacuum. Direct LC/MS analysis (results not shown) demonstrated that this was an imidazolone-containing oligomer that hydrolyzed to oxazolone upon drying and/or storage. At high [ONOO-]: DNA ratios (g10), an additional product was observed at 18.5 min which was identified as the cyanuric acidcontaining oligodeoxynucleotide (CCACAACQCAAA, where Q is cyanuric acid; see Scheme 1). MALDI-TOF mass spectra of these modified oligonucleotides are presented in Figure 2. Since their molecular weights differ by only 1-3 mass units, good mass spectral resolution and mass accuracy were essential. The mass resolution of the MALDI-TOF mass spectrometer used in this study is about 4000. By careful desalting of DNA
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Scheme 2. Cleavage of CCACAACXCAAA by Hot Piperidine or Fpg Repair Enzyme Treatment
samples, application of the lowest possible laser energy, and internal mass calibration using ions bracketing the molecular ion of each analyte, we were able to distinguish between the oxazolone oligomer (MW ) 3571.4), cyanuric acid oligodeoxynucleotide (MW ) 3570.4), and the oxaluric acid oligomer (MW ) 3573.4). ESI- mass spectra of the same samples were consistent with the MALDITOF results (data not shown). Further proof of product identity was obtained by partial digestion of oligonucleotides with 3′ f 5′ phosphodiesterase, followed by MALDI-TOF MS analysis of the resulting DNA ladders. The mass differences between CCACAACX and CCACAAC corresponded to the molecular weights of the oxidized mononucleotides and had values of 307.4, 308.2, and 310.3 for cyanuric acid, oxazolone, and oxaluric acid oligomers, respectively. Complete enzymatic digestion of the oligomers to 2′-deoxyribonucleosides followed by LC/ MS analysis showed the presence of dA, dC, and dX in a ratio of 6:5:1 where dX is cyanuric acid (M ) 245.2), oxazolone (M ) 246.2), and oxaluric acid (M ) 248.1). The same products, although in lower yield, were observed when a double-stranded version of CCACAACXCAAA (X being 8-oxo-G) was treated with ONOO-, indicating that the same transformations take place in both single-stranded and double-stranded DNA. Piperidine Cleavage of Oligodeoxynucleotides Containing ONOO--Induced Secondary Oxidation Products. Piperidine-induced cleavage of chemically modified DNA includes a nucleophilic displacement of the modified nucleobase with piperidine, followed by strand scission at the abasic site through β-δ elimination. This leaves behind phosphorylated 5′ and 3′ fragments of the
Figure 3. MALDI-TOF mass spectra of CCACAACXCAAA following piperidine cleavage: (a) X being oxazolone, (b) X being oxaluric acid, and (c) X being cyanuric acid.
Table 1. Fpg- and Hot Piperidine-induced Cleavage of Oligonucleotides CCACAACXCAAA Containing Secondary Oxidation Products of Guanine (X) % cleavage oxazolone oxaluric acid cyanuric acid
Fpg enzyme
hot piperidine
70 100
