Synthesis of DNA Oligonucleotides Containing Site-Specifically

Milton S. Hershey Medical Center, Pennsylvania State University College of .... Markus Christmann , Barbara Verbeek , Wynand P. Roos , Bernd Kaina...
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Chem. Res. Toxicol. 1999, 12, 127-131

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Communications Synthesis of DNA Oligonucleotides Containing Site-Specifically Incorporated 6 O -[4-Oxo-4-(3-pyridyl)butyl]guanine and Their Reaction with O6-Alkylguanine-DNA Alkyltransferase Lijuan Wang,†,‡ Thomas E. Spratt,§ Anthony E. Pegg,| and Lisa A. Peterson*,†,⊥ Division of Carcinogenesis and Molecular Epidemiology and Division of Toxicology and Pathology, American Health Foundation, 1 Dana Road, Valhalla, New York 10595, and Department of Cellular and Molecular Physiology, Milton S. Hershey Medical Center, Pennsylvania State University College of Medicine, Hershey, Pennsylvania 17033 Received November 17, 1998

DNA pyridyloxobutylation occurs during the metabolic activation of the tobacco-specific nitrosamines, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) and N′-nitrosonornicotine (NNN). This pathway contributes significantly to the carcinogenic and mutagenic activity of these nitrosamines. In general, the chemical structure of pyridyloxobutyl adducts are not well understood. Recently, an AGT reactive pyridyloxobutyl adduct was identified as O6-[4-oxo-4(3-pyridyl)butyl]guanine (O6-pobG). To better understand the importance of this adduct to the biological activity of pyridyloxobutylating agents, we developed a method for site-specifically incorporating O6-pobG into DNA oligonucleotides. They were synthesized using the phosphoramidite of the precursor 2′-deoxy-O6-{3-[2-(3-pyridyl)-1,3-dithian-2-yl]propyl}guanosine. The dithiane group was oxidatively removed with N-chlorosuccinimide in a final postoligomerization reaction to generate the desired product. Human AGT with a polyhistidine tag was able to repair the O6-pobG-containing DNA oligonucleotide, generating unmodified oligonucleotide. These results are consistent with an alkyl group transfer mechanism for the repair of O6-pobG by AGT.

Introduction The tobacco-specific nitrosamines, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK)1 and N′-nitrosonornicotine (NNN), are two of the most active carcinogens present in tobacco products and tobacco smoke. NNK is a potent pulmonary carcinogen in laboratory animals (13). It also induces liver, nasal cavity, and pancreatic tumors (4). NNN is an esophageal and nasal cavity * To whom correspondence should be addressed. Phone: (612) 6260164. Fax: (612) 626-5135. † Division of Carcinogenesis and Molecular Epidemiology, American Health Foundation. ‡ Current address: T8-019 VRT, Department of Biomedical Sciences, Cornell University, Ithaca, NY 14853. § Division of Toxicology and Pathology, American Health Foundation. | Pennsylvania State University College of Medicine. ⊥ Current address: University of Minnesota Cancer Center, Box 806 Mayo, 420 Delaware St. SE, Minneapolis, MN 55455. 1Abbreviations: AGT, O6-alkylguanine-DNA alkyltransferase; DTT, dithiothreitol; HPB, 4-hydroxy-1-(3-pyridyl)-1-butanone; NCS, N-chlorosuccinimide; NNK, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone; NNKOAc, 4-(acetoxymethylnitrosamino)-1-(3-pyridyl)-1-butanone; NNN, N′-nitrosonornicotine; O6-pdbdG, 2′-deoxy-O6-{3-[2-(3-pyridyl)-1,3dithian-2-yl]propyl}guanosine; O6-pdbG, 2′-deoxy-O6-[4-oxo-4-(3-pyridyl)butyl]guanosine; O6-pobG, O6-[4-oxo-4-(3-pyridyl)butyl]guanine; O6-mG, O6-methylguanine.

carcinogen in rats and a respiratory tract carcinogen in mice and hamsters (4). DNA alkylation is believed to initiate the carcinogenic process. NNK can both methylate and pyridyloxobutylate DNA (5-8). NNN pyridyloxobutylates DNA (9-11). The available experimental evidence suggests that DNA pyridyloxobutylation by NNK or NNN contributes to the carcinogenic and mutagenic activity of these nitrosamines (4). Recently, a pyridyloxobutyl adduct, O6-[4-oxo-4-(3pyridyl)butyl]guanine (O6-pobG), was detected in DNA reacted with the model pyridyloxobutylating agent, 4-(acetoxymethylnitrosamino)-1-(3-pyridyl)-1-butanone (NNKOAc; 12). This adduct could significantly contribute to the mutagenic effects seen with pyridyloxobutylating agents since other O6-alkylguanine adducts are mutagenic bases (13-18). Our studies on A/J mice suggest that the pyridyloxobutylation pathway contributes to the lung tumorigenicity of NNK by increasing the levels and persistence of O6-methylguanine (O6-mG) formed from the DNA methylation pathway of NNK (7). The protein O6-alkylguanine-DNA alkyltransferase (AGT) repairs O6-mG in a stoichiometric methyl transfer reaction that inactivates the protein (19). Significantly, pyridyloxobutylated DNA

10.1021/tx980251+ CCC: $18.00 © 1999 American Chemical Society Published on Web 01/20/1999

128 Chem. Res. Toxicol., Vol. 12, No. 2, 1999

can block O6-mG repair in vitro (20, 21). Since O6-pobG is one of the AGT substrate adducts present in pyridyloxobutylated DNA (12), this adduct could contribute to the carcinogenic activity of NNK by decreasing the rate of O6-mG repair. To determine how O6-pobG contributes to the mutagenic and carcinogenic activity of pyridyloxobutylating agents, it was necessary to develop a method for sitespecifically incorporating this adduct into DNA oligonucleotides. In this work, we report the synthesis of DNA oligonucleotides containing O6-pobG. In addition, we demonstrate that O6-pobG is repaired by AGT when presented in a double-stranded DNA oligonucleotide.

Experimental Procedures Materials. 2′-Deoxy-O6-{3-[2-(3-pyridyl)-1,3-dithian-2-yl]propyl}guanosine (O6-pdbdG) and 2′-deoxy-O6-[4-oxo-4-(3-pyridyl)butyl]guanosine (O6-pobdG) were prepared as previously described (12). Human AGT with a polyhistidine tail was expressed and purified as previously reported (22). T4 polynucleotide kinase and [γ-32P]ATP (6000 mCi/mmol) were purchased from Amersham Life Science (Arlington Heights, IL). Instrumental Analyses. NMR spectra were acquired with a Bruker model AM360 WB spectrometer and reported in ppm relative to an external standard. UV spectra were collected on a Hewlett-Packard 8425A diode array spectrophotometer, which was computer controlled by HP 89530 MS-DOS UV/visible operating software. HPLC analyses were carried out with a Waters 510 system (Millipore, Waters Division, Milford, MA) with a Shimadzu SPD-UV-visible detector. Electrospray MS analyses were obtained on either a Finnigan model TSQ 700 or a SCIEX API III tandem quadrupole mass spectrometer. N2-(Phenoxyacetyl)-O6-{3-[2-(3-pyridyl)-1,3-dithian-2-yl]propyl}-2′-deoxyguanosine. Published methods were modified for the protection of the N2-amino group (23, 24). Chlorotrimethylsilane (0.39 mL, 3 mmol) was added to a suspension of dry O6-pdbdG (300 mg, 0.60 mmol) in dry pyridine (6 mL) at 0 °C. The reaction mixture was warmed to room temperature and stirred for 25 min. This solution was cooled with ice and added to a solution containing dry 1-hydroxybenzotriazole (135 mg, 0.9 mmol) and phenoxyacetyl chloride (0.126 mL, 0.9 mmol) in 0.9 mL of acetonitrile and 0.9 mL of pyridine at 0 °C in a nitrogen environment. After the mixture was stirred overnight at 4 °C, water (0.6 mL) was added and the mixture stirred for 15 min. NH4OH (2 N, 5 mL) was subsequently added and the mixture stirred for 15 min. After concentration under reduced pressure, the residue was dissolved in H2O (150 mL) and washed with ether. The product was extracted from the aqueous layer with ethyl acetate (5 × 150 mL). The combined ethyl acetate fractions were dried over MgSO4, filtered, and concentrated under reduced pressure. The product was isolated by silica gel flash chromatography using a gradient from 2% MeOH in CH2Cl2 to 10% MeOH in CH2Cl2. The pure compound eluted with 10% MeOH in CH2Cl2 (230 mg, 0.36 mmol, 60% yield, >98% pure): 1H NMR (CDCl3) δ 9.13 (s, 1H, 2-pyridyl), 8.95 (s, 1H, CON2H-), 8.47 (d, J ) 3.9 Hz, 1H, 6-pyridyl), 8.24-8.21 (m, 1H, 4-pyridyl), 7.97 (s, 1H, C8-H), 7.36-7.26 (m, 3H, 5-pyridyl, ArH), 7.06-7.00 (m, 3H, ArH), 6.80 (br s, 1H, 5′-OH), 6.35-6.31 (m, 1H, 1′-H), 4.95 (t, J ) 2.5 Hz, 1H, 3′-H), 4.70 (s, 2H, phenyl-OCH2CONH), 4.49-4.44 (m, 2H, O6-CH2), 4.15 (d, J ) 2.3 Hz, 1H, 5′-Ha), 3.94 (dd, J ) 12.5, 2.1 Hz, 1H, 4′-H), 3.82 (dd, J ) 12.5, 2.1 Hz, 1H, 5′-Hb), 3.02-2.94 (m, 1H, 2′-Ha), 2.72-2.58 (m, 4H, SCH2CH2CH2S), 2.43-2.37 (m, 1H, 2′-Hb), 2.24-2.20 (m, 2H, O6-CH2CH2CH2), 1.99-1.84 (m, 4H, O6-CH2CH2, SCH2CH2CH2S); positive ESI-MS/MS m/z (relative intensity) 639 (M + 1, 77), 523 (M - 2′-deoxyribose, 36), 238 [M - N2(phenoxyacetyl)-2′-deoxyguanosine, 100]. 5′-O-(4,4′-Dimethoxytrityl)-N2-(phenoxyacetyl)-O6-{3-[2(3-pyridyl)-1,3-dithian-2-yl]propyl}-2′-deoxyguanosine. A modification of a published procedure was used in this step (25).

Communications 4,4′-Dimethoxytrityl chloride (82 mg, 0.230 mmol) was added to dry N2-(phenoxyacetyl)-O6-{3-[2-(3-pyridyl)-1,3-dithian-2-yl]propyl}-2′-deoxyguanosine (120 mg, 0.188 mmol) in anhydrous pyridine (2 mL) at 0 °C. After the mixture was stirred overnight at 4 °C, MeOH (0.13 mL) was added and the reaction mixture was concentrated under reduced pressure. The residue was dissolved in CH2Cl2 (20 mL) and washed sequentially with 5% NaHCO3 (2 × 5 mL) and brine (20 mL). The organic layer was dried over anhydrous Na2SO4, filtered, and concentrated under reduced pressure. The pure product was obtained following silica gel flash chromatography eluting with 100:2:0.5 CH2Cl2/MeOH/ triethylamine (150 mg, 0.160 mmol, 85% yield, >95% pure): 1H NMR (CDCl3) δ 9.16 (d, J ) 2.3 Hz, 1H, 2-pyridyl), 8.78 (bs, 1H, CON2H), 8.51-8.49 (m, 1H, 6-pyridyl), 8.22-8.20 (m, 1H, 4-pyridyl), 7.97 (s, 1H, C8-H), 7.39-7.14 (m, 12H, 5-pyridyl, ArH), 7.07-6.99 (m, 3H, ArH), 6.78-6.74 (m, 4H, ArH), 6.55 (t, J ) 6.5 Hz, 1H, 1′-H), 4.80-4.77 (m, 1H, 3′-H), 4.68 (bs, 2H, phenyl-O-CH2CONH), 4.46 (t, J ) 6.6 Hz, 2H, O6-CH2), 4.164.15 (m, 1H, 5′-Ha), 3.75 (s, 6H, ArOCH3), 3.46-3.42 (m, 1H, 4′-H), 3.34-3.30 (m, 1H, 5′-Hb), 2.77-2.52 (m, 6H, 2′-H, SCH2CH2CH2S), 2.25-2.20 (m, 2H, O6-CH2CH2CH2), 1.98-1.88 (m, 4H, O6-CH2CH2, SCH2CH2CH2S); positive ESI-MS/MS m/z (relative intensity) 941 (M + 1, 8), 303 [M - N2-(phenoxyacetyl)O 6 -{3-[2-(3-pyridyl)-1,3-dithian-2-yl]propyl}-2′-deoxyguanosine, 100], 238 [M - 5′-O-(4,4′-dimethoxytrityl)-N2-(phenoxyacetyl)-2′-deoxyguanosine, 2]. 5′-O-(4,4′-Dimethoxytrityl)-N2-(phenoxyacetyl)-O6-{3-[2(3-pyridyl)-1,3-dithian-2-yl]propyl}-2′-deoxyguanosine-3′O-(2-cyanoethyl)-N,N-diisopropylamide-O-phosphite. Using a modification of a published procedure (25), N,N-diisopropylethylamine (100 mg, 139 µL, 0.795 mmol) was added to dry 5′-O-(4,4′-dimethoxytrityl)-N2-(phenoxyacetyl)-O6-{3-[2-(3pyridyl)-1,3-dithian-2-yl]propyl}-2′-deoxyguanosine (150 mg, 0.160 mmol) in dry THF (8 mL) at 0 °C. Cyanoethyl N,Ndiisopropylchlorophosphoramidite (60.5 mg, 56 µL, 0.238 mmol) was added, and the reaction mixture was stirred at room temperature for 1 h. The mixture was concentrated under reduced pressure. The residue was dissolved in ethyl acetate (25 mL) and washed with brine (3 × 25 mL). The organic layer was dried over anhydrous Na2SO4, filtered, and concentrated under reduced pressure. The product was purified using silica gel flash chromatography eluting with 20:80:10 hexane/ethyl acetate/triethylamine (112 mg, 0.098 mmol, 62% yield, >95% pure): 1H NMR (CDCl3) δ 9.15 (d, J ) 2.3 Hz, 1H, 2-pyridyl), 8.69 (bs, 1H, CON2H), 8.51-8.49 (m, 1H, 6-pyridyl), 8.23-8.20 (m, 1H, 4-pyridyl), 7.99 (2s, 1H, C8-H), 7.39-7.14 (m, 12H, 5-pyridyl, ArH), 7.08-6.99 (m, 3H, ArH), 6.78-6.74 (m, 4H, ArH), 6.44-6.40 (m, 1H, 1′-H), 4.79-4.69 (m, 3H, 3′-H, phenylO-CH2CONH), 4.49-4.46 (m, 2H, O6-CH2), 4.29-4.25 (m, 1H, 4′-H), 3.86-3.54 (m, 9H, ArOCH3, 5′-H, POCH2), 3.40-3.31 (m, 2H, Me2CHN), 2.84-2.57 (m, 7H, 2′-Ha, SCH2CH2CH2S, CH2CN), 2.45-2.42 (m, 1H, 2′-Hb), 2.25-2.21 (m, 2H, O6-CH2CH2CH2), 1.99-1.86 (m, 4H, O6-CH2CH2, SCH2CH2CH2S), 1.31-1.08 (m, 12H, CH3). DNA Oligonucleotide Synthesis. DNA oligonucleotides of the sequence 5′GGCGCTXGAGGCGTG in which X is O6-{3-[2(3-pyridyl)-1,3-dithian-2-yl]propyl}guanine (O6-pdbG) were prepared using previously published methods (24). They were synthesized on a Millipore Cyclone Plus DNA synthesizer on a 0.2 µmol scale. When it was time for the incorporation of the modified nucleoside, the machine was stopped and the column disconnected. With two gastight syringes, one on each end of the column, 100 µL of 0.5 M tetrazole and 20 mg of the O6pdbdG phosphoramidite in 100 µL of acetonitrile were passed over the resin by slowly pushing the solution from one syringe to the other. This was performed for 10 min. The column was reattached to the synthesizer, and the remaining nucleotides were added. The DNA oligonucleotide was removed from the resin by treating the column with concentrated ammonium hydroxide for 1.5 h. Heating at 55 °C for 6 h deprotected the DNA oligonucleotide. The solution was concentrated under reduced

Communications pressure. The residue was dissolved in 1 mL of 100 mM triethylammonium acetate (pH 7) and loaded onto a SepPak C-18 cartridge that had been preequilibrated in the same buffer. The cartridge was then washed sequentially with 3% ammonium hydroxide (3 mL), 100 mM triethylammonium acetate (pH 7, 3 mL), 2% trifluoroacetic acid (3 mL), 100 mM triethylammonium acetate (pH 7, 3 mL), and water (5 mL). The fully deprotected DNA oligonucleotide containing O6-{3-[2-(3-pyridyl)1,3-dithian-2-yl]propyl}-2′-deoxyguanosine was eluted off the cartridge using 30% acetonitrile in water. The DNA oligonucleotide was further purified by reverse-phase HPLC using a 3.9 mm × 300 mm Bondclone C18 column (Phenomenex, Torrence, CA). The column was eluted with solvent A [100 mM triethylammonium acetate (pH 7)] and solvent B (95% acetonitrile) using a linear gradient from 90% solvent A and 10% solvent B to 80% solvent A and 20% solvent B over the course of 15 min, followed by a 10 min linear gradient to 60% solvent A and 40% solvent B (1 mL/min). The DNA oligonucleotide eluted at approximately 18.7 min under these conditions. The DNA oligonucleotide containing O6-pobG was obtained by oxidatively removing the dithiane group with either a 4- or 10-fold excess of N-chlorosuccinimide (NCS) in 50% acetonitrile (100 µL) at room temperature. Similar results were obtained with either treatment. After 20 min, 100 mM triethylammonium acetate (800 µL) was added and the entire reaction mixture was injected onto a Bondclone C18 column. The column was eluted with a 40 min linear gradient from 100% solvent A to 60% solvent A and 40% solvent B. The product eluted at 24 min. The fractions containing the product were further purified on the same system using a 40 min linear gradient from 90% solvent A and 10% solvent B to 80% solvent A and 20% solvent B. The DNA oligonucleotide was further purified on a Hypersil ODS column (Keystone Scientific, Inc., Bellfonte, PA) eluting with solvent C [20 mM sodium phosphate (pH 7)] and solvent D (95% MeOH) using a 30 min linear gradient from 90% solvent C and 10% solvent D to 80% solvent C and 20% solvent D. The sample was desalted using the triethylammonium acetate HPLC system. The resulting DNA oligonucleotide was concentrated under vacuum. Enzyme Hydrolysis. The DNA oligonucleotides were incubated with snake venom phosphodiesterase in 10 mM Tris (pH 7) containing 5 mM MgCl2 for 30 min prior to the addition of alkaline phosphatase. The incubation was continued for an additional 30 min prior to HPLC analysis. The components of the hydrolysates were eluted from a Phenomenex Prodigy C18 column using a 30 min linear gradient from 100% C to 50% C and 50% D. See the Supporting Information for HPLC traces. Mass Spectral Analysis. Electrospray analyses of the DNA oligonucleotides were obtained on a SCIEX API III triplequadrupole instrument. The unmodified DNA oligonucleotide and the DNA oligonucleotide containing O6-pdbG were dissolved in 20% water/80% acetonitrile with 5% triethylamine (20 pmol/ µL), whereas the DNA oligonucleotide containing O6-pobG was dissolved in 50% MeOH/50% triethylammonium acetate (100 mM; rate of 25 pmol/µL). The DNA oligonucleotide solutions were infused into the electrospray source via a transfer line using a Harvard Apparatus pump at a rate of 3 µL/min. When the DNA oligonucleotides were in 20% water/80% acetonitrile/ 5% triethylamine, the ion voltage was -3800 V and the oriface voltage was -85 V. When the solvent was 50% MeOH/50% triethylammonium acetate (100 mM), the ion voltage was -6000 V and the oriface voltage was -125 V. The interface temperature was maintained at 52 °C. A total of 20 scans were averaged with a step size of 0.2 amu over the measured mass range of m/z 600-1800. Molecular masses were determined using the Hypermass method. The instrument was calibrated in the negative ion mode using the unmodified DNA oligonucleotide. Reaction of Modified DNA Oligonucleotides with AGT. The DNA oligonucleotide containing O6-pobG (100 pmol) was 32P-end-labeled with [32P]ATP (45 µCi) and T kinase (14 units) 4 in 50 mM Tris-HCl (pH 7.5) containing 10 mM MgCl2 and 10 mM 2-mercaptoethanol for 30 min at 37 °C. The solution was

Chem. Res. Toxicol., Vol. 12, No. 2, 1999 129 heated for 2 min at 100 °C, and the DNA oligonucleotide was purified via C18 spin column chromatography. A portion of the 32P-end-labeled DNA oligonucleotide was combined with a 10fold excess of the complementary DNA oligonucleotide and the mixture heated at 80 °C and slowly cooled to room temperature. AGT (1.6-3.7 pmol) was incubated with 32P-end-labeled DNA oligonucleotide containing O6-pobG (1.5-2.5 pmol) in 50 mM Tris (pH 7.8) containing 0.1 mM EDTA, 5 mM DTT, and 12.520% glycerol at 37 °C (total volume of 30 µL). Controls were performed in the absence of AGT for 30-60 min. At 0, 10, 20, 30, 45, and 60 min, aliquots (5 µL) were added to a solution (5 µL) containing 95% formamide, 5% 100 mM Na4EDTA, and 0.025% (w/v) bromophenol blue. The extent of reaction was determined by denaturing PAGE in 20% acrylamide (19:1 acrylamide/N,N′-methylene bisacrylamide) and 7 M urea in 1x TBE buffer (89 mM Tris, 89 mM boric acid, and 2 mM EDTA). The gel (40 cm × 33 cm × 0.4 cm) was run at 2000 mV for 2 h. The amount of radioactivity associated with each gel band was determined with a Bio-Rad GS 250 Molecular Imager. The extent of repair was calculated by dividing the amount of radioactivity in the product band by the sum of the radioactivity in the substrate and product bands. See the Supporting Information for the graph.

Results and Discussion Synthesis of O6-pobG-Containing DNA Oligonucleotides. The objective of this work was the preparation of DNA oligonucleotides containing site-specifically incorporated O6-pobG using phosphoramidite chemistry. The DNA oligonucleotides will be used in biochemical studies. Preliminary studies indicated that O6-pobdG was not stable with respect to the conditions of solid-phase DNA oligonucleotide synthesis. However, the dithianeprotected precursor, O6-pdbdG, was stable under these conditions. In a previous report, we demonstrated that this compound generates O6-pobdG upon reaction with N-chlorosuccinimide in the presence of silver ion (12). Therefore, we proposed to prepare site-specifically modified DNA oligonucleotides using the phosphoramidite of O6-pdbdG and then treat the resulting DNA oligonucleotide with NCS in the presence of silver ion to generate the desired product (Scheme 1). The phosphoramidite of O6-pdbdG was prepared using modifications of published methods (23-25). The N2position of this guanine derivative was protected with a phenoxyacetyl group. This protecting group can be removed under milder conditions than can an isobutyryl group. This avoids the formation of 2,6-diaminopurine and its subsequent conversion into 2′-deoxyguanosine as observed for other O6-alkylguanine derivatives (25, 26). DNA oligonucleotides of the sequence 5′GGCGCTXGAGGCGTG (X is O6-pdbG) were prepared using standard automated phosphoramidite chemistry. The resulting DNA oligonucleotide was then deprotected by 28% ammonium hydroxide at 55 °C for 6 h. The composition of the DNA oligonucleotide was confirmed by HPLC analysis following enzymatic digestion to the corresponding nucleosides. The presence of a peak coeluting with O6-pdbdG indicated that this nucleoside had survived the DNA synthesis and deprotection conditions. Electrospray mass spectral analysis of the oligonucleotide indicated a molecular weight of 4910 ( 3. This is 238 mass units higher than that of the unmodified oligonucleotide (MW ) 4672 ( 5), consistent with the presence of the 3-[2-(3pyridyl)-1,3-dithian-2-yl]propyl modification. The desired product, the DNA oligonucleotide containing O6-pobG, was generated upon treatment with NCS.

130 Chem. Res. Toxicol., Vol. 12, No. 2, 1999

Communications

Scheme 1. Synthetic Route to DNA Oligonucleotides Containing O6-pobGa

a

PAC, phenoxyacetyl; DMT, dimethoxytrityl; NCS, N-chlorosuccinimide.

Figure 1. Reaction between AGT and O6-pobG in doublestranded oligonucleotides. AGT (3.7 pmol) was incubated with 32P-end-labeled DNA oligonucleotide containing O6-pobG (2.5 pmol) at 37 °C. Controls were performed in the absence of AGT for 60 min. The unmodified oligonucleotide was separated from the oligonucleotide containing O6-pobG using PAGE (see Experimental Procedures for details).

This reaction was performed in the absence of silver ion since silver inhibited the reaction. NCS did not react with the unmodified DNA oligonucleotide (data not shown). Enzymatic digestion and HPLC analysis of the resulting DNA oligonucleotide indicated the presence of O6-pobdG and the absence of O6-pdbdG. These results were confirmed by electrospray MS analysis. The experimental molecular weight of this oligonucleotide was 4818 (expected, 4820). This is consistent with the replacement of the 1,3-dithiane group with oxygen. Reaction of O6-pobG-Containing DNA Oligonucleotides with Human AGT. Previously, we had shown that O6-pobG in DNA was a substrate for AGT whereas the nucleoside derivative was not (12). To determine whether this DNA oligonucleotide containing O6-pobG was a substrate for AGT, it was end-labeled with 32P and incubated with human recombinant AGT with a polyhistidine tag. PAGE separated the product of the reaction, the unmodified DNA oligonucleotide, from the modified oligonucleotide (Figure 1). Formation of the unmodified DNA oligonucleotide required active protein. The repair reaction was completed in less than 10 min. These results confirm that O6-pobG in DNA is a substrate for AGT. The formation of unmodified DNA oligonucleotide is consistent with a repair mechanism involving transfer of the alkyl group from the O6-position of guanine to the active site of AGT (27). Future studies will characterize the AGT reaction product.

Acknowledgment. The majority of this work was performed at the American Health Foundation. The Instrument Facility at the American Health Foundation is partially supported by National Cancer Institute Cancer Center Support Grant CA-17613. Dr. Jinsong Ni performed mass spectral analyses at the American Health Foundation. Ms. Nancy Raha and Dr. Leo Bonilla performed the electrospray mass spectral analyses of the DNA oligonucleotides at the University of Minnesota Cancer Center. We thank Dr. James McCloskey for his valuable advice regarding electrospray analysis of DNA oligonucleotides. This research was supported by Grants CA-59887 (L.A.P.) and CA-18137 (A.E.P.) from the National Cancer Institute. Supporting Information Available: HPLC traces for the enzymatic hydrolysis of the DNA oligonucleotides and a graph of the AGT-mediated repair of O6-pobG in double-stranded oligonucleotides. This material is available free of charge via the Internet at http://pubs.acs.org.

References (1) Hecht, S. S., Chen, C. B., Ohmori, T., and Hoffmann, D. (1980) Comparative carcinogenicity in F344 rats of the tobacco specific nitrosamines, N′-nitrosonornicotine and 4-(N-methyl-N-nitrosamino)-1-(3-pyridyl)-1-butanone. Cancer Res. 40, 298-302. (2) Hecht, S. S., and Hoffmann, D. (1988) Tobacco-specific nitrosamines: an important group of carcinogens in tobacco and tobacco smoke. Carcinogenesis 9, 875-884. (3) Hecht, S. S., Morse, M. A., Amin, S. G., Stoner, G. D., Jordan, K. G., Choi, C.-I., and Chung, F.-L. (1989) Rapid single dose model for lung tumor induction in A/J mice by 4-(methylnitrosamino)1-(3-pyridyl)-1-butanone and the effect of diet. Carcinogenesis 10, 1901-1904. (4) Hecht, S. S. (1998) Biochemistry, Biology, and Carcinogenicity of tobacco-specific N-nitrosamines. Chem. Res. Toxicol. 11, 560603. (5) Hecht, S. S., Trushin, N., Castonguay, A., and Rivenson, A. (1986) Comparative tumorigenicity and DNA methylation in F344 rats by 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone and N-nitrosodimethylamine. Cancer Res. 46, 498-502. (6) Hecht, S. S., Spratt, T. E., and Trushin, N. (1988) Evidence for 4-(3-pyridyl)-4-oxobutylation of DNA in F344 rats treated with the tobacco specific nitrosamines 4-(methylnitrosamino)-1-(3pyridyl)-1-butanone and N′-nitrosonornicotine. Carcinogenesis 9, 161-165.

Communications (7) Peterson, L. A., and Hecht, S. S. (1991) O6-Methylguanine is a critical determinant of 4-(methylnitrosamino)-1-(3-pyridyl)-1butanone tumorigenesis in A/J mouse lung. Cancer Res. 51, 55575564. (8) Spratt, T. E., Trushin, N., Lin, D., and Hecht, S. S. (1989) Analysis for N2-(pyridyloxobutyl)deoxyguanosine adducts in DNA of tissues exposed to tritium labeled 4-(methylnitrosamino)-1-(3-pyridyl)1-butanone and N′-nitrosonornicotine. Chem. Res. Toxicol. 2, 169173. (9) Brittebo, E. B., Castonguay, A., Furuya, K., and Hecht, S. S. (1983) Metabolism of tobacco specific nitrosamines by cultured rat nasal mucosa. Cancer Res. 43, 4343-4348. (10) Murphy, S. E., Heiblum, R., and Trushin, N. (1990) Comparative metabolism of N′-nitrosonornicotine and 4-(methylnitrosamino)1-(3-pyridyl)-1-butanone by cultured rat oral tissue and esophagus. Cancer Res. 50, 4685-4691. (11) Hecht, S. S., Reiss, B., Lin, D., and Williams, G. M. (1982) Metabolism of N′-nitrosonornicotine by cultured rat esophagus. Carcinogenesis 3, 453-456. (12) Wang, L., Spratt, T. E., Liu, X. K., Hecht, S. S., Pegg, A. E., and Peterson, L. A. (1997) Pyridyloxobutyl adduct O6-[4-oxo-4-(3pyridyl)butyl]guanine is present in 4-(acetoxymethylnitrosamino)1-(3-pyridyl)-1-butanone-treated DNA and is a substrate for O6alkylguanine-DNA alkyltransferase. Chem. Res. Toxicol. 10, 562567. (13) Ellison, K. S., Dogliotti, E., Connors, T. D., Basu, A. K., and Essigmann, J. M. (1989) Site-specific mutagenesis by O6-alkylguanines located in the chromosomes of mammalian cells: influence of the mammalian O6-alkylguanine-DNA alkyltransferase. Proc. Natl. Acad. Sci. U.S.A. 86, 8620-8624. (14) Loechler, E. L., Green, C. L., and Essigmann, J. M. (1984) In vivo mutagenesis by O6-methylguanine built into a unique site in a viral genome. Proc. Natl. Acad. Sci. U.S.A. 81, 6271-6275. (15) Bhanot, O. S., and Ray, A. (1986) The in vivo mutagenic frequency and specificity of O6-methylguanine in øX174 replicative form DNA. Proc. Natl. Acad. Sci. U.S.A. 83, 7348-7352. (16) Pauly, G. T., Hughes, S. H., and Moschel, R. C. (1995) Mutagenesis in Escherichia coli by three O6-substituted guanines in double-stranded or gapped plasmids. Biochemistry 34, 89248930. (17) Kamiya, H., Miura, K., Ohtomo, N., Nishimura, S., and Ohtsuka, E. (1991) Transforming activity of a synthetic c-Ha-ras gene

Chem. Res. Toxicol., Vol. 12, No. 2, 1999 131

(18)

(19) (20)

(21)

(22)

(23)

(24)

(25)

(26)

(27)

containing O6-methylguanine in codon 12. Jpn. J. Cancer Res. 82, 997-1002. Pauly, G. T., Hughes, S. H., and Moschel, R. C. (1998) Comparison of mutagenesis by O6-methyl- and O6-ethylguanine and O4methylthymine in Escherichia coli using double-stranded and gapped plasmids. Carcinogenesis 19, 457-461. Pegg, A. E., and Byers, T. L. (1992) Repair of DNA containing O6-alkylguanine. FASEB J. 6, 2302-2310. Peterson, L. A., Liu, X.-K., and Hecht, S. S. (1993) Pyridyloxobutyl DNA adducts inhibit the repair of O6-methylguanine. Cancer Res. 53, 2780-2785. Liu, X. K., Spratt, T. E., Murphy, S. E., and Peterson, L. A. (1996) Pyridyoxobutylation of guanine residues by 4-(acetoxymethylnitrosamino)-1-(3-pyridyl)-1-butanone generates substrates of O6alkylguanine-DNA alkyltransferase. Chem. Res. Toxicol. 9, 949953. Edara, S., Kanugula, S., Goodtzova, K., and Pegg, A. E. (1996) Resistance of the human O6-alkylguanine-DNA alkyltransferase containing arginine at codon 160 to inactivation by O6-benzylguanine. Cancer Res. 56, 5571-5575. Schulhof, J. C., Molko, D., and Teoule, R. (1987) The final deprotection step in oligonucleotide synthesis is reduced to a mild and rapid ammonia treatment by using labile base-protecting groups. Nucleic Acids Res. 15, 397-416. Spratt, T. E., and Campbell, C. R. (1994) Synthesis of oligodeoxynucleotides containing analogs of O6-methylguanine and reaction with O6-alkylguanine-DNA alkyltransferase. Biochemistry 33, 11364-11371. Smith, C. A., Xu, Y.-Z., and Swann, P. F. (1990) Solid-phase synthesis of oligodeoxynucleotides containing O6-alkylguanine. Carcinogenesis 11, 811-816. Gaffney, B. L., Marky, L. A., and Jones, R. A. (1984) Synthesis and characterization of a set of four dodecadeoxyribonucleoside undecaphosphates containing O6-methylguanine opposite adenine, cytosine, guanine, and thymine. Biochemistry 23, 56865691. Pegg, A. E. (1990) Properties of mammalian O6-alkylguanine-DNA transferases. Mutat. Res. 233, 165-175.

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