Analysis of the Effect of Bulk at N2-Alkylguanine DNA Adducts on Catalytic Efficiency and Fidelity of the Processive DNA Polymerases Bacteriophage T7 Exonuclease- and HIV-1 Reverse Transcriptase
2004; Elsevier BV; Volume: 279; Issue: 18 Linguagem: Inglês
10.1074/jbc.m313759200
ISSN1083-351X
AutoresJeong‐Yun Choi, F. Peter Guengerich,
Tópico(s)Advanced biosensing and bioanalysis techniques
ResumoThe N-2 atom of guanine (G) is susceptible to modification by various carcinogens. Oligonucleotides with increasing bulk at this position were analyzed for fidelity and catalytic efficiency with the processive DNA polymerases human immunodeficiency virus, type 1, reverse transcriptase (RT), and bacteriophage T7 exonuclease- (T7-). RT and T7- effectively bypassed N2-methyl(Me)G and readily extended primers but were strongly blocked by N2-ethyl(Et)G, N2-isobutylG, N2-benzylG, and N2-methyl(9-anthracenyl)G. Steady-state kinetics of single nucleotide incorporation by RT and T7- showed a decrease of 103 in kcat/Km for dCTP incorporation opposite N2-MeG and a further large decrease opposite N2-EtG. Misincorporation frequency was increased 102-103-fold by a Me group and another ∼103-fold by an Et group. dATP was preferentially incorporated opposite bulky N2-alkylG molecules. N2-MeG attenuated the pre-steady-state kinetic bursts with RT and T7-, and N2-EtG eliminated the bursts. Large elemental effects with thio-dCTP(αS) were observed with N2-EtG (6- and 72-fold decreases) but were much less with N2-MeG, indicating that the N2-Et group may affect the rate of the chemistry step (phosphodiester bond formation). Similar values of Kd(dCTP) and Kd(DNA) and koff rates of DNA substrates from RT and T7- indicate that ground-state binding and dissociation rates are not considerably affected by the bulk. We conclude that even a Me group at the guanine N-2 atom can cause a profound interfering effect on the fidelity and efficiency; an Et or larger group causes preferential misincorporation and strong blockage of replicative polymerases, probably at and before the chemistry step, demonstrating the role of bulk in DNA lesions. The N-2 atom of guanine (G) is susceptible to modification by various carcinogens. Oligonucleotides with increasing bulk at this position were analyzed for fidelity and catalytic efficiency with the processive DNA polymerases human immunodeficiency virus, type 1, reverse transcriptase (RT), and bacteriophage T7 exonuclease- (T7-). RT and T7- effectively bypassed N2-methyl(Me)G and readily extended primers but were strongly blocked by N2-ethyl(Et)G, N2-isobutylG, N2-benzylG, and N2-methyl(9-anthracenyl)G. Steady-state kinetics of single nucleotide incorporation by RT and T7- showed a decrease of 103 in kcat/Km for dCTP incorporation opposite N2-MeG and a further large decrease opposite N2-EtG. Misincorporation frequency was increased 102-103-fold by a Me group and another ∼103-fold by an Et group. dATP was preferentially incorporated opposite bulky N2-alkylG molecules. N2-MeG attenuated the pre-steady-state kinetic bursts with RT and T7-, and N2-EtG eliminated the bursts. Large elemental effects with thio-dCTP(αS) were observed with N2-EtG (6- and 72-fold decreases) but were much less with N2-MeG, indicating that the N2-Et group may affect the rate of the chemistry step (phosphodiester bond formation). Similar values of Kd(dCTP) and Kd(DNA) and koff rates of DNA substrates from RT and T7- indicate that ground-state binding and dissociation rates are not considerably affected by the bulk. We conclude that even a Me group at the guanine N-2 atom can cause a profound interfering effect on the fidelity and efficiency; an Et or larger group causes preferential misincorporation and strong blockage of replicative polymerases, probably at and before the chemistry step, demonstrating the role of bulk in DNA lesions. High fidelity of DNA polymerization is essential for preservation of genomic integrity and survival of organisms. When a DNA polymerase inserts an incorrect nucleotide, mutations can result (1Friedberg E. Walker G.C. Siede W. DNA Repair and Mutagenesis. American Society for Microbiology, Washington, D. C.1995: 1-83Google Scholar). Mutation can be an advantage for bacteria, but it can lead to detrimental effects in humans, including aging and cancer. Although having intrinsic high fidelity with unmodified DNA bases (10-3 to 10-6 error rate per base), DNA polymerases often have potent miscoding abilities (altered base pairing with incoming dNTPs) opposite modified DNA adducts, thus inducing mutations during replication (2Echols H. Goodman M.F. Annu. Rev. Biochem. 1991; 60: 477-511Google Scholar, 3Kunkel T.A. Bebenek K. Annu. Rev. Biochem. 2000; 69: 497-529Google Scholar). DNA adducts act as core sources of misincorporation because they are inevitably formed by endogenous sources and exogenous mutagens in cellular DNA (4Searle C.E. Chemical Carcinogens. American Chemical Society, Washington, D. C.1984Google Scholar). Some adducts that escape repair are usually present in replicating DNA, and therefore, the ability of polymerases to incorporate the appropriate base partner in the presence of a modification to the base in DNA is critical to preserving genetic information. Moreover, some DNA adducts can cause stalling of DNA polymerases at those sites during DNA replication, which in turn can lead to cellular death, depending on the extent. Therefore, DNA polymerases play key roles in both misincorporation and blockage during DNA replication when DNA adducts are present. Depending on the DNA adduct, different DNA polymerases show different degrees of misincorporation and blockage (1Friedberg E. Walker G.C. Siede W. DNA Repair and Mutagenesis. American Society for Microbiology, Washington, D. C.1995: 1-83Google Scholar, 2Echols H. Goodman M.F. Annu. Rev. Biochem. 1991; 60: 477-511Google Scholar, 3Kunkel T.A. Bebenek K. Annu. Rev. Biochem. 2000; 69: 497-529Google Scholar). Some DNA adducts are small (e.g. abasic sites and oxidative adducts), but others range in size to very bulky adducts, e.g. pyrimidine dimers, photoproducts, large carcinogen-bound adducts, and cross-links (4Searle C.E. Chemical Carcinogens. American Chemical Society, Washington, D. C.1984Google Scholar). The molecular size of DNA adducts might be a key differentiating factor in the misincorporation and blockage of DNA polymerases, which have confined active site pockets. The bulkiness of DNA adducts may be more important in mutagenesis by environmental xenobiotics, because many of these are larger than "endogenous" DNA adducts such as 8-oxo-7,8-dihydroG. 1The abbreviations used are: G, guanine; T7-, bacteriophage DNA polymerase T7 (exonuclease deficient); dGuo, 2′-deoxyguanosine; 3′-dGuo (or 3′-dG), 3′-deoxyguanosine; Me2SO, dimethyl sulfoxide; FAM, 6-carboxyfluorescein; N2-CH2(9-anthracenyl), N2-methyl(9-anthracenyl); dCTPαS, 2′-deoxycytidine 5′-O-(1-thiotriphosphate); RT, human immunodeficiency virus-1 reverse transcriptase; MS, mass spectrometry; MALDI-TOF, matrix-assisted laser desorption ionization time-of-flight; HPLC, high pressure liquid chromatography. 1The abbreviations used are: G, guanine; T7-, bacteriophage DNA polymerase T7 (exonuclease deficient); dGuo, 2′-deoxyguanosine; 3′-dGuo (or 3′-dG), 3′-deoxyguanosine; Me2SO, dimethyl sulfoxide; FAM, 6-carboxyfluorescein; N2-CH2(9-anthracenyl), N2-methyl(9-anthracenyl); dCTPαS, 2′-deoxycytidine 5′-O-(1-thiotriphosphate); RT, human immunodeficiency virus-1 reverse transcriptase; MS, mass spectrometry; MALDI-TOF, matrix-assisted laser desorption ionization time-of-flight; HPLC, high pressure liquid chromatography. We have studied some details of misincorporation and blockage with the model replicative polymerases T7- and RT in work focused on small modifications of guanine at the C-8 (8-oxo-7,8-dihydroG) and O-6 atoms (O6-methylG, O6-benzylG) of guanine (5Furge L.L. Guengerich F.P. Biochemistry. 1997; 36: 6475-6487Google Scholar, 6Woodside A.M. Guengerich F.P. Biochemistry. 2002; 41: 1027-1038Google Scholar, 7Woodside A.M. Guengerich F.P. Biochemistry. 2002; 41: 1039-1050Google Scholar). These adducts are bypassed fairly readily, and the misincorporations are considerable. We have also found that large groups (glutathionylethylene) at the O-6 and N-2 atoms are very blocking to several polymerases (8Kim M.-S. Guengerich F.P. Chem. Res. Toxicol. 1998; 11: 311-316Google Scholar). In this work we decided to evaluate systemically the effect of varying bulk at the guanine N-2 atom. The N-2 atom of guanine is susceptible to modification by various potential carcinogens including formaldehyde (9Yasui M. Matsui S. Ihara M. Laxmi Y.R. Shibutani S. Matsuda T. Nucleic Acids Res. 2001; 29: 1994-2001Google Scholar), acetaldehyde (10Terashima I. Matsuda T. Fang T.-W. Suzuki N. Kobayashi J. Kohda K. Shibutani S. Biochemistry. 2001; 40: 4106-4114Google Scholar) (a metabolite of ethanol and also produced endogenously), styrene oxide (11Forgacs E. Latham G. Beard W.A. Prasad R. Bebenek K. Kunkel T.A. Wilson S.H. Lloyd R.S. J. Biol. Chem. 1997; 272: 8525-8530Google Scholar), and the oxidation products of various polycyclic aromatic hydrocarbons, e.g. benzo[a]pyrene (12Meehan T. Straub K. Nature. 1979; 277: 410-412Google Scholar, 13Cheng S.C. Hilton B.D. Roman J.M. Dipple A. Chem. Res. Toxicol. 1989; 2: 334-340Google Scholar). Benzo[a]pyrene diol epoxide N2-G adducts have been studied extensively; they are mutagenic and generate G to T transversions in Escherichia coli and simian kidney (COS7) cells and are readily bypassed by DNA polymerase κ, depending upon the base sequence context (14Fernandes A. Liu T. Amin S. Geacintov N.E. Grollman A.P. Moriya M. Biochemistry. 1998; 37: 10164-10172Google Scholar, 15Rechkoblit O. Zhang Y. Guo D. Wang Z. Amin S. Krzeminsky J. Louneva N. Geacintov N.E. J. Biol. Chem. 2002; 277: 30488-30494Google Scholar, 16Huang X. Kolbanovskiy A. Wu X. Zhang Y. Wang Z. Zhuang P. Amin S. Geacintov N.E. Biochemistry. 2003; 42: 2456-2466Google Scholar). N2-EthyldGuo has been detected in granulocyte and lymphocyte DNA of alcoholic patients and in human urine (17Fang J.L. Vaca C.E. Carcinogenesis. 1997; 18: 627-632Google Scholar, 18Matsuda T. Terashima I. Matsumoto Y. Yabushita H. Matsui S. Shibutani S. Biochemistry. 1999; 38: 929-935Google Scholar). Even the relatively small N2-methylG and N2-ethylG adducts have been reported to be miscoding. The Klenow fragment (of E. coli DNA polymerase I) incorporated mainly dCTP, along with some dTTP, opposite N2-methylG in an oligonucleotide (9Yasui M. Matsui S. Ihara M. Laxmi Y.R. Shibutani S. Matsuda T. Nucleic Acids Res. 2001; 29: 1994-2001Google Scholar). The same enzyme (exonuclease- form) incorporated dGTP and dCTP to similar extents opposite N2-ethylG and also efficiently incorporated N2-ethylGTP opposite a template C (10Terashima I. Matsuda T. Fang T.-W. Suzuki N. Kobayashi J. Kohda K. Shibutani S. Biochemistry. 2001; 40: 4106-4114Google Scholar). In this study, we prepared site-specifically modified oligonucleotides containing N2-methylG, N2-ethylG, N2-isobutylG, N2-benzylG, and N2-CH2(9-anthracenyl)G, with gradually increasing bulk at the guanine N2-alkyl adduct (Fig. 1), and we used these with the replicative DNA polymerases T7- and RT, which have been good models of replicative polymerases (19Einolf H.J. Guengerich F.P. J. Biol. Chem. 2000; 275: 16316-16322Google Scholar) and have advantages over E. coli polymerase I (and Klenow fragment) (20Johnson K.A. Annu. Rev. Biochem. 1993; 62: 685-713Google Scholar). We investigated steady-state and pre-steady-state kinetics and also the features of DNA substrate binding to polymerase. With both polymerases, we found that even N2-methylG exerted a strong effect, and a further dramatic difference on both misinsertion and blockage was seen in increasing the bulk from N2-methylG to N2-ethylG. The results are considered in the context of knowledge about replicative polymerases and their kinetic behavior. Materials—Unlabeled dNTPs, dCTPαS, and dATPαS were purchased from Amersham Biosciences. [γ-32P]ATP (specific activity 3000 Ci/mmol) was purchased from PerkinElmer Life Sciences. T4 polynucleotide kinase was purchased from New England Biolabs (Beverly, MA). Bio-spin columns were purchased from Bio-Rad. Most chemicals used for synthesis were purchased from Aldrich. Synthesis of 9-(Aminomethyl)anthracene—The amine was prepared by a modified Gabriel procedure. 9-(Chloromethyl)anthracene (Aldrich, 9.0 g, 13 mmol) was stirred with potassium phthalimide (2.5 g, 13.5 mmol) in 80 ml of N,N-dimethylformamide at 60 °C for 3 h. The mixture was diluted with 100 ml of H2O, and the product (9-[N-phthalimido-(methyl)]anthracene) was extracted with three 200-ml portions of CHCl3. The organic layers were combined, dried with Na2SO4, and concentrated in vacuo (2.35 g, 57% yield). The product was dissolved in 450 ml of C2H5OH and heated with 1.0 ml of NH2NH2·H2O (21 mmol) under reflux for 4 h. The solvent was removed in vacuo, and the resulting 9-(aminomethyl)anthracene was purified by chromatography on a 2 × 25-cm silica gel column, eluting with CH2Cl2-CH3OH (98-2, v/v) (1.22 g, 84% yield): MS (electron impact) m/z 207 (relative abundance 100, M+), 206 (57, M - 1), 191 (49, M - 16), 178 (80, M - 29); 1H NMR (CDCl3) δ 4.82 (s, 2H, -C H2NH2), 5.28 (s, -NH2), 7.54 (m, 4H, H-2, H-3, H-6, and H-7), 8.03 (d, 2H, H-1 and H-8), 8.33 (d, 2H, H-4 and H-5), 8.39 (s, 1H, H-10). Enzymes—RT and T7- were expressed and purified as described previously (5Furge L.L. Guengerich F.P. Biochemistry. 1997; 36: 6475-6487Google Scholar) by using stock plasmids provided by S. Hughes (RT, Frederick Cancer Facility, Frederick, MD) (21Lunn C.A. Kathju S. Wallace B.J. Kushner S.R. Pigiet V. J. Biol. Chem. 1984; 259: 10469-10474Google Scholar, 22Patel S.S. Wong I. Johnson K.A. Biochemistry. 1991; 30: 511-525Google Scholar) and K. A. Johnson (T7- and thioredoxin, University of Texas, Austin, TX) (23Le Grice S.F.J. Cameron C.E. Benkovic S.J. Methods Enzymol. 1995; 262: 130-144Google Scholar). The T7- expression and purification procedures were as modified by Zang et al. 2H. Zang, T. M. Harris, and F. P. Guengerich, submitted for publication. T7- was reconstituted with thioredoxin immediately prior to use as described (22Patel S.S. Wong I. Johnson K.A. Biochemistry. 1991; 30: 511-525Google Scholar). Protein concentrations were determined using ϵ280 values of 144 mm-1 cm-1 for T7-, 13.7 mm-1 cm-1 for thioredoxin, and 522 mm-1 cm-1 for RT (24Furge L.L. Guengerich F.P. Biochemistry. 1999; 38: 4818-4825Google Scholar). Oligodeoxynucleotides—18-FAM-mer, 24 (3′-dGuo)-mer, and the unmodified 24-mer and 36-mer (Table I) were purchased from Midland Certified Reagent Co. (Midland, TX). Four 36-mers, each containing a guanine N2-adduct (e.g. N2-methylG, N2-ethylG, N2-isobutylG, or N2-benzylG), were synthesized on an Expedite 8909 DNA synthesizer (PerSeptive Biosystems, Framingham, MA) from tert-butylphenoxyacetyl-protected cyanoethyl phosphoramidites and the adducted phosphoramidites (see below) by using standard DNA synthesis protocols. N2-Alkyl adducts of dGuo were synthesized from dGuo or 2-fluoro-(O6-trimethylsilylethyl)-2′-deoxyinosine (25Decorte B.L. Tsarouhtsis D. Kuchimanchi S. Cooper M.D. Horton P. Harris C.M. Harris T.M. Chem. Res. Toxicol. 1996; 9: 630-637Google Scholar) as described below, and the correct structures and molecular masses were confirmed by 1H NMR spectroscopy and electrospray MS. The 5′-O-dimethoxytrityl-3′-phosphoramidite derivatives of dGuo N2-alkyl adducts were prepared by standard procedures (26Meyer R.B. Agrawal S. Protocols for Oligonucleotide Conjugates. Humana Press Inc., Totowa, NJ1994: 73-91Google Scholar, 27Nechev L.V. Kozekov I.D. Brock A.K. Rizzo C.J. Harris T.M. Chem. Res. Toxicol. 2002; 15: 607-613Google Scholar) with minor modification and then introduced into oligonucleotides. We found that protection of the N-2 atom was not needed in the preparation of the phosphoramidites used for synthesis, due to the effect of the added bulk (even with N2-methyldGuo). DNA oligonucleotides were purified by HPLC and denaturing PAGE (see below). MALDI-TOF MS was used to confirm correct mass/charge ratios (m/z) of the oligonucleotides (see Supplemental Material). Purity was analyzed by capillary gel electrophoresis on a Beckman P/ACE 2000 instrument as described previously (6Woodside A.M. Guengerich F.P. Biochemistry. 2002; 41: 1027-1038Google Scholar). We estimated the purity of the oligonucleotides used here to be ∼99% (see Supplemental Material). The extinction coefficients for the oligonucleotides, estimated by the Borer method (28Borer P.N. Fasman G.D. Handbook of Biochemistry and Molecular Biology. 3rd Ed. CRC Press, Inc., Cleveland, OH1975: 589-590Google Scholar), were as follow: 18-FAM-mer, ϵ260 = 196 mm-1 cm-1; 24-mer, ϵ260 = 224 mm-1 cm-1; and 36-mer, ϵ260 = 310 mm-1 cm-1.Table IOligodeoxynucleotides used in this study18-FAM-mer5′ (FAM)-AGCCAGCCGCAGACGCAG24-mer5′-GCCTCGAGCCAGCCGCAGACGCAG24-(3′-dGuo)mer5′-GCCTCGAGCCAGCCGCAGACGCA (3′-dG)36-mer3′-CGGAGCTCGGTCGGCGTCTGCGTCG*CTCCTGCGGCT Open table in a new tab Synthesis of N2-MethyldGuo (29Sako M. Kawada H. Hirota K. J. Org. Chem. 1999; 64: 5719-5721Google Scholar)—dGuo (479 mg, 1.79 mmol) and NaBH3CN (700 mg, 11.1 mmol) were dissolved in 100 ml of 50% aqueous CH3OH (v/v) under an argon atmosphere. Formaldehyde (840 μl, 11.2 mmol) was added, and the sample was kept at room temperature for 2 days. The pH was then lowered to 4 by using 1 n HCl. After several days the solvent was removed, and the sample was redissolved in H2O with a small amount of CH3OH. The N2-methyldGuo was purified using a semi-preparative octadecylsilane (C18) HPLC column (5 μm, 10 × 250 mm, Phenomenex, Torrance, CA) with a 4.0 ml/min gradient composed of 10 mm NH4HCO3 (solvent A) and CH3OH (solvent B) as follows: 90% A and 10% B at 0 min, 90% A and 10% B at 5 min, and 70% A and 30% B at 30 min (all v/v). The total yield after purification was 48 mg (9.5%). MS: m/z 282 (MH+); 1H NMR (Me2SO-d6): δ 2.20 (m, 1H, H2″), 2.60 (m, 1H, H2′), 2.80 (m, 1H, -CH3), 3.50 (m, 2H, H5′ and H5″), 3.80 (m, 1H, H4′), 4.36 (m, 1H, H3′), 4.85 (bs, 1H, 5′-OH), 5.3 (bs, 1H, 3′-OH), 6.15 (t, 1H, H1′), 6.47 (bs, 1H, 2-NH), 7.89 (s, 1H, H8), and 10.2 (bs, 1H, 1-NH). Synthesis of N2-EthyldGuo—2-Fluoro-(O6-trimethylsilylethyl)-2′-deoxyinosine (52 mg, 0.14 mmol) (25Decorte B.L. Tsarouhtsis D. Kuchimanchi S. Cooper M.D. Horton P. Harris C.M. Harris T.M. Chem. Res. Toxicol. 1996; 9: 630-637Google Scholar) and C2H5NH2 (25 mg, 0.55 mmol) were dissolved in a mixture of dry Me2SO (300 μl) and diisopropylethylamine (75 μl), and the reaction mixture was stirred at 60 °C for 15 h, according to procedures published previously (25Decorte B.L. Tsarouhtsis D. Kuchimanchi S. Cooper M.D. Horton P. Harris C.M. Harris T.M. Chem. Res. Toxicol. 1996; 9: 630-637Google Scholar, 30Nechev L.V. Zhang M.Z. Tsarouhtsis D. Tamura P.J. Wilkinson A.S. Harris C.M. Harris T.M. Chem. Res. Toxicol. 2001; 14: 379-388Google Scholar) with some modification. The solvent was removed in vacuo, and 5% (v/v) CH3CO2H (5 ml) was added, and the mixture was stirred at room temperature for 2 h. The solvent was removed, and the dry residue was purified by flash column chromatography on silica gel using CH3CN/H2O/NH4OH, 90:5:5 (v/v/v), as the eluent. The total yield after purification was 38 mg (92%). MS: m/z 296 (MH+); 1H NMR (Me2SO-d6): δ 1.10 (t, 1H, CH2CH3), 2.20 (m, 1H, H2″), 2.61 (m, 1H, H2′), 3.53 (m, 2H, H5′ and H5″), 3.80 (m, 1H, H4′), 4.36 (m, 1H, H3′), 4.85 (t, 1H, 5′-OH), 5.25 (d, 1H, 3′-OH), 6.14 (t, 1H, H1′), 6.34 (bs, 1H, 2-NH), 7.88 (s, 1H, H8), and 10.48 (bs, 1H, 1-NH). Synthesis of N2-IsobutyldGuo—2-Fluoro-(O6-trimethylsilylethyl)-2′-deoxyinosine (52 mg, 0.14 mmol) (25Decorte B.L. Tsarouhtsis D. Kuchimanchi S. Cooper M.D. Horton P. Harris C.M. Harris T.M. Chem. Res. Toxicol. 1996; 9: 630-637Google Scholar) and isobutylamine (36 mg, 0.49 mmol) were dissolved in a mixture of dry Me2SO (300 μl) and diisopropylethylamine (75 μl), and the reaction mixture was stirred at 60 °C for 15 h, according to procedures published previously (25Decorte B.L. Tsarouhtsis D. Kuchimanchi S. Cooper M.D. Horton P. Harris C.M. Harris T.M. Chem. Res. Toxicol. 1996; 9: 630-637Google Scholar, 30Nechev L.V. Zhang M.Z. Tsarouhtsis D. Tamura P.J. Wilkinson A.S. Harris C.M. Harris T.M. Chem. Res. Toxicol. 2001; 14: 379-388Google Scholar) with slight modification. The solvent was evaporated under vacuum, and 5% (v/v) CH3CO2H (5 ml) was added. The mixture was stirred at room temperature for 2 h. The solvent was removed, and the dry residue was purified by flash column chromatography on silica gel using CH3CN/H2O/NH4OH, 90:5:5 (v/v/v), as the eluent. The yield after purification was 40 mg (88%). MS: m/z 324 (MH+); 1H NMR (Me2SO-d6): δ 0.91 (d, 6H, CH(CH3)2), 1.83 (m, 1H, CH(CH3)2), 2.20 (m, 1H, H2″), 2.60 (m, 1H, H2′), 3.10 (d, 2H, CH2CH), 3.52 (m, 2H, H5′ and H5″), 3.78 (m, 1H, H4′), 4.34 (m, 1H, H3′), 4.84 (t, 1H, 5′-OH), 5.26 (d, 1H, 3′-OH), 6.13 (t, 1H, H1′) 6.39 (bs, 1H, 2-NH), 7.88 (s, 1H, H8), and 10.34 (bs, 1H, 1-NH). Synthesis of N2-BenzyldGuo—To prepare N2-benzyldGuo, dGuo (302 mg, 1.13 mmol) and NaBH3CN (454 mg, 7.22 mmol) were dissolved in 70 ml of 50% aqueous CH3OH (v/v) under an argon atmosphere (29Sako M. Kawada H. Hirota K. J. Org. Chem. 1999; 64: 5719-5721Google Scholar). Benzylaldehyde (4 ml) was added, and the solution was kept at 50 °C for 2 days. Residual benzaldehyde was removed by extraction with diethyl ether. The product was purified using a Sep-Pak Vac C18 column (Millipore, Bedford, MA) by applying the sample and eluting sequentially with 50 ml each of 0, 10, 20, 30, 40, and 50% CH3OH in H2O (v/v). The product was eluted in the 30 and 40% CH3OH (v/v) fractions. The total yield after purification was 60 mg (15%). MS: 358 (MH+); 1H NMR (Me2SO-d6): δ 2.15 (m, 1H, H2″), 3.50 (m, 2H, H5′, and H5″), 3.78 (m, 1H, H4′), 4.31 (m, 1H, H3′), 4.49 (s, 2H, CH2Ph), 4.83 (t, 1H, 5′-OH), 5.25 (d, 1H, 3′-OH), 6.11 (t, 1H, H1′), 6.84 (bs, 1H, 2-NH), 7.27 (5H, benzyl protons), 7.90 (s, 1H, H8), and 10.57 (bs, 1H, 1-NH) (the H2′ proton was hidden under the Me2SO signal at 2.5 ppm). Synthesis of 5′-O-Dimethoxytrityl-3′-phosphoramidite Derivatives of dGuo N2-Adducts and Site-specifically Modified 36-mers—Dimethoxytritylation and phosphitylation of dGuo N2-alkyl adducts were performed according to standard methods (26Meyer R.B. Agrawal S. Protocols for Oligonucleotide Conjugates. Humana Press Inc., Totowa, NJ1994: 73-91Google Scholar, 27Nechev L.V. Kozekov I.D. Brock A.K. Rizzo C.J. Harris T.M. Chem. Res. Toxicol. 2002; 15: 607-613Google Scholar) with minor modification. Briefly, for the dimethoxytritylation of each dGuo N2-adduct, the N2-dGuo adduct (∼50 mg, ∼0.2 mmol) was dried with anhydrous pyridine (3 × 10 ml). The sample was redissolved in 6 ml of pyridine. 4,4′-Dimethoxytrityl chloride (∼70 mg) was added, and the mixture was stirred at room temperature for ∼5 h, until the disappearance of starting material, as judged by TLC (CH2Cl2/CH3OH/(C2H5)3N, 95:4:1, v/v/v) during the course of the reaction. The solvent was removed under vacuum, and the residue was purified by flash column chromatography on silica gel (CH2Cl2/CH3OH/(C2H5)3N, 98:1.5:0.5 to 90:9.5:0.5, v/v/v). The identities of products were confirmed by 1H NMR spectroscopy and electrospray MS. For the further phosphitylation, dimethoxytrityl derivatives of dGuo N2-adducts (∼100 mg) were dried with anhydrous pyridine (3 × 10 ml) and placed under vacuum overnight (vacuum pump, <0.1 mm Hg). Dry CH2Cl2 (∼2.5 ml), tetrazole (∼10 mg), and 2-cyanoethyl-N,N,N′,N′-tetraisopropyl phosphoramidite (∼60 μl) were added, and the mixture was stirred at room temperature for 4 h. The solvent was removed, and the residue was purified by flash column chromatography on silica gel (CH2Cl2/CH3OH/(C2H5)3N, 98:1.5:0.5 to 90:9.5:0.5, v/v/v). The 36-mer oligonucleotides containing dGuo N2-adducts were synthesized on an Applied Biosystems DNA synthesizer on a 1-μmol scale using the corresponding phosphoramidites and a standard DNA synthesis protocol. After overnight deprotection in aqueous 0.1 n NaOH, the beads were pelleted, and the solution was neutralized. The supernatant was filtered and lyophilized before oligonucleotide purification. Synthesis of N2-CH2(9-anthracenyl)dGuo-containing 36-mer—The 36-mer oligonucleotide containing N2-CH2(9-anthracenyl)dGuo adduct was prepared according to the post-oligomerization methodology developed by Harris and co-workers (25Decorte B.L. Tsarouhtsis D. Kuchimanchi S. Cooper M.D. Horton P. Harris C.M. Harris T.M. Chem. Res. Toxicol. 1996; 9: 630-637Google Scholar, 31Harris C.M. Zhou L. Strand E.A. Harris T.M. J. Am. Chem. Soc. 1991; 113: 4328-4329Google Scholar). (An alternative approach had been used by Casale and McLaughlin (32Casale R. McLaughlin L.W. J. Am. Chem. Soc. 1990; 112: 5264-5271Google Scholar).) A 2-fluoro-O6-[2-(p-nitrophenyl)ethyl]-2′-deoxyinosine-containing 36-mer was synthesized on an Applied Biosystems DNA synthesizer on a 1-μmol scale using the phosphoramidite of 2-fluoro-O6-[2-(p-nitrophenyl)ethyl]-2′-deoxyinosine and a standard DNA synthesis protocol. After synthesis, oligonucleotide-bound beads were dried and reacted with 9-(aminomethyl)anthracene (100 mg, see above) in a mixture of anhydrous Me2SO (350 μl) and diisopropylethylamine (100 μl) for 24 h at 65 °C. Beads were washed three times with Me2SO and then CH3CN. The dried beads were reacted with 1 m 1,8-diazabicyclo(5,4,0)undec-7-ene in CH3CN (1 ml) at room temperature for 4 h to remove the p-nitrophenylethyl protecting group. Beads were washed three times with CH3OH and then CH3CN. After overnight deprotection of the oligonucleotide in aqueous 0.1 n NaOH, the beads were pelleted, and the solution was neutralized. The supernatant was filtered and taken to dryness by lyophilization prior to oligonucleotide purification. The presence of N2-CH2(9-anthracenyl)G in the oligonucleotide was confirmed using MALDI-TOF MS: m/z calculated for [MH]+ 11244.4 and found 11244.2. Purification of Oligonucleotides—Oligonucleotides were purified on a Zorbax Oligo HPLC column (mixed ion-exchange and reversed-phase chromatography, 9.4 × 250 mm, Agilent Technologies, Palo Alto, CA) using the following gradient with the solvents A (20% CH3CN, 80% 20 mm NH4CH3CO2 (pH 7.0), v/v) and B (20% CH3CN, 80% 20 mm NH4CH3CO2, 1 m NaCl (pH 7.0), v/v): 0 - 40% B (all v/v) over 15 min; 40-55% B over 30 min; 55-60% B over 5 min, and 60-100% B over 10 min, at a flow rate of 2 ml/min. The column was heated to 45 °C to increase the resolution. The fractions were collected, concentrated by lyophilization, and desalted on a Sephadex G-10 column (1.5 × 40 cm, Amersham Biosciences) using only H2O as solvent. For the further purification of the 36-mer, gel electrophoresis was also done using gels containing 8.0 m urea and 13% acrylamide (w/v) as described previously (6Woodside A.M. Guengerich F.P. Biochemistry. 2002; 41: 1027-1038Google Scholar). Reaction Conditions for Enzyme Assays—Unless indicated otherwise, standard DNA polymerase reactions were performed in 50 mm Tris-HCl (pH 7.5) buffer containing 50 mm NaCl, 1 mm dithiothreitol, 100 μg of bovine serum albumin/ml (w/v), and 10% glycerol (v/v) with 100 nm primer-template at 37 (for RT) or 22 °C (for T7-). Primers were 5′-end-labeled using T4 polynucleotide kinase with [γ-32P]ATP and annealed with template (36-mer) as described previously (6Woodside A.M. Guengerich F.P. Biochemistry. 2002; 41: 1027-1038Google Scholar). All reactions were initiated by the addition of dNTP and MgCl2 (12.5 mm final concentration) to preincubated enzyme/DNA mixtures. Full-length Extension Assay with All Four dNTPs—A 32P-labeled primer, annealed to either an unmodified or adducted template, was extended in the presence of all four dNTPs (100 μm each) for 30 min. Reaction mixtures (8 μl) were quenched with 2 volumes of a solution of 20 mm EDTA in 95% formamide (v/v). Products were resolved on a 16% polyacrylamide (w/v) gel system containing 8 m urea and visualized using a Bio-Rad Molecular Imager FX and Quantity One software (Bio-Rad). Steady-state Reactions—A 32P-labeled primer, annealed to either an unmodified or adducted template, was extended in the presence of increasing concentrations of a single dNTP. The molar ratio of primer-template to enzyme was at least 10:1 except for the 36-mer template containing N2-CH2(9-anthracenyl)G (molar ratio of 2-4:1). Enzyme concentrations and reaction times were chosen so that maximal product formation would be ∼20% of the substrate concentration (33Goodman M.F. Creighton S. Bloom L.B. Petruska J. Crit. Rev. Biochem. Mol. Biol. 1993; 28: 83-126Google Scholar). The unmodified primer-template was extended in the presence of 0.1-0.8 nm enzyme for 5 min. For the primer-template with G N2-modification (or when using unmodified template with a dNTP other than dCTP), the reaction was done in the presence of 0.5-10 nm enzyme (or 25-50 nm enzyme with the 36-mer containing N2-CH2(9-anthracenyl)G) for 5-30 min. All reactions (8 μl) were done at nine dNTP concentrations (in duplicate) and quenched with 2 volumes of a solution of 20 mm EDTA in 95% formamide (v/v). Products were resolved on a 16% polyacrylamide (w/v) gel containing 8 m urea and quantitated by PhosphorImaging analysis using a Bio-Rad Molecular Imager FX instrument and Quantity One software. Graphs of rates versus dNTP concentration were fit using nonlinear regression (hyperbolic fits) in GraphPad Prism version 3.0 (San Diego, CA) for the determination of kcat and Km values. Pre-steady-state Reactions—Rapid quench experiments were performed using a model RQF-3 KinTek Quench Flow Apparatus (KinTek Corp., Austin, TX). Reactions were initiated by rapid mixing of 32P-primer/template/polymerase mixtures (12.5 μl) with the dNTP-Mg2+ complex (10.9 μl) and then quenched with 0.3 m EDTA after times varying from 5 ms to 5 s. Reactions were mixed with 450 μl of formamide dye solution (20 mm EDTA, 95% formamide, 0.5% bromphenol blue (w/v), and 0.05% xylene cyanol (w/v)), run on a denat
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