HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS		QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

J. Biol. Chem., Vol. 279, Issue 18, 19217-19229, April 30, 2004

This Article Abstract Full Text (PDF) Supplemental Data An addition or correction has been published All Versions of this Article: 279/18/19217    most recent M313759200v1 Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Choi, J.-Y. Articles by Guengerich, F. P. Search for Related Content PubMed PubMed Citation Articles by Choi, J.-Y. Articles by Guengerich, F. P. Social Bookmarking                      What's this?

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^*

Jeong-Yun Choi and F. Peter Guengerich

From the Department of Biochemistry and Center in Molecular Toxicology, Vanderbilt University School of Medicine, Nashville, Tennessee 37232-0146

Received for publication, December 16, 2003 , and in revised form, February 22, 2004.

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
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 k_cat/K_m 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(S) 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 K_d(dCTP) and K_d(DNA) and k_off 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.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
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 (1). 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 (2, 3). DNA adducts act as core sources of misincorporation because they are inevitably formed by endogenous sources and exogenous mutagens in cellular DNA (4). 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 (1-3).

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 (4). 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.¹ 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 (5-7). 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 (8). 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 (9), acetaldehyde (10) (a metabolite of ethanol and also produced endogenously), styrene oxide (11), and the oxidation products of various polycyclic aromatic hydrocarbons, e.g. benzo[a]pyrene (12, 13). 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 , depending upon the base sequence context (14-16). N²-EthyldGuo has been detected in granulocyte and lymphocyte DNA of alcoholic patients and in human urine (17, 18). 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 (9). 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 (10).

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 (19) and have advantages over E. coli polymerase I (and Klenow fragment) (20). 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.

View larger version (12K): [in this window] [in a new window]   FIG. 1. N2-Alkyl guanine derivatives used in this work.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

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 x 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) 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 (5) by using stock plasmids provided by S. Hughes (RT, Frederick Cancer Facility, Frederick, MD) (21, 22) and K. A. Johnson (T7^- and thioredoxin, University of Texas, Austin, TX) (23). The T7^- expression and purification procedures were as modified by Zang et al.² T7^- was reconstituted with thioredoxin immediately prior to use as described (22). Protein concentrations were determined using ₂₈₀ 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 (24).

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 (25) 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 (26, 27) 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 (6). 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 (28), were as follow: 18-FAM-mer, ₂₆₀ = 196 mM^-1 cm^-1; 24-mer, ₂₆₀ = 224 mM^-1 cm^-1; and 36-mer, ₂₆₀ = 310 mM^-1 cm^-1.

Synthesis of N²-EthyldGuo—2-Fluoro-(O⁶-trimethylsilylethyl)-2'-deoxyinosine (52 mg, 0.14 mmol) (25) 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 (25, 30) 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): 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) (25) 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 (25, 30) 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): 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 (29). 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): 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 (26, 27) 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 x 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 x 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 (25, 31). (An alternative approach had been used by Casale and McLaughlin (32).) 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 x 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 x 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 (6).

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 [-³²P]ATP and annealed with template (36-mer) as described previously (6). 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 (33). 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 k_cat and K_m 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 denaturing gel, and quantitated as described for the steady-state reactions. Pre-steady-state experiments were fit with the burst equation y = A(1 - e^-kpt) + k_sst, where A is burst amplitude; k_p is pre-steady-state rate of nucleotide incorporation; t is time, and k_ss is steady-state rate of nucleotide incorporation (not normalized for enzyme concentration in the equation) (22, 34), using nonlinear regression analysis in GraphPad Prism version 3.0 (San Diego, CA). The % burst of incorporation was determined by dividing the burst amplitude of product by the estimated concentration of enzyme.

Phosphorothioate Analysis—With the ³²P-primer, annealed to either an unmodified or N²-methylG-containing template, reactions were initiated by rapid mixing of ³²P-primer/template/polymerase mixtures (12.5 µl) with dCTPS-Mg²⁺ complex (or dCTP-Mg²⁺, 10.9 µl) and then quenched with 0.3 M EDTA after reaction times varying from 5 ms to 5 s. Products were analyzed as described for the pre-steady-state reactions. With the ³²P-primer annealed to an N²-ethylG-containing template, reactions were initiated by the manual mixing of ³²P-primer/template/polymerase mixtures with the dNTPS-Mg²⁺ complex (or dNTP-Mg²⁺) and then quenched with 2 volumes of a solution of 20 mM EDTA in 95% formamide (v/v) after 1-10 min (or after 5-60 min when using T7^- and dNTPS-Mg²⁺).

Pre-steady-state Kinetics with Excess Unlabeled DNA ("Trap" or "Single Cycle" Experiments)—DNA trap experiments were initiated in the rapid quench-flow apparatus by mixing pre-equilibrated polymerase-DNA complex (RT, 90 nM; primer/template 100 nM) in syringe A with 200 µM dCTP-Mg²⁺ and unlabeled 24-mer/36-G-mer (3.75 µM) in syringe B and then quenched with 0.3 M EDTA at time intervals from 0.01-60 s as described previously (7). Products were quantitated by gel analysis. A graph of nM product versus time was fit with a double-exponential equation: y = E_tA₁(1 - e^-k1t) + E_tA₂(1 - e^-k2t), where E_t is enzyme concentration; A₁ is burst amplitude of fast phase; k₁ is observed fast phase rate; A₂ is burst amplitude of slow phase; k₂ is observed slow phase rate; and t is time, using nonlinear regression analysis in GraphPad Prism version 3.0.

Estimation of K_d by Fluorescence Quenching—Ground-state binding (K_d) of the DNA substrate to RT was estimated by fluorescence titration as described previously (7). Varying concentrations of RT (2.5-320 nM) were added to a solution of 100 nM 18-FAM-mer/36-mer in the standard reaction buffer containing 12.5 mM MgCl₂. Fluorescence was monitored with a Varian SF-330 spectrofluorometer (Varian, Walnut Creek, CA) using an excitation wavelength of 492 nm and emission wavelength of 516 nm. The data were fit to a fluorescence quadratic equation in GraphPad Prism using the equation: E·DNA = F₀ + (A/D_t)(0.5)((K_d + E_t + D_t) - ((K_d + E_t + D_t)² - 4 E_tD_t)^1/2), where F₀ is initial observed fluorescence; A is amplitude; E_t is enzyme concentration; K_d is DNA dissociation constant from the E·DNA complex; and D_t is DNA concentration (35).

K_d Estimation of dCTP Binding to RT·DNA and T7^-·DNA by Fluorescence Quenching—The equilibrium dissociation constant for dCTP binding to RT or T7·3'dGuo-terminated primer-template complexes was determined by intrinsic tryptophan fluorescence quenching as described previously (35). Varying concentrations of dCTP (1-1024 nM) were added to a solution of 100 nM 24 (3'-dGuo)-mer terminated primer (to prevent phosphodiester bond formation)/36-mer complex and 40 nM RT (or T7^-) in the standard reaction buffer containing 12.5 mM MgCl₂. Fluorescence measurements were made in a Varian SF-330 spectrofluorometer using an excitation wavelength of 290 nm and emission wavelength of 338 nm. The resulting data were fit to a fluorescence quadratic equation in GraphPad Prism (35) to estimate K_d.

DNA Dissociation from Enzyme k_off Measurements—DNA dissociation rates from the polymerase-oligonucleotide complex (E·DNA) were determined using the rapid quench-flow apparatus (7). Preincubated solutions of RT or T7^- (350 nM) and unlabeled target DNA (24-mer/36-mer, 50 nM) were rapidly mixed with ³²P-labeled 24-mer/36-G-mer (450 nM) for 0.05 to 30 s, and then polymerization was initiated with 200 µM dNTP-Mg²⁺ from the central drive syringe for a constant reaction time of 0.25 s. Expelled samples from the apparatus were rapidly mixed into tubes containing 500 µl of 0.3 M EDTA (in 50% formamide, v/v) to stop the reaction. Products were quantitated by gel analysis. A graph of nM product versus time was fit using the equation: y = E_f + E₀(1 - e^-kt), where E_f is free enzyme concentration; E₀ is DNA-bound enzyme concentration; k_(off) is dissociation rate DNA from E·DNA; and t is time (7, 36), using nonlinear regression in GraphPad Prism version 3.0.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Synthesis of Site-specifically Modified Oligodeoxynucleotides with Various dGuo N²-Adducts—The substrate 24-mer/36-mer duplex in Table I was based on previous work in this laboratory (5, 35) and contains a long duplex region to sufficiently accommodate the DNA-binding sites of T7^- (37) and RT (38). We systematically increased bulk at guanine N²-alkyl adducts of the 36-mers by substitution of a methyl, ethyl, isobutyl, benzyl, or CH₂(9-anthracenyl) group by two different synthetic methods. One involved an aldehyde NaBH₃CN/dGuo reaction (29) at the pre-oligomerization step; the other was a 2-fluorodeoxyinosine/RNH₂ reaction at the pre- or post-oligomerization step (25). All N²-dGuo-modified 36-mers, except for that containing N²-CH₂(9-anthracenyl)G, were prepared from the phosphoramidites of dGuo N²-alkyl adducts. N²-MethyldGuo and N²-benzyldGuo were synthesized using the RCHO/NaBH₃CN/dGuo reaction, and N²-ethyldGuo and N²-isobutyldGuo were synthesized using the 2-fluorodeoxyinosine/RNH₂ reaction. Yields of dGuo N²-adducts were better with the 2-fluorodeoxyinosine/RNH₂ reaction than the aldehyde NaBH₃CN/dGuo reaction. For the synthesis of the bulkiest adduct, N²-CH₂(9-anthracenyl)dGuo, we used a post-oligomerization synthesis strategy because the large CH₂(9-anthracenyl) group provided for good separation of the product oligonucleotide from the remaining reactants and hydrolyzed by-products in denaturing gel electrophoresis. The purity of the oligonucleotides used in this study was 99% as judged by capillary gel electrophoresis and MS (see Supplemental Material), and any trace impurities do not affect our major conclusions.

Full-length Extension in the Presence of All Four dNTPs—Processive polymerization of RT and T7^- at and beyond various N²-alkylG adducts was analyzed using 24-mer/36-mer duplexes containing G and each of five different N²-alkylG-adducts (Fig. 1) at position 25 of the template (Fig. 2). Increasing concentrations of RT and T7^- were used in 30-min incubations with each of six primer-template complexes in the presence of all four dNTPs, permitting full-length synthesis. Both polymerases readily extended the primer annealed to the unmodified G template to the full length of the 36-mer template, with the blunt-end addition of one or two bases (bluntend addition has been detected with RT and T7^- previously (39, 40)). Polymerization with the N²-methylG derivative yielded some full-length product but was retarded compared with unmodified DNA substrate, indicating that the polymerases inserted opposite the N²-methylG adduct and extended with some difficulty. However, the polymerization of the N²-ethylG-containing oligonucleotide yielded only trace 1-base extension even with 20 nM enzyme concentrations, indicating that both polymerases were strongly blocked at N²-ethylG. The larger G adducts N²-isobutylG, N²-benzylG, and N²-CH₂(9-anthracenyl)G also strongly blocked polymerization. Thus, there were two major changes in the attenuated ability of the polymerases to incorporate opposite N²-G adducts, from G to N²-methylG and from N²-methylG to N²-ethylG, the latter being more prominent. The lack of a "ladder" of polymerized intermediates in the gels (i.e. partially extended oligonucleotides occurring from the resulting polymerase release from oligonucleotides containing N²-methylG) contrasts with O⁶-methylG and O⁶-benzylG, which yielded substantial amounts of these intermediates (6), indicating apparently higher processivity of both polymerases with oligonucleotides containing N²-methylG than with O⁶-methylG.

View larger version (38K): [in this window] [in a new window]   FIG. 2. Extension of a 32P-labeled 24-mer primer opposite G, N2-methylG, N2-ethylG, N2-isobutylG, N2-benzylG, or N2-CH2(9-anthracenyl)G by RT and T7- in the presence of all four dNTPs. Primer was annealed with each of the six different 36-mer templates (Table I) containing an unmodified G or N2-modified G placed at the 25th position from the 3'-end. Reactions were done for 30 min with increasing concentrations of polymerases (0-20 nM) and a constant concentration of DNA substrate (100 nM primer/template) as indicated. The reaction products were analyzed by denaturing gel electrophoresis with subsequent PhosphorImaging analysis. A, RT; B, T7-. N2-MeG, N2-methylG; N2-EtG, N2-ethylG; N2-IbG, N2-isobutylG; N2-BzG, N2-benzylG; and N2-AnthG, N2-CH2(9-anthracenyl)G.

Ground-state Binding of DNA for RT and T7^-, K_d—Fluorescence titrations (addition of RT or T7^- to a solution of 18-FAM-mer/36-mer) were used to estimate the ground state binding of DNA substrates to the polymerases. The environment of the fluorescent group in an 18-mer is sensitive to the movement of RT as it binds DNA substrate in the active site (42); the same applies to T7^-. These fluorescence changes were monitored (excitation 492 nm, emission 516 nm), and data were fit to a quadratic equation to determine K_d. The ground-state binding of unmodified DNA substrate (18-FAM-mer/36-mer) was not significantly different from any of the N²-alkylG-adducted DNA substrates (Table III), indicating that the initial binding of DNA to either polymerase is not significantly affected by the presence of N²-alkylG adducts.

View larger version (19K): [in this window] [in a new window]   FIG. 3. Pre-steady-state burst kinetics of incorporation opposite G, N2-methylG, and N2-ethylG by RT and T7-. A, RT (40 nM) was incubated with 100 nM 24-mer/36-mer primer-template complex in the rapid quench-flow instrument and mixed with dNTP (and MgCl2) to initiate reactions: 200 µM dCTP for the 24-mer/36-G-mer (), 2 mM dCTP for the 24-mer/36-N2-methylG-mer (), or 3 mM dCTP () or dATP () for the 24-mer/36-N2-ethylG-mer (Table I). B, T7- (40 nM) was incubated with 100 nM 24-mer/36-mer primer-template complex in the rapid quench-flow instrument and mixed with dNTP to initiate reactions: 200 µM dCTP for the 24-mer/36-G-mer (), 2 mM dCTP for the 24-mer/36-N2-methylG-mer (), 3 mM dCTP () or dATP () for the 24-mer/36-N2-ethylG-mer. All polymerization reactions were quenched with 0.3 M EDTA at various time intervals from 0.05-5 s. The data were fit to the burst equation, y = A(1 - e-kpt) + ksst, as described under "Experimental Procedures" (without normalization of kss for enzyme concentration in the equation). Pre-steady-state rates (kp) occurring in the burst phase are indicated in the figure. The following kss values were estimated: A, G-mer, 0.070 ± 0.008 s-1; N2-methylG-mer, 0.078 ± 0.005 s-1; N2-ethylG-mer, 0.005 s-1. B, G-mer, 0.16 ± 0.02 s-1; N2-methylG-mer, 0.16 ± 0.01 s-1; N2-ethylG-mer, 0.005 s-1.

View larger version (25K): [in this window] [in a new window]   FIG. 4. Phosphorothioate analysis of pre-steady-state kinetics of nucleotide incorporation by RT and T7-. RT (A-C)or T7- (D-F) (40 nM) was incubated with 100 nM 24-mer/36-mer primer-template complex in the rapid quench-flow instrument and mixed with dCTP () or dCTPS () to initiate reactions: 200 µM dCTP (or dCTPS) for the 24-mer/36-G-mer (A and D),2mM dCTP (or dCTPS) for the 24-mer/36-N2-methylG-mer (B and E), and 3 mM dCTP (or dCTPS) for the 24-mer/36-N2-ethylG-mer (C and F). For the 24-mer/36-N2-ethylG-mer, reaction times were also extended to 1-60 min by the manual (conventional) mixing of 32P-primer/template/polymerase mixtures with the dNTPS-Mg2+ complex (or dNTP-Mg2+) for accurate measurement. The pre-steady-state rates were determined from the burst equation and are indicated in the figure. The solid lines represent the best fits to the burst equation (A, B, D, and E) or a linear equation (C and F). The following kss values were estimated: A, dCTP, 0.070 ± 0.008 s-1; dCTPS, 0.070 ± 0.008 s-1; B, dCTP, 0.078 ± 0.005 s-1; dCTPS, 0.045 ± 0.003 s-1; D, dCTP, 0.16 ± 0.02 s-1; dCTPS, 0.15 ± 0.02 s-1; E, dCTP, 0.066 ± 0.018 s-1; dCTPS, 0.0125 ± 0.005 s-1. The kss rates are shown on the graphs for C and F.

View larger version (22K): [in this window] [in a new window]   FIG. 5. Pre-steady-state kinetics of RT and T7- in the presence of excess unlabeled DNA trap (single cycle experiments). RT (A and B) or T7- (C and D) (90 nM) was incubated with 100 nM 24-mer/36-G-mer () or 24-mer/36-N2-methylG-mer () primer-template complex in the rapid quench-flow apparatus and then mixed with a combination of (pre-mixed) 200 µM dCTP and excess unlabeled 24-mer/36-G-mer. Reactions were quenched with 0.3 M EDTA at various time intervals ranging from 0.01 to 75 s. Graphs of nM product versus time were plotted (A and C), and the points were fit to a double-exponential equation in the Prism program, y = EtA1(1 - e-k1t) + EtA2(1 - e-k2t), as described under "Experimental Procedures." The semilogarithmic plots (insets B and D) of remaining DNA substrate (nM) versus time clearly indicate the double exponential nature of kinetics. The best-fit values opposite G with RT were A1 = 0.57, k1 = 23 s-1, A2 = 0.14, k2 = 0.14 s-1; opposite N2-methylG with RT, A1 = 0.08, k1 = 6 s-1, A2 = 0.40, k2 = 0.14 s-1; opposite G with T7-, A1 = 0.32, k1 = 110 s-1, A2 = 0.33, k2 = 0.04 s-1; opposite N2-methylG with T7-, A1 = 0.15, k1 = 10 s-1, A2 = 0.25, k2 = 0.04 s-1.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

The relatively small bulk of some adducts at the G N-2 atom had a critical effect on the catalytic efficiency of correct dCTP incorporation by polymerases RT and T7^-. A plot of k_cat/K_m versus the molecular volume of the substituent at the guanine N-2 atom (Fig. 6) indicates that even a small increase of bulk due to an added methyl group (21.2 ų), compared with hydrogen (6.1 ų), caused a profound decrease (about 3 orders of magnitude). Further increase of the bulk to an ethyl group (37.5 ų) caused another large decrease (about 3 orders of magnitude) in the catalytic efficiencies of RT and T7^- (Fig. 6 and Table II). This pattern is compatible with the effects on full-length extension (Fig. 2) and the burst kinetics (Fig. 3). The methyl group at guanine N-2 slowed full-length extension (Fig. 2) and also reduced the burst rate (and burst amplitude) in pre-steady-state kinetic studies (Fig. 3). The ethyl group at the guanine N-2 atom strongly blocked polymerization and eliminated the pre-steady-state state burst. An isobutyl or larger group caused a greater decrease of catalytic efficiency (about 10-fold), but it was not much more than the ethyl group. Most interesting, a "reversal" of the phenomenon was also observed (Table II), with higher catalytic efficiency of dCTP incorporation opposite N²-benzylG rather than N²-ethylG by T7^- but not by RT. This result might be attributed to chemical interactions of benzyl group and the active site of the polymerase. Thus, factors other than the interfering bulk may also play other roles in polymerase efficiency, depending on the nature of the polymerase.

View larger version (16K): [in this window] [in a new window]   FIG. 6. Effect of the volume of adduct at the guanine N-2 atom on catalytic efficiency (kcat/Km) in dCTP incorporation opposite guanine N2-alkyl adducts by RT and T7-. The molecular volumes (Å3) of adducts at the guanine N-2 atom were calculated using the program Chem3D (version 7.0) based on the Connolly surface algorithm (48) and plotted against kcat/Km values (Table II) for dCTP (, ) and dATP (, ) incorporations against various guanine N2-adducts by RT (, , and solid lines) and T7- (, , and broken lines). Me, methyl; Et, ethyl; Ib, isobutyl; Bz, benzyl; CH2-Anth, -CH2(9-anthracenyl).

The results with N²-methylG are interpreted as evidence for the presence of an inactive complex during polymerization, including (i) the reduced burst amplitude (Fig. 3) and (ii) the DNA trap (single cycle) experiments (Fig. 5). A modified kinetic mechanism (Fig. 7) with an inactive complex in equilibrium with productive complexes is needed for proper fitting of partial burst kinetics, as demonstrated in several other studies on 8-oxo-7,8-dihydroG, O⁶-methylG, O⁶-benzylG, and other adducts (7, 24, 47), as well as some other situations with unmodified DNA (e.g. hairpins (36)). A minimal mechanism (50), devoid of step 8 (Fig. 7), fails to adequately explain the altered kinetics of dCTP incorporation opposite N²-methylG due to the partial burst. In the case of N²-ethylG, either a minimal or the modified mechanism (with step 8 added, Fig. 7) could be used because of lack of the burst, but the modeling would not necessarily discriminate. As in the cases of 8-oxo-7,8-dihydroG, O⁶-methylG, and O⁶-benzylG, modeling cannot distinguish whether an alternate E·DNA·dNTP complex or an E·DNA binary complex (47) should be placed in the alternate kinetic mechanism because either can be used in fitting (24); furthermore, alternate placements of the position of the ternary complex are possible (24). Although we were not able to obtain direct evidence for the existence of an inactive complex in the case of N²-ethylG, the work presented here with N²-methylG and previous work with small DNA adducts (7, 24) and the cisplatin-DNA adduct (47) argue that this is probably a general phenomenon with replicative polymerases. However, the possibility also exists that small adducts may favor inactive complexes that can re-enter the catalytic cycle, but large adducts may not be so conformationally reversible.

View larger version (11K): [in this window] [in a new window]   FIG. 7. Kinetic mechanism for DNA polymerase incorporation. Individual steps are numbered. E, polymerase; Dn, DNA substrate; E*, conformational change in polymerase; E, nonproductive conformation of polymerase; and Dn+1, DNA extended by 1 base. A minimal kinetic mechanism (50) is modified by the addition of step 8 to yield an alternate kinetic mechanism of polymerization for oligonucleotides containing various DNA adducts (e.g. 8-oxo-7,8-dihydroG, O6-methylG, O6-benzylG) (7, 24). An additional, nonproductive ternary complex (E·Dn·dNTP) is formed in step 8, which is catalytically incompetent but can re-enter the cycle in step -8.