pyrene Adducts by Escherichia coli DNA Polymerase I and Sulfolobus

12 Apr 2017 - We examined two polymerases, Escherichia coli DNA polymerase I (Kf) and Sulfolobus solfataricus DNA polymerase IV (Dpo4), as models of a...
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Mutagenic Replication of N2‑Deoxyguanosine Benzo[a]pyrene Adducts by Escherichia coli DNA Polymerase I and Sulfolobus solfataricus DNA Polymerase IV A. S. Prakasha Gowda,† Jacek Krzeminski,‡ Shantu Amin,‡ Zucai Suo,§ and Thomas E. Spratt*,† †

Department of Biochemistry and Molecular Biology, Milton S. Hershey Medical Center, Pennsylvania State University College of Medicine, Hershey, Pennsylvania 17033, United States ‡ Department of Pharmacology, Milton S. Hershey Medical Center, Pennsylvania State University College of Medicine, Hershey, Pennsylvania 17033, United States § Department of Chemistry and Biochemistry, The Ohio State University, Columbus, Ohio 43210, United States S Supporting Information *

ABSTRACT: Benzo[a]pyrene, a potent human carcinogen, is metabolized in vivo to a diol epoxide that reacts with the N2position of guanine to produce N2-BP-dG adducts. These adducts are mutagenic causing G to T transversions. These adducts block replicative polymerases but can be bypassed by the Y-family translesion synthesis polymerases. The mechanisms by which mutagenic bypass occurs is not well-known. We have evaluated base pairing structures using atomic substitution of the dNTP with two stereoisomers, 2′-deoxy-N-[(7R,8S,9R,10S)7,8,9,10-tetrahydro-7,8,9-trihydroxybenzo[a]pyren-10-yl]guanosine and 2′-deoxy-N-[(7S,8R,9S,10R)-7,8,9,10-tetrahydro7,8,9-trihydroxybenzo[a]pyren-10-yl]guanosine. We have examined the kinetics of incorporation of 1-deaza-dATP, 7-deazadATP, 2′-deoxyinosine triphosphate, and 7-deaza-dGTP, analogues of dATP and dGTP in which single atoms are changed. Changes in rate will occur if that atom provided a critical interaction in the transition state of the reaction. We examined two polymerases, Escherichia coli DNA polymerase I (Kf) and Sulfolobus solfataricus DNA polymerase IV (Dpo4), as models of a high fidelity and TLS polymerase, respectively. We found that with Kf, substitution of the nitrogens on the Watson−Crick face of the dNTPs resulted in decreased rate of reactions. This result is consistent with a Hoogsteen base pair in which the template N2-BPdG flipped from the anti to syn conformation. With Dpo4, while the substitution did not affect the rate of reaction, the amplitude of the reaction decreased with all substitutions. This result suggests that Dpo4 bypasses N2-BP-dG via Hoogsteen base pairs but that the flipped nucleotide can be either the dNTP or the template.

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most abundant PAHs that is carcinogenic in humans,4 producing primarily GC to TA mutations.5−9 BP is metabolized to (+)-7R,8S-dihydrodiol-9R,10R-epoxy-7,8,9,10tetrahydrobenzo[a]pyrene ((+)-anti-BPDE) as shown in Figure 1.10,11 The N2-position of guanine in DNA reacts primarily via a trans-attack on the epoxide to form (+)-N2-BP-dG.10 Tobaccoassociated lung cancer in humans may be related, at least in part, to mutation of the p53 tumor suppressor gene by benzo[a]pyrene products.12−14 In mammalian cells, the N2-BP-dG adduct is blocking to replicative polymerases, and accurate bypass is accomplished by polymerase κ.15 The high reactivity of the incorporation of dCTP opposite (+)-N2-BP-dG16 and the ternary pol κ crystal structure17 indicate that correct insertion occurs via a Watson− Crick hydrogen bonding scheme. The BP moiety is in a cavity

INTRODUCTION DNA is constantly damaged by exogenous and endogenous electrophiles. While the cell can repair much of this damage, bulky DNA adducts that remain as the cell enters S-phase inhibit high fidelity polymerases, stall replications forks, and lead to cell death. One pathway by which the cell is able to respond to DNA damage during S-phase is via translesion DNA synthesis (TLS) polymerases.1−3 In this pathway, TLS polymerases are recruited to the stalled replication fork to bypass the damage. Depending on which TLS polymerase replaces the high fidelity polymerase, accurate or inaccurate replication occurs. Understanding the mechanisms by which individual polymerases bypass damage can enable us to predict the effectiveness of the TLS process and the fate of the cell under endogenous and exogenous stress. Polycyclic aromatic hydrocarbons (PAHs) are widespread environmental contaminants present in tobacco smoke, food, and automobile exhaust.4 Benzo[a]pyrene (BP) is one of the © 2017 American Chemical Society

Received: December 30, 2016 Published: April 12, 2017 1168

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Figure 1. Metabolism of benzo[a]pyrene and reaction with dG.42 The primary compound formed in vivo is (+)-N2-BP-dG.

of pol κ and consequently does not deform the structure of the polymerase. The catalytic site of the polymerase is positioned as with undamaged DNA, and the enzyme retains its catalytic activity.18 The mechanism underlying mutagenic bypass is less understood. Cellular knockdown experiments indicate that pol η and ζ are involved in mutagenic bypass.19−21 The mechanism(s) of error prone bypass of N2-alkyl-dG adducts have been examined with several polymerases including the prokaryotic A-family polymerases, which are models for the high fidelity polymerases, and Sulfolobus solfataricus P2 DNA polymerase IV (Dpo4), an archaeal DinB-like DNA polymerase that is a model for human TLS polymerases. A-family polymerases react slowly with N2-BP-dG; however, some bypass is observed in vitro.22,23 The structure of N2-BP-dG paired with dC was observed with Bacillus stearothermophilus pol I (BF).24 However, the position of the BP-group distorted the positions of critical active site residues, and consequently, this structure was concluded to be unreactive. Capture of a structure that can illuminate the mechanism of mutagenic bypass has proved elusive. Molecular modeling experiments provided insight for the base-pairing structure of (+)-N2-BP-dG bound to dNTPs with the A-family T7 DNA polymerase (T7 pol).25,26 The authors concluded that a stable complex can exist with (+)-N2-BP-dG, in which the deoxyribose-purine bond was in the syn-conformation with the BP-moiety pointing toward the major groove and the Hoogsteen hydrogen bonding face pointing toward the incoming dNTP. The computations predict that the relative stability of the complexes are not dependent on interstrand hydrogen bonds but through hydrophobic interactions between the BP moiety and the polymerase. In contrast, a more recent computational study using BF as the polymerase indicated that Hoogsteen

interstrand hydrogen bonds (Figure 2C and D) form to stabilize the structure.27

Figure 2. Potential hydrogen-bonding interactions. The left-side base is the DNA template, while the right-side base is the dNTP. R is a proton or the BP moiety.

The Dpo4 catalyzed bypass of N2-BP-dG can provide insight into the mechanisms by which pol η bypasses this adduct. The DNA binding site of Dpo4 is relatively open, and modeling experiments have suggested that the ribose−purine bond of the template N2-BP-dG can be in either the anti- or synconformations and still form base pair interactions with the incoming dNTPs (Figure 2).28,29 Kinetic, protein engineering, and modeling experiments have suggested that correct base pair formation occurs with Watson−Crick hydrogen bonding with the BP moiety in the minor groove. In contrast, mispair formation with purines occurs with the N2-BP-dG in the syn1169

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the HOAc was evaporated, the oligodeoxynucleotides were redissolved in 1 mL of 100 mM Et3N-HOAc (pH 7.0) and purified by HPLC with an analytical 4 μm Jupiter Proteo 90 Å (250 × 4.6 mm) column. The gradient was 15 to 50% acetonitrile over 35 min in 100 mM Et3NHOAc (pH 7.0) at 1 mL/min at a temperature of 60 °C (Figure S1). Typically, six peaks eluted off the column from 15−20 min, four of which contained the N2-BP-dG adducts. The peaks that contained the N2-BP-dG adducts were initially identified by UV spectroscopy from the band at 340 nm and then confirmed by MALDI-TOF MS (Supporting Information). The two major peaks and were attributed to the trans-opening of the epoxide during synthesis. The oligodeoxynucleotides were enzymatically hydrolyzed by treatment with DNase I, snake venom phosphodiesterase I, and alkaline phosphatase.39 The peak containing the BP group was isolated, and the stereochemistry of the adduct was determined by CD (Figure S2). The concentrations of oligodeoxynucleotides were determined from the absorbance at 260 nm, using the method of Borer40 in which it was assumed that the spectroscopic properties of the modified nucleotides were identical to the unmodified nucleotides. The primer was 32Plabeled with γ -[32P]ATP in a reaction catalyzed by T4-polynucleotide kinase. The oligomer was separated from low molecular weight impurities with a spin column containing Sephadex G-25, and the primer was annealed with a 50% excess of the template as previously described.41 Polymerase Kinetics. The reactions were initiated by the addition of equal volumes of dNTP in 50 mM Tris-HCl (pH 7.8), 10 mM MgCl2, and DNA−enzyme solution in 50 mM Tris-HCl (pH 7.8), 0.1 mM EDTA, 5 mM DTT, and 200 μg/mL BSA at 25 °C. Rapid reactions were performed with a KinTek-3 rapid quench instrument. The composition of the buffer during the reaction was 50 mM TrisHCl (pH 7.8), 5 mM MgCl2, 2.5 mM, 50 μM EDTA, 1 mM DTT, and 100 μg/mL BSA. Typically, the DNA concentration was 25 nM, and the polymerase concentration was 250 nM. The concentration of dNTPs varied from 0 to 2000 μM. The reactions were quenched by the addition of 300 mM EDTA. Product Analysis by PAGE. The progress of the reaction was analyzed by denaturing PAGE in 15% acrylamide (19:1, acrylamideN,N′-methylene bis(acrylamide)) and 7 M urea in 1×X TBE buffer (0.089 M Tris, 0.089 M boric acid, and 0.002 M Na2EDTA). The size of the gel was 40 × 33 × 0.4 cm3 and was run at 2000 V for 2−2.5 h. The radioactivity on the gel was visualized with a Typhoon 9200 PhosphorImager. The progress of the reaction was quantitated by dividing the total radioactivity in the product band(s) by the radioactivity in the product and reactant bands. Multiple product bands appeared when the incorrect dNTP was added to the reaction. Data Analysis. Data were fitted by nonlinear regression using the program Prism version 5 for Windows (GraphPad Software, San Diego, CA, www.graphpad.com). Data from the reactions were fitted to eq 1, where P is the product formed, A is the total amount of DNA reacted, and k is the first order rate for the dNTP incorporation. The k values for these experiments were fitted to eq 2, where kpol is the maximum rate of dNTP incorporation, and Kd is the equilibrium dissociation constant for the interaction of dNTP with the polymerase−DNA complex. Equation 2 was also used to analyze the [dNTP]-dependence on the amplitude (A), in which case Amax and KA parameters were obtained.

conformation with the BP in the major groove of the DNA (Figure 2C and D).30−32 In this article, we examined the mechanism by which the exonuclease deficient Escherichia. coli DNA polymerase I (Klenow fragment) (Kf) and Dpo4 catalyzed purine insertion opposite (+)- and (−)-N2-BP-dG (Figure 1) using atomic substitution on the dNTPs, with the nucleotide analogues 1deaza-dA, 7-deaza-dA, dI, and 7-deaza-dG (Figure 3). Specific

Figure 3. Structures of nucleotide analogues. Hypoxanthine is the name of the base of deoxyinosine.

nitrogen atoms were replaced by carbon or eliminated to probe whether these atoms were important to replication. If these atoms participate in critical hydrogen bonds, then a reduction in rate will be observed.33−35 A decrease in kpol would indicate that the substituted nitrogen is involved in a hydrogen bond that is more important in the transition state of the polymerization reaction than in the preceding ground state intermediate. If the hydrogen bond were important in binding of the dNTP, then we would observe an increase in Kd. In this work with Kf, our results support a mechanism in which the N2-BP-dG is in the syn-conformation and forms a Hoogsteen base pair with the incoming dNTP as shown in Figure 2C and D. In contrast with Dpo4, our results support a model in which N2-BP-dG binds to Dpo4 in either the anti- or syn-conformations.

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EXPERIMENTAL PROCEDURES

Reagents. [32P]ATP was purchased from PerkinElmer at 6000 Ci/ mmol. T4 polynucleotide kinase and the Klenow fragment of E. coli DNA polymerase I with the exonuclease activity inactivated (KF−) were obtained from United States Biologicals (Salem MA), and DNase I, snake venom phosphodiesterase, and alkaline phosphatase were purchased from Sigma-Aldrich. dNTPs (ultrapure grade) were purchased from Amersham Biosciences. 7-Deaza-dATP, 7-deazadGTP, and 2′-deoxyinsoine 5′-triphosphate (dITP) were purchased from TriLink Biotechnologies. 1-Deaza-2′-deoxyadenosine triphosphate was synthesized as described.36 The concentrations were determined by UV absorbance.37 Oligodeoxynucleotide Synthesis. 5′-DMT-N2-BP-dG-3′-Ophosphoramidites were synthesized as described by Johnson et al.,38 and the oligodeoxynucleotides were prepared by the phosphoramidite method.38 The sequences are shown in Chart 1, in which X is (+) or (−)-N2-BP-dG. The oligodeoxynucleotides were removed from the resin and purified by a semiprep 4 μm Jupiter Proteo 90 Å (250 × 10 mm) column. The gradient was 20 to 60% acetonitrile over 15 min in 100 mM Et3N-HOAc (pH 7.0) at 4 mL/min. The failure sequences eluted at 7 min, while the DMT-protected sequences eluted at 22 min. The DMT-protected oligodeoxynucleotide fractions were evaporated and treated with 80% HOAc for 30 min at room temperature. After

P = A(1 − e−kt)

(1)

k = k pol[dNTP]/([dNTP] + Kd)

(2)

Chart 1. Sequences of the Template Strand Containing N2-BP-dG

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Figure 4. Kf catalyzed insertion of dATP and 1-deaza-dATP opposite (+)-N2-BP-dG in sequence 1. A and B, time course for the addition of dATP (A) and 1-deaza-dATP (B). The dNTP concentrations were 15 (diamond), 25, (open circle), 50 (solid circle), 100 (open square), 300 (solid triangle), 500 (open triangle), and 1000 (solid square) μM. The lines are the best fit to the first-order equation product = A(1 − exp(−kobst)). (C) Determination of kpol and Kd. Fit of kobs to hyperbolic equation kobs = kpol[dNTP]/(Kd + [dNTP]) for dATP (solid square) and 1-deaza-dATP (open square). (D) Determination of Amax and KA. Fit of A to hyperbolic equation A = Amax × [dNTP]/(KA + [dNTP]) for dATP(solid square) and 1deaza-dATP (open square). Data are the mean ± standard error of three replicates.

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RESULTS Synthesis of Oligodeoxynucleotides. The oligodeoxynucleotides containing the N2-BP-dG adducts were prepared by total synthesis as described by Johnson.38 In cells, BP is metabolized to (+)-anti-BPDE that reacts with the N2-position of guanine. The reaction is primarily via the trans-opening to form (+)-N2-BP-dG, but in addition, a small amount of cisopening occurs. During the synthetic procedure, a key intermediate is the racemic anti-BPDE. This compound was reacted with ammonia to form primarily the (+)- and (−)-trans-amino-triols with a small amount of the cis-aminotriols. The stereoisomers were not isolated, and consequently, the 5′-DMT-N2-BP-dG-3′-O-phosphoramidites consisted of four BP-stereoisomers, with the trans-isomers in excess over the cis-isomers. (Scheme S1). This phosphoramidite mixture was used in the oligodeoxynucleotide synthesis to produce four diasteriomeric oligodeoxynucleotides that were separated by HPLC after removal of the DMT-group. Two major peaks, which corresponded to the trans-opening of the epoxide, were isolated by HPLC (Figure S1). The oligodeoxynucleotides were enzymatically hydrolyzed and the N2-BP-dG nucleosides separated by HPLC. The assignment of the stereochemistry of the N2-BP-dG was made based upon the CD spectrum (Figure S2).38,42 The cis-isomers were isolated in low yield and were not used in this study. Kf Catalyzed Bypass via Hoogsteen Base Pairs. Modeling studies with T7 pol and BF, two A-family polymerases, predict that dGTP and dATP will be inserted opposite N2-BP-dG via a Hoogsteen base pair (Figure 2C and D).25−27 The interstrand hydrogen bonds were probed with dATP and dGTP analogues, 1-deaza-2′-deoxyadenosine (1deaza-dA), 7-deaza-2′-deoxyadenosine (7-deaza-dA), 2′-deoxynosine (dI), and 7-deaza-2′-deoxyguanosine (7-deaza-dG) as

shown in Figure 3. These compounds differ from adenine and guanine in that a nitrogen, on the Watson−Crick or Hoogsteen hydrogen bonding face is replaced by carbon or hydrogen. If that atom was involved in a critical hydrogen bond, rates of reaction will decrease. The time course for the Kf catalyzed incorporation of the dNTPs were conducted with 25 nM DNA, 250 nM polymerase, and a variable amount of dNTP. Control experiments were conducted with 2′-deoxyguanosine as template, and kinetic constants were similar to those we previously obtained.43 Eight DNA substrates containing N2-BP-dG were investigated, four sequences (Chart 1) in combination with the (+)- or (−) N2BP-dG stereochemistry (Figure 1). Figure 4A and B show the time courses for the incorporation of dATP and 1-deaza-dATP, opposite (+)-N2-BP-dG in sequence 1. These graphs show that 1-deaza-dATP is incorporated more slowly than dATP. The time courses were fit to a first order equation and the rate constants and amplitudes fitted to the hyperbolic equation. It should be noted that although the reactions are carried out with enzyme in excess, we do not observe a burst of product formation that is associated with the presteady-state. The kpol is a complex parameter that is dependent on phosphodiester bond formation and most likely a variety of equilibria involving conformational changes associated with nonproductive binding complexes. Figure 4C shows that the 1-deaza-dATP is incorporated more slowly than dATP due to a lower kpol parameter, while the apparent Kd was unaffected. Figure 4D demonstrates that the amplitude was not affected by the substitution. The values for Kf are presented in Table S1 and summarized in Figure 5. For each of the eight DNA substrates, modification of the Watson−Crick hydrogen bonding site produced a greater decrease in kpol than modification of a Hoogsteen hydrogen1171

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between the O6-guanine position of N2-BP-dG and the N6position of dATP.27 However, we found that the 1-deaza-dATP substitution caused a reduction in kpol. With this result, we predict that there is a protonated hydrogen bond as shown in Figure 2D. No significant effects were found for the apparent Kd values. During the initial binding of the dATP or dGTP, the affinity may be determined by factors not involving a hydrogen bond to the N1 or NH2 positions. Perhaps the dNTP is mostly bound via hydrophobic stacking and electrostatic interactions through the triphosphate. The stereochemistry of the BP adduct did not have a large effect on kpol. The ratio of the kpol for (+)-N2-BP-dG to the kpol of (−)-N2-BP-dG only varied from 0.8 to 2 with all sequences and dNTP analogues. Similarly, the effect of the atomic substitution was constant with respect to the stereochemistry. As observed in Figure 5, the largest change occurred with sequence 1 (5′-A). With (+)-N2-BPdG, the 1-deaza-dATP substitution decreased the kpol 16-fold. However, with (−)-N2BP-dG, the substitution caused a reduction of 3-fold. Thus, the change in stereochemistry cause a 5-fold reduction in effect of the nitrogen substitution. However, with the other DNA sequences, the changes in rate caused by the substitution were minimal. The sequence of the template plays a small role in reactivity. In particular, the substrates 2, 3, and 4, with C, G, and T 5′- to N2-BP-dG, reacted slightly differently than the 5′-A sequence. With 1, the 7-deaza-dA and 7-deaza-dG substitutions did not alter the kpol. However, with 2, 3, and 4 the 7-deaza-dG substitution increased kpol up to 3-fold, and the 7DA substitution reduced the kpol up to 3-fold. Crystallographic experiments have shown that the 5′-template base is positioned outside of the active site during the insertion step of BF-

Figure 5. Effect of dNTP substitution on the Kf catalyzed insertion of purine analogues opposite (+) or (−) N2-BP-dG with DNA sequences 1−4 with M = A, C, G, and T. The Y-axis is the ratio of the kpol for the analogue divided by that for dATP or dGTP.

bonding participant (Figure 5). For example with (+)-N2-BPdG and A as the 5′-template base, the 1-deaza-dA substitution (solid red) produced a 16-fold reduction in kpol, while the 7deaza-dA substitution (solid blue) produced a 14% increase in kpol. Similarly, deletion of the N2-amino group with dITP (open red) produced a 7-fold reduction in kpol, while the 7-deaza-dG substitution (open blue) produced a 40% increase in kpol. This trend exists with each of the other seven DNA substrates. This result is consistent with the structures in Figure 2C and D, in which the Watson−Crick face of the incoming dNTP makes a critical contact with the DNA/polymerase complex. The base pair between dATP and (+)- and (−)-N2-BP-dG are predicted to be stabilized by a single hydrogen bond

Figure 6. Dpo4. Catalyzed insertion of dATP and 1-deaza-dATP opposite (+)-N2-BP-dG in sequence 1. (A and B) Time course for the addition of dATP (A) and 1-deaza-dATP (B). The dNTP concentrations were 25 (solid square), 50 (open circle), 100 (solid triangle), 200 (open square), 400 (diamond), 600 (open triangle), and 1000 (solid circle) μM. Data are the mean ± standard deviation of three replicates. The lines are the best fit to the first-order equation product = A(1 − exp(−kobst)). (C) Determination of kpol and Kd. kobs vs [dNTP] was fitted to the hyperbolic equation kobs = kpol[dNTP]/(Kd + [dNTP]). (D) Determination of Amax and KA. A vs [dNTP] was fitted to hyperbolic equation for dATP (solid square), and 1deaza-dATP (open square). 1172

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Chemical Research in Toxicology catalyzed undamaged DNA replication.44 Modeling studies with BF predict a similar structure with (+)-N2-BP-dG as template.27 One explanation is that the 5′-base induces subtle changes in structure that affect the rate of reaction. Alternative mechanisms by which a sequence would affect rates is via a slip-template mechanism. In this mechanism, a deletion occurs when the incoming dNTP pairs with the nucleotide 5′ to the adduct. However, if this were the mechanism, then we would expect the dNTP that is complementary to the 5′-base to be incorporated most rapidly. With DNA substrates, 2 and 4, the 5′-bases are C and T, respectively. Under the slip-template mechanism, dGTP should be the best substrate for 2 and dATP for 4. However, as shown in Table S1, this is not the case. dATP is always the better substrate with both DNA sequences. Therefore, we have no evidence that the slip-template mechanisms occurs. Dpo4 Catalyzed Bypass via Multiple Hoogsteen Base Pairs. Dpo4 is a low fidelity Y-family polymerase of the DinBsubfamily, and it has been intensively studied as a model for Yfamily TLS polymerases. In contrast to replicative polymerases, Dpo4 shows high catalytic activity for the insertion of dCTP opposite a series of N2-alkyl-dG, ranging in size from methyl to anthrecenylmethyl.45 However, when the size of the N2-alkyl group increases to the BP group the reactivity drops 1000-fold. While, Dpo4 can insert dCTP opposite N2-anthrcenylmethyldG with a kpol of 2.2 s−1, the pseudo-first order rate constant decreases to 8.5 × 10−5 s−1 for N2-BP-dG.32,45 We found kpol values of up to 2.7 × 10−3 s−1 for the incorporation of dCTP opposite N2-BP-dG. In contrast, the rates of dATP and dGTP incorporation remain relatively constant, and with N2-BP-dG, purine triphosphates are preferred cosubstrates. Computer modeling studies predict that (+)-N2-BP-dG can bind to Dpo4 in either the syn- or anti-conformation.29 We probed these interactions using dNTP analogues. The time courses were fit to a first order equation as shown in Figure 6A and B. From these graphs, it is evident that the 1-deaza-dATP reactions produce less product over the similar time periods. The resulting rate constants were plotted against the dNTP concentration and fitted to a hyperbolic equation to obtain kpol and Kd values. As illustrated in Figure 6C, the curves resulting from dATP and 1-deaza-dATP are very similar indicating that the kpol and Kd values were not affected by the dATP to 1deaza-dATP substitution. The amplitudes were plotted against [dNTP], and the curves were again fitted to the hyperbolic equation as shown in Figure 6D. Here, we found that the maximum amplitude (Amax) was reduced by the 1-deaza-dA substitution. The kinetic parameters are listed in Table S2 and summarized in Figure 7. The dATP to 1-deaza-dATP and 7-

deaza-dATP, as well as the dGTP to dITP and 7-deaza-dGTP, substitutions did not affect the kpol and Kd parameters (Figure 7A) but caused a decrease in the maximum amplitude (Figure 7B). These results differ from those obtained with Kf in two regards. First, the amplitude is reduced by Dpo4, while the rate of reaction is reduced by Kf. Second, for Kf, modification on the Watson−Crick face of the dNTP reduced the kpol. However, with Dpo4, substitution on both the Watson−Crick and Hoogsteen hydrogen bonding faces reduced the amplitude of reactions. We interpret these results to indicate that this reaction occurs via multiple mechanisms, as illustrated in Scheme 1. DNA containing N2-BP-dG can bind to Dpo4 in two configurations in which the dG is in the syn-configuration (top) or anticonfiguration (bottom). The dGTP will bind to either complex via Hoogsteen hydrogen bonding. However, with 7-deazadGTP, the top pathway will be inhibited, but the bottom pathway will proceed. In contrast, with dITP, the bottom pathway will be in inhibited, but the top pathway will proceed. In this manner, while the rate of the reaction will not be affected, the amplitude of the reaction will be decreased. The dual mechanisms of dCTP and dGTP incorporation were previously proposed.32

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DISCUSSION Benzo[a]pyrene is a potent human carcinogen, forming (+)-anti-BPDE that reacts with the N2-position of guanine, primarily via the trans-opening of the epoxide to form (+)-N2BP-dG (Figure 1). Computer modeling with polymerases T7 pol, BF, and Dpo4 predict that mutagenic bypass of (+)-N2BaP-dG with dATP and dGTP occurs via Hoogsteen base pairing as shown in Figure 2.27,29 We tested these hypotheses by examining specific interactions using atomic substitution. Our results with Kf support the role of Hoogsteen base pairs in which N2-BP-dG is in the syn-conformation and the incoming dNTP is in the anti-conformation. This orientation of the N2BP-dG will allow the bulky BP moiety to be positioned in the major groove of the DNA, where there are fewer clashes with polymerase residues. Dpo4 has a more open architecture, and our results agree with mechanisms in which the BP group can be positioned in either the major or minor groove. From the present results, we could speculate that pol η bypasses N2-BPdG via multiple mechanisms as with Dpo4. However, there are many differences between these enzymes, and the base pair structures for each polymerase must be investigated. This study demonstrates that we have tools to investigate potential hydrogen-bonding interactions during insertion and extension past the N2-BP-dG adducts by both pol η and ζ. The stereochemistry of anti-BPDE plays a role in turmorgenicity, with (+)-anti-BPDE being more tumorigenic than (−) anti-BPDE.46,47 The differences in mutagenicity can be due to multiple factors including the differential location/ base specificity of adduct formation, repair, and mutagenesis. (+)-N2-BP-dG and (−)-N2-BP-dG exist in different conformations in duplex DNA. While the BP moiety of both isomers lie in the minor groove, the orientation is reversed. The pyrene moiety of (+)-N2-BP-dG points to the 5′-side of the modified guanine, while the pyrene moiety of (−)-N2-BP-dG points toward the 3′-side of the nucleotide.48,49 This difference in structure is manifested in the differential binding to XPC/ HR23B, in which (−)-N2-BP-dG is bound more effectively than (+)-N2-BP-dG.50 However, difference in excision rates are not

Figure 7. Effect of the atomic substitution on Dpo4-catalyzed insertion of purine analogues opposite 1 containing (+)- and (−)-N2-BP-dG. 1173

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Chemical Research in Toxicology Scheme 1. Mechanism for the Dpo4-Catalyzed Insertion of Purine dNTPs Opposite N2-BP-dG

observed,51 indicating that differential repair is unlikely to be the cause of the differential tumorigenic potency of the isomers. During DNA replication, the polymerase initially interacts with N2-BP-dG as a single-strand template; thus, the difference in structure observed in duplex DNA may not be transmitted to the polymerase/DNA structure. Neither pol κ nor η shows a significance difference in reactivity or dNTP selectivity between (+)-and (−)-N2-BP-dG.19,52 Thus, differences in the efficiency of translesion bypass of (+)- or (−)-N2-BP-dG by mammalian pol κ or η do not account for their differential tumorigenicity. In our experiments, we found that the kinetic parameters for both Kf and Dpo4 were minimally affected by the stereochemistry of the BP-group. Likewise, the effect of the atomic substitutions were not influenced by the stereochemistry of the adduct. Our results support previous studies in that translesion bypass is not a major factor in the increased mutagenicity of (+)-anti-BPDE over (−)-anti-BPDE.

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Notes

The Pennsylvania Department of Health specifically disclaims responsibility for any analyses, interpretations, or conclusions. The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The oligodeoxynucleotide synthesis and MS analysis were performed in the Macromolecular Core facility at the PSU College of medicine. The NMR spectra were recorded in the solution NMR core facility. Core Facility services and instruments used in this project were funded, in part, under a grant with the Pennsylvania Department of Health using Tobacco Settlement Funds.

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ABBREVIATIONS BF, large fragment of Bacillus stearothermophilus DNA polymerase I; BP, benzo[a]pyrene; BPDE, 7R,8S-dihydrodiol-9R,10Repoxy-7,8,9,10-tetrahydrobenzo[a]pyrene; Dpo4, Sulfolobus solfataricus DNA polymerase IV; Kf, Klenow fragment of E. coli DNA polymerase I with the proofreading exonuclease inactivated; PAH, polycyclic aromatic hydrocarbon; T7 pol, T7 DNA polymerase; TLS, translesion DNA synthesis

ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.chemrestox.6b00466. Purification and stereochemical assignment of the N2-BPdG adducts, MALDI-TOF MS characterization of the modified oligodeoxynucleotides, and tables of kinetic constants (PDF)

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REFERENCES

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AUTHOR INFORMATION

Corresponding Author

*Pennsylvania State University College of Medicine, Department of Biochemistry and Molecular Biology, H171, 500 University Drive, Hershey, PA 17033-0850, USA. Tel: 717-5314623. Fax: 717-531-7072. E-mail: [email protected]. ORCID

A. S. Prakasha Gowda: 0000-0002-0055-7103 Zucai Suo: 0000-0003-3871-3420 Thomas E. Spratt: 0000-0002-6805-3729 Funding

This project was funded under NIH grant no. ES021762. 1174

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