deoxyguanosine Using Nucleotide Analogues - American Chemical

Oct 15, 2012 - Department of Chemistry, University of Richmond, Gottwald B-100, .... Spectrometry Facility at the University of California at Riversid...
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Biochemical Investigations into the Mutagenic Potential of 8‑Oxo-2′deoxyguanosine Using Nucleotide Analogues Michelle L. Hamm,* Kelly A. Crowley, Michael Ghio, Maria A. M. Lindell, Emily J. McFadden, Jordan S. L. Silberg, and Amelia M. Weaver Department of Chemistry, University of Richmond, Gottwald B-100, Richmond, Virginia 23173, United States S Supporting Information *

ABSTRACT: 8-Oxo-2′-deoxyguanosine (OdG) is an abundant DNA lesion produced during oxidative damage to DNA. It can form relatively stable base pairs with both dC and dA that mimic natural dG:dC and dT:dA base pairs, respectively. Thus, when in the template strand, OdG can direct the insertion of either dCTP or dATP during replication, the latter of which can lead to a dG → T transversion. The potential for OdG to cause mutation is dependent on the preference for dCTP or dATP insertion opposite OdG, as well as the ability to extend past the resulting base pairs. The C2-amine and C8oxygen could play major roles during these reactions since both would lie outside the Watson−Crick cognate base pairs shape in the major groove when OdG base pairs to dA and dC, respectively, and both have the ability to form strong interactions, like hydrogen bonds. To gain a more generalized understanding of how the C2-amine and C8-oxygen of OdG affect its mutagenic potential, the incorporation opposite and extension past seven analogues of dG/OdG that vary at C2 and/or C8 were characterized for three DNA polymerases, including an exonuclease-deficient version of the replicative polymerase from RB69 (RB69), human polymerase (pol) β, and polymerase IV from Sulfolobus solfataricus P2 (Dpo4). Based on the results from these studies, as well as those from previous studies with RB69, pol β, Dpo4, and two A-family polymerases, the influence of the C2amine and C8-oxygen during each incorporation and extension reaction with each polymerase is discussed. In general, it appears that when the C2-amine and the C8-oxygen are in the minor groove, they allow OdG to retain interactions that are normally present during insertion and extension. However, when the two groups are in the major groove, they each tend to form novel active site interactions, both stabilizing and destabilizing, that are not present during insertion and extension with natural DNA.

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INTRODUCTION Reactive oxygen species (ROS), which include hydroxyl radicals (•OH), peroxides (R2O2), and superoxides (O3•−), are created by chemical carcinogens and radiation from the sun, as well as during metabolic respiration.1−3 They can readily react with 2′deoxyguanosine (dG) at the C8 position to create 8-oxo-2′deoxyguanosine lesions (OdG; Figure 1), which are formed at the rate of approximately 1000−10000 per day within a mammalian cell.4,5 The addition of the C8-oxygen, which predominates as the C8-keto tautomer at physiological pH,6,7 alters the sterics and electronics within the imidazole ring and allows OdG to form stable base pairs to both 2′-deoxycytidine (dC) and 2′-deoxyadenosine (dA; Figure 2). When part of an OdG:dC base pair, OdG is in the anti conformation [OdG(anti)], and its Watson−Crick (W−C) face is used for hydrogen bonding to dC.8,9 However, when part of an OdG:dA mismatch, OdG utilizes the syn conformation [OdG(syn)], allowing the Hoogsteen face N7-hydrogen, which is not present in dG, to hydrogen bond with N1 of dA.10,11 These two base pairs structurally mimic the natural base pairs dG:dC and dT:dA, respectively, and show comparable overall stability.12 Thus, during replication, when a polymerase comes across an OdG in the template strand, either a dCTP or a dATP can be © 2012 American Chemical Society

added to the growing daughter strand. If dCTP is inserted, no mutation should result; however, incorporation of dATP would lead to a dG → T transversion after the next round of replication.13,14 It is mutations such as these that are thought to be responsible for the known link between OdG and aging, as well as many diseases, including cancer.15−18 To better understand the mutagenic potential of OdG, and thus the ability for it to promote aging and disease, the activity of dCTP insertion relative to dATP insertion (summarized as the dCTP/dATP incorporation ratio) opposite OdG has been determined for many polymerases.19−24 It has been shown that some polymerases have a large dCTP/dATP incorporation ratio opposite OdG and will more often insert dCTP, thus lowering the mutagenic potential of OdG. However, other polymerases have a dCTP/dATP incorporation ratio opposite OdG near or below 1, resulting in more insertion of dATP and a higher mutagenic potential. Additionally, because the ability for a polymerase to extend past the resulting base pairs, OdG:dC and OdG:dA, can also influence the overall mutagenic potential of OdG, extensions past OdG have been an area of Received: August 28, 2012 Published: October 15, 2012 2577

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be of significance since each can participate in strong intermolecular interactions like hydrogen bonding, and both would lie outside the cognate W−C shape when in base pairs to dC and dA, respectively, potentially generating clashing interactions in the major groove of a polymerase active site.29 Thus, we have focused on testing, with various polymerases, the activity of incorporation opposite and extension past analogues of dG/OdG that vary in size and/or electronics at the C8 position and/or remove the C2-amine. These analogues, which have been purchased or previously synthesized, include 8chloro-2′-deoxyguanosine (CldG), 8-bromo-2′-deoxyguanosine (BrdG), 9-deaza-2′-deoxyguanosine (CdG), 8-thio-2′-deoxyguanosine (SdG), 2′-deoxyinosine (dI), 8-oxo-2′-deoxyinosine (OdI), and 8-thio-2′-deoxyinosine (SdI; Figure 1). Through determination of the various incorporation and extension activities with these analogues, a more nuanced understanding of the effects of the C8-oxygen and C2-amine on OdG mutagenicity can be gained. For example, by comparing the efficiencies of incorporation opposite and extension past dG, CldG, BrdG, OdG, and SdG, as well as dI, OdI, and SdI, the importance of size and/or electronics at the C8 position can be tested. Alternately, by comparing the efficiency of incorporation opposite and extension past dG, OdG, and SdG to that of dI, OdI, and SdI, respectively, the importance of the C2-amine can be accessed. In a recent study, we sought to better understand the influence of the C2-amine and C8-oxygen on the mutagenic potential of OdG with two replicative A-family polymerases, exonuclease-deficient Klenow Fragment (KF-exo) and the large fragment from Bacillus stearothermophilus Pol I (BF), which have opposite incorporation preferences.27 In the work described herein, we have expanded our focus to include polymerases from three additional families. These new polymerases include an exonuclease-deficient version of the B-family replicative DNA polymerase from bacteriophage RB69 (RB69) and the bypass Y-family DNA polymerase IV from the archaea Sulfolobus solfataricus P2 (Dpo4), which both have large dCTP/dATP incorporation ratios opposite OdG,21−23 as well as the X-family repair polymerase, human DNA polymerase (pol) β, which has a much smaller dCTP/dATP incorporation ratio opposite OdG.20 These enzymes were chosen because they all lack any proofreading exonuclease activity (which could complicate analysis), they have been used previously as models for their class of polymerase, and there are crystal structures available with OdG:dCTP and OdG:dATP (or an equivalent) available for each.21−23,26,28 By furthering our understanding of the roles of the C2 and C8 positions during incorporation opposite and extension past OdG with each of these enzymes, we can also gain a more general understanding of the overall importance of these two sites to OdG mutagenicity and thus the potential for OdG to cause disease.

Figure 1. Structures and abbreviations of nucleotides used in this study.

Figure 2. Structures of OdG:dC and OdG:dA base pairs.

significant inquiry as well.22−27 For example, slow extension past an OdG:dA base pair could allow time for the proofreading site present in some polymerases to remove the dA from the newly created mismatch, potentially lowering the mutagenic potential of OdG. Similar to dCTP and dATP incorporation opposite OdG, the efficiencies of extension past OdG:dC relative to OdG:dA base pairs can vary considerably between different polymerases. Our lab uses organic chemistry and biochemistry to gain a deeper understanding of the atomic properties of OdG that influence its mutagenic potential. The three sites that we have concentrated on include the C8-oxygen and N7-hydrogen (the two sites that differ between dG and OdG), as well as the C2exocyclic amine. Previous studies have already established that the N7-hydrogen is essential for efficient OdG:dA base pairing,12 dATP incorporation opposite OdG, and extension past OdG:dA base pairs.23,26−28 However, the influences of the C8-oxygen and C2-amine on the incorporation opposite and extension past OdG are not as well understood. Both sites may

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EXPERIMENTAL PROCEDURES

Pol β was purchased from Trevigen. WT and Y567A RB69 (both mutated to abolish the inherent exonuclease activity)28 and WT and R332A Dpo430 were gifts from William Konigsberg and F. Peter Guengerich, respectively. Oligonucleotide synthesis was carried out by The Midland Certified Reagent Company (Midland, TX). MALDITOF mass spectrometry analysis was carried out at the Mass Spectrometry Facility at the University of California at Riverside. Preparative RP-HPLC was performed using a Beckman Ultrasphere ODS C18 column (10 mm × 250 mm) run at 3 mL/min; HPLC solvents A and B were 0.1 M triethylammonium acetate (TEAAC), pH 7, and acetonitrile, respectively. 2578

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Oligonucleotide Synthesis and Purification. Template, primer, and complement oligonucleotides used during the reactions with Dpo4 and RB69, as well as pol β, are summarized in Figure 3.

Radiolabeling of Primers. Primers were radiolabeled using Optikinase (USB) and γ-32P-ATP (MP Biomedical) and purified using mini Quick Spin Oligo Columns (Roche) for a final concentration of 4 μM. Steady-State Insertion and Extension with the Polymerases. Solutions containing buffer, template (T), complement (for pol β only), and primer (P) were heated at 90 °C for 3 min before slow cooling to room temperature. Polymerase was then added to make a 2×T/P solution that was incubated at the reaction temperature (25 °C for RB69 and 37 °C for pol β and Dpo4) for 5 min. Five microliters of the 2×T/P solution was then added to 5 μL of a 2×dNTP solution that had also been incubated for 5 min at the reaction temperature. After the appropriate reaction time, reactions were stopped with 20 μL of a solution containing 95% formamide, 20 mM EDTA, and 0.0025% each of bromophenol blue and xylene cyanol before heating at 90 °C for 5 min. RB69. The 2×T/P solution contained 50 mM Tris-HCl, pH 7.5, 0.2 μM template, 0.2 μM 32P- radiolabeled primer (10mer for insertion or 11mer for extension; Figure 3), and 5 nM WT or Y567A RB69 (except for extension past SdI:dA with WT RB69, which contained 50 nM WT RB69) in 10% glycerol, while the 2×dNTP solution contained 50 mM Tris-HCl, pH 7.5, 10 mM MgCl2, and the appropriate dNTP concentration (dATP or dCTP for insertion and dGTP for extension). Pol β. The 2×T/P solution contained 50 mM Tris-HCl, pH 8, 5 mM MgCl2, 10 mM NaCl, 4 mM DTT, 0.4 mg/mL BSA, 0.2 μM template, 0.2 μM complement, 0.2 μM 32P-radiolabeled primer (10mer for insertion or 11mer for extension; Figure 3), and 0.6 nM Pol β (except for dATP incorporation opposite CdG, which contained 6 nM Pol β) in 10% glycerol, while the 2×dNTP solution contained 50 mM Tris-HCl, pH 8, 10 mM NaCl, and the appropriate dNTP concentration (dATP or dCTP for insertion and dGTP for extension). Dpo4. The 2×T/P solution contained 100 mM Tris-HCl, pH 7.8, 5 mM MgCl2, 100 mM NaCl, 10 mM DTT, 100 μg/mL BSA, 0.2 μM template, 0.2 μM 32P-radiolabeled primer (10mer for insertion or 11mer for extension; Figure 3), and WT or R332A Dpo4 (5 nM for incorporation and 10 nM for extension) in 2% glycerol, while the 2×dNTP solution contained 5 mM MgCl2 and the appropriate dNTP concentration (dATP or dCTP for insertion and dGTP for extension). Separation of Product and Michaelis−Menten Kinetics. Product oligonucleotides were separated from starting oligonucleotides using 20% denaturing PAGE. The resulting gel was dried, exposed to a storage phosphor screen (Amersham) overnight, and visualized using a Storm 860 Phosphorimager (Amersham). Steadystate kinetic experiments were run under initial conditions where ≤20% of the reaction had progressed and included seven different

Figure 3. DNA oligonucleotide substrates used in this study, where X (in the Tn site during incorporation and the Tn−1 site during extension) is dG, CldG, BrdG, CdG, OdG, SdG, dI, OdI, or SdI, and Y (in the Pn−1 site during extension) is dC or dA. The asterisk indicates a 32 P-radiolabeled phosphate, and a lowercase p indicates a nonradiolabeled phosphate. Unmodified oligonucleotides were purchased from IDT DNA, while those containing OdG and BrdG were purchased from Midland. Oligonucleotides containing CdG,31 SdG,32 CldG,33 OdI,34 and SdI27 were synthesized as previously described. All oligonucleotides were purified by 20% denaturing PAGE and HPLC as previously described.27 Oligonucleotide Characterization. Template oligonucleotides used during the experiments with RB69 and Dpo4 have been characterized previously,27 while those used for the experiments with pol β were characterized by HPLC (only one peak was present for each) and MALDI-TOF. Template containing CldG: MALDI-TOF [M + 3K+]; calculated, 8114; actual, 8112. Template containing CdG: MALDI-TOF [M + K+]; calculated, 8001; actual, 8002. Template containing SdG: MALDI-TOF [M + Na+]; calculated, 8018; actual, 8019. Template containing OdI: MALDI-TOF [M + H+]; calculated, 7962; actual, 7965. Template containing SdI: MALDI-TOF [M + H+]; calculated, 7980; actual, 7980.

Table 1. Kinetic Parameters for Steady-State Incorporations and Extensions with RB69a incorporation with RB69 Tn:dNTPb

kcat (min−1)

dG:dCTP CldG:dCTP BrdG:dCTP CdG:dCTP CdG:dATP OdG:dCTP OdG:dATP SdG:dCTP SdG:dATP dI:dCTP OdI:dCTP OdI:dATP SdI:dCTP SdI:dATP

6.8 3.9 4.0 6.4 0.48 6.8 4.4 5.2 0.18 2.0 1.5 1.1 0.64 1.0

± ± ± ± ± ± ± ± ± ± ± ± ± ±

1.2 0.4 0.3 0.9 0.08 0.4 0.8 0.8 0.02 0.4 0.3 0.1 0.08 0.2

Km dNTP (μM)a 0.087 0.39 0.50 0.30 81 3.2 58 42 77 0.23 12 7.7 100 140

± ± ± ± ± ± ± ± ± ± ± ± ± ±

0.018 0.07 0.09 0.05 10 0.3 9 5 15 0.04 2 1.9 17 30

kcat/Km (min−1 μM−1) 78 10 8.0 21 0.0059 2.1 0.076 0.12 0.0023 8.7 0.12 0.15 0.0064 0.0071

± ± ± ± ± ± ± ± ± ± ± ± ± ±

21 2 1.6 5 0.0012 0.2 0.018 0.02 0.0005 2.2 0.03 0.04 0.0014 0.0005

extension with RB69 dCTP/ dATPc

3559 28 52

0.8 0.9

Tn−1:Pn−1d dG:dC CldG:dC BrdG:dC CdG:dC CdG:dA OdG:dC OdG:dA SdG:dC SdG:dA dI:dC OdI:dC OdI:dA SdI:dC SdI:dA

kcat (min−1) 5.2 4.4 4.8 1.8 2.4 1.7 1.0 1.6 3.0 1.8 0.68 2.5 0.10 2.1

± ± ± ± ± ± ± ± ± ± ± ± ± ±

0.8 0.8 0.4 0.3 0.4 0.3 0.1 0.1 0.3 0.3 0.12 0.4 0.02 0.4

Km dNTP (μM)a 0.14 37 75 0.33 29 180 0.63 73 24 0.39 280 39 47 160

± ± ± ± ± ± ± ± ± ± ± ± ± ±

0.03 7 13 0.05 5 20 0.12 10 4 0.06 40 4 7 30

kcat/Km (min−1 μM−1) 37 0.12 0.064 5.6 0.083 0.010 1.5 0.022 0.13 4.7 0.0024 0.064 0.0021 0.013

± ± ± ± ± ± ± ± ± ± ± ± ± ±

10 0.03 0.012 1.3 0.019 0.0029 0.3 0.003 0.02 1.0 0.0006 0.013 0.0005 0.004

The average ± standard deviation was calculated from three or more experiments. bTn is the template nucleotide in the insertion site. cdCTP/dATP = (kcat/Km)dCTP/(kcat/Km)dATP. dTn−1 and Pn−1 are the template and primer nucleotides, respectively, in the extension site.

a

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Table 2. Kinetic Parameters for Steady-State Incorporations and Extensions with pol βa incorporation with pol β Tn:dNTPb dG:dCTP CldG:dCTP BrdG:dCTP CdG:dCTP CdG:dATP OdG:dCTP OdG:dATP SdG:dCTP SdG:dATP dI:dCTP OdI:dCTP OdI:dATP SdI:dCTP SdI:dATP

kcat (min−1) 110 50 43 16 2.7 170 150 60 47 87 93 103 53 40

± ± ± ± ± ± ± ± ± ± ± ± ± ±

20 7 7 2 0.3 30 30 7 7 17 20 17 7 7

Km dNTP (μM)a 15 150 200 180 720 54 80 52 180 14 52 23 63 48

± ± ± ± ± ± ± ± ± ± ± ± ± ±

1 20 20 30 90 6 12 7 10 3 6 4 10 7

kcat/Km (min−1 μM−1) 7.3 0.33 0.22 0.09 0.0037 3.2 1.9 1.2 0.26 6.2 1.8 4.5 0.85 0.83

± ± ± ± ± ± ± ± ± ± ± ± ± ±

1.4 0.06 0.04 0.02 0.006 0.6 0.4 0.2 0.04 1.8 0.04 1.1 0.017 0.18

extension with pol β dCTP/ dATPc

24 1.7 4.6

0.4 1.0

Tn−1:Pn−1d dG:dC CldG:dC BrdG:dC CdG:dC CdG:dA OdG:dC OdG:dA SdG:dC SdG:dA dI:dC OdI:dC OdI:dA SdI:dC SdI:dA

kcat (min−1) 43 47 50 22 28 73 43 70 20 77 53 87 47 24

± ± ± ± ± ± ± ± ± ± ± ± ± ±

Km dNTP (μM)a kcat/Km (min−1 μM−1)

10 7 10 3 7 10 10 10 2 13 7 20 07 4

12 11 14 14 170 16 37 15 91 19 16 51 47 61

± ± ± ± ± ± ± ± ± ± ± ± ± ±

1 2 3 3 30 2 2 2 13 3 3 11 5 11

3.6 4.2 3.6 1.6 0.17 4.6 1.2 4.7 0.22 4.0 3.3 1.7 1.0 0.39

± ± ± ± ± ± ± ± ± ± ± ± ± ±

0.9 1.0 1.0 0.4 0.05 0.8 0.3 0.9 0.9 0.9 0.8 0.5 0.2 0.10

The average ± standard deviation was calculated from three or more experiments. bTn is the template nucleotide in the insertion site. cdCTP/dATP = (kcat/Km)dCTP/(kcat/Km)dATP. dTn−1 and Pn−1 are the template and primer nucleotides, respectively, in the extension site.

a

Table 3. Kinetic Parameters for Steady-State Incorporations with WT and R332A Dpo4a WT Dpo4 Tn:dNTPb dG:dCTP CldG:dCTP BrdG:dCTP CdG:dCTP CdG:dATP OdG:dCTP OdG:dATP SdG:dCTP SdG:dATP dI:dCTP OdI:dCTP OdI:dATP SdI:dCTP SdI:dATP

kcat (min−1) 17 23 28 8.8 3.6 11 8.4 15 3.1 22 22 14 20 8.0

± ± ± ± ± ± ± ± ± ± ± ± ± ±

4 7 3 1.6 1.0 3 2.0 4 0.2 3 2 2 2 1.2

Km dNTP (μM)a 6.7 19 39 43 246 1.2 223 12 300 41 11 34 103 87

± ± ± ± ± ± ± ± ± ± ± ± ± ±

2.0 6 8 11 58 0.1 79 3 100 9 3 6 6 19

R332A Dpo4

kcat/Km (min−1 μM−1) 2.6 1.2 0.72 0.20 0.015 9.3 0.038 1.2 0.010 0.55 2.0 0.4 0.19 0.09

± ± ± ± ± ± ± ± ± ± ± ± ± ±

1.0 0.5 0.2 0.06 0.005 2.8 0.02 0.5 0.004 0.1 0.6 0.1 0.02 0.02

dCTP/ dATPc

13 245 120

5.0 2.1

kcat (min−1) 98 65 56 12 19 43 25 38 14 31 28 12 15 6.0

± ± ± ± ± ± ± ± ± ± ± ± ± ±

10 17 13 4 4 5 6 12 3 7 3 2 1 1.2

Km dNTP (μM)a 11 44 86 200 140 20 96 17 148 42 32 41 84 171

± ± ± ± ± ± ± ± ± ± ± ± ± ±

3 8 14 40 30 3 22 4 17 10 2 7 7 15

kcat/Km (min−1 μM−1) 8.9 1.5 0.65 0.058 0.14 2.2 0.26 2.2 0.092 0.73 0.88 0.29 0.18 0.035

± ± ± ± ± ± ± ± ± ± ± ± ± ±

2.6 0.5 0.18 0.021 0.04 0.4 0.08 0.9 0.022 0.24 0.10 0.06 0.019 0.008

dCTP/ dATPc

0.41 8.5 24

3.0 5.1

The average ± standard deviation was calculated from three or more experiments. bTn is the template nucleotide in the insertion site. cdCTP/dATP = (kcat/Km)dCTP/(kcat/Km)dATP.

a

dNTP concentrations. Reagent and product bands were quantified using ImageQuant 5.0. Michaelis−Menten curves were generated using values obtained from the averaging of at least three experiments and were repeated at least three times to ensure accuracy. kcat and Km values ± standard deviation were obtained directly from the Michaelis−Menten curves using SigmaPlot 9.0. The overall activity was provided by the specificity constant (kcat/Km), and the overall error was determined by propagation of the individual errors for kcat and Km.

in the Supporting Information). However, because Dpo4 is a low-fidelity Y-family polymerase35 and the incorporation of dTTP is likely due to the formation of a dG:dT wobble pair, only the incorporations of dATP and dCTP were studied further with the three polymerases. Additionally, because the analogues lacking an N7-hydrogen (dG, CldG, BrdG, and dI) cannot base pair to dA in the same manner as OdG and there was little incorporation of dATP opposite these nucleotides, dATP incorporations were not quantified opposite these analogues. Thus, for each polymerase, single insertion steadystate Michaelis−Menten kinetics were used to determine the kinetic parameters for each of the remaining 14 incorporation reactions as well as the extension reactions past the resulting base pairs. While steady-state (multiple turnover) conditions can be problematic with highly processive enzymes, leading to underestimation of the specificity constant (kcat/Km),36 the three purified polymerases used in this study are not highly processive,37−39 and the specificity constants can be used, with

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RESULTS RB69, pol β, and Dpo4 were first tested qualitatively for their ability to incorporate each of the four natural dNTPs opposite dG, OdG, and the seven analogues. Duplex DNA can be used as the substrate for experiments with RB69 and Dpo4; however, pol β, which is used during cellular short patch base excision repair, requires a gapped substrate for high activity (Figure 3).20 While there was little incorporation of dGTP or dTTP opposite OdG and the analogues with both RB69 and pol β, there was some dTTP incorporation observed with Dpo4 (Figures S1−S3 2580

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Table 4. Kinetic Parameters for Steady-State Extensions with WT and R332A Dpo4a WT Dpo4 Tn−1:Pn−1b

kcat (min−1)

dG:dC CldG:dC BrdG:dC CdG:dC CdG:dA OdG:dC OdG:dA SdG:dC SdG:dA dI:dC OdI:dC OdI:dA SdI:dC SdI:dA

5.2 2.6 1.6 5.4 1.2 8.0 2.8 1.2 2.0 3.8 5.6 6.4 0.82 5.0

± ± ± ± ± ± ± ± ± ± ± ± ± ±

1.0 0.4 0.2 0.4 0.3 1.8 0.6 0.3 0.2 0.8 0.8 1.0 0.16 1.0

Km dNTP (μM) 92 150 180 150 210 31 150 68 240 83 25 150 100 280

± ± ± ± ± ± ± ± ± ± ± ± ± ±

3 30 30 10 20 5 30 15 20 19 6 30 10 60

R332A Dpo4 kcat/Km (min−1 μM−1) 0.06 0.017 0.0091 0.036 0.0058 0.26 0.019 0.017 0.0083 0.046 0.22 0.043 0.0082 0.018

± ± ± ± ± ± ± ± ± ± ± ± ± ±

0.01 0.004 0.002 0.004 0.0014 0.07 0.005 0.005 0.0011 0.014 0.06 0.011 0.0018 0.005

kcat (min−1) 4.2 1.7 1.5 2.8 0.86 3.2 14 0.54 7.2 3.8 3.8 11 0.38 6.0

± ± ± ± ± ± ± ± ± ± ± ± ± ±

0.08 0.2 0.3 0.4 0.08 0.4 2 0.12 0.8 0.8 0.6 2 0.02 0.8

Km dNTP (μM) 132 79 77 95 170 150 140 40 170 170 130 130 110 200

± ± ± ± ± ± ± ± ± ± ± ± ± ±

22 9 11 15 20 20 10 8 20 10 10 20 20 10

kcat/Km (min−1 μM−1) 0.032 0.021 0.019 0.029 0.0051 0.021 0.10 0.014 0.042 0.022 0.029 0.086 0.0035 0.030

± ± ± ± ± ± ± ± ± ± ± ± ± ±

0.008 0.004 0.004 0.006 0.0008 0.004 0.02 0.004 0.007 0.005 0.005 0.020 0.0007 0.004

The average ± standard deviation was calculated from three or more experiments. bTn−1 and Pn−1 are the template and primer nucleotides, respectively, in the extension site. a

relative to dG (by 8−10-fold), the activities opposite OdG and SdG, which contain similarly sized or smaller atoms off C8, were much more reduced (37−631-fold; Table 1). Thus, the size of the atom off C8 cannot alone account for the reduction observed during dCTP incorporation opposite OdG versus dG, and it seems that electronics may play a role as well. Because the 8-keto tautomer predominates with both OdG and SdG,6,7,41,42 the electronics of the imidazole ring would be opposite that of dG, CldG, and BrdG,27 and additional negative interactions within the active site could result. Removal of the C2-amine also led to a significant drop in activity during dCTP incorporation with RB69; reactions opposite dI, OdI, and SdI were 9−19-fold less efficient than opposite dG, OdG, and SdG, respectively (Table 1). This result mirrors base pair stability studies, which show that dI:dC, OdI:dC, and SdI:dC base pairs (which all lack the C2-amine) are significantly less stable than dG:dC, OdG:dC, and SdG:dC base pairs, respectively.27 Thus, the drops in activity observed with the dI analogues during dCTP incorporation with RB69 are likely due, at least in part, to the loss of a hydrogen bond between the template base and the incoming dCTP. Interestingly, the reductions in dCTP incorporation efficiency produced through removal of the C2amine are enough to eliminate the strong preference for dCTP incorporation opposite OdG; the dCTP/dATP incorporation ratios opposite OdI and SdI are near 1 (Figure 4). During dATP incorporation with RB69, the most efficient reactions occurred opposite OdG and OdI, with significant decreases in efficiency (13−32-fold) observed opposite CdG, SdG, and SdI (Table 1). Because the only similarity between CdG, SdG, and SdI that differs from OdG and OdI is their reduced (SdG and SdI)43,44 or nonexistent (CdG) hydrogen bond-accepting ability at C8, these results suggest that there is an important minor groove hydrogen bond to the C8-oxygen of OdG(syn) during dATP incorporation. The efficiencies of dATP incorporation opposite OdI and SdI were about 2-fold higher than those opposite OdG and SdG, respectively, indicating that the C2-amine plays only a limited role during dATP incorporation with RB69. Substantial differences in efficiency were found during extensions past base pairs containing dC and one of the analogues (XdG:dC) with RB69 (Table 1). Substitution of chlorine and bromine off C8 of dG led to 308- and 578-fold

some caution, to compare the overall efficiencies of the various reactions.40 The results with all three polymerases reported herein for insertion opposite and extension past dG and OdG were similar to those previously reported. For example, herein, the dCTP/ dATP incorporation ratios for RB69, pol β, and Dpo4 were found to be 28 (Table 1), 1.7 (Table 2), and 244 (Table 3), respectively, while in previous studies, ratios of 20,21 1.8,20 and 91,23 respectively, were reported. There were also similarities in the relative efficiencies of dCTP incorporation opposite OdG as compared to dG. The studies herein showed RB69 and pol β inserted dCTP opposite OdG with 37- (Table 1) and 2.3-fold (Table 2) lower efficiencies than opposite dG, whereas past studies reported decreases of 45-21 and 3.5-fold,20 respectively. Both the current and the past studies with Dpo4 found dCTP incorporation to be more efficient opposite OdG than dG but by 3.6- (Table 3) and 6.8-fold,23 respectively. With respect to extension past OdG:dC and OdG:dA base pairs, previous studies and those herein both observed greater activity past OdG:dC base pairs than past OdG:dA base pairs with Dpo4 (Table 4),23 but extension past OdG:dA base pairs was more efficient than past OdG:dC base pairs with RB69 (Table 1).21 There was a difference in activity observed during extension with pol β; extensions past OdG:dC base pairs were 4-fold more efficient than past OdG:dA base pairs herein (Table 2), but there was no preference observed in a previous study.26 Incorporation Opposite and Extension Past OdG and the Analogues with RB69. The specificity constants for incorporations opposite and extensions past the dG/OdG analogues were determined with each polymerase to characterize the influence, if any, of the C2-amine and C8-oxygen on each reaction. We first focused on RB69 and the factors that lead to the roughly 40-fold decrease in dCTP incorporation efficiency observed opposite OdG with this polymerase. It has been found previously that large atoms off C8 can destabilize the anti conformation of dG as well as OdG:dC base pairs where the 8-oxoguanine base is in the anti conformation.6,33 We wanted to test if the size of the C8 atom is the main influencing factor for the reduced activity of dCTP incorporation opposite OdG or if other properties at C8 play a role. While dCTP incorporation opposite CldG and BrdG, which both contain large atoms off C8, did show decreased efficiency 2581

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Incorporation Opposite and Extension Past OdG and the Analogues with pol β. The dCTP incorporation results opposite the analogues with pol β were quite different to those observed with RB69 and, consistent with a previous study,46 show much larger decreases in efficiency opposite CldG and BrdG (22−33-fold) than opposite OdG and SdG (2−3-fold; Table 2). Thus, it appears that pol β is more tolerant of a C8oxygen or -sulfur in the major groove than RB69, while still being relatively intolerant of a C8-chlorine or -bromine, and suggests that the opposite electronics in the imidazole ring of OdG and SdG, as compared to dG, likely play a neutral or even slightly positive role during dCTP incorporation opposite OdG with pol β. Removal of the C2-amine had a much smaller effect during dCTP incorporation with pol β than with RB69, producing less than 2-fold reductions in kcat/Km opposite dI, OdI, and SdI, relative to dG, OdG, and SdG, respectively. During dATP incorporation, removal of the C8-oxygen from OdG (CdG) led to a 514-fold reduction in efficiency, while its replacement with sulfur (SdG or SdI) led to 5−7-fold reductions in efficiency. Similar to what was reasoned with RB69, these results suggest the possibility of a minor groove hydrogen bond to the C8-oxygen of OdG(syn) during dATP incorporation. The kcat/Km values for dATP incorporation opposite OdI and SdI were only 2−3-fold higher than opposite OdG and SdG with RB69. Thus, it appears that the C2-amine influences dATP incorporation opposite OdG only modestly, similar to what was observed during dCTP incorporation. Still, these modest changes were enough to remove any preference for dCTP incorporation opposite SdI and switch to a preference for dATP incorporation opposite OdI (Figure 5).

Figure 4. Graphical representations, with a logarithmic y-axis, of the specificity constants determined with RB69.

reductions in activity, respectively, while substitution with oxygen or sulfur at C8 of dG or dI led to 1958- to 3700-fold less efficient reactions. Thus, similar to the dCTP incorporation reactions, RB69 appears more sensitive to a C8-oxygen or -sulfur than a C8-chlorine or -bromine during extension past XdG:dC base pairs, indicating that both the steric and the electronic properties of the major groove C8-oxygen are important factors during extension past OdG:dC base pairs. Also similar to the dCTP insertion results, extensions past OdI:dC and SdI:dC were less efficient (4−11-fold) than past OdG:dC and SdG:dC base pairs, most likely due, once again, to the loss of a hydrogen bond within the XdG:dC base pair. We next turned our attention to extensions past base pairs containing dA and each of the analogues (XdG:dA) with RB69. Similar to dATP incorporation reactions, extension past an OdG:dA base pair was the most efficient, with 18- and 12-fold reductions in kcat/Km past CdG:dA and SdG:dA base pairs, respectively. Thus, it appears there may also be an important minor groove hydrogen bond to the C8-oxygen during extension past OdG:dA base pairs. Previous biochemical and structural work with RB69 has suggested the presence of a minor groove water-mediated hydrogen bond between Tyr567 and the template base (at Tn−1) during extension.45 To further test for this interaction, extensions past our analogues with a Y567A RB69 mutant were also investigated. Those experiments showed that removal of Tyr567 eliminates the increased activity previously observed during extensions past OdG:dA base pairs relative to CdG:dA and SdG:dA base pairs (Figure S4 in the Supporting Information and unpublished results). As a control, dCTP and dATP incorporations opposite the analogues with Y567A RB69 were also examined, and it was found that the higher activity of dATP incorporation previously observed opposite OdG, as compared to opposite CdG and SdG, is retained upon removal of Tyr567 (Figure S5 in the Supporting Information and unpublished results). Extensions past OdG:dA and SdG:dA base pairs were much more efficient (10−23-fold) than past OdI:dA and SdI:dA base pairs, suggesting an important role for the C2-amine during extension past OdG:dA base pairs with RB69.

Figure 5. Graphical representations, with a logarithmic y-axis, of the specificity constants determined with pol β.

During extensions past XdG:dC base pairs with pol β, the kcat/Km values for almost all of the reactions were within 2-fold (Table 2), suggesting that pol β is not significantly selective at the C2 or C8 positions during these reactions. More significant activity differences with pol β were observed during extensions past XdG:dA base pairs. Extensions past OdG:dA and OdI:dA base pairs were 5−7-fold more efficient than when the C8oxygen was removed (CdG:dA) or replaced with sulfur 2582

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Figure 6. Graphical representations, with a logarithmic y-axis, of the specificity constants determined with WT and R332A Dpo4.

were 7−9-fold more efficient with R332A Dpo4 than WT Dpo4, and once Arg332 was removed, there was no longer any benefit in removing the C2-amine (dATP incorporations opposite OdG and OdI, as well as SdG and SdI, had similar efficiencies with R332A Dpo4). Previous structural work has suggested extensions past OdG:dC and OdG:dA base pairs may also be influenced by interactions between Arg332 and the C8-oxygen and C2-amine, respectively.23,47 Therefore, extensions past the analogues with both WT and R332A Dpo4 were quantified (Table 4). During extensions past XdG:dC base pairs with WT Dpo4, the most efficient reactions (by at least 4-fold) were those past OdG and OdI, but deletion of Arg332 removed this preference. During extensions past most of the XdG:dA base pairs, removal of either the C2-amine or Arg332 led to increased efficiencies, but once Arg332 was mutated, there was no increase in activity upon removal of the C2-amine. Thus, the results for extensions past XdG:dC and XdG:dA base pairs mirror those found during dCTP and dATP incorporation, respectively. It is interesting to note that unlike during extensions past OdG:dC and SdG:dC base pairs, and during dATP incorporation opposite CdG, there was no increase in efficiency observed for extensions past CdG:dA base pairs with R332A Dpo4 as compared to WT Dpo4 (Figure 6). Finally, removal or replacement of the C8oxygen led to only slightly decreased kcat/Km values during dATP incorporation and extension past XdG:dA base pairs (as little as 2.5- and 2.4-fold, respectively), suggesting a limited role for this group during these reactions.

(SdG:dA and SdI:dA), once again suggesting the presence of a minor groove hydrogen bond to the C8-oxygen, but this time during extension past OdG:dA. Incorporation Opposite and Extension Past OdG and the Analogues with Dpo4. Unlike any other polymerase studied, the most efficient reactions during dCTP incorporation with Dpo4 occur opposite OdG and OdI, with at least 4-fold decreases in efficiency past the other dG- and dI-based analogues, respectively (Table 3). These results are consistent with previous Dpo4:OdG:dCTP ternary structure studies, which suggest that the side chain of Arg332 may be involved in a major groove hydrogen bond (either direct or water mediated) with the C8-oxygen of OdG(anti) during dCTP incorporation.22,23 To further investigate the presence of this interaction, the activities of dCTP incorporation opposite our analogues with an R332A Dpo4 mutant were studied. While the overall efficiencies during dCTP incorporation increased or remained similar between WT and R332A Dpo4 opposite almost all of the other analogues, the efficiencies opposite OdG and OdI decreased. Thus, removal of Arg332 also removes the comparatively higher activity of dCTP incorporation opposite OdG and OdI. It should be noted that with both R332A and WT Dpo4, as well as with pol β, the kcat/Km values for dCTP incorporation opposite CdG were even smaller than opposite CldG and BrdG despite no large atom being present off the C8 position of CdG. During dATP incorporations with Dpo4, reactions opposite OdI and SdI were 9−11-fold more efficient than opposite OdG and SdG, respectively (Table 3), indicating that removal of the C2-amine leads to increased activity. Previous work with R332A Dpo4 has suggested the potential for a clashing interaction between OdG and Arg332 in the insertion site,30 and we wondered if this clashing could be due to steric and/or electronic repulsion between the C2-amine of OdG(syn) and the side chain of Arg332. Hence, dATP insertion reactions opposite the N7-hydrogen-containing analogues were also tested with R332A Dpo4. Consistent with the proposed major groove clash, dATP insertions opposite CdG, OdG, and SdG

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DISCUSSION Previous results with our analogues and two A-family polymerases, KF-exo and BF, revealed that despite these two enzymes having contrasting incorporation preferences opposite OdG, they show similar overall activity trends.27 Both polymerases are sensitive to groups that extend outside the cognate W−C base pair shape in the major groove, including large and electronegative atoms off C8 of dG during dCTP incorporation and extension past the resulting base pair, as well 2583

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this case may provide half the stabilization of a standard hydrogen bond.28,50 Thus, it appears that the ability of OdG to retain this interaction is important for the capability of RB69 to insert dATP opposite OdG and possibly create a mutation. The influence of the C2-amine during dATP incorporation appears more modest, especially relative to the A-family polymerases, and may be due, once again, to the tighter and more rigid major groove of the A-family polymerases as compared to those in the B-family. The mutagenic potential of OdG with RB69 is likely increased by the relative efficiency of extension past OdG:dA mismatches and inefficiency of extension past OdG:dC base pairs. Inefficient extension past OdG:dC base pairs can lead to the removal of dC by the exonuclease site present in RB69 and recreate the possibility for dATP insertion. Alternatively, efficient extension past OdG:dA base pairs can hide this mismatch from the exonuclease proofreading site, allowing it to persist and lead to a permanent mutation. Similar to dCTP incorporation, extension past an OdG:dC base pair appears to be hindered by the size and electronics of the C8-oxygen, although to a much larger extent. This result is also similar to the A-family polymerases27 and is likely due to clashing between the C8-oxygen and the DNA backbone. Unlike in the insertion site, where there is a kink in the 5′-backbone that limits potential clashing with a large and/or electronegative atom at C8, ternary structural studies have shown that in the extension site of RB69, the 5′-bridging phosphate lies in close proximity to the C8 position (within 3.6 Å based on PDB 1IG9 and 4DU1). Two modeling studies with RB69 have confirmed that the C8-oxygen of OdG(anti) would clash with the adjacent phosphate, likely leading to a destabilizing perturbation in the DNA backbone.21,28 An analogous backbone distortion has been previously observed in a ternary structure of BF that contained an OdG:dC base pair in the extension site.19 Also similar to dCTP incorporation, extension past an OdG:dC base pair (at Tn−1:Pn−1) site appears to be aided by the presence of the C2-amine and the additional hydrogen bond it can make within the base pair. As for extension past OdG:dA base pairs with RB69, the relatively high efficiency of this reaction appears to be due, at least in part, to the ability of the C8-oxygen and C2-amine of OdG to hydrogen bond in the minor and major grooves, respectively. Past biochemical (with 3DA and Y567A RB69) and crystallographic studies with RB69 support the presence of a minor groove water-mediated hydrogen bond between the template base (at Tn−1) and the highly conserved Tyr567,45 and our work with the analogues as well as the preliminary data with the Y567A RB69 mutant provides further evidence for this interaction. As mentioned above, an analogous minor groove hydrogen bond has also been found in the A-family polymerases between the template base (at Tn−1) and a highly conserved glutamine.19 With respect to the C2-amine, its removal during extensions past XdG:dA base pairs led to a significant decrease in activity. This result is also similar to those with the A-family polymerases and is likely due to the loss of a hydrogen bond between the C2-amine and the 5′-bridging phosphate.27 Consistent with this possibility, previous modeling studies have proposed that, similar to the C8-oxygen during extension past OdG:dC base pairs, the C2-amine would be in close proximity to the adjacent phosphate during extension past OdG:dA base pairs.21,28 Thus, it appears that the presence of the C8-oxygen and the C2-amine allows OdG to make or retain

as the C2-amine during dATP incorporation. These results are consistent with much previous work that indicates A-family polymerases have tight and rigid active sites that are sensitive to steric perturbations.29,48 The contrasting dCTP/dATP incorporation ratios opposite OdG between the two polymerases appear to derive from the different extents of this sensitivity. BF is much less tolerant of changes at C8 during dCTP incorporation, leading to a dCTP/dATP incorporation ratio of less than 1. With both polymerases, two stabilizing interactions that appear important for efficient extension past OdG:dA pairs, allowing for these pairs to persist, are a minor groove hydrogen bond between the C8-oxygen and a highly conserved glutamine and a possible major groove hydrogen bond between the C2-amine and the 5′-bridging phosphate. It was the goal of the work reported herein to expand these studies to additional polymerases, including those from other families, to gain insights into the importance of the C2-amine and C8-oxygen during incorporation opposite and extension past OdG for each polymerase and, thus, a more general understanding of how these two sites may influence the mutagenic potential of OdG. Influence of the C2 and C8 Positions to the Mutagenic Potential of OdG with RB69. On the basis of previous results and those within this study, RB69 appears to have a relatively low mutagenic potential in that dCTP incorporation is much preferred over dATP incorporation opposite OdG. We first set about using the analogues to better understand the role of the C8-oxygen and C2-amine during dCTP incorporation with RB69. Although this reaction is much preferred over dATP incorporation opposite OdG, its efficiency is significantly reduced as compared to dCTP incorporation opposite dG, thus helping to create some possibility for mutation. The results of dCTP incorporation with RB69, a Bfamily replicative polymerase, are similar to those previously found with the A-family of replicative and repair polymerases.27 Both sets of these high-fidelity polymerases show sensitivity to the size and electronics of the C8-oxygen during dCTP incorporation opposite OdG, thus decreasing the activity of this reaction (Figure 4). RB69 does appear to be a bit less sensitive to atomic changes off C8 during dCTP incorporation, however, and consistent with this result, biochemical evidence has suggested that the insertion sites of B-family polymerases are less rigid than those of the A-family.49 Furthermore, evidence from the crystal structure of a ternary complex and modeling studies both indicate that the C8 position of dG at Tn is less restricted in RB69 than in A-family polymerases, like KF-exo and BF, where protein side chains are found within close proximity (within 3.5 Å) of the C8 position.27 In keeping with the similar dCTP incorporation results between RB69 and the A-family polymerases, they are all sensitive to the loss of the C2-amine and, presumably, the third hydrogen bond that it allows between the template nucleotide (Tn) and the incoming dCTP. The results with the OdG analogues also reveal the possibility of a minor groove hydrogen bond to the C8-oxygen during dATP incorporation opposite OdG with RB69. Consistent with this finding, the presence of a nonstandard hydrogen bond between the C8-oxygen and the Cα-H of the universally conserved Gly568 has been suggested based on biochemical studies with 3-deaza-2′-deoxyadenosine (3DA), as well as crystallographic data from a mutant enzyme ternary complex.28 Hydrogen bonds with a Cα-H have been characterized previously in transmembrane proteins and in 2584

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OdG(anti)] and dATP [to the C8-oxygen of OdG(syn)] incorporation; thus, the loss of Arg283 would be deleterious to both reactions. Consistent with a more open active site and flexible DNA backbone in pol β, the C2-amine does not appear to play a major role during dATP incorporation opposite OdG. Turning to the extension reactions with pol β, of note is the similarity in efficiencies during extension past XdG:dC base pairs (Figure 5). Crystallographic studies are consistent with this result and, unlike in the A- and B-family polymerases, show the 5′-phosphate turned away from the base at Tn−1,26 allowing for the presence of large and electronegative atoms off C8 with no large effect on structure or activity. Results with the analogues indicate an important interaction with the C8-oxygen during extensions past XdG:dA base pairs with pol β, and consistent with this finding, previous structural studies have observed a minor groove hydrogen bond (direct or water mediated) between Tn−1 and Arg283.26,54 It appears that the presence of the C8-oxygen allows this interaction to remain, leading to more efficient extension past an OdG:dA mismatch and an increased potential for its permanence. Influence of the C2 and C8 Positions to the Mutagenic Potential of OdG with Dpo4. On the basis of in vitro insertion and extension studies, OdG has a very low mutagenic potential with Dpo4, a Y-family bypass polymerase. Not only does Dpo4 have the highest known dCTP/dATP incorporation ratio opposite OdG known, but it also extends past OdG:dC and OdG:dA base pairs with very high and low efficiencies, respectively (Figure 6). Although Dpo4 has no inherent exonuclease activity, it does have a very active pyrophosphorolysis reaction (the back reaction of insertion) that can lead to primer degradation in the presence of mismatched base pairs.55 Thus, the inefficient extension past OdG:dA mismatches observed with Dpo4 could activate a type of proofreading activity that would serve to lower the possibility of an OdG-generated mutation. On the basis of results herein and structural results previously published,22,23,30,47 it appears that the C8-oxygen and C2-amine of OdG are key to both the strong preference for dCTP incorporation opposite OdG and the much higher extension efficiency past OdG:dC base pairs relative to OdG:dA base pairs observed with Dpo4. The C8oxygen of OdG forms a major groove hydrogen bond to the side chain of Arg332 during both dCTP incorporation and extension past an OdG:dC base pair, increasing the activity of these reactions. Alternatively, the C2-amine clashes in the major groove with the side chain of Arg332 during both dATP incorporation and extension past an OdG:dA base pair, limiting their activity. Consistent with this theory, substitution of Arg332 with alanine drops the dCTP/dATP incorporation ratio opposite OdG from 245 to 9, opposite SdG from 120 to 24, and opposite OdI from 5 to 3, as well as reverses the increased activity for extension past OdG:dC and SdG:dC as compared to OdG:dA and SdG:dA. When OdG is not present, Arg332 is believed to interact with the bridging phosphate between Tn and Tn−1, consistent with it having access to both the insertion and the extension sites of Dpo4.23 It should be noted that one Dpo4 ternary structural study showed the possibility of OdG:dA being held in an alternate OdG(anti):dA(syn) structure in the extension site of Dpo4, allowing the C8oxygen of OdG(anti) to hydrogen bond to Arg332, much like when OdG:dC is present in the extension site.47 Our work with the analogues does not support the presence of this alternate conformation of OdG:dA during extension since removal of Arg332 leads to increased activity during extension past

important interactions during extension past OdG:dA base pairs, increasing the potential for a permanent mutation. Influence of the C2 and C8 Positions to the Mutagenic Potential of OdG with pol β. The mutagenic potential of OdG is quite different with pol β than RB69 and stems from its much smaller dCTP/dATP incorporation ratio (1.7 vs 28). Likewise, the C2-amine and C8-oxygen appear to influence dCTP and dATP incorporation opposite OdG differently with pol β than RB69. As compared to the A- and B-family polymerases, pol β, a low-fidelity X-family polymerase, is more tolerant of a C8-oxygen or -sulfur in the major groove during dCTP incorporation (Figure 5). Several crystallographic studies with a ternary pol β:OdG:dCTP equivalent complex have shown that the DNA template makes few enzyme contacts; thus, it is flexible and able to reorient so as to avoid major groove clashing between the 5′-bridging phosphate and the C8-oxygen of OdG.26,51 Additionally, the active site of pol β appears less constrained; the only protein side chain in the vicinity of the major groove is Lys280, which is believed to stack on templating purine bases.52 It is possible that the higher efficiency of dCTP incorporation observed opposite OdG and SdG, especially relative to that opposite CldG and BrdG, may arise due to Lys280. Its side chain is very flexible, and upon comparison of available pol β ternary structures, shows great variation in its orientation and to where the ζ-NH2 hydrogen bonds.26 Thus, it is possible that more optimal placement and/ or interactions of Lys280 during dCTP incorporation opposite OdG (and SdG) led to the observed results. A similar argument has been made to explain the slight preference for dCTP insertion over dATP insertion opposite OdG with pol β.26 One surprising result with both pol β and Dpo4 is the relatively low activity of dCTP incorporation opposite CdG (Figures 5 and 6), despite CdG having no large or electronegative atoms off C8. CdG is a C-nucleotide where the sugar is attached to the base through a carbon, and this atomic change can lead to distinct structural changes and conformational preferences as compared to N-nucleotides.31,53 It is possible that the C-nucleotide character of CdG alters its ability to adopt the appropriate orientations at Tn with pol β and Dpo4 and is responsible for the significant reductions in efficiency observed during incorporation opposite CdG with these polymerases. However, the activity decreases opposite CdG observed during dCTP incorporation do not appear to translate to the extension site since there are only minimal decreases in activity during extensions past CdG:dC base pairs as compared to dG:dC with pol β and Dpo4. Work with the OdG analogues also reveals that the relatively high efficiency of dATP incorporation opposite OdG, and thus the increased mutagenic potential for OdG observed with pol β, is likely due, at least in part, to a minor groove hydrogen bond to the C8-oxygen. Consistent with these findings, a recent crystallography study with a ternary pol β complex has indicated the presence of a hydrogen bond between the C8oxygen of OdG(syn) and the side chain of Arg283.26 The loss of this interaction, and possibly the C-nucleotide character of CdG, help to explain the over 500-fold decrease in efficiency observed as dATP is incorporated opposite CdG relative to OdG. It should be noted that a previous study tested the incorporation of dCTP and dATP opposite OdG with a R283A mutant of pol β and found no significant change in the dCTP/ dATP incorporation ratio.20 This result is not unexpected since a minor groove hydrogen bond between Arg283 and the template base would occur during both dCTP [to the N3 of 2585

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CdG:dA, OdG:dA, and SdG:dA base pairs, but not past OdI:dA and SdI:dA base pairs, and there are minimal differences in activity between extensions past CdG:dA, OdG:dA, and SdG:dA with WT Dpo4. Arg332 is not the only influencing factor in the insertion site of Dpo4. Y-family polymerases, like Dpo4, are known to have flexible and loose active sites that are generally tolerant of variations that lie outside the W−C cognate base pair shape.49,56 Furthermore, similar to pol β, the DNA backbone in Dpo4 is also flexible and able to reorient itself to avoid potential clashing in the insertion site.22 The ternary structural studies with Dpo4 also suggest the presence of a C(H)3···π interaction between Ala42 and OdG(anti) during dCTP incorporation.23 This interaction is not present during dATP incorporation when OdG is in the syn conformation and may explain why Dpo4 retains a preference for dCTP over dATP incorporation opposite the OdG analogues even in the absence of Arg332. Finally, unlike during extensions past OdG:dA, SdG:dA, OdI:dA, and SdI:dA base pairs, the activity during extensions past CdG:dA base pairs did not increase when Arg332 was removed. This result is surprising but may be explained by a previous R332A Dpo4 ternary structure that contained an OdG:dA base pair in the extension site. That study suggests the possibility of a minor groove water-mediated hydrogen bond between the C8-oxygen of OdG(syn) (at Tn−1) and Tyr12, an interaction that has not been observed in any WT Dpo4 structure.30 It is possible that this hydrogen bond is an artifact of the R332A mutant, and the inability of CdG to form this insular interaction leads to the significantly reduced extension efficiency past CdG:dA base pairs found only with R332A Dpo4. It should be noted that we did consider other factors that could influence activity during incorporation opposite and extension past OdG and/or the analogues with each of the polymerases. These factors included desolvation and base stacking effects with all of the analogues,27 as well as halogen bonding with CldG and BrdG.57 We found little evidence that any of these factors play a significant role in the reactions studied; there were no clear correlations between lower activity and an increased electrostatic potential, between higher activity and increased stacking ability, as judged by surface area and polarizability,58 or between higher activity and the presence of a C8-chlorine or bromine.

preserve minor groove hydrogen bonds that are present in the insertion and extension sites of RB69 and pol β, as well as the extension sites of the A-family polymerases. During incorporation of dCTP and extension past OdG:dC base pairs, the presence of the C2-amine can help to keep activity high by retaining the third hydrogen bond between dG/ OdG:dC base pairs. During dATP incorporation and extension past OdG:dA base pairs, the C2-amine also resides outside the W−C cognate shape; thus, its presence can also lead to novel interactions in the major groove that alter activity. These can include clashing interactions in the very tight insertion sites of A-family polymerases or when specific residues, like Arg332 of Dpo4, are nearby, as well as stabilizing hydrogen bonds, like those between the C2-amine and the 5′-adjacent phosphate during extension past OdG:dA base pairs with RB69 and the Afamily polymerases. Each polymerase has its own unique active site; thus, OdG interacts with each a bit differently, leading to differences in incorporation and extension preferences and, consequently, the overall mutagenic potential of OdG.

CONCLUSION Through the work with the OdG analogues, not only can previously suggested interactions with OdG be further supported, but new interactions and more subtle information on those interactions can be determined. Additionally, by combining the analogue data from the polymerases in this study as well as those of the previous study with the two A-family polymerases,27 the roles of the C8-oxygen and C2-amine during OdG mutagenicity can be more generalized. During dCTP incorporation and extension past OdG:dC base pairs, the presence of the C8-oxygen, which lies outside the W−C cognate base pair shape, can lead to novel interactions in the major groove, whether that be clashing interactions in the more rigid insertion sites of A- and B-family polymerases that reduce activity or a specific hydrogen bond, like that to Arg332 of Dpo4, which increases activity. During dATP incorporation and extension past OdG:dA base pairs, the presence of the C8oxygen can keep activity relatively high by allowing OdG to

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ASSOCIATED CONTENT

S Supporting Information *

Qualitative gels for dNTP incorporations opposite the analogues with the three polymerases. Qualitative gels for extension past and incorporation opposite the analogues with WT and Y567A RB69. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*Tel: 1-804-287-6327. Fax: 1-804-287-1897. E-mail: mhamm@ richmond.edu. Funding

This work was supported by the University of Richmond, the Howard Hughes Medical Institute, the Arnold and Mabel Beckman Foundation, and the NSF-RUI (CHE0956474) program. M.L.H. is a Henry Dreyfus Teacher Scholar. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We thank F. Peter Guengerich and William Konigsberg for the gifts of WT and R332A Dpo4 and WT and Y567A RB69, respectively.

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ABBREVIATIONS OdG, 8-oxo-2′-deoxyguanosine; BrdG, 8-bromo-2′-deoxyguanosine; CldG, 8-chloro-2′-deoxyguanosine; CdG, 9-deaza-2′deoxyguanosine; SdG, 8-thio-2′-deoxyguanosine; OdI, 8-oxo2′-deoxyinosine; SdI, 8-thio-2′-deoxyinosine; RB69, the DNA polymerase from bacteriophage RB69 which contains D222A and D327A mutations to remove the 3′-exonuclease activity; pol β, human DNA polymerase β; Dpo4, DNA polymerase IV from Sulfolobus solfataricus P2; BF, the large fragment from Bacillus stearothermophilus polymerase I which lacks 3′exonuclase activity; KF-exo, the 3′-exonuclease deficient version of Klenow Fragment

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