Basis of Miscoding of the DNA Adduct N²,3-Ethenoguanine by Human Y-family DNA Polymerases

Background: The miscoding of N²,3-etheno(ϵ)guanine(G) is of interest regarding cancer.

Results: N²,3-ϵG:T mispairing was found with Y-family human DNA polymerases, and crystal structures of polymerase ι revealed Hoogsteen base pairing.

Conclusion: Structural similarity for N²,3-ϵG:C and N²,3-ϵG:T underlies similar catalytic efficiencies for polymerase ι.

Significance: The structural basis of N²,3-ϵG miscoding is revealed.

Abstract

N²,3-Ethenoguanine (N²,3-ϵG) is one of the exocyclic DNA adducts produced by endogenous processes (e.g. lipid peroxidation) and exposure to bioactivated vinyl monomers such as vinyl chloride, which is a known human carcinogen. Existing studies exploring the miscoding potential of this lesion are quite indirect because of the lability of the glycosidic bond. We utilized a 2′-fluoro isostere approach to stabilize this lesion and synthesized oligonucleotides containing 2′-fluoro-N²,3-ϵ-2′-deoxyarabinoguanosine to investigate the miscoding potential of N²,3-ϵG by Y-family human DNA polymerases (pols). In primer extension assays, pol η and pol κ replicated through N²,3-ϵG, whereas pol ι and REV1 yielded only 1-base incorporation. Steady-state kinetics revealed that dCTP incorporation is preferred opposite N²,3-ϵG with relative efficiencies in the order of pol κ > REV1 > pol η ≈ pol ι, and dTTP misincorporation is the major miscoding event by all four Y-family human DNA pols. Pol ι had the highest dTTP misincorporation frequency (0.71) followed by pol η (0.63). REV1 misincorporated dTTP and dGTP with much lower frequencies. Crystal structures of pol ι with N²,3-ϵG paired to dCTP and dTTP revealed Hoogsteen-like base pairing mechanisms. Two hydrogen bonds were observed in the N²,3-ϵG:dCTP base pair, whereas only one appears to be present in the case of the N²,3-ϵG:dTTP pair. Base pairing mechanisms derived from the crystal structures explain the slightly favored dCTP insertion for pol ι in steady-state kinetic analysis. Taken together, these results provide a basis for the mutagenic potential of N²,3-ϵG.

Introduction

The integrity of DNA is continually challenged by environmental factors (e.g. UV irradiation and radiation), exogenous and endogenous chemicals, and suboptimal repair processes (1). DNA damage produces modified DNA bases (i.e. DNA lesions or DNA adducts), abasic sites, DNA inter- and intrastrand cross-links, and DNA-protein cross-links that, if not properly repaired, can lead to genomic instability and ultimately disease (e.g. cancer).

DNA polymerases (pols)² are crucial in maintaining genome integrity. Fifteen human DNA pols, varying in their functions in replication, repair, and tolerance of DNA damage, are known (2). The Y-family DNA polymerases (pol η, pol ι, pol κ, and REV1) are specialized in translesion synthesis (3, 4). For example, pol η is known for its unique role in correctly bypassing UV irradiation-induced cyclobutane pyrimidine dimer (5, 6). Pol ι, on the other hand, is unable to copy past cyclobutane pyrimidine dimer but can proficiently insert T or C opposite adducted purines that are impaired in their capability of forming Watson-Crick base pairs (7–9). Pol κ has a specialized role in bypassing bulky N²-G adducts (10) and interstrand cross-links (11) and is distinct in its moderate processivity, extending beyond the lesion, possibly due to the use of its N-clasp domain. REV1 is highly selective for inserting C opposite normal (12) and adducted template G (10, 13). Crystal structures of Y-family pols provide insight into their diverse functions in bypassing normal and adducted templates (14). Pol ι adopts an induced fit mechanism by flipping template purines into the syn conformation, forming Hoogsteen base pairs (7, 8, 15, 16). REV1 features pairing between dCTP and template G but uses its G-loop to hydrogen bond with the template G and an Arg in another segment (N-digit) to ensure the incorporation of dCTP (12). A high degree of functional and structural differences underlies the diverse but specialized roles in lesion bypass by Y-family human DNA polymerases (17).

Etheno (ϵ) DNA adducts comprise a series of exocyclic adducts, including 1,N⁶-ethenoadenine (1,N⁶-ϵA), 3,N⁴-ethenocytidine (3,N⁴-ϵC), N²,3-ethenoguanine (N²,3-ϵG), and 1,N²-ethenoguanine (1,N²-ϵG) (Fig. 1). These were first identified as reaction products of nucleobases with chloroacetaldehyde (18) and were used as fluorescent analogs in biochemical studies and as probes for nucleic acid structures (19–21). The ϵ DNA adducts were subsequently recognized as reaction products of DNA with reactive metabolites of several genotoxic chemicals, including the carcinogens vinyl chloride and vinyl carbamate (an oxidation product of urethane). The detection of etheno DNA adducts in various tissues of unexposed rodents (22) and humans (23) led to the discovery of the endogenous pathways of formation (e.g. via reaction with trans-4-hydroperoxy-2-nonenal, a lipid peroxidation product (24)). In livers of unexposed rats or humans, the steady-state amounts of N²,3-ϵG, 1,N²-ϵG, and 1,N⁶-ϵA have been estimated to be ∼36, 30, and 12 lesions/cell, respectively (25).

FIGURE 1.

Etheno DNA adduct structures.

The mutagenic potentials of etheno adducts have been established in in vitro bypass assays (26–30) and site-specific mutagenesis in bacteria (31, 32), Chinese hamster ovary cells (33), and simian kidney cells (34). N²,3-ϵG has been less well studied in terms of its replication and repair mechanisms because of the lability of its glycosidic bond. In a polyribo(G/N²,3-ϵG) template, both C and T were incorporated opposite N²,3-ϵG by avian myeloblastosis virus reverse transcriptase (35). N²,3-ϵ-Deoxyguanosine triphosphate was reported to be inserted opposite T by Escherichia coli DNA polymerase I (Klenow fragment), Drosophila melanogaster polymerase α-primase complex, and human immunodeficiency virus-I reverse transcriptase (30). An indirect assay in E. coli showed an estimated mutation frequency of 13% for N²,3-ϵG, resulting in G to A transitions (32). The long half-life of N²,3-ϵG in rat liver and lung (150 days) and in rat kidney (75 days) in vinyl chloride-exposed rats suggests inefficient repair of this lesion. In human glycosylase assays (in vitro), N²,3-ϵG was released at a much slower rate compared with 1,N⁶-ϵA and 3,N⁴-ϵC (36). The mutagenicity and persistence of N²,3-ϵG suggest a high miscoding potential in vivo. N²,3-ϵG is generally considered to contribute to the carcinogenesis of vinyl chloride and inflammation-driven malignancies (37). The dominance of GC to AT transitions in five of six K-ras (oncogene) tumors found in vinyl chloride workers (25) suggests the importance of G adducts, and the miscoding pattern of 1,N²-ϵG is not consistent with this transition (26–35).

We recently investigated the miscoding of N²,3-ϵG using a stabilized 2′-fluoro-substituted analog, 2′-fluoro-N²,3-ϵ-2′-deoxyarabinoguanosine (2′-F-N²,3-ϵdG) (38). The presence of the electronegative fluorine atom destabilizes the transition state leading to an oxocarbenium-like intermediate and hydrolysis of the glycosidic bond. This analog was site-specifically incorporated into oligonucleotides; the stability of 2′-F-N²,3-ϵdG permitted steady-state kinetics, primer extension assays, and crystallographic studies. Catalytic insertions opposite 2′-F-N²,3-ϵdG were examined using five DNA polymerases, including Sulfolobus solfataricus P2 DNA polymerase IV (Dpo4), the replicative bacteriophage pol T7 DNA exonuclease⁻, (E. coli pol I) Klenow fragment exonuclease⁻, yeast pol η, and human DNA pol κ, where a consistent miscoding pattern (2′-F-N²,3-ϵdG:T) was found. Crystal structures of Dpo4 with 2′-F-N²,3-ϵdG paired with dCTP showed a Watson-Crick-like structure, whereas the complex with 2′-F-N²,3-ϵdG:T revealed a “sheared” base pair (38).

To further understand the miscoding potential of N²,3-ϵG by Y-family human DNA polymerases, which are highly relevant to translesion synthesis, we carried out a series of primer extension and steady state-kinetic analyses using human pol ι, human pol η, and human REV1 with a template containing 2′-F-N²,3-ϵdG. The extension products formed by pol ι were identified using LC-MS/MS. A consistent mispairing pattern was observed (2′-F-N²,3-ϵdG:T), and base pairing mechanisms were revealed in two pol ι crystal structures with either dCTP or dTTP paired with 2′-F-N²,3-ϵdG but with individual differences.

EXPERIMENTAL PROCEDURES

Materials

All commercial chemicals were of the highest quality available and were used without further purification. Unlabeled dNTPs, T4 polynucleotide kinase, uracil-DNA glycosylase, and restriction endonucleases were from New England Biolabs (Ipswich, MA). [γ-³²P]ATP (specific activity, 3 × 10³ Ci mmol⁻¹) was purchased from PerkinElmer Life Sciences. Biospin columns were from Bio-Rad. Unmodified oligonucleotides were from Midland Certified Reagents (Midland, TX). 2-Amino-9-(2-deoxy-2-fluoro-β-d-arabinofuranosyl)guanine was from Metkinen (Kuopio, Finland). Modified oligonucleotides containing 2′-F-N²,3-ϵdG were synthesized as described earlier (38) followed by HPLC purification and desalting with Sephadex G-25 (Sigma-Aldrich). The modified 23-mer template used for extension and kinetic assays was 5′-TCATXGAATCCTTACGAGCCCCC-3′ where X = 2′-F-N²,3-ϵdG (MALDI-TOF MS (3-hydroxypicolinic acid) m/z calculated for [M − H]⁻, 6986.5; found, 6985.6) or 2′-fluoro-2′-deoxyarabinoguanosine (2′-F-dG) (MALDI-TOF MS (3-hydroxypicolinic acid) m/z calculated for [M − H]⁻, 6962.5; found, 6963.5). The 18-mer oligomer used for crystallographic studies was 5′-TCT(2′-F-N²,3-ϵdG)GGGTCCTAGGACC(ddC)-3′ (ddC, dideoxy-CMP) (MALDI-TOF MS (3-hydroxypicolinic acid) m/z calculated for [M − H]⁻, 5514.9; found, 5515.2). Human DNA pol ι catalytic fragments (residues 1–420) (16), pol η (39), and pol κ (38) were purified following protocols described previously.

Preparation of Recombinant Catalytic Core of Human REV1

The gene fragment covering the catalytic core (residues 330–833) (12) of wild-type human REV1 was obtained by PCR amplification from the vector pET-22b(+)/hREV1 (13) as template using Pfu DNA polymerase (Stratagene, La Jolla, CA) with a pair of primers (5′-GGATCCATGTCTACGTTTAGCAAGGCAG-3′ and 5′-GCGGCCGCTTATGTGGAAGGGTTCAGATTAG-3′). The resulting PCR product of the 1.5-kb hREV1 fragment was cloned into the vector pSC-B-Amp/Kan (Stratagene). Following sequence confirmation, the hREV1 gene fragment was cloned into the BamHI and NotI sites of the vector pBG101 (obtained from the Center for Structural Biology, Vanderbilt University) to generate the cleavable glutathione S-transferase (GST)-tagged protein. The GST-tagged hREV1(330–833) was expressed in E. coli BL21 (DE3) cells, which were grown at 37 °C and 220 rpm to an OD₆₀₀ of 0.6 and then induced with isopropyl β-d-1-thiogalactopyranoside (0.2 mm) for 12 h at 16 °C. The harvested pellets were resuspended in lysis buffer containing 50 mm Tris-HCl (pH 7.4), 500 mm NaCl, 10% (v/v) glycerol, 5 mm β-mercaptoethanol, 1 mg ml⁻¹ lysozyme, and protease inhibitor mixture (Roche Applied Science). Suspensions were sonicated, and the cell lysate was clarified by centrifugation at 4 × 10⁴ × g for 60 min at 4 °C. The resulting supernatants were loaded onto a 1-ml GSTrap 4B column (GE Healthcare), and the column was washed with 20 ml of Buffer A (50 mm Tris-HCl (pH 7.4) containing 150 mm NaCl, 10% (v/v) glycerol, and 5 mm β-mercaptoethanol). The GST-tagged REV1(330–833) bound on the column was cleaved with Prescission protease (GE Healthcare) for 14 h at 4 °C. Cleaved REV1(330–833) was eluted with Buffer A, and the purity was analyzed by SDS-polyacrylamide gel electrophoresis with Coomassie Brilliant Blue R-250 staining. A typical yield was ∼760 μg from 1 liter of culture.

Primer Extension and Steady-state Kinetic Assays

An 18-mer oligomer (5′-GGGGGCTCGTAAGGATTC-3′) was 5′ [γ-³²P]ATP end-labeled and annealed to a 23-mer template (5′-TCATXGAATCCTTACGAGCCCCC-3′ where X = 2′-F-N²,3-ϵdG, 2′-F-dG, or 2′-deoxyguanosine (dG)). Primer extension experiments were performed in 50 mm Tris-HCl buffer (pH 7.5) containing 60 nm primer·template complex, 250 μm dNTPs, 20 nm polymerase, 5% (v/v) glycerol, 5 mm DTT, 50 mm NaCl, 5 mm MgCl₂, and 50 μg ml⁻¹ bovine serum albumin (BSA) at 37 °C. Steady-state kinetic experiments were carried out under the same conditions except using 5–20 nm pol, varying dNTP concentrations, and 2–10-min incubation times. Reactions were quenched with 9 μl of 20 mm EDTA (pH 9.0) in 95% (v/v) formamide. Products were separated using 20% (w/v) acrylamide electrophoresis gels, and results were visualized using a phosphorimaging system (Bio-Rad, Molecular Imager® FX) and analyzed by Quantity One software as described (38).

LC-MS Analysis of Full-length Extension Products

An 18-mer primer (5′-GGGGGCTCGTAAGGAT(dU)C-3′) was annealed to the same 23-mer oligomer as described above at a 1:1 molar ratio. Reaction conditions were similar to those used in steady-state kinetic assays except that the final concentrations were as follows: 10 μm pol ι, 12.5 μm primer·template complex, and 2% (v/v) glycerol in a total volume of 80 μl. Reactions were carried out in the presence of four dNTPs (10 mm each) for 3.5 h at 37 °C. The reactions were terminated by spin column separations to extract dNTPs and Mg²⁺, and the resulting product was treated with 50 units of uracil-DNA glycosylase and then 0.25 m hot piperidine (40). LC-MS/MS analyses were performed using an ACQUITY ultraperformance liquid chromatography (UPLC) system (Waters Corp.) connected to a Finnigan LTQ mass spectrometer (Thermo Scientific Corp., San Jose, CA) operating in the electrospray ionization negative ion mode and using an ACQUITY UPLC system BEH octadecylsilane (C₁₈) column (1.7 μm; 1.0 × 100 mm). UPLC conditions were as described (38).

Crystallization of Pol ι·2′-F-N²,3-ϵdG·DNA Ternary Complexes

The sequence of the 18-mer oligomer used for co-crystallization with pol ι, 5′-TCT(2′-F-N²,3-ϵdG)GGGTCCTAGGACC(ddC)-3′, was designed based on previous studies (15, 16). Crystals were obtained under conditions similar to those described previously (15, 16). Specifically, 210 μm pol ι, 253 μm annealed DNA, 10 mm MgCl₂, and 20 mm dCTP (or dTTP) were mixed and preincubated on ice. Droplets (a 1:1 (v/v) mixture of pol·DNA complex mixture and the reservoir solution) were equilibrated against a reservoir solution containing 0.10 m 2-(N-morpholino)ethanesulfonic acid (MES) (sodium salt, pH 6.5), 0.3 m (NH₄)₂SO₄, and 15% (w/v) polyethylene glycol 5000 for pol ι-1 (pol ι·2′-F-N²,3-ϵdG:dCTP) or 17% (w/v) polyethylene glycol 5000 for pol ι-2 (pol ι·2′-F-N²,3-ϵdG:dTTP). Crystals were grown using the hanging drop vapor diffusion method at 4 °C and mounted at 4 °C with step soaking in mother liquor solutions containing 0–25% (w/v) glycerol prior to flash freezing in liquid nitrogen.

Structure Determination and Refinement

X-ray diffraction data were collected at the Advanced Photon Source (Argonne National Laboratory, Argonne, IL) on the 21-ID-F and 21-ID-G (Life Sciences Collaborative Access Team) beam lines. All data sets were recorded from cryoprotected crystals using a single wavelength at 100 K. Data were indexed and scaled with the program HKL2000 (41). Both crystal types belonged to space group P6₅22. X-ray diffraction data collection and processing statistics are listed in Table 3 (see below). Phases were calculated using MOLREP as a part of the CCP4 program suite (42, 43) based on a previously refined model (Protein Data Bank code 3OSN) (16). Refinements were performed using Refmac 6.0 with restrained and rigid body refinement (44, 45). Repeated cycles of manual rebuilding were performed in Coot (46). Structural images were generated in PyMOL (47).

TABLE 3.

Crystal data collection and refinement statistics for the ternary complexes pol ι-1 (pol ι·2′-F-N²,3-ϵdG:dCTP, Protein Data Bank code 4FS2) and pol ι-2 (pol ι·2′-F-N²,3-ϵdG:dTTP, Protein Data Bank code 4FS1)

	Pol ι-1 (2′-F-N2,3-ϵdG:dCTP)	Pol ι-2 (2′-F-N2,3-ϵdG:dTTP)
Data collection
Beamline	21-ID-F	21-ID-G
Space group	P6522	P6522
Unit cell (a, b, c) (Å)	97.30, 97.30, 202.91	97.47, 97.47, 203.54
Unit cell (α, β, γ) (°)	90.0, 90.0, 120.0	90.0, 90.0, 120.0
Resolution (Å)a	2.05 (2.05–2.09)	2.49 (2.49–2.53)
No. of measured reflections	36,517	23,120
No. of unique reflections	36,444	23,074
Percent possible (%)	99.8 (98.4)	99.8 (100)
Redundancy	7.8 (6.3)	6.9 (6.8)
Rlinearb	0.051 (0.581)	0.067 (0.505)
Signal to noise (I/σI)	32.5 (2.5)	23.0 (3.2)
Coordinate composition (asymmetric unit)
No. of protein molecules	1	1
No. of amino acid residues	383	383
No. of water molecules	179	65
No. of Mg2+ ions	3	2
No. of template nucleotides	8	9
No. of primer nucleotides	7	7
No. of dCTP	1	0
No. of dTTP	0	1
Refinement
Resolution range (Å)	30.00–2.05	30.00–2.50
Reflections	34,557	19,448
Rwork (%)c	21.4	21.5
Rfree (%)d	26.0	27.0
Root mean square deviation bond length (Å)	0.018	0.014
Root mean square deviation bond angle (°)	2.05	1.99
Mean B-factor	51.3	56.7
Wilson B-factor	42.8	49.4
Ramachandran summary
In preferred regions	354 (93.40%)	350 (92.35%)
In allowed regions	16 (4.22%)	23 (6.07%)
Outliers	9 (2.37%)	5 (1.58%)

^a Values for highest resolution bin are given in parentheses.

^b R_linear = Σ|I − 〈I〉|ΣI where I is the integrated intensity of a given reflection.

^c R_work = Σ‖F_observed − F_calculated‖/Σ|F_observed|.

^d R_free was calculated using 5% test size with random selection.

RESULTS

Primer Extensions by Human Y-family Pols

Primer extension experiments were performed using a template containing stabilized 2′-F-N²,3-ϵdG and compared with those using templates containing 2′-F-dG and unmodified dG (Fig. 2). Among the four pols, pol η appeared to be most efficient in terms of producing full-length products (5 bases extended). Although similar to pol η in efficiency, pol κ only produced products with 4 or fewer bases extended in a 20-min reaction. REV1 and pol ι extended the primer by 1 base within the same time frame. Quantitation of the extension products showed that the pol η-extended products constituted 93, 96, and 83% of the substrate for dG, 2′-F-dG, and 2′-F-N²,3-ϵdG templates, respectively, upon 20-min incubation. The corresponding respective values were 73, 76, and 50% for REV1; 19, 10, and 10% for pol ι; and 94, 94, and 70% for pol κ. The resistance to extension observed here is consistent with the known low processivities of pol ι and REV1 (9, 13).

FIGURE 2.

³²P-labeled primer extension in the presence of four dNTPs by different Y-family human DNA pols. A, primer and template sequences where X is dG, 2′-F-dG, or 2′-F-N²,3-ϵ-dG. B, pol η. C, REV1. D, pol ι. E, pol κ. Reactions were carried out for increasing times (as indicated) in 50 mm Tris-HCl buffer (pH 7.5) containing 60 nm primer·template complex, 250 μm dNTPs, 20 nm pol, 5% (v/v) glycerol, 5 mm DTT, 50 mm NaCl, 5 mm MgCl₂, and 50 μg ml⁻¹ BSA at 37 °C.

Steady-state Kinetics of Nucleotide Incorporation

To determine the catalytic efficiency of incorporation of each dNTP catalyzed by different pols, steady-state kinetic analysis was performed. The catalytic efficiency (i.e. k_cat/K_m) and misinsertion frequency (i.e. f = (k_cat/K_m(dNTP))_incorrect/(k_cat/K_m(dCTP))) are two useful parameters for comparing different polymerase reactions (Table 1). For the template containing 2′-F-N²,3-ϵdG, all three pols preferred to insert dCTP. Pol ι and pol η had relatively high misincorporation frequencies for T because of their marginally lower catalytic efficiencies compared with those for C insertion. For REV1, the order of preference for dNTP misinsertion opposite the lesion was G > T > A (based on k_cat/K_m) but with much lower misincorporation frequencies compared with the three other pols (Table 1). When comparing the k_cat/K_m values for dCTP insertion opposite the lesion with those obtained for the dG template, pol η and pol ι showed ∼40-fold attenuation followed by about 10-fold attenuation for REV1, suggesting that the presence of the lesion did not significantly affect the deoxycytidyltransferase activity of REV1. The catalytic efficiencies for different dNTPs were similar in magnitude for the 2′-F-dG and dG templates, indicating that 2′-F modification did not dramatically perturb pol recognition. Among all four pols examined (including pol κ (38)), pol κ exhibited the highest relative efficiency (0.24) of nucleotide (dCTP) incorporation opposite 2′-F-N²,3-ϵdG (compared with dCTP incorporation opposite unmodified dG) followed by REV1 (0.11), pol η (0.027), and pol ι (0.026) (Table 1). Pol η showed the highest absolute value of catalytic efficiencies for all three types of template, consistent with results seen in product extension experiments (see above).

TABLE 1.

Steady-state kinetic analysis of polymerase-catalyzed single base insertion

The insertion was opposite X in the template sequence of 3′-CCCCCGAGCATTCCTAAGXTACT-5′ where X is 2′-F-N²,3-ϵdG, 2′-F-dG, or dG.

Polymerase/template	dNTP	kcat	Km(dNTP)	kcat/Km(dNTP)	fa	Relative efficiencyb
		min−1	μm	min−1 μm−1
pol ι
2′-F-N2,3-ϵdG	C	0.26 ± 0.03	153 ± 58	0.0017		0.026
	T	0.33 ± 0.02	280 ± 36	0.0012	0.71
	A	0.016 ± 0.001	956 ± 83	0.000017	0.010
	G	0.007 ± 0.001	223 ± 71	0.000031	0.018
2′-F-dG	C	0.079 ± 0.1	14 ± 4	0.0056		0.085
	T	0.049 ± 0.005	156 ± 46	0.00031	0.055
	A	0.0067 ± 0.0011	88 ± 47	0.000076	0.014
	G	0.0040 ± 0.0006	44 ± 25	0.000091	0.016
dG	C	0.093 ± 0.001	1.4 ± 0.5	0.066		1
	T	0.63 ± 0.18	1090 ± 510	0.00058	0.0088
	A	0.0052 ± 0.0007	111 ± 53	0.000047	0.00071
	G	0.0098 ± 0.0009	365 ± 69	0.000027	0.00041
Pol η
2′-F-N2,3-ϵdG	C	0.88 ± 0.04	11 ± 2	0.08		0.027
	T	1.3 ± 0.06	26 ± 6	0.05	0.63
	A	0.40 ± 0.03	63 ± 21	0.0063	0.079
	G	0.30 ± 0.02	45 ± 9	0.0067	0.084
2′-F-dG	C	1.7 ± 0.06	0.51 ± 0.12	3.3		1.1
	T	0.70 ± 0.05	49 ± 12	0.014	0.0042
	A	0.96 ± 0.07	15 ± 6	0.064	0.019
	G	0.63 ± 0.05	18 ± 7	0.035	0.011
dG	C	2.3 ± 0.14	0.76 ± 0.23	3.0		1
	T	0.51 ± 0.03	57 ± 11	0.0089	0.0030
	A	0.88 ± 0.03	74 ± 9	0.012	0.0040
	G	0.51 ± 0.03	50 ± 9	0.010	0.0033
REV1
2′-F-N2,3-ϵdG	C	0.083 ± 0.005	4.8 ± 1.4	0.017		0.11
	T	0.024 ± 0.003	24 ± 2	0.0010	0.059
	A	0.012 ± 0.001	32 ± 8	0.00038	0.022
	G	0.060 ± 0.018	44 ± 13	0.0013	0.080
2′-F-dG	C	0.17 ± 0.01	1.6 ± 0.5	0.11		0.73
	T	0.37 ± 0.02	90 ± 13	0.0041	0.037
	A	0.043 ± 0.004	27 ± 10	0.0016	0.015
	G	0.89 ± 0.03	10 ± 2	0.0089	0.081
dG	C	0.12 ± 0.01	0.81 ± 0.37	0.15		1
	T	0.024 ± 0.001	7.8 ± 4.2	0.0031	0.021
	A	0.043 ± 0.004	27 ± 11	0.0016	0.011
	G	0.49 ± 0.03	130 ± 14	0.0037	0.025
Pol κc
2′-F-N2,3-ϵdG	C	1.6 ± 0.1	73 ± 13	0.022		0.24
	T	0.90 ± 0.04	111 ± 14	0.0082	0.37	0.091
	A	0.063 ± 0.002	55 ± 7	0.0011	0.05
	G	0.22 ± 0.01	210 ± 22	0.0010	0.045
2′-F-dG	C	1.9 ± 0.1	2.8 ± 0.3	0.68
dG	C	1.8 ± 0.1	20 ± 1	0.090		1

^a Misinsertion frequency: f = (k_cat/K_m(dNTP))_incorrect/(k_cat/K_m(dCTP)).

^b Relative efficiency calculated as the ratio of the k_cat/K_m for dNTP insertion opposite the adduct to the k_cat/K_m for dCTP insertion opposite dG.

^c From Ref. 38.

Analysis of Pol ι Primer Extension Products by LC-MS/MS

Because the highest misinsertion frequency was observed for pol ι, we examined the extension products of pol ι reactions using LC-MS/MS. Previous procedures were followed using a uracil-containing primer (Fig. 3A), and the product was cleaved using uracil-DNA glycosylase to simplify the sequencing results obtained with collision-induced dissociation fragmentation. The most abundant species (−2 or −3 charge) were chosen for collision-induced dissociation analysis, and the identity of the product was established by matching the fragmentation pattern to the theoretical pattern obtained from a program linked to the mass spectrometry group at the University of Utah (48). By using a longer incubation time (3.5 h) and higher enzyme and substrate concentrations compared with those in the primer extension gel analyses, a greater portion of primer was extended by pol ι, and the products could be identified. As shown in Fig. 3, B–D, three products were identified with C, T, and A incorporated opposite 2′-F-N²,3-ϵdG. Two additional products containing C and T opposite the lesion (Fig. 3, E and F, respectively) were identified as having an extra A at the end. The confirmed fragment ions are illustrated in the spectra with fragmentation patterns in the insets.

FIGURE 3.

Collision-induced dissociation spectra from LC-MS/MS analysis of the full-length extension assays using pol ι and a 23-mer oligomer template containing 2′-F-N²,3-ϵdG in the presence of all four dNTPs. A, template and primer sequences. The confirmed product sequences with the fragmentation patterns shown are 5′-CCATGA-3′ (B), 5′-CTATGA-3′ (C), 5′-CAATGA-3′ (D), 5′-CCATGAA-3′ (E), and 5′-CTATGAA-3′ (F). The reaction contained 12.5 μm DNA complex, 10 mm dNTPs, 10 μm pol ι, 5 mm DTT, 50 mm NaCl, 5 mm MgCl₂, and 50 μg ml⁻¹ BSA and were incubated at 37 °C for 3.5 h. Underlined U indicates the cutting site by uracil-DNA glycosylase after DNA polymerase reactions.

The relative yields of various products were calculated based on the peak areas of extracted LC-MS chromatograms (data not shown). The sum of the peak areas was used for the product, which existed in more than one charge state. Consistent with the low efficiency of pol ι seen in the primer extension gel analysis, the amount total of extended products (based on the total peak areas; data not shown) only accounted for 6% of the total products by Dpo4 formed under the same conditions (38). The major extension products were those containing T and C opposite 2′-F-N²,3-ϵdG, and the other three are minor products (Table 2). Pol ι produced similar yields of extension products with C (41%) and T (52%) incorporated opposite the lesion. In addition, pol ι readily extended the 2′-F-dG and dG templates in an error-free manner. The base insertion pattern obtained from LC-MS/MS analysis agrees with the steady-state kinetic analysis (Table 1) with T insertions being the major misincorporation events for pol ι-catalyzed bypass.

TABLE 2.

Summary of pol ι extension products from LC-MS/MS analysis

The results were obtained using template · primer complexes containing 2′-F-N²,3-ϵdG, 2′-F-dG, or dG. Underlined nucleotides indicate the base incorporated opposite the lesion.

Crystal Structures of Pol ι with Oligonucleotides Containing 2′-F-N²,3-ϵdG and dCTP or dTTP

To understand the base pairing mechanisms for dCTP and dTTP observed above, we conducted co-crystallization experiments with pol ι, a template containing 2′-F-N²,3-ϵdG, and dCTP or dTTP. Two types of crystals were obtained, i.e. pol ι-1 (pol ι·2′-F-N²,3-ϵdG:dCTP; Protein Data Bank code 4FS2) and pol ι-2 (pol ι·2′-F-N²,3-ϵdG:dTTP; Protein Data Bank code 4FS1). Structures of these two ternary complexes were determined by molecular replacement using a previously refined model (Protein Data Bank code 3OSN) (16) without the lesion and the incoming nucleotide (Table 3). Clear electron densities around the 2′-F-N²,3-ϵdG and incoming nucleotide facilitated the unbiased determination of the base pairing conformations at the active site. Similar to several structures seen previously for template native purines (7, 15) and adducted purines (8, 9, 16), the electron density around 2′-F-N²,3-ϵdG indicated that the lesion was rotated from the anti to the syn conformation (Fig. 4, A and C). This substrate-induced conformational change of template purine is thought to be dictated by the rigid active site of pol ι (7).

FIGURE 4.

Crystal structures of ternary complexes pol ι-1 (pol ι·2′-F-N²,3-ϵdG:dCTP, Protein Data Bank code 4FS2) and pol ι-2 (pol ι·2′-F-N²,3-ϵdG:dTTP, Protein Data Bank code 4FS1). A, pol ι-1 active site with template 2′-F-N²,3-ϵdG pairing with the incoming dCTP. Green spheres show the two observed Mg²⁺ ions. B, 2′-F-N²,3-ϵdG in syn conformation in pol ι-1 with one hydrogen bond formed between the O6 atom of 2′-F-N²,3-ϵdG and the N4 atom of dCTP (2.5 Å); the other potential hydrogen bond formed is indicated with a dashed line between the N7 atom of 2′-F-N²,3-ϵdG and the N3 atom of dCTP (3.0 Å). C, pol ι-2 active site with template 2′-F-N²,3-ϵdG pairing with incoming dTTP. The green sphere shows the observed single Mg²⁺ ion. D, 2′-F-N²,3-ϵdG in the syn conformation in pol ι-2 with one hydrogen bond formed between the O6 atom of 2′-F-N²,3-ϵdG and the N3 atom of dTTP (2.8 Å). The quality of the data is demonstrated using the Fourier 2F_o − F_c sum electron density map displayed (blue mesh) at 1.0 σ in A and C.

In both the pol ι-1 (Fig. 4A) and pol ι-2 (Fig. 4C) structures, the incoming nucleotide served as a donor in hydrogen bonds with the Hoogsteen edge of 2′-F-N²,3-ϵdG (i.e. the O6 and N7 atoms). When 2′-F-N²,3-ϵdG was paired with dCTP (Fig. 4B), one hydrogen bond was observed between the N4 atom of dCTP and O6 atom of 2′-F-N²,3-ϵdG as indicated by a 2.5-Å distance. The possibility of a second hydrogen bond cannot be ruled out based on the distance (3.0 Å) between the N3 atom of dCTP and the N7 atom of 2′-F-N²,3-ϵdG provided that the N3 atom of dCTP is protonated. This mechanism was proposed in previous studies for dCTP paired with G (15) or adducted G (16).

In the case of 2′-F-N²,3-ϵdG-paired dTTP (Fig. 4D), it is likely that only one hydrogen bond exists between the O6 atom of 2′-F-N²,3-ϵG and the N3 atom of dTTP with a distance of 2.8 Å. Although a distance of 3.2 Å between N7(2′-F-N²,3-ϵdG) and N3(dTTP) can also be interpreted as a potential hydrogen bonding distance, the asymmetry between the two distances (2.8 versus 3.2 Å) makes it unlikely that a bifurcated hydrogen bonding structure is present. The slightly longer distance (2.8 Å) in the case of 2′-F-N²,3-ϵdG:T hydrogen bond may be an indication of its weaker strength compared with the 2.5-Å distance seen in the 2′-F-N²,3-ϵdG:C pair, although the resolution limit of 2.5 Å does not permit a firm conclusion in this respect. Together with the possibility of two hydrogen bonds in the 2′-F-N²,3-ϵdG:C pair, the base pair modes observed here are consistent with the slightly favorable insertion of C observed in the steady-state kinetic analysis.

Irrespective of the incoming nucleotide, the pol ι-1 and pol ι-2 structures are quite similar with a root mean square deviation value of 0.27 Å for all atom pairs upon superimposition. The superimposition of the incoming nucleotides suggested a movement of 2′-F-N²,3-ϵdG toward the minor groove for the 2′-F-N²,3-ϵdG:T base pair (Fig. 5A). The pol ι-1 structure superimposes with the native G:C complex (Protein Data Bank code 2ALZ; Ref. 15) with a root mean square deviation value of 0.27 Å (Fig. 5B), indicating that the presence of the lesion (2′-F-N²,3-ϵdG) did not significantly affect the conformations of the protein and the nucleic acid. The conformation of the 2′-F-N²,3-ϵdG:C pair also resembles that of an N²-ethylguanine:C pair crystallized with pol ι (9) (Fig. 5C). When the conformation of the 2′-F-N²,3-ϵdG:T mispair is compared with an O⁶-methylguanine:T base pair (16) (because of the lack of a structure with the pol ι G:T pair in the Protein Data Bank), the conformations of both base pairs are similar except that the lesion is slightly moved toward the minor groove in the case of the 2′-F-N²,3-ϵdG:T pair (Fig. 5D, green). This shift could be due to the bulkier size of 2′-F-N²,3-ϵdG compared with O⁶-methylguanine. Overall, pol ι appears to be able to accommodate the 2′-F-N²,3-ϵdG pair rather well at the active site without significant protein and nucleic acid conformational changes. The structures showed that both C and T pair with the lesion in a similar fashion in line with the observation that pol ι promoted both error-free and error-prone bypass in steady-state kinetic and LC-MS/MS analyses.

FIGURE 5.

Comparisons of base pair positions at the active site of pol ι complexes based on the superimposition of incoming nucleotides. A, shearing of 2′-F-N²,3-ϵdG toward the minor groove for the 2′-F-N²,3-ϵdG:dTTP pair (green) compared with the 2′-F-N²,3-ϵdG:dCTP pair (red). B, similarity between the 2′-F-N²,3-ϵdG:dCTP pair (red) and the native G:dCTP pair (blue; Protein Data Bank code 2ALZ). C, structural similarity between the 2′-F-N²,3-ϵdG:dCTP pair (red) and the N²-ethylguanine:dCTP pair (gray; Protein Data Bank code 3EPG) crystallized with pol ι; D, shearing of 2′-F-N²,3-ϵdG towards the minor groove for the 2′-F-N²,3-ϵdG:dTTP pair (green) compared to O⁶-methylguanine:dTTP pair (16) (orange, Protein Data Bank code 3OSN).

DISCUSSION

The DNA adduct N²,3-ϵG is a ubiquitous modification produced from endogenous processes (e.g. lipid peroxidation) or exposure to environmental pollutants (e.g. vinyl chloride or urethane). We recently developed an isostere approach to incorporate the stabilized analog (2′-F-N²,3-ϵdG) into oligonucleotides and investigated the miscoding potential of N²,3-ϵG using several prokaryotic and eukaryotic DNA pols (38). In the present work, we extended our previous investigation into the other three human Y-family DNA pols and provided the structural basis of the most error-prone bypass enzyme, pol ι.

Primer extension gel analysis generated a qualitative comparison of the capability of bypassing 2′-F-N²,3-ϵdG by Y-family pols (Fig. 1). The order of bypassing efficiency (from the percentage of total product extended) is pol η > pol κ > REV1 ≈ pol ι. Compared with pol κ and pol ι, the higher activity of pol η copying past N²,3-ϵG observed here is similar to that seen previously for other etheno adducts, i.e. 1,N²-ϵG (29), 1,N⁶-ϵA (26, 49), and 3,N⁴-ϵC (50). With regard to DNA polymerases, the extension pattern is particularly similar to that of bypass of 1,N²-ϵG; i.e. pol η readily extended the primer into full-length products, whereas pol ι and pol κ showed some single base incorporation (29).

Steady-state kinetic analysis established the preferred base incorporated opposite the lesion and provided a kinetic rationale for primer extension experiments (Table 1). For all four human Y-family DNA pols, the correct base C is marginally preferred opposite 2′-F-N²,3-ϵdG with similar relative efficiencies in comparison with the insertion of C opposite a regular G (Table 1). The misinsertion of T is consistent for all four human Y-family DNA pols as well as for several other prokaryotic and eukaryotic DNA polymerases (38). The highest absolute value of catalytic efficiency (k_cat/K_m) seen (for pol η) is in line with primer extension results, which may be partly explained by the more open active site of pol η compared with other polymerases (51). The pattern of fidelity for pol η bypassing different etheno lesions is similar: both error-free and error-prone syntheses have been observed. Pol η inserted a C opposite N²,3-ϵG in a marginally error-free manner with a misinsertion frequency of 0.63 for T (Table 1). Similarly, pol η copied past 1,N⁶-ϵA in the order of preference T > C > A > G (49). The order was G > A > C for 1,N²-ϵG (29) and A ≈ G > C ≈ T for 3,N⁴-ϵC (50). Pol ι has the highest misincorporation frequency (although C is preferred 1.5-fold compared with T), which is consistent with the view that pol ι generally catalyzes error-prone bypass (3). The incorporation patterns seen for pol ι bypassing other etheno DNA adducts are as follows: pol ι somewhat prefers to incorporate C opposite 1,N⁶-ϵA (8) and inserts both C and T opposite 1,N²-ϵG with almost the same catalytic efficiencies (29). The fact that REV1 prefers to catalyze dCTP insertion is not surprising in that REV1 utilizes its G-loop to hydrogen bond with template G and an Arg in another segment (N-digit) to ensure the incorporation of dCTP (12). When comparisons are made with the catalytic efficiency of dCTP insertions opposite native G in the template, the order of relative efficiency is pol κ (0.24) > REV1 (0.11) > pol η (0.027) ≈ pol ι (0.026) (Table 1), suggesting that 2′-F-N²,3-ϵdG affects the DNA syntheses of the four Y-family pols to a similar extent.

LC-MS/MS analysis of the primer extension products by pol ι provided further insight into the nature of the bases inserted beyond the lesion in these error-prone reactions. With pol ι, approximately half of the products contained T with a high fidelity extension beyond the lesion (Fig. 3 and Table 2). The observation of almost equal amounts of products containing C and T opposite 2′-F-N²,3-ϵdG (with LC-MS/MS analysis) is in line with results from kinetic analysis (Table 1). The much lower amount of total extended products (6%) compared with Dpo4 (38) agrees with the low bypass efficiency of pol ι seen in the primer extension gel analysis (Fig. 2). These extension products are similar to the products generated by Dpo4 (38); however, the pattern of miscoding is considerably different from that generated by 1,N²-ϵG, which yields mainly products with G inserted by human pol η (29) and −1 deletion products by Dpo4 (28).

The hydrogen bonding patterns of 2′-F-N²,3-ϵdG:C and 2′-F-N²,3-ϵdG:T base pairs seen in the crystal structures provided molecular explanations for the error-free and error-prone bypass of pol ι. The distance of 2.5 Å is a clear indication that a hydrogen bond is established between the O6 atom of 2′-F-N²,3-ϵdG and the N4 atom of dCTP. The possibility of a second hydrogen bond also exists, i.e. between the N7 atom of 2′-F-N²,3-ϵdG and the N3 atom of dCTP, provided that the N3 atom of dCTP is protonated. The tendency for protonation of the N3 atom of dCTP has been discussed in several other pol ι·DNA structures with both native and adducted purines in the templates (8, 15, 16). Although the N3 atom of free cytosine has a pK_a ∼4.5, the local molecular environment could elevate the pK_a to 6.2–7.2 at a terminal position or >8.5 at an internal position in DNA triple helices (52, 53). Nair et al. (8) suggested that an elevation of the pK_a of dCTP could be due to the base-stacking and long range electrostatic interactions of the active site residues Asp-126 and Glu-127. The one hydrogen bond observed in the 2′-F-N²,3-ϵdG:T pair is an indication that the 2′-F-N²,3-ϵdG:T pair might be less stable compared with the 2′-F-N²,3-ϵdG:C pair. Our crystallization attempts are consistent with this view in that pol ι-1 type crystals (with dCTP) grew more easily and diffracted to higher resolution than the pol ι-2 crystal (with dTTP). Collectively, the difference in hydrogen bonding may explain a slightly higher catalytic efficiency for dCTP by pol ι.

The typical strategy that pol ι uses a Hoogsteen base pairing mechanism to accommodate native and adducted purines was once again demonstrated in both the pol ι-1 and pol ι-2 structures albeit with different hydrogen bonding schemes. The similarity of the two structures consists of their use of the Hoogsteen edge of 2′-F-N²,3-ϵdG to hydrogen bond with the incoming nucleotide. The conformation of 2′-F-N²,3-ϵdG:C also resembles G:dCTP (15) and N²-ethylG:dCTP (9) at the pol ι active site.

However, the base pair conformations seen here are quite different from what has been observed at the active site of Dpo4 (38). Specifically, the 2′-F-N²,3-ϵdG:C pair adopts a Watson-Crick-like conformation, and the 2′-F-N²,3-ϵdG:T structure contains a sheared base pair at the Dpo4 active site (Fig. 6, C and D, and Ref. 38). That 2′-F-N²,3-ϵdG was observed to be positioned in the anti conformation by Dpo4 is likely due to the relatively open active site compared with pol ι (17) (surface view shown in Fig. 6, A and B). Particularly, the residues adjacent to the template base are bulkier (Leu-62, Val-64, and Gln-59) in pol ι compared with Dpo4 (Ala-42, Ala-44, and Val-32) (54). In the Dpo4 structure, hydrophobic interactions are likely to exist between Val-32 and the imidazole ring of 2′-F-N²,3-ϵdG (anti). Conversely, residues (Leu-62, Val-64, and Gln-59) may force 2′-F-N²,3-ϵdG to rotate into the syn conformation, which would otherwise clash with these residues if the lesion were positioned in the anti conformation. Despite these conformational differences, similar extents of T misinsertion are observed in both cases.

FIGURE 6.

A comparison of the active site conformations of pol ι (A) and Dpo4 (B) (protein shown in surface view) is shown. The conformations of 2′-F-N²,3-ϵdG paired with incoming nucleotides at the Dpo4 active site are shown in C (dCTP) and D (dTTP) (from Ref. 38).

As mentioned in the Introduction, Singer and co-workers (30, 32, 35) reported three studies on the miscoding of N²,3-ϵG more than 20 years ago. These studies were limited by the general methods available for studying miscoding at the time as well as the inherent lability of the glycosidic bond of N²,3-ϵdG. The uncorrected mutation frequency for N²,3-ϵdG inserted into an M13 phage system was only 0.5%, but in that study (32), an in vitro study with a polyribo(G/N²,3-ϵG) template and a reverse transcriptase (35), and a study on “reverse” incorporation of N²,3-ϵdG triphosphate (30), the general pattern was pairing of N²,3-ϵG with T and C. This pattern, despite any deficiencies in the earlier work, is similar to those seen in our own studies (Ref. 38 and the present work). The N²,3-ϵG:T wobble pairing proposed in that early work (35) had no experimental basis and has not been observed in our crystal structures with Dpo4 (38) or human pol ι (Fig. 4).

More recently, theoretical studies (55) have predicted that G should be the base most likely to pair with N²,3-ϵG followed by T > A > C, a prediction that is clearly inconsistent with the results obtained with all DNA polymerases thus far (Tables 1 and 2) (38). The pairing patterns predicted in the theoretical study (55) are also inconsistent with our N²,3-ϵG:C and N²,3-ϵG:T structures observed in the Dpo4 (38) and human pol ι (Fig. 4) crystals.

As mentioned in the Introduction, the goal of the 2′-fluoro substitution was to stabilize the glycosidic bond by destabilizing the transition state leading to an oxocarbenium-like intermediate in hydrolysis. The substitution was clearly successful in stabilizing the residue in oligonucleotides (38). Although miscoding by N²,3-ϵG (specifically, 2′-F-N²,3-ϵdG) was clearly demonstrated relative to both dG and 2′-F-dG (Table 1), it should be noted that the substitution of fluorine for hydrogen at the C2′ sugar position is not without effect; i.e. the substitution caused up to a 12-fold change in k_cat/K_m (primarily in the K_m parameter) among four Y-family DNA polymerases: an ∼8-fold decrease of k_cat/K_m with pol κ and a ∼12-fold increase of k_cat/K_m with pol ι but no changes with pol η or REV1. Therefore, 2′-fluoro substitution seems to slightly interfere with pol ι activity but to facilitate pol κ activity, which might be related to a possible stabilizing effect of 2′-fluorine to exert a (intra- and/or inter-residual) pseudo-hydrogen bonding interaction with purine H8 as shown previously with 2′-fluoroarabinonucleic acid (56–58). Such a conformational effect (preferentially to an anti conformation) by 2′-F at dG might affect catalysis differently with the various polymerases by interfering with the (syn-anti) Hoogsteen base pairing adopted by pol ι but facilitating the (anti-anti) Watson-Crick base pairing utilized by pol κ (albeit not with pol η). Nevertheless, these points regarding the influence of the fluorine do not affect our conclusions about the miscoding properties of N²,3-ϵG reported here.

In conclusion, we have utilized a recently developed stabilized analog, 2′-F-N²,3-ϵdG, to discern the mutation potential of a ubiquitous but unstable DNA lesion, N²,3-ϵdG. Kinetic and extension analyses allow qualitative and quantitative assessments of the miscoding pattern of this lesion for Y-family DNA polymerases, which are particularly relevant to translesion synthesis. Structural insights provided the molecular bases of error-free and error-prone synthesis by pol ι. The consistency of T misinsertion with all polymerases studied thus far underscores the miscoding potential of N²,3-ϵG. The miscoding for T suggests the relevance of N²,3-ϵG to vinyl chloride-induced angiosarcomas in which prevailing GC to AT transition mutations were found in the second base of codon 13 of the K-ras gene (59). Our study supports the hypothesis that N²,3-ϵG may contribute to the carcinogenesis of vinyl chloride and inflammation-driven malignancies (25, 37).