Base-displaced intercalation of the 2-amino-3-methylimidazo[4,5-f]quinolone N2-dG adduct in the NarI DNA recognition sequence.

2-Amino-3-methylimidazo[4,5-f]quinolone (IQ), a heterocyclic amine found in cooked meats, undergoes bioactivation to a nitrenium ion, which alkylates guanines at both the C8-dG and N2-dG positions. The conformation of a site-specific N2-dG-IQ adduct in an oligodeoxynucleotide duplex containing the iterated CG repeat restriction site of the NarI endonuclease has been determined. The IQ moiety intercalates, with the IQ H4a and CH3 protons facing the minor groove, and the IQ H7a, H8a and H9a protons facing the major groove. The adducted dG maintains the anti-conformation about the glycosyl bond. The complementary dC is extruded into the major groove. The duplex maintains its thermal stability, which is attributed to stacking between the IQ moiety and the 5'- and 3'-neighboring base pairs. This conformation is compared to that of the C8-dG-IQ adduct in the same sequence, which also formed a 'base-displaced intercalated' conformation. However, the C8-dG-IQ adopted the syn conformation placing the Watson-Crick edge of the modified dG into the major groove. In addition, the C8-dG-IQ adduct was oriented with the IQ CH3 group and H4a and H5a facing the major groove. These differences may lead to differential processing during DNA repair and replication.

INTRODUCTION

Although browning meats during cooking imparts flavor, it also leads to the formation of heterocyclic amines (n class="Chemical">HCAs) such as 2-amino-3-methylimidazo[4,5-f]quinoline (IQ) (1–5). IQ has been produced in cooked meats at ppb levels (6,7) and has also been detected in tobacco smoke (8). HCAs and their metabolites have been isolated from human urine (9). Human exposures to HCAs, estimated to be ∼60 ng/day (10), are modest, but are likely to be involved in cancer etiology (11,12).

IQ is a potent mutagen (13). It is less prevalent than 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (n class="Chemical">PhIP) (14), but it is 200-fold more mutagenic in Salmonella reversion (Ames) assays (3) and it is an order of magnitude more mutagenic than aflatoxin B1. In these assays (15–18), HCAs such as IQ are active in point and frameshift tester strains (19). In bacteria, mutations occur primarily at G:C base pairs (20,21). IQ is a potent inducer of two-base frameshifts in CG repeats. Similar levels of mutations are seen in mammalian hprt (22) and ef-2 (23) gene assays. Base-pair substitutions are the predominant mutations observed in mammalian cells (24–26). Sister chromatid exchange has been observed in rodent cells (27–29). IQ induces tumors in the organs of rodents and in the livers of monkeys (30–33). Liver, forestomach and lung tumors have been observed in IQ treated mice (34), whereas liver, intestine, zymbal gland, clitoral gland, skin (35), mammary glands, liver and ear ducts tumors have been observed in exposed rats (36). The TD50 values for IQ are 0.7 and 14.7 mg/kg/day in rats and mice, respectively (37). Human exposures to HCAs have been associated with pancreatic (38), colon (39), prostate (40) and breast cancers (41,42).

The genotoxicity of IQ derives primarily from its oxidation by CYP P450 1A2 to an n class="Chemical">N-hydroxylamine (43–47) although extra-hepatic CYP P450s oxidize HCAs with lower efficiencies (Scheme 1) (48). The N-hydroxylamine is acetylated by N-acetyl transferases, particularly NAT2 (49–51). In humans, the NAT2 fast acetylator polymorphism correlates with increased genotoxicity and cancer (52–54). The nitrenium ion resulting from solvolysis of N-acetoxy-IQ is the ultimate electrophile (26,48). It reacts predominately at the C8 atom of guanine, while a minor alkylation product is formed at the N2 atom of guanine (55–57). In addition, IQ can be converted to a reactive and genotoxic N-nitrosamine, which shows similar regioselectivity for DNA alkylation (58,59).

Scheme 1.

The bioactivation of IQ. Cytochrome P450-mediated N-hydroxylation followed by N-acetylation of the hydroxylamine and deacetylation forms an electrophilic aryl nitrenium ion. The aryl nitrenium ion alkylates guanine in DNA either via the IQ amine nitrogen to form the C8-dG-IQ adduct, or alternatively, alkylates DNA via the C5 position of the IQ ring to form the N2-dG-IQ adduct.

The bioactivation of IQ. Cytochrome P450-mediated N-hydroxylation followed by n class="Chemical">N-acetylation of the hydroxylamine and deacetylation forms an electrophilic aryl nitrenium ion. The aryl nitrenium ion alkylates guanine in DNA either via the IQ amine nitrogen to form the C8-dG-IQ adduct, or alternatively, alkylates DNA via the C5 position of the IQ ring to form the N2-dG-IQ adduct.

Levels of C8 and N2-dG-IQ adducts have been measured in n class="Species">rat and primate tissues using mass spectrometry (60,61) and 32P post-labeling methodology. Turesky et al. (62) have monitored C8-dG-IQ adduct formation in human hepatocytes using tandem liquid chromatography-electrospray ionization mass spectrometry. Levels range from 7 to 26 adducts per 107 bases. While less abundant, the N2-dG-IQ adduct is more persistent than is the C8-dG-IQ adduct, suggesting that it is repaired less efficiently (63). The N2-dG-IQ adduct may therefore play a significant role in the genotoxicity of IQ.

We have synthesized phosphoramidite reagents of the C8- and n class="Chemical">N2-dG-IQ adducts in which the Buchwald–Hartwig palladium-catalyzed N-arylation was the key C-N bonding-forming step (64–67). These adducts have been site-specifically incorporated into the NarI restriction sequence, 5′-d(CG1G2CX3CC)-3′, using automated solid-phase synthesis. The G3 position is a hot spot for two-base frameshift deletions in bacterial mutagenesis assays, while the G1 position is not (68–71). In addition, human DNA polymerase (hpol) η produces two-base deletions when replicating past the N2-dG-IQ adduct at position G3, in vitro (72). Thus, this sequence provides a platform for investigating sequence-specific conformational perturbation of DNA structure by IQ adducts, in relationship to their biological processing (68–71,73,74). Previously, we determined the conformation of the C8-dG-IQ adduct at the G3 position of the NarI sequence, which exhibited a base-displaced intercalated conformation (75).

Presently, we have determined the conformation of the N2-dG-IQ adduct in 5′-d(C1T2C3G4G5C6X7n class="CellLine">C8C9A10T11C12)-3′:5′-d(G13A14T15G16G17C18G19C20C21G22A23G24)-3′; X = N2-dG-IQ adduct (Chart 1). This duplex contains the recognition sequence of the NarI restriction endonuclease, in which the G3 nucleotide (X3 in the NarI sequence and X7 in this study) represents a hot spot for two-base deletions. The IQ moiety intercalates, with the IQ H4a and CH3 protons facing the minor groove, and the IQ H7a, H8a and H9a protons facing the major groove. The adducted nucleotide maintains the anti-conformation about the glycosyl bond. The complementary dC is extruded into the major groove. Nevertheless, the duplex maintains its thermal stability. This is attributed, in part, to stacking between the IQ moiety and the 5′- and 3′-neighboring base pairs. The base-displaced intercalated conformation of the N2-dG-IQ adduct differs from that of the C8-dG-IQ adduct, and may also be compared with that of the N2-dG AAF adduct (76), providing insight as to the persistence of the N2-dG-IQ adduct (63) and its processing during replication and repair events.

Chart 1.

(A) Structure of IQ. (B) Structure of the N2-dG-IQ adduct, showing the numbering of guanine base and IQ protons. (C) Structure of the C8-dG-IQ adduct. (D) Structure of the N2-dG-AAF adduct. (E) The duplex containing the NarI sequence, showing the numbering of the nucleotides. The N2-dG-IQ adduct is positioned at X7, which corresponds to the G3 frameshift-prone position of the NarI sequence.

(A) Structure of IQ. (B) Structure of the N2-dG-IQ adduct, showing the numbering of n class="Chemical">guanine base and IQ protons. (C) Structure of the C8-dG-IQ adduct. (D) Structure of the N2-dG-AAF adduct. (E) The duplex containing the NarI sequence, showing the numbering of the nucleotides. The N2-dG-IQ adduct is positioned at X7, which corresponds to the G3 frameshift-prone position of the NarI sequence.

MATERIALS AND METHODS

Sample preparation

The N2-dG-IQ-adducted n class="Chemical">oligodeoxynucleotide 5′-d(CTCGGCXCCATC)-3′ was synthesized as described (67). The complement strand 5′-d(GATGGCGCCGAG)-3′ was synthesized by Midland Certified Reagents Co. (Midland, TX, USA) and purified by anion exchange chromatography. HPLC chromatographic utilized a Supelcosil LS-18-DB analytical base-deactivated C-18 column (Sigma-Aldrich Inc., St. Louis, MO, USA), using a gradient from 5% to 12% acetonitrile in ammonium formate buffer (pH 7), over 35 min, or a gradient from 5% to 30% acetonitrile in NaH2PO4 buffer (pH 5), over 25 min. The oligodeoxynucleotides were characterized by negative mode MALDI-TOF spectrometry in a hydroxypicolinic acid matrix. The oligodeoxynucleotides were annealed at 1:1 ratio at room temperature in 180 µl buffer containing 10 mM NaH2PO4, 100 mM NaCl and 5 µM Na2EDTA (pH 7).

Thermal melting experiments

UV melting temperatures were collected on Cary 100 Bio UV spectrometer using 0.5 OD of duplex in 1 ml of solution containing 0.1 M n class="Chemical">NaCl, 10 mM NaH2PO4 and 0.05 mM Na2EDTA (pH 7.0). The temperature was increased from 25 to 75°C at a rate of 1°C per min.

NMR spectroscopy

The N2-dG-IQ modified and the unmodified duplexes were prepared at concentn class="Species">rations of 570 and 810 µM, respectively, and placed into 3 mm diameter micro NMR sample tubes (77). The samples were prepared in 0.1 M NaCl, 50 µM Na2EDTA and 10 mM NaH2PO4 (pH 7.0). To observe non-exchangeable protons, the samples were exchanged with D2O. 1H NMR spectra were recorded at 600 or 800 MHz. The spectra were collected at 15°C; NOESY experiments were conducted at mixing times of 150, 200 and 250 ms with a relaxation delay of 1.8 s. Additional experiments were conducted with a longer relaxation delay to evaluate NOE distances arising from the adenine H2 protons, which typically exhibit longer T1 relaxation values. The data were collected with 512 points in the t1 dimension and 2048 points in the t2 dimension. Chemical shifts were referenced to water. For the observation of exchangeable protons, the samples were dissolved in 9:1 H2O:D2O. 1H NMR spectra were recorded at 600 or 800 MHz at 15°C. The data were collected with 512 points in the t1 dimension and 2048 points in the t2 dimension. A mixing time of 250 ms was used. Water suppression was performed using the WATERGATE pulse sequence (78). The spectra were processed using the TOPSPIN software (Bruker Biospin Inc., Billerica, MA, USA).

NMR experimental restraints

The spectral data were evaluated using the program SPARKY (79). The intensities of NOE cross peaks were measured by volume integn class="Species">rations. The bounds for overlapped peaks were optimized manually. Noise was assigned half the intensity of the weakest peak, and motion was assumed to be isotropic. Experimental intensities were combined with intensities obtained from complete relaxation matrix analysis (CORMA) of starting model to generate a hybrid intensity matrix (80,81). The intensities were converted to distances with the program MARDIGRAS (82), which refined the hybrid intensity matrix. Calculations were performed using 150, 200 and 250 ms mixing time data and 2, 3 and 4 ns isotropic correlation times. Evaluation of the resulting distance data allowed creation of upper and lower bound distance restraints that were used in restrained molecular dynamics (rMD) calculations.

Restrained molecular dynamics calculations

An unmodified B-DNA model (83) was used as a starting structure. The n class="Chemical">guanine at position G7 was replaced by the N2-dG-IQ adduct using the program INSIGHT II (Accelrys Inc., San Diego, CA, USA). Partial charges for the N2-dG-IQ adduct were calculated with the B3LYP/6-31 G* basis set in GAUSSIAN (84). The starting structure was energy minimized for 1000 cycles. Simulated annealing protocols (85) used for the rMD calculations were conducted with the parm99 force field (86), using the program AMBER (87). Force constants of 32 kcal/mol/Å2 were applied for distance and torsion angle restraints. The generalized Born model (88) was used for solvation. The salt concentration was 0.1 M. The molecule was coupled to the bath to control the temperature during simulated annealing (89). First, calculations were performed for 20 ps (20 000 steps) and recording data every ps by the following protocol: during steps 0–1000, the system was heated from 0 to 600 K with a coupling of 0.5 ps. During steps 1001–2000, the system was kept at 600 K. The system was then cooled from 600 to 100 K during steps 2001–18 000 with a coupling of 4 ps. Further cooling from 100 to 0 K occurred during steps 18 001–20 000 with a coupling of 1 ps. After initial cycles of refinement a longer 100 ps (100 000 steps) calculation was performed by the following protocol: During steps 0–5000, the system was heated from 0 to 600 K with a coupling of 0.5 ps. During steps 5001–10 000, the system was kept at 600 K. The system was cooled from 600 to 100 K during steps 10 001–90 000 with a coupling of 4 ps. Additional cooling from 100 to 0 K occurred during steps 90 001–100 000 with a coupling of 1 ps. Structure coordinates were saved after each cycle and were subjected to potential energy minimization. CORMA (80,81) was used to compare intensities calculated from these emergent structures with the distance restraints. Helicoidal analysis was performed using the CURVES+ web server (90,91).

RESULTS

Oligodeoxynucleotide containing the N-dG-IQ adduct

The N2-dG-IQ adduct was incorpon class="Species">rated into 5′-d(CTCGGCXCCATC)-3′ using automated solid-phase synthesis (67). The position of the N2-dG-IQ adduct was located at position X7, corresponding to position G3 in the NarI sequence. The modified oligodeoxynucleotide was purified by C18 reverse phase HPLC and characterized by MALDI-TOF mass spectrometry in negative ion mode [m/z 3777.7, calcd for (M – H), 3776.6]. Thermal melting (Tm) profiles of 0.5 A260 units of the IQ-modified duplex were monitored at 100 mM NaCl (1 ml volume) as a function of temperature by absorbance at 260 nm. An unmodified duplex was evaluated under the same conditions to provide a basis of comparison. The Tm of the modified duplex was 63°C, within experimental error of the unmodified duplex (Supplementary Figure S1). Thus, the N2-dG-IQ adduct did not reduce the stability of this oligodeoxynucleotide. This result differed from our previous report, which had indicated that this adduct destabilized this duplex (67). Subsequent analysis of the previous sample by mass spectrometry revealed that the complement strand was not correct, accounting for the discrepancy. Table 1 lists the correct Tm values of the N2-dG-IQ adduct at the three positions of the NarI sequence.

Table 1.

Thermal melting temperatures (Tm measurements) of NarI duplexes containing the N2-dG-IQ adducts

NarI N2-dG-IQ modified duplex	Tm (°C)	ΔTm* (°C)
5′-CTCXGCGCCATC-3′	62	−1
3′-GAGCCGCGGTAG-5′
5′-CTCGXCGCCATC-3′	64	+1
3′-GAGCCGCGGTAG-5′
5′-CTCGGCXCCATC-3′	63	0
3′-GAGCCGCGGTAG-5′

Thermal melting temperatures (Tm measurements) of n class="Chemical">NarI duplexes containing the N2-dG-IQ adducts

NMR

The modified duplex yielded well-resolved NMR spectra with narrow line shapes for the non-exchangeable protons at 15°C. The best spectral quality for the exchangeable protons was obtained at 5°C.

Non-exchangeable DNA protons

The base aromatic and deoxyribose anomeric protons were assigned using established procedures (Figure 1) (92,93). The intensity of the X7 H8 to X7 H1′ n class="Chemical">NOE was not changed in the presence of the adduct, indicating minimal change in the conformation of the glycosyl torsion angle. In the complementary strand, the intensity of the NOE between C18 H1′ and G19 H8 was weakened. The N2-dG-IQ adduct did not induce breaks in the sequential pattern of NOEs between the aromatic base protons and the anomeric protons. With the exception of the adduct site, the internucleotide NOEs were characteristic of a B-type duplex. The adenine H2 protons were assigned based upon NOEs to the thymine imino protons of the respective A:T base pairs. With the deoxyribose H1′ assignments in hand, the remainder of the deoxyribose protons was assigned from a combination of NOESY and COSY data. The assignments of the non-exchangeable DNA protons are summarized in Supplementary Table S1.

Figure 1.

Expanded plot of the 250 ms NOESY spectrum showing NOEs between the base aromatic and deoxyribose anomeric protons of the N2-dG-IQ modified duplex. (A) Bases C1 to C12 of the modified strand. (B) Bases G13 to G24 of the complementary strand. The spectrum was acquired at 800 MHz at 15°C.

Expanded plot of the 250 ms NOESY spectrum showing n class="Chemical">NOEs between the base aromatic and deoxyribose anomeric protons of the N2-dG-IQ modified duplex. (A) Bases C1 to C12 of the modified strand. (B) Bases G13 to G24 of the complementary strand. The spectrum was acquired at 800 MHz at 15°C.

Exchangeable DNA protons

The imino and amino proton regions of the NOESY spectrum are shown in Figure 2. The assignments were made using established methods (94). The n class="Chemical">N2-dG-IQ adduct perturbed Watson–Crick hydrogen bonding. At the X7:C18 base pair, the X7 imino proton resonance was broadened, probably due to an enhanced rate of exchange with water. The amino protons for C18 were not detected. No NOE was observed between the X7 and G19 imino protons, perhaps due to the broadening of the X7 imino proton. The chemical shifts of the X7 and G17 imino protons were almost isochronous. It was not possible to determine if a NOE between these two protons existed. All other base pairs were assigned, with the exception of the two terminal base pairs C1:G24 and C12:G13. The imino protons from the terminal base pairs were exchange broadened. Overall, the data suggested that the duplex maintained Watson–Crick hydrogen bonding, with the exception of the modified base pair (Figure 2B). The assignments of the exchangeable protons are summarized in Supplementary Table S2.

Figure 2.

Expanded plots of the NOESY spectrum, showing the NOEs between the exchangeable imino and amino protons of the N2-dG-IQ-modified duplex. (A) Sequential NOE connectivity for the imino protons of base pairs T2:A23 to T11:A14. (B) NOE connectivity within Watson–Crick base pairs and between the imino protons and the amino protons. The lettered cross-peaks are assigned as follows: a′, X7 N2H →C6 N4Hb; a, X7 N2H →C6 N4Ha; b′, G17 N1H→C8 N4Ha; b, G17 N1H→C8 N4Hb; c′, G19 N1H→C6 N4Hb; c, G19 N1H→C6 N4Ha; d′, G16 N1H→C9 N4Hb; d, G16 N1H→C9 N4Ha; e′, G22 N1H→C3 N4Hb; e, G22 N1H→C3 N4Ha; f′, G5 N1H→C20 N4Hb; f, G5 N1H→C20 N4Ha; g′, G4 N1H→C21 N4Hb; g, G4 N1H→C21 N4Ha; h, T15 N3H→A10 H2; i, T11 N3H→A14 H2; j, T2 N3H→A23 H2; 1, X7 N2H→IQ H4a; 2, IQ H7a→X7 N2H; 3, X19 N1H→IQ H4a. The spectrum was collected at 800 MHz at 5°C.

Expanded plots of the NOESY spectrum, showing the n class="Chemical">NOEs between the exchangeable imino and amino protons of the N2-dG-IQ-modified duplex. (A) Sequential NOE connectivity for the imino protons of base pairs T2:A23 to T11:A14. (B) NOE connectivity within Watson–Crick base pairs and between the imino protons and the amino protons. The lettered cross-peaks are assigned as follows: a′, X7 N2H →C6 N4Hb; a, X7 N2H →C6 N4Ha; b′, G17 N1H→C8 N4Ha; b, G17 N1H→C8 N4Hb; c′, G19 N1H→C6 N4Hb; c, G19 N1H→C6 N4Ha; d′, G16 N1H→C9 N4Hb; d, G16 N1H→C9 N4Ha; e′, G22 N1H→C3 N4Hb; e, G22 N1H→C3 N4Ha; f′, G5 N1H→C20 N4Hb; f, G5 N1H→C20 N4Ha; g′, G4 N1H→C21 N4Hb; g, G4 N1H→C21 N4Ha; h, T15 N3H→A10 H2; i, T11 N3H→A14 H2; j, T2 N3H→A23 H2; 1, X7 N2H→IQ H4a; 2, IQ H7a→X7 N2H; 3, X19 N1H→IQ H4a. The spectrum was collected at 800 MHz at 5°C.

IQ protons

The IQ protons, consisting of the CH3 group, the H4a proton, and the H7a, H8a and H9a spin system, were assigned from a combination of COSY and NOESY data (Figure 3). The CH3 resonance was observed at 3.57 ppm. It displayed an intense n class="Chemical">NOE to the H4a proton, whose resonance was observed at 8.55 ppm. A coupling between the H8a proton (δ 6.55 ppm) and the H9a proton (δ 7.65 ppm) was observed in the COSY spectrum. The H8a proton also exhibited an NOE to the H7a proton (δ 7.6 ppm). The 3J coupling between H8 and H7a exhibited weak intensity in the COSY spectrum. This was attributed to presence of the nitrogen atom in the ring, which broadened the H7a resonance. This effect was also observed for the C8-dG-IQ adduct, for which the COSY cross-peak between H7a and H8a was only observed between 25 and 35°C (95). The IQ amine proton was not assigned.

Figure 3.

Expanded tile plot of the 250 ms NOESY spectrum showing the assignments of the IQ ring protons. (A) The IQ H4a proton was observed at 8.55 ppm. (B) The IQ H7a proton was observed at 7.65 ppm. (C) The IQ H9a proton was observed at 7.6 ppm. (D) The IQ H8a proton was observed at 6.55 ppm. (E) The IQ CH3 protons were observed at 3.57 ppm. The spectrum was collected at 800 MHz at 15°C.

Chemical shift perturbations

The N2-dG-IQ adduct resulted in localized chemical shift perturbations, involving the modified base pair X7:n class="Gene">C18 and the neighboring C6:G19 and C8:G17 base pairs (Figure 4). At the modified X7:C18 base pair, the X7 H8 resonance shifted 0.4 ppm downfield relative to the G7 H8 resonance in the unmodified duplex. In contrast, the C18 H6 and C18 H1′ resonances shifted 1 and 0.8 ppm downfield, respectively. At the 5′-neighbor C6:G19 base pair, the C6 H6 resonance shifted upfield by 0.2 ppm, whereas the C6 H1′ resonance shifted downfield by 0.4 ppm. The G19 H8 and H1′ resonances each shifted upfield by 0.4 ppm. At the 3′-neighbor C8:G17 base pair, the C8 H6 resonance shifted downfield by 0.3 ppm, whereas the C8 H1′ resonance shifted upfield by 0.2 ppm. The G17 H8 resonance shifted downfield by 0.1 ppm. The G17 H1′ resonance showed negligible chemical shift perturbation. The resonances for the remaining base pairs in the duplex also showed negligible chemical shift perturbations. In the imino proton region of the spectrum, the X7 and G17 N1H imino resonances, at 11.57 and 11.59 ppm, respectively, exhibited upfield chemical shifts of >1 ppm from those of the unmodified duplex, at 13.24 and 13.16 ppm, respectively. The X7 N2H amine resonance was observed at 9.5 ppm.

Figure 4.

Chemical shift perturbations of the deoxyribose H1′ protons (gray) and the pyrimidine H6 or purine H8 aromatic protons (black), for the N2-dG-IQ modified duplex. (A) Nucleotides C1–C12 in the modified strand. (B) Nucleotides G13–G24 in the complementary strand. The Δδ (ppm) values were calculated as δmodified duplex–δunmodified duplex. Positive Δδvalues represent upfield chemical shift perturbations. Negative Δδvalues represent downfield chemical shift perturbations.

Chemical shift perturbations of the deoxyribose H1′ protons (gray) and the n class="Chemical">pyrimidine H6 or purine H8 aromatic protons (black), for the N2-dG-IQ modified duplex. (A) Nucleotides C1–C12 in the modified strand. (B) Nucleotides G13–G24 in the complementary strand. The Δδ (ppm) values were calculated as δmodified duplex–δunmodified duplex. Positive Δδvalues represent upfield chemical shift perturbations. Negative Δδvalues represent downfield chemical shift perturbations.

NOEs between IQ and DNA

The CH3, H4a, H7a and H8a protons of IQ exhibited NOEs to the C8, G17, n class="Gene">C18 and G19 bases (Table 2). The pattern of NOEs involving H9a was difficult to establish due to resonance overlap with G17. The CH3 group showed medium strength NOEs to X7 H1′ and C8 H6 and weak NOEs to C8 H1′ and C8 H5. It also showed an NOE to the G19 N1H imino proton. The H4a proton showed a strong NOE to X7 H1′, and medium NOEs to C8 H5, the X7 N2H amine proton and the G19 N1H imino proton and weak NOEs to X7 H2′ and X7 H2′′ The H7a proton exhibited weak NOEs to G17 H1′, and the X7 N2H amine proton. Three NOEs from X7 to C18 were observed; these were of medium strength NOE between H9a and C18 H1′, and H8a and medium strength between C18 H2′ and H2′′. The H8a proton showed a medium strength NOE to G19 H3′ and weak NOEs to G17 H1′ and G19 H8. Some 30% of the NOEs from the IQ ring were to protons in the complementary strand, whereas another 46% were to other IQ protons and protons of the modified base. The remaining 24% of the NOEs were to neighbor bases in the modified strand.

Table 2.

Summary of NOEs observed between N2-dG-IQ(X7) adduct protons and oligodeoxynucleotide protons and their intensities

IQ proton	NOEs to oligodeoxynucleotide protons
CH3	X7 H1′: medium; C8 H6: medium; C8 H1′: weak; C8 H5: weak
H4a	X7 H1′: strong; C8 H5: medium; X7 H2′: weak; X7 H2′′: weak; X7 N2H: medium; G19 N1H: medium
H7a	G17 H1′: weak; X7 H2: weak
H8a	G19 H3′: medium; C18 H2′: medium; C18 H2′′: medium; G17 H1′: weak; G19 H8: weak; G17 H2′′: weak
H9a	C18 H1′: medium

Summary of NOEs observed between n class="Chemical">N2-dG-IQ(X7) adduct protons and oligodeoxynucleotide protons and their intensities

Conformational Refinement

After the unmodified duplex was constructed using B-DNA coordinates (83), the guanine at position G7 was replaced by the N2-dG-IQ adduct. The partial charges for the N2-dG-IQ adduct are provided in Supplementary Figure S2. Potential energy minimization provided an energy minimized starting duplex. A total of 329 distance restraints consisting of 127 inter- and 202 intra-nucleotide distances (Table 3) were obtained using the program MARDIGRAS (81,82), from 15°C NOESY data. Similar distance restraints were obtained if the data were collected at 150, 200 or 250 ms mixing times. These restraints included 16 DNA-IQ distances. A total of 49 Watson–Crick hydrogen-bonding restraints were applied for all of the base pairs except for the modified X7:C18 base pair. An additional 100 phosphodiester backbone and 20 deoxyribose pseudorotation restraints for base pairs not proximal to the site of modification were obtained from canonical values derived from B-DNA (83), consistent with the spectroscopic data indicating that the duplex maintained a B-DNA like structure. A series of rMD calculations were performed using a simulated annealing protocol in which the generalized Born solvation model (88) was used, with a salt concentration of 0.1 M. The emergent structures were subjected to potential energy minimization before further analysis, which involved a 100 ps rMD calculation using the protocol described above, again followed by potential energy minimization.

Table 3.

NMR restraints used for the N2-dG-IQ structure calculations and refinement statistics

NOE restraints
Internucleotide	127
Intranucleotide	202
Total	329
Backbone torsion angle restraints	100
H-bonding restraints	49
Deoxyribose restraints	20
Total number of restraints	498
Refinement statistics
Number of distance restraint violations	56
Number of torsion restraint violations	50
Total distance penalty/maximum penalty (kcal/mol)	2.3/0.187
Total torsion penalty/maximum penalty (kcal/mol)	2.8/0.177
r.m.s. distances (Å)	0.012
r.m.s. angles (°)	2.5
Distance restraint force field (kcal/mol/Å2)	32
Torsion restraint force field (kcal/mol/deg2)	32

NMR restraints used for the n class="Chemical">N2-dG-IQ structure calculations and refinement statistics

The pairwise rmsd analysis of structures emergent from the rMD calculations was used to measure the precision of the structural refinement. Ten structures were chosen based on the lowest deviations from the experimental distance and dihedral restraints (Figure 5). These exhibited an rmsd of 0.012 Å in distances and 2.5° in torsion angles (Table 3). There were 56 distance violations with a maximum penalty of 0.187 kcal/mol and a total distance penalty of 2.3 kcal/mol. There were 50 torsion angle violations with a maximum penalty of 0.177 kcal/mol and a total torsion angle penalty of 2.8 kcal/mol. The maximum pairwise rmsd distances were 1.12 Å. These structures were averaged and subjected to potential energy minimization.

Figure 5.

Superposition of ten potential energy minimized structures emergent from the rMD calculations of the N2-dG-IQ modified duplex, using distance restraints from the 250 ms NOESY data. The positions of the modified X7 nucleotide and the C18 nucleotide in the complementary strand are as indicated. The maximum pairwise rmsd between these 10 structures was 1.12 Å.

Superposition of ten potential energy minimized structures emergent from the rMD calculations of the N2-dG-IQ modified duplex, using distance restraints from the 250 ms n class="Chemical">NOESY data. The positions of the modified X7 nucleotide and the C18 nucleotide in the complementary strand are as indicated. The maximum pairwise rmsd between these 10 structures was 1.12 Å.

The accuracy of the refined structures was assessed by complete relaxation matrix analyses (80,81), which compared intensities calculated from the refined structures with the distance restraints (Figure 6). The sixth root residual R1x value of the average structure was 8.4%, and the individual values for intra-nucleotide restraints (8.5%) and inter-nucleotide restraints (8.3%) were of similar magnitudes. This indicated agreement with the NOE data. n class="Chemical">Nucleotide G19 exhibited a greater R1x value of 17.1%, suggesting that it was not as well-refined. This was attributed to several NOEs involving G19 being overlapped with other resonances. The structural statistics are summarized in Table 4.

Figure 6.

Sixth root residuals (R1x) calculated using complete relaxation matrix calculations from the average of 10 structures emergent from the rMD calculations of the N2-dG-IQ modified duplex. The black bars represent intra-nucleotide sixth root residuals, and the gray bars represent inter-nucleotide sixth root residuals. (A) Nucleotides C1–C12 in the modified strand. (B) Nucleotides G13–G24 in the complementary strand.

Table 4.

Structural statistics for the N2-dG-IQ modified duplex

Average structure (obtained from 10 structures)
RMS pairwise difference between structures		1.12
RMS difference from average structure		0.75
	CORMA analysis for average structurea
	Intranucleotide	Internucleotide	Total
R1xb	0.085	0.083	0.084
Average errorc			0.0037

aThe mixing time was 250 ms.

bR1x is the 6th root R factor: .

cAverage error: , where the Ic are NOE intensities calculated from the refined structure, the Io are experimental NOE intensities.

cAverage error: , where the Ic are NOE intensities calculated from the refined structure, the Io are experimental n class="Chemical">NOE intensities.

Conformation of the N2-dG-IQ Adduct

The modified nucleotide (X7) remained in the anti-conformation about the glycosyl bond. It was displaced toward the major groove. The IQ ring was intercalated and oriented such that the H4a proton and the CH3 group faced into the minor groove, whereas the H7a, H8a and H9a protons faced into the major groove (Figure 7). The IQ ring was angled by ∼15° with respect to the modified guanine, but otherwise remained largely in plane with the damaged base. The helix was unwound between C6 and X7, with a reduced helicoidal twist of 30°. This was partially compensated by an increased twist of 9° between X7 and C8. At base pair X7:n class="Gene">C18, the roll of the X7 purine decreased by 24°. This was compensated at base pair C8:G17, where the roll decreased by 12°. Consequently, the N2-dG-IQ adduct induced a bend of 10° to the duplex. The IQ ring exhibited stacking with the flanking base pairs (Figure 8). IQ was stacked between G17 and G19 of the complementary strand of the C6:G19 and C8:G17 base pairs. The complementary nucleotide, C18, extruded into the major groove and did not exhibit stacking with the neighboring base pairs. The base opening between X7 and C18 increased by 76°. This disrupted Watson–Crick hydrogen bonding. The other base pairs maintained Watson–Crick hydrogen bonding.

Figure 7.

Expanded view of the average structure calculated from 10 structures emergent from the rMD calculations of the N2-dG-IQ(X7) modified duplex, showing base pairs C6:G19, X7:C18 and C8:G17. The view is from the major groove. The modified base pair X7:C18 is shown in magenta, with the IQ moiety shown in blue.

Figure 8.

Expanded views of the average structure calculated from 10 structures emergent from the rMD calculations of the N2-dG-IQ(X7) modified duplex. Base stacking of the modified X7:C18 base pair with the 5′-neighbor and 3′-neighbor base pairs. (A) Stacking of C6:G19 above X7:C18. (B) Stacking of X7:C18 above C8:G17. Expanded views of the average structure of the corresponding C8-dG-IQ adduct at X7. (75) (C) Stacking of C6:G19 above X7:C18. (D) Stacking of X7:C18 above C8:G17. In each instance, the modified base pair X7:C18 is shown in magenta, with the IQ moiety shown in blue.

Expanded view of the average structure calculated from 10 structures emergent from the rMD calculations of the N2-dG-IQ(X7) modified duplex, showing base pairs C6:G19, X7:n class="Gene">C18 and C8:G17. The view is from the major groove. The modified base pair X7:C18 is shown in magenta, with the IQ moiety shown in blue.

Expanded views of the average structure calculated from 10 structures emergent from the rMD calculations of the N2-dG-IQ(X7) modified duplex. Base stacking of the modified X7:n class="Gene">C18 base pair with the 5′-neighbor and 3′-neighbor base pairs. (A) Stacking of C6:G19 above X7:C18. (B) Stacking of X7:C18 above C8:G17. Expanded views of the average structure of the corresponding C8-dG-IQ adduct at X7. (75) (C) Stacking of C6:G19 above X7:C18. (D) Stacking of X7:C18 above C8:G17. In each instance, the modified base pair X7:C18 is shown in magenta, with the IQ moiety shown in blue.

DISCUSSION

The N2-dG-IQ DNA adduct has been of interest following reports that it is more persistent than the C8-dG-IQ adduct in rodents and primates that were fed IQ in their diet (63). The synthesis of this adduct into oligodeoxynucleotides (67) has allowed the conformation of the N2-dG-IQ adduct at the G3 position of this sequence to be determined. This is a hot spot for two-base frameshift deletions in bacterial mutagenesis assays (68–71,73,74). In addition, human DNA polymerase (hpol) η produces two-base deletions when replicating past the N2-dG-IQ adduct at the reiterated G3 position of the NarI sequence, in vitro (72).

Conformation of the N2-dG-IQ adduct

The IQ ring intercalates when the N2-dG-IQ adduct is positioned at the frameshift-prone G3 position of the n class="Chemical">NarI sequence (Figure 7). The strong NOE intensities of the IQ H4a and CH3 protons to the X7 and C8 H1′ protons (Table 1) indicate that these protons face into the minor groove and establish the conformation about the bond between N2-dG and C5 of the IQ moiety. In contrast, NOEs involving the H8a proton of the IQ ring are primarily to bases G17, C18 and G19 of the complementary strand (Table 1). The chemical shifts of the IQ H7a, H8a and H9a protons are observed between 6.5 and 8.0 ppm, which is 1.3–2.0 ppm upfield as compared to the N2-IQ-dG nucleoside. This is consistent with the intercalated conformation and stacking of the IQ ring below the 5′-neighboring G19 of the complementary strand and above the 3′-neighboring C8:G17 base pair (Figure 8). Chemical shift perturbations corroborate the NOE data (Figure 4). The IQ H4a proton resonance, observed at 9.6 ppm, is 0.4 ppm upfield from the resonance observed for the modified N2-dG-IQ nucleoside (67), consistent with its location below G19 and above C8 (Figure 8). The IQ moiety displaces the complementary C18 base from the duplex, and flips it into the major groove. This is supported by smaller perturbations in chemical shifts for the H4a and CH3 protons as compared to the H7a, H8a and H9a aromatic protons of IQ. The displacement of the modified nucleotide X7 toward the major groove (Figure 8) is supported by the downfield chemical shift change of 0.4 ppm for the X7 H8 and H1′ protons of the modified base. The C8 H6 proton resonance also experiences a downfield shift of 0.3 ppm. The stacking interactions of the IQ ring with the flanking bases C8, G17 and G19 are reflected in the thermodynamic analysis of the adduct, in which the thermal melting temperature of 63°C is unchanged from that of the unmodified duplex.

Comparison to the N2-acetylaminofluorene-dG adduct

The other N2-dG n class="Chemical">arylamine adduct that has been subjected to conformational analysis, although not in the NarI sequence of interest herein, is that arising from N-acetylaminofluorene (AAF; Chart 1) (76). The N2-dG-AAF adduct conformation has also been examined using computational approaches (96). Zaliznyak et al. (76) have shown that the AAF moiety resides in the minor grove with its long axis directed toward the 5′-end of the modified strand. This shields the hydrophobic AAF ring from water. Similar to the N2-dG-IQ adduct, the modified nucleotide maintains the anti-conformation about the glycosyl bond. Notably, the N2-dG-AAF adduct increases the stability of the DNA, which has been attributed to a favorable entropic effect (76). The present data reveal that the base-displaced intercalated conformation of the N2-dG-IQ adduct at position G3 of the NarI sequence differs from that of the N2-dG-AAF adduct, suggesting that the conformations of N2-dG arylamine adducts vary rather than following a common motif. At the molecular level, the factors governing whether planar aromatic molecules such as AAF or IQ favor DNA groove binding versus intercalation are not well established, but may be influenced both by their electronic structures and their respective geometries (97). Replication bypass studies have revealed that the N2-dG-AAF adduct largely blocked DNA synthesis, but with some bypass and mis-incorporation of dATP opposite the lesion (98).

Comparison to the C8-dG-IQ adduct

When the C8-dG-IQ adduct was placed into the n class="Chemical">NarI sequence at the frameshift-prone G3 position, the IQ ring also intercalated into the duplex and the complementary C18 base was extruded into the major groove. The conformation of the C8-dG-IQ adduct also was characterized as base-displaced intercalated (75). Thus, at the G3 position within the NarI sequence, both the C8-dG-IQ and N2-dG-IQ adducts share a motif in which the IQ ring intercalates and C18 is extruded into the major groove. However, the two conformations are distinctive. Apart from the difference in the regiochemistry of alkylation (C8 versus N2; Scheme 1), a major difference between the C8-dG-IQ and N2-dG-IQ adducts is that the C8-dG-IQ-modified guanine adopts a syn conformation about the glycosyl bond, whereas the N2-dG-IQ-modified guanine maintains the anti-conformation about the glycosyl bond (Figures 8 and 9). In addition, for the C8-dG-IQ adduct, rotation of the glycosyl bond into the syn conformation places the Watson−Crick hydrogen bonding edge of the modified dG into the major groove. The X7 imino and amino protons are exposed to solvent. For the C8-dG-IQ adduct the orientation of the IQ ring with respect to the base is opposite to that of the N2-IQ adduct, such that the IQ CH3 group and H4a and H5a protons face the major groove rather than the minor groove (75). The orientation of the C8-dG-IQ adduct in the duplex rotates the bulk of the IQ aromatic ring away from the flanking bases, resulting in a loss of base-stacking interactions, as shown in Figure 8. In comparison, the N-dG-IQ adduct appears to have more favorable stacking interactions with G19. These differences may lead to differential processing during both DNA repair and DNA replication.

Structure–activity relationships

The N2-dG-IQ adduct is less efficiently removed from genomic DNA by nucleotide excision repair (63,99). The NER machinery is thought to recognize bulky DNA damage that is destabilizing and distortive to the duplex (100–103). It has been proposed that the thermal stabilization of the N2-dG-AAF adduct hinders NER (76). We observe that the Tm of the N2-dG-IQ adduct at position G3 within the NarI sequence does not destabilize the duplex (Table 1), correcting our original report (67). The Tm of the N2-dG-IQ modified duplex is 63°C, and does not differ significantly from the unmodified duplex. This is remarkable given that the intercalated IQ moiety disrupts Watson–Crick hydrogen bonding and that the complementary C18 base is displaced into the major groove. The stability of the N2-dG-IQ modified duplex likely arises from favorable stacking between the IQ moiety and the neighboring base pairs (Figure 8). It is also interesting to note that unlike the N-dG-IQ adduct, the C8-dG-IQ adduct, which does not stack with the neighboring bases as well at this position (Figure 8) thermally destabilizes the duplex, reducing the Tm by 4°C. Yeo et al. (104) examined AAF and AF C8-dG adducts within the NarI sequence and observed a correlation between the degree of destabilization induced by the lesions, binding affinities to the damage recognition protein XPC-RAD23B and overall NER efficiencies. Likewise, Zaliznyak et al. (76) attributed the increased stability of the N2-dG-AAF adduct to its orientation within the minor groove and the entropy-favored release of waters from the duplex. Similar conclusions were reached by Cai et al. (105) who correlated thermodynamic stabilities and van der Waals interaction energies with repair efficiencies for stereoisomeric intercalated N6-dA PAH adducts. Their studies showed that intercalated adducts with fewer DNA structural distortions and increased van der Waals interactions with neighboring bases correlated with reduced repair efficiencies. The HCA PhIP adduct has been compared with the cis-B[a]P-N2-dG adduct in duplex DNA and in a nucleotide deletion duplex, and it was concluded that local stabilization induced by these adducts governs the ability of the β-hairpins of NER proteins to recognize the damage (106). In summary, it seems plausible that the thermal stability of the N2-dG-IQ adduct may, in part, explain the persistence of the N2-dG-IQ adduct in rats and primates.

If not repaired, the N2-dG-IQ adduct is anticipated to be genotoxic. Indeed, IQ is an order of magnitude more mutagenic than is n class="Chemical">aflatoxin B1 in Ames assays. The mutations occur primarily at G:C base pairs (20,21). The replication of the N2-dG-IQ-adduct within the NarI sequence is influenced by the identity of the DNA polymerase. Because the damaged guanine remains in the anti-conformation about the glycosyl bond (Figures 7 and 8), one might anticipate that the N2-dG-IQ lesion should block Watson–Crick base pairing with incoming dNTPs during lesion bypass. Stover et al. (107) incorporated the N2-dG-IQ-adduct into the G1 and G3 positions of the NarI sequence and examined replication of the oligodeoxynucleotides with Escherichia coli polymerases (pol) I (exonuclease deficient Klenow fragment), exonuclease deficient pol II and the Solfolobus solfataricus P2 DNA polymerase IV (Dpo4), in vitro. At the G3 position, the N2-dG-IQ adduct blocked the E. coli polymerases. Pol II exo− favored correct incorporation of dCTP over dGTP but was unable to extend either of these initial insertion products. In contrast, the Dpo4 polymerase bypassed the N2-dG-IQ adduct and produced an error-free product. The present studies do not necessarily predict the structure of the N2-dG-IQ adduct during trans-lesion bypass. Consequently, it will be of interest to prepare complexes of bypass polymerases with N2-dG-IQ modified template:primers in an effort to determine how the N2-dG-IQ adduct is accommodated during lesion bypass and how polymerases, e.g. the Dpo4 polymerase (108) allow bypass of this lesion.

Bypass of the N2-dG-IQ adduct has been reported to be dependent upon its position in the n class="Chemical">NarI sequence. Choi et al. (72) have demonstrated that the human DNA polymerase (hpol) η can extend primers beyond template N2-dG-IQ adducts. Pol η correctly inserts dCTP and incorrectly inserts dATP. Analyses of hpol η extension products reveal that a −2 bp deletion occurs with the G3 N2-dG-IQ adduct. In contrast, at the G1 position replication past the N2-dG-IQ adduct results in error-free incorporation of dCTP, but further extension is inhibited and the polymerase stalls. In contrast, hpol η does not yield −2 bp deletions with the C8-dG-IQ adduct located at position G3. While further studies will be necessary to probe the basis for these observations, it is of interest to note that the stability of the N2-dG-IQ adduct placed opposite a 2-bp deletion increases as compared to the fully complementary duplex, suggesting that the adduct may stabilize a 2-bp strand slippage intermediate (67). At the G1 position, the N2-dG-IQ adduct is bypassed and extended by the E. coli polymerases and the Dpo4 polymerase, and error-free product is observed. Thus, it will also be of interest to examine the structure of the N2-dG-IQ adduct when positioned at position G1 of the NarI sequence.

Summary

Analysis of the N2-dG-IQ adduct placed at position G3 of the n class="Chemical">NarI sequence (68–71,73,74), where it has been observed to cause −2 bp deletions when bypassed by hpol η (72), reveals that it adopts a base-displaced intercalated conformation in which the H4a and CH3 protons of the IQ ring face the minor groove and the H7a, H8a and H9a protons face the major groove. The IQ ring is shielded from water and stacks with the 5′- and 3′-neighbor base pairs. Remarkably, despite this conformational perturbation, the N2-dG-IQ adduct does not destabilize the duplex, which may correlate with the observation that it is refractory to repair by NER (63,99). In addition, the IQ moiety disrupts the potential for Watson–Crick hydrogen bonding with incoming dNTPs, which perhaps explains why this lesion blocks DNA synthesis by many polymerases.

ACCESSION NUMBERS

The structural coordinates were deposited in the Protein Data Bank (www.rcsb.org): The PDB ID code for the n class="Chemical">N2-dG-IQ duplex is 2MAV.

SUPPLEMENTARY DATA

Supplementary Data are available at NAR Online.

FUNDING

National Institutes of Health (n class="Chemical">NIH) [R01 CA55678 to M.P.S., R01 ES016561 to C.J.R.]; Center [P30 ES000267, P30 CA068485]; instrumentation [S10 RR05805, S10 RR025677]; National Science Foundation Instrumentation [DBI 0922862] and the latter funded by the American Recovery and Reinvestment Act of 2009 (Public Law 111-5); Vanderbilt University assisted with the purchase of NMR instrumentation; K.M.S. and E.K.H. acknowledge support from National Institutes of Health predoctoral traineeship [T32 ES007028]. Funding for open access charge: NIH.

Conflict of interest statement. None declared.