Chem. Res. Toxicol. 2004, 17, 1007-1019
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Stereospecific Structural Perturbations Arising from Adenine N6 Butadiene Triol Adducts in Duplex DNA W. Keither Merritt, Tandace A. Scholdberg, Lubomir V. Nechev,† Thomas M. Harris, Constance M. Harris, R. Stephen Lloyd,‡ and Michael P. Stone* Department of Chemistry, Center in Molecular Toxicology, Vanderbilt-Ingram Cancer Center, Vanderbilt University, Nashville, Tennessee 37235 Received March 22, 2004
Butadiene is oxidized in vivo to form stereoisomeric butadiene diol epoxides (BDE). These react with adenine N6 in DNA yielding stereoisomeric N6-(2,3,4-trihydroxybutyl)-2′-deoxyadenosyl (BDT) adducts. When replicated in Escherichia coli, the (2R,3R)-N6-(2,3,4-trihydroxybutyl)-2′-deoxyadenosyl adduct yielded low levels of AfG mutations whereas the (2S,3S)-N6(2,3,4-trihydroxybutyl)-2′-deoxyadenosyl butadiene triol adduct yielded low levels of AfC mutations [Carmical, J. R., Nechev, L. V., Harris, C. M., Harris, T. M., and Lloyd, R. S. (2000) Environ. Mol. Mutagen. 35, 48-56]. Accordingly, the structure of the (2R,3R)-N6-(2,3,4-trihydroxybutyl)-2′-deoxyadenosyl adduct at position X6 in d(CGGACXAGAAG)‚d(CTTCTTGTCCG), the ras61 R,R-BDT-(61,2) adduct, was compared to the corresponding structure for the (2S,3S)N6-(2,3,4-trihydroxybutyl)-2′-deoxyadenosyl adduct in the same sequence, the ras61 S,S-BDT(61,2) adduct. Both the R,R-BDT-(61,2) and S,S-BDT-(61,2) adducts are oriented in the major groove of the DNA, accompanied by modest structural perturbations. However, structural refinement of the two adducts using a simulated annealing restrained molecular dynamics (rMD) approach suggests stereospecific differences in hydrogen bonding between the hydroxyl groups located at the β- and γ-carbons of the BDT moiety, and T17 O4 of the modified base pair X6‚T17. The rMD calculations predict hydrogen bond formation between the γ-OH and the T17 O4 in the R,R-BDT-(61,2) adduct whereas in the S,S-BDT-(61,2) adduct, hydrogen bond formation is predicted between the β-OH and the T17 O4. This difference positions the two adducts differently in the major groove. This may account for the differential mutagenicity of the two adducts and suggests that the two adducts may interact differentially with other DNA processing enzymes. With respect to mutagenesis in E. coli, the minimal perturbation of DNA induced by both major groove adducts correlates with their facile bypass by three E. coli DNA polymerases in vitro and may account for their weak mutagenicity [Carmical, J. R., Nechev, L. V., Harris, C. M., Harris, T. M., and Lloyd, R. S. (2000) Environ. Mol. Mutagen. 35, 48-56].
Introduction
Scheme 1. Reactive Metabolites of BD
1
1,3-BD (CAS RN 106-99-0) is used in the manufacture of SBR (1, 2); several billion lbs/year are produced in the United States. It is a combustion product from automobile emissions (3) and cigarette smoke (4). BD is genotoxic and is a carcinogen in rodents, particularly in mice (57) and also in rats (8). Recently, BD was classified by the United States Environmental Protection Agency as “carcinogenic to humans by inhalation” (9). The International Agency for Cancer Research lists BD as a “probable human carcinogen” (group 2A) (10-12). Chronic * To whom correspondence should be addressed. † Current address: Transgenomic, Inc., 5555 Airport Blvd. Suite 100, Boulder, CO 80301. ‡ Current address: Center for Research on Occupational and Environmental Toxicology, Oregon Health and Science University, Portland, OR 97239. 1 Abbreviations: BD, butadiene; BDE, 3,4-epoxy-1,2-butanediol; BDO, butadiene monoepoxide (1,2-epoxy-3-butene); BDO2, butadiene diepoxide (1,2:3,4-diepoxybutane); BDT, butadiene triol; CPK, CoreyPauling-Koltun space-filling models; DQF-COSY, double quantumfiltered correlation spectroscopy; HMBC, heteronuclear multiple bond correlation spectroscopy; R1x, sixth root residual; rMD, restrained molecular dynamics; rmsd, root-mean-square deviation; SBR, styrenebutadiene rubber; TOCSY, total correlation spectroscopy; TPPI, time proportional phase increment.
human exposure in the SBR industry may induce genotoxic effects (13-15) and is correlated with an increased risk for leukemia (1, 16-24). BD is epoxidized primarily by cytochrome P450 2E1, but also by cytochrome P450 2A6, to form BDOs (Scheme 1) (25, 26). These may be further oxidized by cytochrome P450 2E1 or 3A4 to form BDO2 (25, 27-31). Hydrolysis of BDO mediated by epoxide hydrolase forms 1,2-dihydroxy-3-butenes (29, 32, 33), which are metabolized by cytochrome P450 to hydroxymethylvinyl ketone (HMVK) (34). Either BDO2 or the 1,2-dihydroxy-3-butenes undergo cytochrome P450-mediated oxidation to form BDEs (29,
10.1021/tx049908j CCC: $27.50 © 2004 American Chemical Society Published on Web 07/13/2004
1008 Chem. Res. Toxicol., Vol. 17, No. 8, 2004 Scheme 2. ras61 Oligodeoxynucleotide (A) and the Chemical Structures of (B) the (2R,3R)-N6(2,3,4-Trihydroxybutyl)-2-deoxyadenosyl and (C) the (2S,3S)-N6-(2,3,4-Trihydroxybutyl)2-deoxyadenosyl Adducts and Nomenclature
32, 35). Thus, proximate electrophiles arising from BD metabolism include BDO, BDO2, and BDE, and potentially HMVK (36). Of these species, BDO2 is highly genotoxic (2, 10, 37), probably due to its potential to form DNA-DNA and DNA-protein cross-links, which were observed in mice (38, 39). The greater sensitivity of mice than rats upon exposure to BD is attributed to their efficient oxidation of BD to BDO2 (40, 41). BDO and BDE are less genotoxic (37, 42, 43); however, BDE is of particular concern because it may represent the most abundant metabolite of BD produced in humans (44). The reaction of DNA with BDE (44, 45) leads to the formation of trihydroxy (BDT) adducts. These are prevalent adducts in humans (46, 47) and also in rodents exposed to BD (44, 45, 48). While BDT adducts at N7dG predominate (44, 45), there is considerable interest in BDT adducts at other sites in DNA, particularly dA (49-51). The BDT dA-N1 adduct was identified in humans exposed occupationally to BD (52). The dA-N6 BDT adduct was identified in Chinese hamster ovary cells (53). The latter likely results from Dimroth rearrangement of the corresponding dA-N1 adduct (54, 55), as opposed to direct alkylation at dA-N6. Analogous chemistry was described for the reactions of styrene oxide with adenine N1 (56, 57). BD-derived BDT adducts form in DNA as stereoisomers because the β- and γ-carbons of the BDT moiety are chiral. Thus, the potential role of stereochemistry in modulating the biological processing of BDT adducts is of considerable interest, particularly in light of the observed stereospecific processing of adducts arising in DNA from diol epoxides of polycyclic aromatic hydrocarbons (58). Evidence that adenine N6 BDT adducts are processed stereospecifically has emerged from site specific mutagenesis experiments conducted in bacteria. Those experiments revealed that the adenine N6 R,R-BDT adduct yielded low levels of AfG transversions, whereas comparable experiments using the S,S-BDT adduct revealed low levels of AfG transitions (59).
Merritt et al.
The solution structure of the first adenine N6 BDT adduct, the S,S-BDT adduct, was reported (60). NMR spectroscopy revealed modest structural perturbations localized to the site of adduction and its nearest-neighbor base pairs. The BDT moiety oriented in the major groove. Relaxation matrix analysis suggested hydrogen bonding between the hydroxyl group located at the β-carbon of the BDT moiety and the T17 O4 of the modified base pair X6‚T17. The minimal perturbation of DNA induced by this major groove adduct correlated with its facile bypass by three Escherichia coli DNA polymerases in vitro and its weak mutagenicity (59). Overall, the structure was consistent with an emerging pattern in which major groove adenine N6 alkylation products that do not strongly perturb DNA structure are not strongly mutagenic. This work characterizes the structural perturbation to the oligodeoxynucleotide caused by the (2R,3R)-N6-(2,3,4trihydroxybutyl)-2′-deoxyadenosyl adduct and compares this adduct with the corresponding (2S,3S)-N6-(2,3,4trihydroxybutyl)-2′-deoxyadenosyl adduct (Scheme 2). NMR experiments indicate that for the R,R-BDT adduct the sugar-phosphate backbone is intact with B-like characteristics. Watson-Crick base pairing is present at the site of adduction. An anti glycosidic torsional angle is observed for all nucleotides, including the R,R-BDT adduct. Molecular dynamics calculations restrained by interproton distances obtained from 1H nuclear Overhauser effects (61) and torsion angle restraints obtained from NMR indicate that the BD moiety is located in the major groove of the DNA. These solution studies suggest the presence of a hydrogen-bonding interaction between the hydroxyl group located at the γ-carbon of the BDT moiety and the T17 O4, the nucleotide in the complementary strand of the DNA opposite the BDT adduct.
Materials and Methods Sample Preparation. The unmodified oligodeoxynucleotide was synthesized by the Midland Certified Reagent Co. (Midland, TX) and purified by anion exchange chromatography. The concentration of the unmodified oligonucleotide (62) was determined from the extinction coefficient of 9.08 × 104 cm-1 at 260 nm (63). The modified oligodeoxynucleotide was annealed with a slight excess of the complementary strand, and the resulting modified duplex was purified using hydroxylapatite. It was desalted by gel filtration. Thermal Melting Experiments. Melting was monitored using UV at 260 nm in a buffer consisting of 1 M NaCl, 10 mM NaH2PO4, and 50 µM Na2EDTA at pH 7.0. Melting temperatures were determined by measuring the absorbance as a function of temperature; the temperature range was 15-75 °C with a 1 °C/min increment. Nuclear Magnetic Resonance. The R,R-BDT-(61,2) modified duplex was at a concentration of 2 mM (0.5 mL) in 10 mM NaH2PO4, 0.1 M NaCl, and 50 µM Na2EDTA at pH 7.0. For the observation of nonexchangeable protons, the duplex was dissolved in 99.96% D2O; experiments were conducted at 25 °C. For the observation of exchangeable protons, the duplex was dissolved in 9:1 H2O/D2O; experiments were conducted at 17 °C. Chemical shifts were referenced to the water resonance. Data were processed using FELIX2000. NOESY spectra were recorded with mixing times of 150, 200, and 250 ms. For the examination of exchangeable protons, solvent suppression used WATERGATE (64). Spectra were recorded at 17 °C and 150 ms mixing time. They were recorded with 1024 real data points in the d1 dimension and 2048 real data points in the d2 dimension. A relaxation delay of 2.0 s was used. TOCSY experiments were performed with mixing times of 90 and 150 ms, utilizing the MLEV17 sequence. DQF-COSY
Adenine N6 Butadiene Triol Adducts in Duplex DNA spectra were recorded using 512 FIDs with 32 scans per spectra. The data in the d1 dimension were zero-filled to give a matrix of 1024 × 2048 real points. A skewed sine-bell square apodization function with a 90° phase shift and a skew factor of 1.0 was used in both dimensions. Structural Refinement. Classical A-DNA and B-DNA were used as reference structures to create starting structures (65). The BD adduct was constructed using the BUILDER module of INSIGHT II (Accelrys, Inc.). The reference structures were energy-minimized by the conjugate gradients algorithm for 200 iterations without experimental restraints to give starting structures IniA and IniB used for subsequent relaxation matrix analysis and MD calculations. The program X-PLOR (66) was used for potential energy minimization. The CHARMM force field was utilized. NOESY cross-peak intensities were determined by volume integration. These were combined with intensities generated from complete relaxation matrix analysis of a starting DNA structure to generate a hybrid intensity matrix (67, 68), which was optimized using MARDIGRAS (69-71). Calculations run at correlation times of 2, 3, and 4 ns for both the sugar and the base protons yielded 18 sets of distances. Average distances and standard deviations calculated from these data were used as bounds for distance restraints. These were divided into classes on the basis of confidence. rMD Calculations. Initial calculations were in vacuo. The electrostatic term used a reduced charge set of partial charges and a distance-dependent dielectric constant of 4.0. The van der Waals term was approximated using the Lennard-Jones potential energy function. The effective energy function included distance and dihedral restraints, which were in the form of square-well potentials (72). Bond lengths involving hydrogen were fixed with the SHAKE algorithm (73). Random velocities fitting a Maxwell-Boltzmann distribution at 2500 K were assigned. The temperature was controlled by coupling to a bath with a coupling constant of 0.05 ps (74). Ten randomly seeded structures were calculated from each starting structure. The calculations were carried out for 30000 steps at 2500 K, then cooled to 300 K over 5000 steps, and continued at 300 K for an additional 16000 steps. An initial force constant of 50.0 kcal mol-1 was used for class 1 distance restraints. Throughout the calculations, the force constants for classes 2, 3, 4, and 5 were set to 90, 80, 70, and 60%, respectively, of the value for class 1. The initial value of the force constant for the base pairing restraints was set at 50.0 kcal mol-1. The force constants were maintained at the initial value for the first 10 000 steps; class 1 force constants were increased to 200 kcal mol-1, and base pairing force constants were increased to 150 kcal mol-1 over the next 10000 steps. The force constants were maintained for 18000 steps, scaled down to 70 and 50 kcal mol-1 for class 1 and base pairing restraints, respectively, over 3000 steps, and remained at these values for the final 10 000 steps. Structure coordinates were archived every 0.1 ps over the final 10 ps. Structure coordinates extracted from the final 4 ps were averaged and energy-minimized for 200 iterations using the conjugate gradients algorithm. A 600 ps MD trajectory including explicit solvent and counterions was calculated using AMBER 7.0 (75). The force field (76) was modified to include the BDT-adducted base. The system was solvated with a rectangular box of TIP3P (77) waters extending approximately 10 Å from the DNA and 18 Na+ ions. The SANDER module was used. The Particle Mesh Ewald (78, 79) method approximated nonbonded interactions. The calculation began with an initial potential energy minimization of the water and counterions, followed by an initial equilibration, two sets of constant pressure equilibrations, a final equilibrium stage, and MD calculations. The restraints included NOE data that were adjusted for confidence by margin of error, phosphate backbone angle restraints, and sugar pucker restraints, as well as base-pairing restraints. Back-calculation of 1H NOE data was performed using CORMA (v. 4.0) (80). Helicodial parameters were examined using 3-DNA (81).
Chem. Res. Toxicol., Vol. 17, No. 8, 2004 1009
Figure 1. Expanded plots of a NOESY spectrum at a mixing time of 200 ms showing sequential NOE connnectivities from aromatic to anomeric protons. (A) Nucleotides C1fG11 of the modified strand of the R,R-BDT-(61,2) duplex. (B) Nucleotides C12fG22 of the R,R-BDT-(61,2) duplex.
Results Sample Properties. The purity of the R,R-BDT-(61,2) sample was assessed using capillary gel electrophoresis. The electropherogram exhibited two peaks in a 1:1 ratio after correction for the respective absorbance coefficients. The identity of the duplex was verified using MALDITOF mass spectrometry. Mass measurement showed two signals that corresponded to mass units of 3500 and 3273. These peaks corresponded to the BD adduct strand 5′d(CGGACXAGAAG)-3′ and to the complementary strand 5′-d(CTTCTTGTCCG)-3′, respectively. The melting temperature of the R,R-BDT-(61,2) duplex as determined by UV spectroscopy was 52 °C, less than the observed 57 °C melting temperature of the unmodified ras61 duplex. The R,R-BDT-(61,2) sample yielded excellent NMR data in the temperature range of 15-25 °C. DNA 1H Resonance Assignments. 1. Nonexchangeable Protons. The sequential NOEs between the aromatic and the anomeric protons of the R,R-BDT-(61,2) oligodeoxynucleotide duplex are displayed in Figure 1. These were assigned using standard methods (82, 83). There were no breaks in connectivity in either the modified strand or its complement. Overall, the 1H spectrum of the R,R-BDT-(61,2) duplex was very similar to that of the unmodified ras61 oligomer (84). This region of the ras61 duplex 1H NMR spectrum characteristically shows prominent upfield-shifted resonance positions of the G8 H1′ proton and the C5 H6 proton, which presumably arise from base stacking interactions unique to this sequence. With assignments of the sugar H1′ protons in hand, the remainder of the deoxyribose sugar protons were assigned from DQF-COSY spectra. With the exception of several of the H5′ and H5′′ protons, assignments of the sugar protons were made unequivocally. Table S1 in the Supporting Information details the complete
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Figure 2. Expanded plot of a NOESY spectrum at a mixing time of 200 ms showing NOE connectivities for the imino protons for the base pairs from G2‚C21 to A10‚T13.
nonexchangeable 1H NMR assignments of the R,R-BDT(61,2) oligodeoxynucleotide duplex. 2. Exchangeable Protons. An expanded region showing the far downfield region of the 1H NMR spectrum, exhibiting cross-peaks between the hydrogen-bonded imino protons, is given in Figure 2. Unequivocal sequential assignments of the imino protons from base pairs G2‚ C21fA10‚T13 were obtained. As compared to the unmodified ras61 duplex (84), the primary change in the 1H NMR spectrum was that the imino proton resonances arising from T16 N3H and T17 N3H were almost superimposed. The NOE between T14 N3H and T13 N3H was very weak and is not observed at the contour level plotted in Figure 2. This NOE cross-peak was also weak in the ras61 spectrum (84) and presumably reflects the effects of strand fraying and resulting rapid exchange of this proton with solvent. Sequential assignment of the amino protons from base pairs G3‚C20fA10‚T13 was obtained. Each of the peaks identified in the amino region was found to have a cross-peak with the appropriate imino proton as expected in Watson-Crick base pairing. 3. BD Protons. The six protons of the BD moiety were observed as separate resonances, with spectral line widths comparable to the oligodeoxynucleotide protons. At 200 ms of mixing time, NOE interactions were observed between all of the protons on the BD moiety as shown in Figure 3. The proton HR resonated at 2.90 ppm. It showed coupling to HR′ at 3.67 ppm and Hβ at 3.83 ppm. HR′ showed a strong NOE to Hβ but was weakly coupled to Hβ. The Hγ proton was almost superimposed with Hβ; it resonated at 3.81 ppm. It showed a strong NOE to Hδ and Hδ′ protons at 3.62 and 3.59 ppm, respectively, but the corresponding COSY cross-peaks were weak. The stereotopic assignment2 of the HR,R′ and Hδ,δ′ proton resonances were elucidated by analysis of 3J coupling constants and NOE intensities, the latter in comparison with predicted intensities generated by complete relaxation matrix analysis (67, 68). 4. BD-DNA NOEs. A tile plot showing some of the 33 NOEs observed between the R,R-BDT-(61,2) adduct 2 The definitions of the prochiral protons at C and C of the BDT R δ moiety are based upon the Cahn, Ingold, and Prelog nomenclature. The proton HR is defined as the pro-R proton at CR; HR′ is defined as the pro-S proton at CR. Likewise, Hδ is the pro-R proton at Cδ; Hδ′ is the pro-S proton at Cδ.
Figure 3. (A) Expanded NOESY spectrum at 200 ms mixing time exhibiting the assignment of BDT protons in the R,R-BDT(61,2) duplex. The experiment was at 800.23 MHz and 25 °C. (B) Expanded eCOSY spectrum exhibiting the coupling pattern BDT protons in the R,R-BDT-(61,2) duplex. The experiment was at 600.23 MHz and 25 °C.
Figure 4. Expanded tile plot showing NOE cross-peaks between BDT and DNA protons. Cross-peaks a, T17 N3HfBDT HR′; b, T16 N3HfBDT HR′; c, T17 N3HfBDT Hδ′′; d, T16 N3HfBDT Hδ′′; e, T17 N3HfBDT HR′′; f, T16 N3HfBDT HR′′; g, T17 N3HfBDT Hβ; h, C5 NH2 (hydrogen bonded)fBDT HR′; i, C5 NH2 (hydrogen bonded)fBDT Hδ′; j, C5 NH2 (hydrogen bonded)fBDT Hβ; k, X6 NHfBDT HR′; l, X6 NHfBDT Hδ′′; m, X6 NHfBDT Hδ′; n, X6 NHfBDT HR′′; o, X6 NHfBDT Hγ; p, X6 NHfBDT Hβ; q, C5 NH2 (nonhydrogen bonded)fBDT HR′; r, C5 NH2 (nonhydrogen bonded)fBDT Hδ′; s, C5 NH2 (nonhydrogen bonded)fBDT Hβ; t, T16 CH3fBDT HR′′; u, T16 CH3fBDT Hδ′; v, T16 CH3fBDT Hδ′′; w, C5 H5fBDT HR′; x, C5 H5fBDT Hδ′′; y, C5 H5fBDT Hδ′; z, C5 H5fBDT Hγ; aa, C5 H6fBDT Hδ′; bb, C5 H6fBDT Hβ; cc, X6 H8fBDT HR′′; and dd, X6 H8fBDT Hγ.
and the DNA protons is shown in Figure 4. These NOE cross-peaks occurred between the BDT moiety and the modified base pair X6‚T17, the 5′-neighbor nucleotide C5, and the 3′-neighbor base pair, A7‚T16. The HR proton exhibited intrastrand NOEs with the C5 H5, C5 NH2, and X6 N6H protons and interstrand NOEs with the T16 CH3,
Adenine N6 Butadiene Triol Adducts in Duplex DNA
Figure 5. Chemical shift differences of protons of the R,R-BDT(61,2) duplex relative to the unmodified ras61 oligodeoxynucleotide. (A) The modified strand of the R,R-BDE-Ade-(61,2) adduct. (B) The complementary strand of the R,R-BDE-Ade(61,2) adduct. Gray bars represent the deoxyribose H1′ protons; black bars represent the purine H8 or pyrimidine H6 protons, respectively.
T16 N3H, and T17 N3H protons. The HR′ proton exhibited intrastrand NOEs with the X6 H8, X6 N6H, and interstrand NOEs with the T16 N3H, T17 N3H protons. The Hβ proton exhibited intrastrand NOEs with the C5 H6, C5 NH2, and X6 N6H protons and an interstrand NOE with the T17 N3H proton. The Hγ proton showed NOEs with the C5 H5, X6 H8, and X6 N6H protons. The Hδ proton showed NOEs with C5 H5, C5 H6, T16 CH3, and the exchangeable A6 NH2, C5 NH2 protons. The Hδ′ proton exhibited cross-peaks with C5 H5, T16 CH3, and the exchangeable A6 NH2, T16 NH, and T17 NH protons. Torsion Angle Measurements. Sugar ring conformations (57) were determined graphically using the sums of 3J 1H coupling constants (58), measured from DQFCOSY spectra. Discrete J1′2′′ and J1′2′ couplings were measured from active and passive couplings, respectively, along the t1 axis of the H2′′ (t2) to H1′ (t1) spectral region. The data were fit to curves relating the coupling constants to the deoxyribose sugar pseudorotation angle (P), sugar pucker amplitude (Φ), and the percentage S type conformation. The sugar pseudorotation angle and amplitude ranges were converted to the five dihedral angles (ν0 to ν4) that encompass the sugar ring. In all cases, the experimentally determined pseudorotation angles were in the C2′-endo range of values. The glycosyl torsion angles of all nucleotides were examined using 1H NOESY experiments. These revealed weak NOEs between the purine H8 or pyrimidine H6 protons and the anomeric H1′ protons of the attached deoxyribose sugars, consistent with glycosyl torsion angles in the anti conformational range, also consistent with a B-form helix (85). Coupling constants measured from 1H-31P HMBC
Chem. Res. Toxicol., Vol. 17, No. 8, 2004 1011
Figure 6. Distribution of NOE restraints in (A) the modified strand of the R,R-BDT-(61,2) duplex and in (B) the complementary strand of the R,R-BDT-(61,2) duplex. The black bars represent intraNOEs while the gray bars represent internucleotide NOEs.
spectra were applied (86, 87) to the Karplus relationship (88) to determine the backbone dihedral angle (C4′C3′-O3′-P), related to the H3′-C3′-O3′-P angle by a 120° shift. The ζ (C3′-O3′-P-O5′) backbone angles were calculated from the correlation between and ζ in B-DNA (60). These data indicated that none of the torsion angles associated with the backbone phosphodiester linkages were perturbed by the presence of the R,R-BDT-(61,2) adduct. Chemical Shift Effects. Figure 5 shows the chemical shift differences between the unmodified ras61 duplex oligodeoxynucleotide and the R,R-BDT-(61,2) adduct. The changes in chemical shift were clustered at the four base pairs C5‚G18fG8‚C15. Of the DNA base aromatic protons, upfield shifts were observed for X6 H8 and A7 H8, on the order of 0.2 and 0.15 ppm, respectively. Changes in the chemical shifts of deoxyribose H1′ protons were observed for X6 H1′ and G8 H1′, for both of which a downfield chemical shift on the order of 0.2 ppm was observed, along with T17 H1′, for which a downfield chemical shift of approximately 0.1 ppm was observed. Both A7 H1′ and T16 H1′ exhibited upfield chemical shifts on the order of 0.2 and 0.15 ppm, respectively. There were no unusually shifted 31P resonances in the duplex. Structural Refinement. The distribution of the NOEbased distance restraints for each nucleotide is summarized in Figure 6. These were evenly distributed over the length of the oligodeoxynucleotide. In total, there were 33 restraints between the BDT protons and the DNA. The presence of the BDT moiety at nucleotide X6 resulted in a significantly greater number of NOE restraints at nucleotides C5 and X6; these served to orient the BDT moiety in the major groove. The structural
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Figure 7. Stereoview of six superimposed structures emergent from the simulated annealing rMD protocol; the structures resulted from randomly seeded calculations.
refinement was based upon 417 experimental distance restraints (these are tabulated in the Supporting Information). In addition to the NOE-based distance restraints, 110 deoxyribose pseudorotation restraints were included in the rMD calculations. The DQF-COSY data were consistent with the C2′-endo sugar ring conformation at all nucleotides (8). Therefore, all deoxyribose rings were restrained to the C2′-endo conformation. There were 71 phosphodiester backbone torsion angle restraints used in the rMD calculations. As there were no unusual chemical shifts or 3J couplings observed in the 31P resonance data, the phosphodiester backbone angles and ζ were restrained at angles of 165 ( 35° and 245 ( 35°, respectively (89). Watson-Crick hydrogen-bonding restraints were used for all base pairs as NOESY data indicated base pairing (90). Fifty empirical base pair planarity restraints were also included in the calculations. Inspection of the structures, which emerged from a series of randomly seeded rMD calculations initiated using either the B-form DNA or the A-form DNA starting structures, suggested that the γ-hydroxyl group of the BDT adduct and T17 O4 were within hydrogen-bonding distance. It was therefore decided to add a restraint for this hydrogen bond in subsequent calculations. The validity of this additional hydrogen-bonding restraint was evaluated by complete relaxation matrix calculations (68). A comparison of R1x values (R1x factors) calculated with a mixing time of 200 ms, using an isotropic molecular correlation time of 2 ns, revealed that addition of this hydrogen-bonding restraint between the γ-hydroxyl group of the BDT adduct and the T17 O4 resulted in a 10% improvement in the R1x residuals for base steps C5fX6 and X6fA7. This modest improvement in the R1x probably was a consequence of the fact that in the
absence of this restraint, the rMD calculations converged to a structure placing the γ-hydroxyl group of the BDT adduct and T17 O4 within hydrogen-bonding distance. To determine if the NOE data distinguished a potential hydrogen-bonding interaction between the γ-hydroxyl group of the BDT adduct from a hydrogen-bonding interaction between the β-hydroxyl group of the BDT adduct and the T17 O4, this exercise was repeated by implementing a hydrogen-bonding interaction between the β-hydroxyl group of the BDT adduct and the T17 O4. When this was done, the R1x at the C5fX6 base step doubled in magnitude, to approximately 16% error. Thus, the NOE data were consistent with a hydrogen-bonding interaction between the γ-hydroxyl group of the BDT and the T17 O4 but not between the β-hydroxyl group of the BDT and the T17 O4. This hydrogen bond between the γ-hydroxyl group of the BDT and the T17 O4 was not observed in the NMR spectrum, probably due to its rapid exchange with solvent. The data did suggest, however, that T17 O4 hydrogen bonded with X6 N6H, the expected WatsonCrick bond. To determine if T17 O4 might alternate between this Watson-Crick hydrogen bond and a hydrogen bond between the γ-hydroxyl group of the BDT adduct, a 600 ps MD trajectory was run using explicit solvent and counterions. The results of this MD simulation did not support the notion that the hydrogen bond involving T17 O4 underwent exchange between the X6 N6H and the BDT γ-OH group. A stereoview of six convergent structures originating from rMD calculations initiated from a B-form starting structure and incorporating the hydrogen bond between the γ-hydroxyl group of the BDT and the T17 O4 can be viewed in Figure 7. The calculations converged to similar structures irrespective of starting structure. Utilizing either A-form or B-form DNA starting structures having
Adenine N6 Butadiene Triol Adducts in Duplex DNA
Chem. Res. Toxicol., Vol. 17, No. 8, 2004 1013
Figure 8. Distribution of R1x values calculated using CORMA between nucleotide units of (A) the modified strand of the R,RBDT-(61,2) duplex and (B) the complementary strand of the R,RBDT-(61,2) duplex. The dark bars represent intranucleotide R1x values. The light bars represent internucleotide R1x values. Table 1. Analysis of the rMD-Generated Structures of the ras61 R,R-BDT-(61,2) Adduct NMR restraints total no. of distance restraints interresidue distance restraints intraresidue distance restraints DNA-BDE distance restraints
417 62 322 33
empirical restraints H-bonding restraints dihedral planarity restraints sugar pucker restraints backbone torsion angle restraints
44 20 110 71
structural statistics NMR R factor (R1x)a-c rmsd of NOE violations (Å) no. of NOE violations > 0.1 Å
9.72 × 10-2 9.38 × 10-2 3
pairwise rmsd (Å) over all atoms IniA vs IniB 5.92 IniA vs rMDAavg 6.16 IniB vs rMDBavg 1.86 rMDA vs rMDB 1.79 rMDA vs rMDA 1.57 rMDB vs rMDB 1.48 〈rMDA〉 vs rMDAavg 0.80 ( 0.25 〈rMDB〉 vs rMDBavg 0.60 ( 0.18 a The mixing time was 200 ms. b R x) ∑|(a ) 1/6 - (a ) 1/6|/ |(a ) 1/6|, 1 o i c i o i where ao and ac are the intensities of observed (nonzero) and c calculated NOE cross-preaks. 〈rMDA〉 represents a group of eight converged structures starting from IniA; 〈rMDB〉 represents a group of eight converged structures starting from IniB. rMDAavg represents the potential energy-minimized average structure of all eight rMD calculations starting with A-form DNA. rMDBavg represents the potential energy-minimized average structure of all eight rMD calculations starting with B-form DNA. The comparisons rMDA vs rMDB, rMDA vs rMDA, and rMDB vs rMDB represent the maximum observed pairwise rmsd over all atoms between these groups.
5.92 Å rmsd, the maximum pairwise rmsd between emergent structures was 1.79 Å. The emergent structures converged toward a B-like helix. The average refined structure showed 1.86 Å rmsd as compared to the B-DNA starting structure, but 6.16 Å rmsd as compared to the A-DNA starting structure.
Figure 9. Comparison of (A) the R,R-BDT-(61,2) duplex and (B) the S,S-BDT-(61,2) duplexes. Views from the major groove of X6‚T17 and its flanking base pairs C5‚G18 and A7‚T16. In each instance, the duplex DNA is shown in blue, the BDT moiety is in red, and the BDT protons are in white. The R,R-BDT-(61,2) adduct induces AfG transitions. The S,S-BDT-(61,2) adduct induces AfC transversions.
The accuracies of the refined structures were evaluated using complete relaxation matrix back-calculations and comparison of the resulting theoretical cross-peak intensities with the NOE data. These yielded R1x values of 9.72 × 10-2 for intranucleotide NOEs and 9.38 × 10-2 for internucleotide NOEs. This sixth root index indicated slightly better agreement with intraresidue NOEs than it did with the interresidue NOEs. Figure 8 shows R1x values as a function of position in the R,R-BDT-(61,2) oligodeoxynucleotide. The residual values were consistent over the length of the modified oligodeoxynucleotide and on the order of 0.10-0.15 at the site of the R,R-BDT(61,2) adduct. Table 1 details the structural refinement parameters. Comparison of the R,R-BDT-(61,2) and S,S-BDT(61,2) Adducts. The prominent difference between the two stereoisomers involved the orientation of the BDT moiety in the major groove (Figure 9). For the R,R-BDT(61,2) adduct, the BDT moiety was oriented in plane with the modified base pair X6‚T17. In contrast, the BDT moiety of the S,S-BDT-(61,2) adduct was tilted out of the X6‚T17 base pairing plane, toward the 3′-direction from the modified nucleotide X6. This was attributed to differential interactions of T17 O4 with the hydroxyl groups of the BDT moiety in the two stereoisomeric adducts. As
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Figure 10. Base stacking orientations of the R,R-BDT-(61,2) duplex and the S,S-BDT-(61,2) duplexes at the lesion site as predicted by rMD calculations, as compared to the unmodified ras61 oligodeoxynucleotide duplex. (A) The R,R-BDT-(61,2) duplex detailing base stacking of the X6 and A7 base pairs. (B) The R,R-BDT-(61,2) duplex detailing base stacking of the C5 and X6 base pairs. (C) The modified S,S-BDT-(61,2) duplex detailing base stacking of the X6 and A7 base pairs. (D) The modified S,S-BDT-(61,2) duplex detailing base stacking of the C5 and X6 base pairs. (E) The unmodified ras61 oligodeoxynucleotide duplex detailing base stacking of the A6 and A7 base pairs. (F) The unmodified ras61 oligodeoxynucleotide duplex detailing base stacking of the C5 and A6 base pairs.
noted above, rMD calculations predicted a hydrogenbonding interaction between the T17 O4 and the γ-OH proton in the R,R-BDT-(61,2) adduct. This hydrogenbonding interaction forced the BDT moiety to remain in plane with base pair X6‚T17. In contrast, T17 O4 interacted with the β-OH proton in the S,S-BDT-(61,2) adduct. In the S,S-BDT-(61,2) adduct, the δ-carbon of the BDT moiety was not restrained to be in plane with the modified base pair.
Except for the adducted base pair, the refined structure remained substantially as in the unmodified ras61 oligodeoxynucleotide (84). Neither stereoisomeric BDT adduct caused major changes in nearest-neighbor base stacking in duplex DNA (Figure 10), although the changes introduced by the S,S-BDT-(61,2) adduct were perhaps somewhat greater than those introduced by the R,R-BDT(61,2) adduct. The effects of differential hydrogen bonding involving T17 O4 may be observed in Figure 10. The
Adenine N6 Butadiene Triol Adducts in Duplex DNA
hydrogen bond between the BDT γ-OH group and the T17 O4 in the R,R-BDT-(61,2) adduct keeps the BDT moiety in plane with base pair X6‚T17, whereas the hydrogen bond between the BDT β-OH group and the T17 O4 in the S,S-BDT-(61,2) adduct allows the BDT moiety to orient in the 3′-direction from X6.
Discussion Several billion lbs/year of BD are produced by chemical industry in the United States, and workplace exposure to this chemical is of epidemiological concern, as BD is recognized as a human carcinogen (2, 10-12). Chronic human exposure in the SBR industry may induce genotoxic effects (13-15) and is correlated with an increased risk for leukemia (1, 16-24). Thus, delineating structureactivity relationships for DNA adducts formed by the oxidative metabolism of BD is of considerable interest. We reported the solution structure of the first oligodeoxynucleotide duplex modified site specifically and stereospecifically at adenine N6 with BDE, the S,S-BDT(61,2) oligodeoxynucleotide (60). Site specific mutagenesis studies conducted on the BDT S,S-BDT-(61,2) and R,RBDT-(61,2) adenine N6 adducts in E. coli revealed stereochemistry-dependent differences in their biological processing. The S,S-BDT-(61,2) adduct generated AfC mutations while the R,R-BDT-(61,2) adduct generated AfG mutations (59). Consequently, a comparative structural study on these two stereoisomeric adducts was of interest. Structure of the R,R-BDT-(61,2) Adenine N6 Adduct. This BDT adduct was oriented in the major groove of the DNA, with the BDT moiety being approximately in plane with the X6‚T17 base pair (Figure 9). This resulted in minimal structural perturbation to the DNA duplex (Figure 10). The NMR spectral data were consistent with this conclusion. No interruptions in DNA sequential NOE connectivities were noted (Figure 1), suggesting that the duplex was intact. The observation of all imino resonances in the spectrum (Figure 2) and their associated NOE connectivities with amino protons supported the conclusion that Watson-Crick hydrogen bonding remained intact. A total of 33 NOEs between the R,R-BDT-(61,2) adduct and the DNA firmly established the orientation of the BDT moiety (Figure 4). There were only minor chemical shift effects at the site of the lesion (Figure 5). The calculations predicted the formation of a hydrogen bond between the γ-OH of the BDT moiety and the T17 O4, which accounted for the in-plane orientation of the BDT moiety with respect to the modified base pair (Figure 10). The observation of one set of NMR resonances for the BDT moiety possessing spectral line widths comparable to that of the DNA anomeric protons (Figure 3) suggested that the correlation times of the BDT protons were similar to those of the DNA protons. This was consistent with the predictions of the rMD calculations. Comparison with the S,S-BDT-(61,2) Adduct. The major conformational difference between the R,R-BDT(61,2) and the S,S-BDT-(61,2) adducts involved the differential interactions of T17 O4 with the hydroxyl groups of the BDT moiety in the two stereoisomers. As noted above, T17 O4 was within hydrogen-bonding distance of the γ-OH group with the R,R-BDT-(61,2) adduct, whereas it was within hydrogen-bonding distance of the β-OH group with the S,S-BDT-(61,2) adduct. This stereo-
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chemical difference in hydrogen-bonding preference resulted in measurable differences in the orientation of the two adducts in the major groove. Formation of a hydrogen bond between the T17 O4 and the γ-OH group positioned the BDT moiety in-plane with the damaged base pair X6‚ T17. In contrast, formation of a hydrogen bond between the T17 O4 and the β-OH group in the S,S-BDT-(61,2) adduct allowed the BDT moiety to orient toward the 3′direction in the major groove. Stereospecific Differences in Processing. The role of stereochemistry in modulating biological processing has been examined in studies of adducts arising from polycyclic aromatic hydrocarbons, the structures of which are strongly modulated by stereochemistry (58). Mao et al. (91) observed stereoselective resistance to digestion of stereoisomeric N2-dG benzo[a]pyrene adducts by phosphodiesterases I and II. Moriya et al. demonstrated that the fidelity of translesion synthesis of such adducts depends on chirality (92), as does mutagenic potential (93). Choi et al. (94, 95) noted differences in T7 polymerase processing. Likewise, Perlow et al. (96) observed stereospecific differences for the interactions of these adducts with RNA polymerase II. Overall, these studies on PAH adducts support the notion that stereochemistry is a major determinant of biological processing by a number of enzymes (97). Consequently, the present results revealing that the R,R-BDT-(61,2) and S,S-BDT(61,2) BDT adducts orient differently in the major groove suggest their potential to exhibit differential interactions with DNA polymerases and other DNA processing enzymes. Site specific mutagenesis studies conducted in E. coli confirm this prediction. The ras61 R,R-BDT-(61,2) and S,S-BDT-(61,2) adducts were ligated into the single-stranded vector M13mp7L2 (98) that was subsequently used to transfect repair deficient AB2480 (uvrA-, recA-) and SOS proficient AB1157 E. coli. The R,R-BDT-(61,2) lesion yielded AfG transitions (0.14%) (59). The corresponding S,SBDT-(61,2) lesion at adenine N6 elicited AfC transversions, exhibiting a mutation frequency of 0.25%. Thus, while neither the R,R-BDT-(61,2) nor the S,S-BDT-(61,2) adducts were strongly mutagenic, those mutations that did occur were dependent upon adduct stereochemistry. The differential mutagenic products observed in E. coli for the R,R-BDT-(61,2) and S,S-BDT-(61,2) adducts illustrate the role of BDT adduct stereochemistry in modulating biological response to DNA damage. Although the R,R-BDT-(61,2) and S,S-BDT(61,2) adducts showed structural differences that appeared to be mirrored by their processing in E. coli, neither adduct was strongly mutagenic in the bacterial system. When ligated into 85-mer templates and examined as to translesion replication in vitro, neither the R,R-BDT-(61,2) nor the S,S-BDT-(61,2) adducts blocked translesion replication by the E. coli polymerases Klenow fragment exo-, polymerase II, or polymerase III (59). These polymerases bypassed either adduct with little or no loss of efficiency or fidelity. The observation that neither adduct induced major structural perturbations into duplex DNA appeared to correlate well with the facile bypass of the two adducts by these polymerases. The results of the translesion replication experiments suggested that DNA replication in E. coli was relatively unperturbed by these adducts and hinted that these adducts should be weakly mutagenic in the bacterial system.
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Biological Implications. With respect to human mutagenesis, the significance of stereospecific structural differences between the stereoisomeric adenine N6 BDT adducts in the ras61 sequence remains to be determined. Mutagenesis studies carried out in vivo with BD using B6C3F1 lacI transgenic mice showed point mutations at dG and dA; the adenine specific mutations were primarily AfT transversions (99-103), but AfG transitions were reported in H-ras codon 61 (104). Thus, the observation that the R,R-BDT-(61,2) adduct induced low levels of AfG transversions in E. coli (59) could be significant. However, because AfT transversions were reported to be the primary dA mutations following exposure to BD (99-103), and site specific mutagenesis studies in bacteria suggested that the R,R-BDT-(61,2) adduct induced only low levels of AfG mutations, not AfT mutations, and the S,S-BDT-(61,2) adduct likewise did not generate AfT transversions (59), there has been speculation that adenine N1 adducts may be of greater biological significance. These are believed to be progenitors of the adenine N6 adducts, via Dimroth rearrangement (54, 55). In contrast to the adenine N6 adducts, adenine N1 adducts are expected to interfere with Watson-Crick hydrogen bonding and introduce greater perturbation into duplex DNA; thus, it seems reasonable that they might be potent genotoxins. The N1 adduct can also undergo rearrangement to the N1-deoxyinosine adduct. Site specific mutagenesis studies of the N1-deoxyinosine adduct arising from BDO in the COS-7 system revealed that it was indeed highly mutagenic and yielded AfG mutations (105, 106). This observation led to a proposal (105) that the N1 adducts may represent the source of H-ras specific AfG mutations observed in vivo (104). In any event, the specific DNA adduct arising from exposure to BD and responsible for inducing AfT transversions (99-103) remains to be determined. It has also been proposed that AfT transversions might result from misincorporation of dA opposite apurinic sites formed by depurination of adenine N3 adducts (107). DNA cross-links arising from bis-alkylation of deoxyadenosine by BDO2 may also prove to be significant sources of BD-mediated genotoxicity (108). BDO2 is highly mutagenic (37, 42, 43), and BDO2-treated cells showed large deletions (109). DNA-DNA and DNAprotein cross-links were observed in mice treated with BD (38, 39), and a guanine N7-guanine N7 cross-link was isolated from salmon sperm DNA (110). Site specific mutagenesis studies have been carried out on intrastrand R,R-BDO2 and S,S-BDO2 adenine N6-adenine N6 crosslinked adducts, and these yielded high levels of mutations in the COS-7 system (105). Similarly, guanine N2guanine N2 cross-links were mutagenic and induced both single base deletions and deletions (111). Structural studies on these additional BD alkylation products of deoxyadenosine are in progress.
Acknowledgment. We acknowledge the contributions of Markus Voehler and Dr. Jaison Jacob, who assisted with NMR spectroscopy and structural refinement. Dr. Jarrod Smith assisted with structural refinement. Dr. J. R. Carmical (The University of Texas Medical Branch, Galveston) provided helpful discussions. This work was supported by NIH Grant ES-05509 (M.P.S.). Funding for the NMR spectrometers was supplied by Vanderbilt University, by NIH Grant RR-05805, and by the Vanderbilt Center in Molecular Toxicology,
Merritt et al.
ES-00267. The Vanderbilt-Ingram Cancer Center is supported by NIH Grant CA-68485. Supporting Information Available: Table S1 showing the 1H chemical shift assignments for the ras61 R,R-BDT-(61,2) adduct; Table S2 showing the NOE restraints utilized in the rMD calculations for the R,R-BDT-(61,2) adduct; and Figure S1 showing force field parameters for the R,R-BDT-(61,2) adduct. This material is available free of charge via the Internet at http://pubs.acs.org.
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