Solution Structure of an Oligodeoxynucleotide Containing the Human

114

Chem. Res. Toxicol. 1996, 9, 114-125

Solution Structure of an Oligodeoxynucleotide Containing the Human N-ras Codon 12 Sequence Refined from 1H NMR Using Molecular Dynamics Restrained by Nuclear Overhauser Effects Irene S. Zegar and Michael P. Stone* Center in Molecular Toxicology and Department of Chemistry, Vanderbilt University, Nashville, Tennessee 37235 Received May 4, 1995X

The structure of d(GGCAGGTGGTG)‚d(CACCACCTGCC), consisting of codons 11, 12 (underlined), and 13 of the human n-ras protooncogene, was refined from 1H NMR data. Patterns of internucleotide NOEs consistent with a B-form helix were observed for each strand. NOE intensities between purine H8 and H1′ protons were small compared to intensities between cytosine H5 and H6 protons, indicative of glycosyl torsion angles in the anti range. Crosspeaks were observed between purine H8 and pyrimidine H5 and CH3 protons on adjacent bases in the direction of purine(5′f3′)pyrimidine, but not in the direction pyrimidine(5′f3′)purine. Watson-Crick hydrogen bonding between bases was intact. A total of 232 experimental distance restraints were obtained. Of these, 143 were intra-residue restraints and 89 were inter-residue restraints. A restrained molecular dynamics/simulated annealing approach yielded 6 MD structures calculated from a B-form starting structure and 6 MD structures from an A-form starting structure. These refined to an average pairwise rms difference of 0.92 Å, with maximum pairwise rmsd of 1.35 Å. Accuracy of the emergent structures was assessed by relaxation matrix back-calculation. The sixth-root residual index of 7.0 × 10-2 measured between the refined structures and the NOE intensity data suggested that the former were in reasonable agreement with the NOE data. The refined solution structures were in the B-family. Similar to the human n-ras codon 61 sequence [Feng, B., & Stone, M. P. (1995) Chem. Res. Toxicol. 8, 821-832], the ras12 sequence contained local variations in B-like conformation which did not confer large structural alterations upon the duplex, but perhaps modulated the reactivity of the first as compared to the second guanine in codon 12.

Introduction Oncogenes have been found in a number of human tumors (1-4); they represent one group of genetic elements sensitive to environmental genotoxins and implicated in human carcinogenesis. Many are members of the ras family (reviewed in refs 5 and 6). These genes are activated by single base pair substitutions, resulting in the alteration of a number of codons including codon 12 (Gly) of the p21 protein (7-10). Activation depends on the identity of the amino acids encoded by a particular DNA sequence and their functional role in the resulting gene product or the altered gene product. This process is anticipated to be modulated by various factors, including the propensity toward DNA adduct formation and repair (11-16) at specific sites. Evidence which suggests that chemical carcinogens induce mutations, and thereby directly activate protooncogenes such as ras, comes from comparison of mutations in carcinogen-induced tumors within a number of tissues to a known or suspected spectrum of adducts induced by specific carcinogens (13, 17-20). Alternatively, tumors might arise from cells with preexisting oncogenic mutations, and not be adduct induced, as proposed for mammary tumors involving the Hras1 gene in rodents thought to have been NMU-induced (21). The latter observation * Author to whom correspondence should be addressed. (615) 3222589; (615) 322-3141 (FAX); [email protected] (Internet). X Abstract published in Advance ACS Abstracts, December 1, 1995.

0893-228x/96/2709-0114$12.00/0

suggests that exposures to chemical mutagens result in secondary mutations or promote tumorigenesis in some other manner. The multistep progression model of adult human tumorigenesis proposes a progressive accumulation of mutations at specific loci within the genome. Statistically, the seemingly low probability that the necessary series of independent mutations would occur within a single cell seems inconsistent with this model (22). This paradox might be explained if the probability of mutations is greater for specific DNA sequences, due to increased probabilities of damage (23-33), or decreased probabilities of repair (13-16), or the existence of mutator phenotypes (34). Substantial evidence supports the notion that DNA adduction by a number of electrophilic species is sequencespecific, with certain nucleotides representing “hot-spots” for adduct formation. The chemical basis for site-specific modulation of reactivity in differing DNA sequences remains poorly understood. The propensity toward DNA adduction by reactive electrophiles could be modulated by sequence-specific changes in DNA conformation (35, 36), electrostatic potential (37, 38), or induced conformational changes of the DNA duplex upon binding of reactive electrophiles (39-42). A combination of these effects could well be involved, depending upon the specific electrophile. Among the polycyclic aromatic hydrocarbons (PAH),1 a distinct bonding preference of benzo[a]© 1996 American Chemical Society

Structure of the N-ras12 Oligodeoxynucleotide Chart 1. The ras12 Oligodeoxynucleotide

pyrene diol epoxide to guanine-rich regions has been documented (23-27, 43, 44). Comparative footprinting analysis of benzo[a]pyrene diol epoxide bonding selectivity revealed a preference for the clustered guanines in and adjacent to the ras codon 12 sequence (28). Aflatoxin B1 also exhibited sequence-dependent effects in chemical reactivity, with runs of guanines being particularly susceptible toward adduction (29-33). Equilibrium conformational studies of oligodeoxynucleotides in solution probe for the existence of sequencespecific modulations of DNA duplex structure. These might modulate adduct formation. Ultimately, detailed structural information can be applied toward the understanding of adduct-induced changes, which may induce mutations during subsequent DNA replication or repair events (45, 46). Interproton distance restraints obtained by relaxation matrix analysis (RMA) of 1H NOEs from high resolution NMR data allow the determination of the three-dimensional solution structure. RMA allows emergent solution structures from restrained molecular dynamics calculations to be assessed as to accuracy, by comparison of calculated with experimentally determined NOE intensities (47-53). In the first phase of an investigation into the specific effects of adduct formation at the exocyclic amino groups of guanine in the human n-ras codon 12 sequence, the ras12 oligodeoxynucleotide d(GGCAGGTGGTG)‚d(CACCACCTGCC)2 was constructed (Chart 1). A combination of NOESY and TOCSY spectra were used to assign the 1 H resonances. The solution structure was refined using restrained molecular dynamics and simulated annealing protocols, based upon NOE-derived distances. The emergent structures refined to an average pairwise rms difference of 0.92 Å, with maximum pairwise rmsd of 1.35 Å, and were compared to the NOE intensity data using complete RMA calculations. The structure which emerges from the calculations is in the B-family. The data predict that the ras12 sequence contains local variations in B-like conformation which are associated with 3 purine-pyrimidine-purine (R-Y-R) steps in the coding strand. These structural alterations in the codon 12 sequence remain within the range of conformations expected for B-like duplexes, but might play a role in modulating the reactivity of the first as compared to the second guanine in codon 12. 1 Abbreviations: NOE, nuclear Overhauser enhancement; NOESY, two-dimensional NOE nuclear Overhauser enhancement experiment; ppm, parts per million; DMT, dimethoxytrityl; DSS, sodium 4,4dimethyl-4-silapentanesulfonate; MD, molecular dynamics; MNDO, modified neglect of diatomic overlap; PAH, polycyclic aromatic hydrocarbon; PEM, potential energy minimization; RMA, relaxation matrix analysis; rMD, restrained molecular dynamics; rmsd, root mean square deviation; SCF, static consistent field; TPPI, time proportional phase increment; TOCSY, total homonuclear correlated spectroscopy; 1D, onedimensional; 2D, two-dimensional. 2 The oligonucleotides discussed in this paper do not have terminal phosphate groupsswe abbreviate the nomenclature for oligonucleotides by leaving out the phosphodiester linkage. A, C, G, and T refer to mononucleotide units. A right superscript refers to numerical position in the oligonucleotide sequence starting from the 5′-terminus of chain A and proceeding to the 3′-terminus of chain A and then from the 5′terminus of chain B to the 3′-terminus of chain B. C2, C5, C6, C8, C1′, C2′, C2′′, etc., represent specific carbon nuclei. H2, H5, H6, H8, H1′, H2′, H2′′, etc., represent the protons attached to these carbons.

Chem. Res. Toxicol., Vol. 9, No. 1, 1996 115

Materials and Methods Sample Preparation. The oligodeoxynucleotides d(GGCAGGTGGTG) and d(CACCACCTGCC) were purchased from the Midland Certified Reagent Co. (Midland, TX). The concentrations of the single stranded oligodeoxynucleotides were determined from the calculated extinction coefficients at 254 nm, 1.09 × 105 and 9.24 × 104 M-1 cm-1, respectively (54). Equal molar amounts were mixed in 10 mM NaH2PO4, 0.1 M NaCl, and 50 µM Na2EDTA at pH 7.4. The annealed duplex was eluted from a hydroxylapatite column using sodium phosphate and desalted by passing through Bio-Gel P-2. All samples were prepared in 0.1 M NaCl, 0.01 M NaH2PO4, and 0.05 mM Na2EDTA (pH 7.4). Nuclear Magnetic Resonance. Samples were repeatedly lyophilized and dissolved in 0.5 mL of 99.996% D2O, giving a 2 mM solution. For assignments of water-exchangeable protons, the samples were dissolved in a 9:1 H2O/D2O buffer. Spectra were recorded at a 1H frequency of 500.13 MHz. The spectra were referenced to the water resonance at 4.72 ppm at 30 °C, or 4.97 ppm at 10 °C. Phase-sensitive NOESY spectra used for resonance assignment were recorded using the TPPI method for phase cycling and a NOE mixing time of 400 ms. A total of 1024 real data points were collected in the d1 dimension with 32 acquisitions per FID and a 1.5 s relaxation delay; 2048 real data points were used in the d2 dimension. The residual water resonance was saturated during the relaxation delay and the mixing period. A sine-bell apodization function with a 90° phase-shift and a skew factor of 0.7 was used in the d1 and d2 dimensions. Data were zero-filled in the d1 dimension to give a matrix of 2K × 2K real points. Phase-sensitive NOESY experiments in 9:1 H2O/D2O buffer were performed using a jump return 1-1 sequence for water suppression as the read pulse (55, 56). Convolution difference was used during processing to minimize the residual water signal (57). The NOE mixing time was 250 ms. A total of 512 data points were collected in the d1 dimension with 64 scans per FID; the relaxation delay was 1.5 s. A total of 2K data points were utilized in the d2 dimension. Phase-sensitive TOCSY spectra were recorded using a 105 ms MLEV17 (58) spin-lock at 2 G for mixing (phase cycling was done according to the TPPI method). The data were transferred to Iris 4D workstations (Silicon Graphics, Inc., Mountain View, CA) and processed using FELIX (Biosym Technologies, San Diego, CA). NMR Distance Restraints. NOESY spectra in deuteriated buffer, at mixing times of 150, 250, and 350 ms, were acquired within a single 3-day period without removing the sample from the spectrometer or changing the experimental conditions. The NOESY pulse program was modified to eliminate artifacts arising from zero-quantum coherence and zz terms observed at short mixing times. A systematically shifted composite 180° pulse was implemented within the mixing period, and composite 90° pulses were used in place of the second and third 90° pulses in the standard pulse sequence (59). Footprints in the 350 ms data were selected manually with FELIX to fit NOE cross-peaks at the contour level chosen, which showed the weak NOEs but not random noise. These footprints were applied to spectra measured at other mixing times. Cross-peak intensities were determined by volume integration of the areas under the footprints. For each of 3 mixing times, and for 4 values of τc (2, 3, 4, and 5 ns), hybrid intensity matrices were constructed. These consisted of the calculated intensities, which were combined as necessary with theoretical intensities generated from RMA of IniB when experimental intensity data were not available. IniB was used as the source of theoretical intensities due to the observation that the NMR data were consistent with a B-form helix. RMA using MARDIGRAS (51) yielded 24 sets of experimental internuclear distances.3 These were averaged, and upper and lower error bounds were calculated to provide the restraints used in subsequent MD calculations. For incompletely resolved cross-peaks, footprints were estimated, and 3 The distances calculated using MARDIGRAS were for experimentally measured intensities only; i.e., the theoretical intensities from IniB were employed only to provide a complete intensity matrix from which the distances were calculated.

116 Chem. Res. Toxicol., Vol. 9, No. 1, 1996

Zegar and Stone

appropriately larger error bounds were assigned to the resulting distances. Molecular Dynamics and Simulated Annealing. A detailed description of the refinement protocol is provided in the supporting information. The Biopolymer module of INSIGHTII was used to build the initial structures and for visualization of calculated structures. A- and B-DNA 11 base pair duplexes (60, 61) were used as initial structures for the refinements. All PEM and restrained MD calculations were performed using X-PLOR (62), derived from CHARMM (63). The empirical energy function was developed for nucleic acids and treated hydrogens explicitly (49). It consisted of the usual energy terms for bonds, bond angles, torsion angles, tetrahedral and planar geometry, hydrogen bonding, and nonbonded interactions, including van der Waals and electrostatic forces. The van der Waals term was approximated using the Lennard-Jones potential energy function. The electrostatic term used the Coulombic function and was based on a full set of charges (-1/nucleotide) and a distancedependent dielectric constant of 4. The nonbonded pair list was updated if any atom moved more than 0.5 Å, and the cutoff radius for nonbonded interactions was 11 Å. The effective energy function was comprised of two terms describing distance and dihedral restraints, which were in the form of a standard square well potential (64). Bond lengths involving hydrogens were kept fixed with the SHAKE algorithm (65) during molecular dynamics calculations. All calculations were performed in vacuo, without explicit counterions. Back-calculation of NMR data was performed using CORMA (50). The refined structures were analyzed using DIALS AND WINDOWS 1.0 (66).

Results NMR Resonance Assignments. 1H NMR spectra were obtained at temperatures from 5 to 55 °C. The assignments detailed in Table S1 in the supporting information were made from NOESY and TOCSY spectra collected at 30 °C. At this temperature, the sequential cross-peaks of the ras12 oligodeoxynucleotide were sharp and reasonably well-resolved. (a) Nonexchangeable Protons. Sequential assignments were determined in the standard manner from the spectral region that showed cross-peaks between the base aromatic protons and the sugar H1′ protons (67, 68). Expanded contour plots of this region, along with the assignment scheme, are shown in Figure 1. In both strands, the aromatic protons of the purine and pyrimidine bases resonated within the expected range of chemical shifts, ranging from 7.1 to 8.4 ppm. For each of the 3 sets of two contiguous guanine nucleotides, the H8 proton of the 5′-neighbor guanine resonated downfield as compared to that from the 3′-neighbor guanine. An overlap problem occurred only for G1 and G2, which were nearly isonchronous, located at 7.85 and 7.86 ppm, respectively. A greater resonance overlap problem was observed for the complementary strand. In this case, the aromatic protons of nucleotides C14 and T19, and of C15, C18, and C21, were nearly isonchronous. As for the three sets of contiguous guanines, the corresponding cytosine aromatic resonances exhibited a characteristic pattern, with the 5′-neighbor cytosine in each case resonating at higher field than the 3′-neighbor cytosine. For both strands, the anomeric protons resonated within the expected range of 5.4-6.4 ppm. C15 H1′ shifted upfield as compared to the other H1′ protons and was observed at 5.43 ppm. The assignments of the H2′ and H2′′ sugar protons were made from their NOEs with the H1′ sugar protons of the same residue and were based on the assumption that, for a B-like DNA, the H1′-H2′′ distances were shorter than the H1′-H2′ distances. Therefore, the stronger cross-peaks were assigned to the H2′′ protons. The assignments of the remainder of the

Figure 1. Expanded plot of a NOESY spectrum at a mixing time of 350 ms showing the sequential NOE connectivities from the aromatic to H1′ protons. (A) Sequential NOE connectivities for nucleotides G1fG11. (B) Sequential NOE connectivities for nucleotides C12fC22. The base positions are indicated at the intranucleotide aromatic to sugar H1′ NOE cross-peak.

deoxyribose sugar protons were determined from TOCSY spectra. The H5′/H5′′ resonances were not separately identified, and moreover, a number of these resonances overlapped, which made unambiguous assignments impossible. The adenine H2 protons were assigned from weak cross-peaks to the H1′ protons of the adjacent 3′nucleotides. Table S1 in the supporting information lists the chemical shifts of the nonexchangeable protons. (b) Exchangeable Protons. Assignments of the imino and amino protons were made from NOESY spectra measured at 10 °C (69). These assignments are listed in Table S2 in the supporting information. The imino proton region of the spectrum is shown in Figure 2. Three well-resolved signals were observed between 13.5 and 14.0 ppm. These were unequivocally assigned to the imino protons of T7, T10, and T19 from cross-peaks to the H2 protons of the complementary adenines A16, A13, and A4. The T10 imino resonance, which arose from the penultimate base pair of the duplex, was not observed upon raising the temperature to 30 °C. The nonhydrogen-bonded amino protons of cytosine (NH2a) were assigned from their connectivities to the H5 of cytosine. The hydrogen-bonded amino protons of cytosine (NH2b) were then assigned from their cross-peaks to (NH2a). Two distinctive resonances for the cytosine amino protons were observed in all instances. Two of the six guanines present in this duplex displayed well-resolved imino proton resonances. Assignments of the partially overlapped guanine imino resonances were made by observation of cross-peaks to the NH2b protons of the comple-

Structure of the N-ras12 Oligodeoxynucleotide

Chem. Res. Toxicol., Vol. 9, No. 1, 1996 117 Table 1. Distribution of Experimental Restraints among Nucleotide Units

Figure 2. Tile plot showing expanded regions of a NOESY spectrum at a mixing time of 350 ms. (a) The sequential NOE connectivities for the imino protons of base pairs G2‚C21fT10‚A13. (b) NOE connectivities between the imino protons of guanines and the amino protons of the complementary cytosines of ras12, where the labels shown represent the amino protons of the cytosines. Also shown are the connectivities between the thymidine imino protons and the H2 of the complementary adenines. Peaks 1-4 represent cross-peaks between the T19 imino proton and the A4 amino protons, A4 H2 and the G20 imino proton, A16 H2 and the G8 imino proton, and A13 H2 and the G9 imino protons, respectively. Peak 4 also represents a cross-peak between the A4 amino protons and the G20 imino proton.

mentary cytosines, shown in Figure 2b. The guanine amino proton resonances were not assigned due to exchange broadening. Internuclear Distances. A total of 334 experimental intensities, supplemented with calculated intensities from IniA and IniB structures, were used in MARDIGRAS calculations. Individual distances calculated by MARDIGRAS were removed if the values were calculated to be greater than 5 Å. Poor distances, usually due to errors in intensity measurements arising from spectral overlap, or from cross-peaks close to the water resonance whose intensities were altered by the water presaturation pulse used during acquisition of NOESY spectra, were removed if they were inconsistent with a reasonable structure. After extracting poor distances, the final experimental set consisted of 232 interproton distances. Of these, 143 were intranucleotide restraints and 89 were internucleotide restraints, summarized in Table 1. Excluding the terminal base pairs, an average of 21 intranucleotide, internucleotide, and empirical distances were obtained for each base pair in ras12. The actual distribution of these distances was unequal, with some base pairs being better characterized than others. This resulted primarily from NMR spectral overlap, which affected some regions of the ras12 oligodeoxynucleotide more than others. Table 1 suggests that base pairs C3‚G20, G6‚C17, and G8‚C15 were underparamatrized, as measured by total experimental restraints for each base pair. G8, C15, C18, and G20 had fewer than 4 intranucleotide restraints. Fewer than 4 internucleotide restraints were available for nucleotides T10, C14, C17, and C21. Structural Refinement. Figure 3 shows the superposition of 6 MD-generated structures based on IniA and IniB and also shows the superposition of the final structures obtained by averaging the 6 IniA- and IniBbased structures followed by PEM. The 6.5 Å rms

nucleotide

intranucleotide restraints

internucleotide restraintsa

total exptl restraints

G1 G2 C3 A4 G5 G6 T7 G8 G9 T10 G11

13 5 5 9 7 5 6 2 12 9 7

0 8 4 5 4 5 5 4 7 0 0

13 13 9 14 11 10 11 6 19 9 7

C12 A13 C14 C15 A16 C17 C18 T19 G20 C21 C22

8 9 6 1 7 6 3 7 2 9 5

0 8 1 5 8 2 8 4 3 0 0

8 17 7 6 15 8 11 11 5 9 5

a

The internucleotide NOEs are listed in the direction nfn+1.

deviation between IniA and IniB shown in Figure 4A was expected as a result of the significant conformational differences evident for these starting structures. Convergence, as illustrated by Figure 4B,C, demonstrated that the emergent structure was in the B family, indicated by rmsd comparisons of 6 Å to IniA and 1.5 Å to IniB, respectively. The average pairwise rms deviation between the structures emergent from both IniA and IniB was 0.85 Å (Figure 4D). This indicated that the experimental restraints converged well to a common structure, when starting from either IniA or IniB. A control set of calculations performed for IniA and IniB without experimental restraints did not converge. Instead, IniA remained A-DNA like and IniB remained B-DNA like, with rms deviation of 6.9 Å. This indicated that the convergence to the final structures shown in Figure 3 was driven by the experimental restraints. Relaxation Matrix Analysis. The NOE cross-peak intensities measured at a mixing time of 250 ms were compared to calculated intensities derived from RMA of the refined structures (50) using the sixth-root residual index (R1x), as shown in Table 2. The IniB structure fit the experimental NOE data much better than the IniA structure, as evidenced by comparative R1x values of 10 and 17 (×10-2), respectively. The refined structures from IniA and IniB fit the experimental data better, evidenced by overall R1x values of 7.0 × 10-2. When the overall fit was broken down into intra- and internucleotide components, the fit to the intranucleotide NOE data was superior. The intranucleotide R1x value reduced to 5.7 × 10-2, while the internucleotide NOE data exhibited a value of 9.1 × 10-2. The RMA calculations indicated reasonable agreement between the calculated structures and the data. Refined Structure. The convergence of the backbone torsion angles, intra-base-pair parameters, and interbase-pair parameters from both IniA and IniB is shown in Figures 5-7. The helicoidal data are documented in DIALS AND WINDOWS format in Figure S1 in the supporting information. (a) Backbone Torsion Angles. The calculated values for the backbone torsion angles R-ζ are shown


Figure 3. Stereo drawings of (a) 6 MD structures which emerged from calculations using IniA as the starting structure, (b) 6 MD structures which emerged from calculations using IniB as the starting structure, and (c) the superposition of the rMDA and rMDB structures. The structure drawn in bold is that of rMDB.

graphically in Figure 5. The torsion angles for β and γ converged outside the canonical range for A- or B-form

Zegar and Stone

DNA, with the β angles being consistently lower whereas the γ angles were consistently greater than expected for the canonical helices. The remaining torsion angles δ, , ζ, and R converged to values consistent with righthanded helices. The pseudorotation angle φ converged well for some nucleotides, whereas for others, the convergence was worse. In the worst instances, dispersions in the calculated values of φ as large as 90° were observed for G5 and C12. The average value of φ remained >100° at all positions with the exceptions of T10, C21, and C22, consistent with predominantly S conformation of the deoxyribose moieties. The lower calculated values for the latter three nucleotides were judged to be artifacts, since inspection of J-coupling data did not reveal evidence for large changes in pseudorotation at these nucleotides. The values of the glycosidic torsion angle χ were consistently greater than 240°, indicative of an anti conformation at each nucleotide. (b) Intra-Base-Pair Helical Parameters. The plots of calculated intra-base-pair helical parameters are shown in Figure 6. The buckle and propeller twist were less converged, as evidenced by greater dispersions of the calculated values for each (Figure S1 in the supporting information). The average values calculated for propeller twist were consistently calculated to be lower than the canonical values for A- or B-form DNA. This was believed to be a consequence of insufficient experimental restraints. In contrast, and despite incomplete convergence, average calculated values for buckle did agree with the canonical value for B-like DNA, with the exception of terminal base pairs C1‚G22 and G11‚C12. The remainder of the averaged intra-base-pair parameters from IniAand IniB-based calculations were in agreement with canonical values for B-like DNA. The IniA- and IniB-based calculations converged to slightly different averaged values, observed in the stretching, opening, propeller twisting, and buckle. These deviations result from insufficient experimental restraints and are comparable to other solution structures of DNA duplexes derived from NOE restraints alone. Table 1 suggests that base pairs C3‚G20, G6‚C17, and G8‚C15 were under-restrained, as measured by total restraints for each. Nucleotides G8, C15, C18, and G20 had fewer than 4 intranucleotide restraints. A 0.8 Å deviation in the shearing parameter at base-pairs C3‚G20 and A4‚T19 was believed to result from insufficient restraints. The terminal base pairs C1‚G22 and G11‚C12 posed particular problems. Poor convergence of y-displacement, tip, inclination, stretch, and opening and unusual values for tip, opening, stagger, and buckle were noted for C1‚G22. For G11‚C12, convergence improved, but unusual values were observed for stagger, propeller twist, and buckle. (c) Inter-Base-Pair Helical Parameters. The calculated averaged inter-base-pair helical parameters are shown in Figure 7. These converged from IniA and IniB with reasonable precision. As for the intra-base-pair parameters, the IniA- and IniB-based calculations converged to similar, but not identical averaged values. Fewer than 4 internucleotide restraints were available for nucleotides T10, C14, C17, and C21. As for the intrabase-pair parameters, the canonical values for A-form and B-form helices were in most cases similar, the exception being the rise parameter between base pairs, which fluctuated about 3.4 Å. The lower number of experimental restraints was reflected in RMA calculations (Table 2), which showed poorer agreement between



Figure 4. Graphical representation of rms differences among the MD-calculated structures and the starting structures. (A) Comparison of IniA vs IniB. (B) Comparison of IniA vs rMD. (C) Comparison of IniB vs rMD. (D) The error bars in the 〈rMD〉 vs rMD graph represent the deviation of all 10 structures calculated at each position. Table 2. Comparison of Sixth-Root Residual Indices R1x for Starting Models and Resulting MD Structuresa-c structure

intra-residue R1x (×10-2)

inter-residue R1x (×10-2)

overall R1x (×10-2)

IniA IniB rMDA rMDB rMDfinal

16 10 6.5 6.9 5.7

17 9.8 9.0 8.5 9.1

17 10 7.0 7.1 7.0

a Only the inner 9 base pairs were used in the calculations to exclude end effects. b R1x ) ∑|(ao)i1/6 - (ac)i1/6|/∑|(ao)i1/6|, where (ao) and (ac) are the intensities of observed (nonzero) and calculated NOE cross-peaks. c IniA, starting energy-minimized A-DNA; IniB, starting energy-minimized B-DNA; rMDA, average of 5 rMD structures starting from IniA; rMDB, average of 5 MD structures starting from IniB; rMDfinal, average of 10 rMD structures starting from IniA and IniB.

experiment and theory in the case of internucleotide R1x values.

Discussion Toxicological interest in the ras12 oligodeoxynucleotide results from the observation that adduct-induced mutations of codon 12 introduce functional changes into the ras protooncogene product, which result in oncogene activation (13, 18, 19). The codon 12 sequences in the ras family of protooncogenes are guanine-rich and are anticipated to represent “hot-spots” for DNA adduction by a number of reactive electrophiles (27). A propensity toward guanine adduction at codon 12 could provide a mechanism for increased rates of mutagenesis and resulting activation of ras. Theoretical studies suggested minor groove binding of PAH diol epoxides might be favored in A-like oligodeoxynucleotide conformations (35) or denatured duplexes (36). These represent large conformational changes which are readily detectable. High resolution structural refinement from NMR data should discern the presence of more subtle conformational changes which may also play a role in modulating chemical reactivity (51, 70, 71). Oligodeoxynucleotide Conformation. This work provides a first detailed view of an oligodeoxynucleotide

sequence containing the coding sequence at and adjacent to codon 12 of the n-ras gene. The results suggest that the ras12 oligodeoxynucleotide forms a B-like duplex in solution. The intensities of the NOEs between the H8/ H6 and the H2′ and H2′′ protons of the same residue and from their estimated distances are consistent with a B-like duplex (72). The greater NOE intensities of the cross-peaks between the H8/H6 of the guanine and cytosine residues and the H2′ and H2′′ of the same residue as compared to the intensities of the cross-peaks between the H8/H6 and the H1′ protons of the attached deoxyribose moieties suggest that these residues adopted the anti conformation about the glycosidic bond (73). Further support for a right-handed helix was reflected in the cross-peaks observed between purine H8 and pyrimidine H5 and CH3 protons on adjacent bases in the direction of purine(5′f3′)pyrimidine, but not in the direction pyrimidine(5′f3′)purine (74). Structural Refinement. The NOE-based structural refinement allowed us to probe for more subtle sequencespecific conformational properties of the ras12 oligodeoxynucleotide. In right-handed oligodeoxynucleotide crystal structures, base pairs typically exhibit positive propeller twisting; this allows for favorable intrastrand stacking interactions, but also leads to steric clashes between adjacent purines at purine-pyrimidine (R-Y) and pyrimidine-purine (Y-R) steps in the helix (75, 76). The ras12 sequence contains 3 R-Y-R steps in the coding strand. These are G2fC3fA4, G6fT7fG8, and G9fT10fG11. The G6fT7fG8 step is of particular interest since it involves the second and third (wobble) nucleotides of codon 12. Upon structural refinement from the NMR data using NOE-based restraints, these R-Y-R steps were reflected in the calculated roll angles, slide, and rise parameters from the refined ras12 structures (Figures 5-7). For each of the R-Y steps G2fC3, G6fT7, and G9fT10 an increased negative roll angle was observed, while for each of the Y-R steps C3fA4, T7fG8, and T10fG11 an increased positive roll angle was observed. Similar perturbations were observed in the slide parameter. For the R-Y steps, the slide parameter was consistently increased nega-


Zegar and Stone

Figure 5. Helicoidal analysis. Backbone torsion angles (R-ζ), glycosidic torsion angles (χ), and sugar pseudorotation angle (φ), plotted for every nucleotide, where 0, O, and b represent helical parameters from 〈rMDA〉, 〈rMDB〉, and rMD, respectively. The dashed line represents the expected value for canonical B-DNA, whereas the dotted line represents the expected value for canonical A-DNA.

tively, while for the Y-R steps, this parameter showed positive perturbation. Increased base-pair rise was observed for the Y-R steps C3fA4 and T7fG8. The value of the rise could not be accurately determined for the T10fG11 step, due to its position at the terminus of the molecule. These calculated perturbations relieved purine-purine clashes in the major and minor grooves, as predicted (75). We were unable to determine whether the effect of the purine-purine clashes in the ras12 oligomer were relieved by alterations in propeller twisting (75). The MD calculations consistently imparted negative propeller twist at each position of the oligodeoxynucleotide (Figure 6), a common feature of structures refined from NMR data (77-81). The large negative propeller twist is inconsistent with the NMR data, which supports a B-like structure. The trinucleotide R-Y-R repeats at G6fT7fG8, and G9fT10fG11 probably should appear as a repetitive structural unit in ras12 oligodeoxynucleotide. At the present level of refinement, reasonable agreement was observed between these sequences, although the second of the two was perturbed at the terminal base pairs as compared to the interior sequence.

The calculated sequence-specific structural features within the ras12 oligodeoxynucleotide are small as compared with transformations from B- to A- or Z-form DNA, or helix denaturation, which could be expected to modulate chemical reactivity at guanine N2 in codon 12, as observed for aflatoxin binding at guanine N7 (82). The extent to which the calculated conformational changes for the G6fT7fG8 base steps in codon 12 might alter the reactivity of nucleotide G5, as compared to G6, is unknown. Altered rise and roll angles may modulate intercalation of planar aromatic electrophiles, which in turn might modulate adduction at G5 as compared to G6. Conversely, small sequence-specific structural features may exert less influence upon chemical reactivity, due to the inherent dynamic fluctuations of B-form DNA in solution (83). Alternatively, observed differences in reactivity may be a consequence of localized induced conformational change upon carcinogen binding. The evaluation of this possibility represents a challenge, since low binding affinities of aflatoxins and PAH, combined with low aqueous solubilities, make NMR-based refinement of DNA conformation under equilibrium binding conditions problematical (84-86).



Figure 6. Helicoidal analysis. Intra-base-pair helicoidal parameters, where 0, O, and b represent helical parameters from 〈rMDA〉, 〈rMDB〉, and rMD, respectively. The dashed line represents the expected value for canonical B-DNA, whereas the dotted line represents the expected value for canonical A-DNA. A representation of each parameter is provide next to each graph.

The high temperature dynamics allowed the trajectory of the ras12 oligodeoxynucleotide to overcome local energy barriers and, hence, to sample more conformational space (49). In comparing IniB and IniA from the MD-emergent structures, there was an overall better fit for IniB, supporting the notion that the duplex is B-like. The initiation of MD calculations from IniA and IniB

monitored the precision with which the available restraints could define a single set of structures. Since the coding strand of the ras12 sequence was guanine-rich (7 of 11 nucleotides are guanine, with 6 of the guanines occurring as three sets of two contiguous guanines, and the opposite situation being the case in the complementary strand), the potential for spectral overlap was of


Zegar and Stone

Figure 7. Helicoidal analysis. Inter-base-pair helicoidal parameters, where 0, O, and b represent helical parameters from 〈rMDA〉, 〈rMDB〉, and rMD, respectively. The dashed line represents the expected value for canonical B-DNA, whereas the dotted line represents the expected value for canonical A-DNA. A representation of each parameter is provide next to each graph.

concern. The ras12 sequence refined to a similar degree of precision as attained for the ras61 sequence (87). This suggested that while spectral overlap restricted the number of restraints available for the ras12 sequence, it did not prevent reasonable convergence to a common structure. Equilibrium Structures and DNA Adduction at Codon 12. The role of the equilibrium structure of the ras12 oligodeoxynucleotide in modulating the rate at which specific electrophiles such as PAH epoxides or aflatoxin B1-8,9-exo-epoxide react at G5 or G6 in codon 12 remains to be determined. DNA adduction mechanisms may involve a first or early step for binding of the electrophile to the double helix, and a subsequent or late step in which adduction occurs to form the covalent linkage. If formation of the bound complex is rate limiting, the rate of adduct formation should be influenced by the equilibrium structure of DNA. Aflatoxin B1-8,9-exo-epoxide reacts with strong regio- and stereospecificity at guanine N7. The epoxide intercalates into B-DNA above the 5′-face of deoxyguanosine, which correctly orients the electrophile for SN2 reaction, predominantly at N7 of guanine (82, 85, 88, 89). Aflatoxin, once oriented above the 5′-face of guanine, reacts rapidly to form the adduct, presumably because the binding geometry of aflatoxin orients the exo-epoxide for facile attack at guanine N7 (89).

The precise mechanisms and the rate-limiting steps for adduction by specific PAH diol epoxides at guanine N2 are less established. The pyrenyl moieties of benzo[a]pyrene diol epoxides may first intercalate within the DNA bases, orienting the electrophilic site on BPDE in close proximity to the N2 position of guanine (39, 90). The successful synthesis of site-specifically and stereospecifically benzo[a]pyrene modified oligodeoxynucleotides from single-stranded oligomers suggests that while stacking of the electrophile between nucleotides may be important, duplexed DNA is not prerequisite (91). This contrasts with aflatoxin B1-8,9-exo-epoxide, for which duplex B-form DNA is necessary for efficient adduct formation (82). Other Refined Oligodeoxynucleotide Structures. An increasing number of oligodeoxynucleotide structures have now been refined from solution NMR data. These have variously incorporated restriction endonuclease sites (78, 92, 93), promoter regions (80, 94, 95), a conserved sequence from the HIV-1 genome (96, 97), and the octamer motif of immunoglobulin genes (98, 99). These structures exhibited sequence-dependent variations in B-form geometry, including variations in groove width (94, 99, 100), base-pair roll (99), base stacking and sugar conformations (94), intrastrand distances between sugar and aromatic protons (71), and base pair inclination, rise, and twist (101).


The present work represents the first refined structure obtained for a ras protooncogene codon 12 sequence. Several oligodeoxynucleotides containing ras protooncogene codon 61 sequences have been studied. A 15-mer DNA sequence from the mouse c-Ha-ras protooncogene, spanning the portion around codon 61, and modified either with 4-aminobiphenyl or 2-aminofluorene, was examined by NMR, but not refined (45, 46). From NOE data and observation of Watson-Crick base pairing, it was concluded that the unmodified c-Ha-ras duplex was B-like (45). A 2-aminofluorene adduct was also studied in the context of a model human c-H-ras1 protooncogene sequence, but the refined structure of the corresponding unmodified sequence was not reported (102, 103). In our laboratory, the structure of d(CGGACAAGAAG)‚d(CTTCTTGTCCG), which contained the coding sequence (underlined) at and adjacent to the human n-ras codon 61, refined to an average rms difference of 1.2 Å, using restrained molecular dynamics/simulated annealing protocols based upon NOE-derived distances (87). The calculated structures agreed well with the NOE data and demonstrated that the ras61 sequence adopted a conformation in solution which is close to that of canonical B-DNA. It was particularly pleasing that the G-rich ras12 oligodeoxynucleotide provided well-resolved spectra at 30 °C. A striking sequence similarity, the 5′-GGT-3′ repeat motif, was observed in comparing the ras12 sequence with a thrombin-binding DNA aptamer which formed a unimolecular quadruplex (104). Many G-rich sequences form unusually stable G-quartet structures (104-109). Under the conditions in which this structure of the ras12 duplex was determined, evidence of quadruplex formation, such as upfield shifts and slow solvent exchange rates for guanine N1 resonances, or sharp and wellresolved guanine amino proton resonances, was not observed. Summary. The ras12 sequence contains sequencespecific structural features related to a series of R-Y-R nucleotide steps. These represent small sequence-specific modulations of the B-like DNA duplex and could be important in modulating the reactivity of specific electrophiles in the ras codon 12 sequence. Refinement of the ras12 oligodeoxynucleotide structure will also be essential in understanding adduct-induced structural changes, which may induce mutations during DNA replication or repair (45, 46). Subsequent experiments will examine the perturbations of the solution structure of the guanine-rich ras12 oligodeoxynucleotide sequence in the presence of sequence-specific and stereospecific adducts at the exocyclic amino groups of the two guanines located in codon 12.

Acknowledgment. This research was supported by a grant from the NIH, ES-05355 (M.P.S.). Funding for the AMX-500 NMR spectrometer was supplied by a grant from the NIH shared instrumentation program, RR005805, and the Vanderbilt Center in Molecular Toxicology, ES-00267. We thank Dr. James G. Moe, Mr. Jason P. Weisenseel, and Mr. Markus Voehler for assistance with NMR spectroscopy and structural refinement. Ms. Randi Tinkham assisted with the preparation of the manuscript. Supporting Information Available: The supporting information consists of a description of the molecular dynamics protocol; details of 1H resonance assignments, and experimental distances and classes of restraints (Tables S1-S3); and DIALS

Chem. Res. Toxicol., Vol. 9, No. 1, 1996 123 AND WINDOWS output (Figure S1) (18 pages). Ordering information is given on any current masthead page.

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Solution Structure of an Oligodeoxynucleotide Containing the Human

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