Mispairing of a Site Specific Major Groove (2S,3S)-N6-(2,3,4

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Mispairing of a Site Specific Major Groove (2S,3S)-N6-(2,3,4-Trihydroxybutyl)-2′-deoxyadenosyl DNA Adduct of Butadiene Diol Epoxide with Deoxyguanosine: Formation of a dA(anti)‚dG(anti) Pairing Interaction Tandace A. Scholdberg,§ Lubomir V. Nechev,§,† W. Keither Merritt,§ 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, and Center for Research on Occupational and Environmental Toxicology, Oregon Health and Science University, Portland, Oregon 97239 Received August 12, 2004

The (2S,3S)-N6-(2,3,4-trihydroxybutyl)-2′-deoxyadenosyl (BDT) adduct arising from alkylation of adenine N6 by butadiene diol epoxide (BDE) was placed opposite a mismatched deoxyguanosine nucleotide in the complementary strand of the oligodeoxynucleotide 5′-d(CGGACXAGAAG)-3′‚5′-d(CTTCTGGTCCG)-3′. This oligodeoxynucleotide contains codon 61 (underlined) of the human N-ras protooncogene. The BDT adduct was at the second position of codon 61, and this was named the ras61 S,S-BDT-(61,2) A‚G adduct. NMR spectroscopy revealed the presence of two conformations of the adducted mismatched duplex. In the major conformation, the mismatched base pair X6‚G17 was oriented in a “face-to-face” orientation, in which both the modified nucleotide X6 and its complement G17 were intrahelical and in the anti conformation about the glycosyl bond. Hydrogen bonding was suggested between X6 N1 and G17 N1H and between X6 N6H and G17 O6. The presence of the BDT moiety allowed formation of a stable A‚G mismatch pair. The identity of the minor conformation could not be determined. If not repaired, the resulting mismatch pair would generate AfC mutations, which have been associated with this adenine N6 BDT adduct [Carmical, J. R., Nechev, L. N., Harris, C. M., Harris, T. M., and Lloyd, R. S. (2000) Env. Mol. Mutagen. 35, 48-56].

Introduction 1,3-Butadiene (CAS RN 106-99-0) (BD)1,2 is used in the manufacture of styrene-butadiene rubber (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 (5-7) 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 (IARC) lists BD as a “probable human carcinogen” (group 2A) (10). Chronic human * To whom correspondence should be addressed. E-mail: [email protected]. telephone: 615-322-2589. § Vanderbilt University. ‡ Oregon Health and Science University. † Current address: Alnylam Pharmaceuticals, Inc., 790 Memorial Drive Suite 202, Cambridge, MA 02139, 617-252-0700. 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); DQF-COSY, double quantumfiltered correlation spectroscopy; HMBC, heteronuclear multiple bond correlation spectroscopy; R1x, sixth root residual; rMD, restrained molecular dynamics; SBR, styrene-butadiene rubber; TOCSY, total correlation spectroscopy. 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.

exposure in the SBR industry may induce genotoxic effects (11-13) and is correlated with an increased risk for leukemia (1, 14-22). BD is epoxidized primarily by cytochrome P450 2E1, but also by cytochrome P450 2A6, to form 1,2-epoxy-3butenes (BDO) (Scheme 1) (23, 24). These may be further oxidized by cytochrome P450 2E1 or 3A4 to form 1,2:3,4diepoxybutanes (BDO2) (23, 25-29). Hydrolysis of BDO mediated by epoxide hydrolase forms 1,2-dihydroxy-3butenes (27, 30, 31), which are metabolized by cytochrome P450 to hydroxymethylvinyl ketone (HMVK) (32). Either the BDO2 or the 1,2-dihydroxy-3-butenes undergo cytochrome P450-mediated oxidation to form 1,2-dihydroxy-3,4-epoxybutanes (BDE) (27, 30, 33). Thus, proximate electrophiles arising from BD metabolism include BDO, BDO2, and BDE, and potentially, HMVK (34). Of these species, BDO2 is highly genotoxic (2, 10, 35), probably due to its potential to form DNA-DNA (36) and DNA-protein cross-links, which were observed in mice (37, 38). The greater sensitivity of mice than rats upon exposure to BD is attributed to their efficient oxidation of BD to BDO2 (39, 40). BDO and BDE are less genotoxic (35, 41, 42); however, BDE is of particular concern because it may represent the most abundant metabolite of BD produced in humans (43). The reaction of DNA with BDE (43, 44) leads to the formation of trihydroxy (BDT) adducts. These represent

10.1021/tx049772p CCC: $30.25 © 2005 American Chemical Society Published on Web 01/20/2005

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Chem. Res. Toxicol., Vol. 18, No. 2, 2005 Scheme 1. Reactive Metabolites of BD

the most prevalent adducts isolated from humans (45, 46) and also from rodents exposed to BD (43, 47, 48). While BDT adducts at N7-dG predominate (43, 47), 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 dAN6. Analogous chemistry was described for the reactions of styrene oxide with adenine N1 (56, 57). Previously, the (2S,3S)-N6-(2,3,4-trihydroxybutyl)-2′deoxyadenosyl adenine N6 BDT adduct was site specifically incorporated into the ras61 oligodeoxynucleotide (58). The modified oligodeoxynucleotide, 5′-d(CGGACXAGAAG)-3′‚5′-d(CTTCTTGTCCG)-3′, contained the N6-dA BDT adduct at the second position of codon 61. This was the N6-dA S,S-BDT-(61,2) lesion. Structural refinement from NMR data demonstrated that the BDT moiety was located in the major groove and caused minimal localized structural perturbation. A series of restrained molecular dynamics (rMD) calculations suggested that the S,S-BDT-(61,2) adduct was stabilized by the formation of a hydrogen bond between the β-hydoxyl group of the BDT moiety and T17 O4 (59). The ras61 S,S-BDT-(61,2) adduct was ligated into the single-stranded vector M13mp7L2 (60, 61) that was subsequently used to transfect repair deficient AB2480 (uvrA, recA) and SOS proficient AB1157 Escherichia coli (62). The S,S-BDT-(61,2) adduct did not result in a significant block to the replication of site specifically modified bacteriophage in vivo, as evidenced by no decrease in the plaque-forming ability of phage-infected bacteria. This was consistent with the results of replication studies carried out in vitro, indicating that all three primary DNA polymerases in E. coli readily bypassed this adduct. Site specific mutagenesis (62) revealed low levels of mutations. The predominant mutations, exhibiting a mutation frequency of 0.25%, were AfC transversions. The corresponding R,R-BDT-(61,2) lesion at adenine N6 elicited a different mutational spectrum. It yielded primarily AfG transitions (0.14%) (62). Thus, while neither the S,S-BDT-(61,2) nor the R,R-BDT-(61,2) adduct was strongly mutagenic, those mutations that did occur were dependent upon adduct stereochemistry. To determine if there existed a structural basis for the low levels of AfC mutations induced by the (2S,3S)-N6(2,3,4-trihydroxybutyl)-2′-deoxyadenosyl adenine N6-BDT adduct, it was site specifically incorporated into the ras61 oligodeoxynucleotide (58), opposite a mismatched dG in the complementary strand opposite the adducted dA. The resulting modified oligodeoxynucleotide, 5′-d(CGGACXAGAAG)-3′‚5′-d(CTTCTGGTCCG)-3′, contained the mis-

Scholdberg et al. Scheme 2. A‚G Mismatched ras61 Oligodeoxynucleotide, the Chemical Structure of the (2S,3S)-N6-(2,3,4-Trihydroxybutyl)2′-deoxyadenosyl Adduct, and Nomenclature

matched N6-dA BDT adduct at the second position of codon 61. This was named the N6-dA S,S-BDT-(61,2) A‚ G adduct (Scheme 2). The key conformational feature of the S,S-BDT-A‚G oligodeoxynucleotide was the formation of an A‚G mismatched base pair in which both mismatched bases were intrahelical and in the anti conformation about the glycosyl bond and in which the BDT moiety was in the major groove.

Materials and Methods Oligodeoxynucleotide Synthesis. Unmodified oligodeoxynucleotides were synthesized by the Midland Certified Reagent Co. (Midland, TX) and purified by anion exchange chromatography. The concentration of the single-stranded unmodified oligonucleotide was determined from the extinction coefficient of 9.08 × 104 cm-1 at 260 nm (63). An excess of unmodified strand was annealed with the S,S-BDT-(61,2) modified strand (58), in a buffer consisting of 10 mM NaH2PO4, 0.1 M NaCl, and 50 µM Na2EDTA at pH 7.0. The solution was heated at 95 °C for 5 min and then allowed to cool slowly to room temperature. The mismatched duplex was eluted from DNA Grade Biogel hydroxylapatite (Bio-Rad Laboratories, Hercules, CA) with a gradient from 10 to 200 mM NaH2PO4, pH 7.0. The duplex was desalted on a Sephadex G-25 column. The sample was lyophilized. NMR Spectroscopy. The S,S-BDT-(61,2) A‚G modified duplex was prepared at a concentration of 2 mM in 0.5 mL of 10 mM NaH2PO4, 0.1 M NaCl, and 50 µM Na2EDTA at pH 7.0. For observation of nonexchangeable protons, the sample was dissolved in 99.96% D2O. For observation of exchangeable protons, the sample was dissolved in 9:1 H2O/D2O. 1H spectra were recorded at 800.23 MHz. The temperature was controlled at 20 ( 0.5 °C. Chemical shifts were referenced to the water resonance at 4.71 ppm at 20 °C. For examination of exchangeable protons, NOESY experiments were carried out using a field gradient Watergate pulse sequence (64). The spectra were recorded at 20 °C and a mixing time of 150 ms. These experiments 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. Total correlation spectroscopy (TOCSY) experiments were performed with mixing times of 90 and 150 ms, utilizing the MLEV17 sequence (65). The data in the d1 dimension were zero-filled to give a matrix of 1024 × 2048 real points. A sine-bell squared apodization with a 90° phase shift and a skew factor of 1.0 was

Formation of a dA(anti)‚dG(anti) Pairing Interaction

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used in both dimensions. Data were processed using FELIX (Accelrys, Inc., San Diego, CA) on Octane workstations (Silicon Graphics, Inc., Mountain View, CA). Molecular Modeling. B-DNA was used as a reference structure (66). The BD adduct was constructed using the BUILDER module of INSIGHT II (Accelrys, Inc.). The reference structure was energy-minimized by the conjugate gradients algorithm for 200 iterations. The program X-PLOR (67) was used for rMD and potential energy minimization calculations. The CHARMM force field was utilized (68).

Results pH Dependence of the 1H NMR Spectrum. Experiments performed using the nonadducted ras61 oligodeoxynucleotide confirmed that the conformation of the A6‚ G17 mismatch was dependent upon pH (69, 70). At pH 7, a sharp and well-resolved 1H NMR spectrum was obtained, suggestive of a single ordered conformation. At pH 5.5, the nonadducted oligodeoxynucleotide containing the A6‚G17 mismatch showed evidence of conformational disorder, possibly associated with formation of a protonated A‚G mismatch in which G17 was in the syn conformation about the glycosyl bond, observed by NMR (69, 70) and crystallographic (71, 72) analyses of oligodeoxynucleotides containing A‚G mismatches. Conformational Interconversion of the S,S-BDTA‚G Mismatch. The S,S-BDT-A‚G duplex differed from the unmodified ras61 oligodeoxynucleotide containing the A6‚G17 mismatch in that it was not possible to obtain a sharp and well-resolved 1H NMR spectrum within a reasonable range of pH. At pH 7, the 1H spectrum was similar to that observed for the corresponding unadducted ras61 oligodeoxynucleotide containing the A6‚G17 mismatch. However, as observed in Figure 1, there was spectral broadening at base pairs C5‚G18, X6‚G17, and A7‚ T16. This suggested the presence of conformational disorder induced by the presence of the N6-dA BDT moiety, at the adducted mispair and at its nearest neighbor base pairs. Several unidentified broad cross-peaks were observed, which apparently arose from the presence of a small amount of a second conformation of the adducted oligodeoxynucleotide. Assignments of Nonexchangeable Protons. Despite the presence of the conformational disorder, the spectral assignment for the major conformation of the S,S-BDT-A‚G duplex at pH 7 was accomplished using standard protocols (73, 74) and by comparison to the corresponding unadducted ras61 oligodeoxynucleotide containing A6‚G17 mismatch. Figure 1 shows the sequential NOE connectivities for the modified and complementary strands of the duplex, respectively. In the modified strand, the sequential NOE X6 H1′fA7 H8 was not observed. This NOE was also not observed for the unadducted ras61 oligodeoxynucleotide containing the A6‚ G17 mismatch. In the complementary strand, the sequential NOE G17 H1′fG18 H8 was not observed. This NOE had been observed for the unadducted ras61 oligodeoxynucleotide containing the A6‚G17 mismatch. The adenine H2 protons were identified on the basis of NOEs to the H1′ protons of the same nucleotide and on the basis of NOEs to hydrogen-bonded thymine imino protons in the complementary strand. At the adducted X6‚G17 base pair, X6 H2 resonated at 7.61 ppm. The complete assignment of the deoxyribose H2′, H2′′, H3′, and H4′ protons was achieved. Because of spectral overlap, a partial set of assignments was made for the deoxyribose H5′ and H5′′ protons.

Figure 1. Expanded plots of a NOESY spectrum of the S,SBDT-A‚G oligodeoxynucleotide at a mixing time of 250 ms showing sequential NOE connectivities from the aromatic to anomeric protons. (A) Nucleotides C1fG11. (B) Nucleotides C12fG22. The base positions are indicated at the intranucleotide cross-peak of the aromatic proton to its own anomeric proton.

Figure 2. Expanded plot of a NOESY spectrum of the S,SBDT-A‚G oligodeoxynucleotide showing the NOE connectivites of the imino protons of base-pair G2‚C21fA10‚T13.

Exchangeable Protons. An expanded contour plot of the imino resonance region of the NOESY spectrum from ∼12-15 ppm is shown in Figure 2. The imino proton resonances arising from base pairs C1‚G22, G2‚C21, G3‚ C20, and A4‚T19 were sharp. Likewise, imino proton resonances from base pairs G8‚C15, A9‚T14, and A10‚T13 were sharp. This contrasted with base pairs C5‚G18, X6‚ G17, and A7‚T16. A broad resonance was observed for T16 N3H, which was superimposed on the sharp resonance for T14 N3H. Thus, the T16 N3HfG8 N1H NOE was weak. Two weak resonances were identified for G17 N1H, suggesting slow exchange between two conformations on the NMR time scale, accompanied by rapid exchange with solvent. These were located at 12.9 and 13.2 ppm, respectively. At the 5′-neighbor C5‚G18 base pair, two

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broad resonances were observed for G18 N1H, also suggestive of slow exchange between two conformations. These observations were consistent with the notion that there was disorder at the mismatch X6‚G17 base pair and the 5′- and 3′-neighboring base pairs. The imino proton of the terminal G11‚C12 pair was not detected, presumably due to fraying at the ends of the helices (75). The assignments of the thymine imino resonances were confirmed by the identification of cross-peaks to adenosine H2 protons of each A‚T pair. At the adducted base pair, the expected NOE from G17 N1H to X6 H2 was not observed, presumably due to rapid exchange of the imino proton with water. BDT Protons. Two sets of BDT resonances were observed, indicative of slow exchange between two conformations. The BDT protons of the major conformation were assigned as follows. BDT Hβ and Hγ were superimposed at 3.81 ppm. BDT HR′ was at 3.58 ppm, and HR was at 3.12 ppm. BDT Hδ and Hδ′ were superimposed at 3.27 ppm. These assignments were consistent with the assignments of the BDT protons in the fully complementary ras61 oligodeoxynucleotide (59). The second set of BDT resonances (the minor conformation) was weaker, and the resonances could not be definitively assigned. There were no NOEs observed between the BDT moiety and the DNA. Torsion Angle Measurements. A series of 1H double quantum filtered correlation spectroscopy (DQF-COSY) and E-COSY experiments suggested that in the major conformation, each of the deoxyribose sugars in the S,SBDT-(61,2) A‚G oligodeoxynucleotide existed predominantly in the C2′-endo conformation (76). The 31P spectrum showed no unusual chemical shifts, suggesting that the phosphodiester backbone was not significantly perturbed by the presence of the S,S-BDT-A‚G moiety. This was confirmed by a 1H-31P heteronuclear multiple bond correlation spectroscopy (HMBC) experiment using an IBURP-shaped pulse (77) in the 1H dimension. The 3J couplings between H3′ and 31P were consistent with backbone torsion angles in the normal range for B type duplexes (78). The glycosyl torsion angles of all nucleotides were examined using 1H NOESY experiments (Figure 3). 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 (79). Chemical Shift Perturbations. The chemical shift differences between the S,S-BDT-A‚G oligodeoxynucleotide and the unadducted A‚G mismatch sample were compared, as detailed in Figure 4. This comparison revealed a similarity between the chemical shifts of the S,S-BDT-A‚G oligodeoxynucleotide and the unadducted A‚G oligodeoxynucleotide, indicating that the presence of the N6-dA BDT adduct did not account for significant perturbations in chemical shift and suggesting that the presence of the S,S-BDT adduct in the S,S-BDT-A‚G oligodeoxynucleotide did not substantially perturb the A‚ G mismatch in the unadducted A‚G oligodeoxynucleotide. The largest chemical shift perturbation was observed at G17, which is the site of the mismatched guanine. Only minimal perturbation was observed in the remainder of the nucleobases of the complementary strand, as well as in the ras61 strand. Small chemical shift perturbations were observed when the S,S-BDT-A‚G oligodeoxynucleotide was compared to

Scholdberg et al.

Figure 3. Comparison of intensities for the H6/H8fH1′ interactions of the aromatic and anomeric protons of the (A) modified strand and (B) complementary strand. Black bars represent intraresidue cross-peaks. Gray bars represent interresidue cross-peaks.

the unmodified ras61 oligodeoxynucleotide (80). The observed chemical shifts were clustered at the modified base pair X6‚G17, and its nearest neighbor base pairs in either direction, C5‚G18 and A7‚T16. The largest of these was on the order of 0.4 ppm and was apparent at the mismatched G17 residue. The remainder of the base pairs in the S,S-BDT-AG oligodeoxynucleotide showed minimal chemical shift perturbations as compared to the unmodified ras61 oligodeoxynucleotide. Molecular Modeling. The presence of an unidentified minor conformation of the S,S-BDT-AG oligodeoxynucleotide and the fact that no NOEs were observed between the BDT moiety and the DNA precluded structural refinement of this oligodeoxynucleotide from NMR data. However, the suggestion that the major conformation of the S,S-BDT-A‚G oligodeoxynucleotide was similar to the dA(anti)‚dG(anti) conformation of the unmodified mismatched ras61 oligodeoxynucleotide, evidenced by similar DNA NOEs and chemical shift values, led to the notion that the unmodified mismatched ras61 oligodeoxynucleotide might provide a basis for molecular modeling of the effect of the N6-dA BDT moiety on the X6‚G17 mismatch. The molecular modeling was conducted in two stages. In the first stage, the structure of the unmodified mismatched ras61 oligodeoxynucleotide was refined using a standard simulated annealing protocol, in which the molecular dynamics calculations were restrained by experimental NOEs. These calculations yielded the expected structure in which both A6 and G17 were intrahelical and in the anti conformation about the glycosyl bond as previously predicted. In the second stage of modeling, the N6-dA BDT moiety was added to the unmodified A6‚



Figure 4. Chemical shift differences of aromatic (black) and anomeric (gray) protons of the unadducted A‚G oligodeoxynucleotide relative to the S,S-BDT-A‚G oligodeoxynucleotide. (A) The modified strand of the S,S-BDT-A‚G adduct. (B) The complementary strand of the A‚G adduct.

G17 base pair. The resulting oligodeoxynucleotide containing the X6‚G17 base pair was then subjected to potential energy minimization using the conjugate gradients algorithm. The molecular modeling results shown in Figure 5, indicated that in the S,S-BDT-A‚G oligodeoxynucleotide, the N6-dA BDT moiety was positioned in the major groove with minimal disruption of the X6‚G17 mispairing interaction.

Discussion Adenine N6 BDT adducts arise from butadiene diol epoxides, the most abundant electrophiles formed from human metabolism of BD (43). They are believed to result as a consequence of Dimroth rearrangement of initially formed N1 adducts (54, 55); the BDT adenine N1 adduct has been identified in human cells (52). Site specific mutagenesis experiments carried out in E. coli revealed that the S,S-(61,2) BDT lesion was weakly mutagenic. The mutation rate was 0.25%, with AfC mutations predominating (62), suggesting that the mutations might arise from mis-incorporation of dGTP opposite this adduct during DNA replication in the bacterial system. The incorporation of the S,S-(61,2) BDT lesion opposite a dG mismatch provided a model in which to study conformational perturbations introduced into duplex DNA resulting from incorrect incorporation of dGTP opposite the S,S-(61,2) BDT lesion. N6-dA BDT Adduct Allows Formation of a dA(anti)‚dG(anti) Mismatch. Molecular modeling yielded the energy-minimized structure for the major conforma-

Figure 5. Stacking interactions of the S,S-BDT-A‚G oligodeoxynucleotide. (A) Stacking of base pair C5‚G18 (yellow; protons white) above base pair X6‚G17 (blue; BDT moiety in red; protons white). (B) Stacking of base pair X6‚G17 (blue; BDT moiety in red; protons white) above base pair A7‚T16 (green; protons white).

tion of the S,S-BDT-A‚G oligodeoxynucleotide shown in Figure 5. In this structure, both purine rings at the mismatch site were in the anti conformation at the glycosyl bond and were intrahelical. This conclusion was corroborated by the weak NOEs observed between X6 H8fX6 H1′ and G17 H8fG17 H1′. The magnitudes of these NOEs were comparable to the other nucleobase aromatic proton to anomeric proton NOEs in the S,SBDT-A‚G oligodeoxynucleotide (Figure 3). The interruption of sequential connectivities between the modified base X6fA7, as well as between the mismatched T16fG17 and G17fG18 (Figure 1), was attributed to the localized distortion in the DNA duplex necessary to accommodate the purine-purine mismatch. Similar disruptions in sequential connectivity at an A‚G mismatch have been noted (81). The small chemical shift perturbations between the unadducted ras61 oligodeoxynucleotide containing an A6‚G17 mismatch and the adducted S,S-BDTA‚G oligodeoxynucleotide were consistent with the conclusion that the BDT moiety was extruded into the major groove and the fact that no NOEs were observed between the BDT moiety and the DNA. The guanine imino N1H and carbonyl O6 groups and the adenine N1

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Scheme 3. “Face-to-Face” A‚G Mismatch Base Pairsa

a (A) The umodified A‚G mismatch. (B) The N6-dA A‚G mismatch.

and N6 NH2 groups, respectively, were within hydrogen bonding distance. Comparison with Other A‚G Mismatch Structures. The A‚G mismatch has been studied in a number of DNA sequence contexts. At neutral pH, others (8291) observed that the A‚G mismatch existed in the dA(anti)‚dG(anti) conformation as reported here; Carbonneux et al. (70) suggested the possibility of bifurcated hydrogen bonding at the mismatch site. This has also been described as a “face-to-face” mismatch (89) (Scheme 3). Below pH 7, rearrangement of the mismatched A‚G base pair into the dA(anti)‚dG(syn) conformation, allowing formation of hydrogen bonds between protonated dA N1H and dG N7 and between dA N6H and dG O6, was reported (69, 70). This low pH structure was also observed in crystals grown at acidic pH (71, 72). We note that NOESY spectra obtained at pH 5.5 on the nonadducted ras61 oligodeoxynucleotide containing an A‚G mismatch revealed broadening of the cross-peaks resulting from C5, A7, G3, and G17. This probably reflected a similar pH-dependent conformational transition (69, 70). The solution structure of the low pH form of the S,SBDT-A‚G mismatched oligodeoxynucleotide was not pursued further. A second type of A‚G mispair, termed “edgeto-edge” pairing (89), was observed for tandem A‚G mispairs in DNA (89-91). This motif was characterized by rotation of dA into the syn conformation about the glycosyl bond and formation of hydrogen bonds between N7 dA and N2 dG and between N6 dA and N3 dG. In the present instance, the possibility of edge-to-edge pairing was inconsistent with the observation that both the mismatched X6 and G17 existed in the anti conformation about the glycosyl bond. In addition, the presence of the dA N6 BDT moiety would be expected to interfere sterically with formation of an edge-to-edge base pair. Presence of a Minor Conformation of the S,SBDT-A‚G Oligodeoxynucleotide. It was unfortunately

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not possible to identify the minor conformation of the S,SBDT-A‚G oligodeoxynucleotide. The possibility was considered that the presence of the N6-dA BDT moiety at the mismatch site altered the pKa of the conformational transition between dA(anti)‚dG(anti) and protonated dA(anti)‚dG(syn) conformations (69, 70) and that the minor structure reflected a small amount of the protonated structure at neutral pH in the presence of the BDT moiety. Several lines of evidence argued against this conclusion. The chemical shift differences for the two sets of exchanging imino proton resonances at base pairs C5‚ G18, X6‚G17, and A7‚T16 were small, and both G17 resonances were observed in the region of 12.9 ppm, consistent with a dA(anti)‚dG(anti) pairing, whereas in the syn conformation, the nonhydrogen-bonded imino resonance of G17 would have been expected to be observed at higher field. Also, the presence of even a small amount of the syn conformation for G17 would be expected to be accompanied by observation of a strong NOE between G17 H8 and G17 H1′, which was not observed. It seems likely that the minor conformation involves a second conformation of the N6-dA BDT moiety, perhaps mediated by hydrogen bonding interactions with the BDT hydroxyl groups. In this regard, it is of interest that the refined structure of the properly paired S,S-BDT-(61,2) oligodeoxynucleotide suggested the possibility of bifurcated hydrogen bond formation involving the β-hydroxyl group of the BDT moiety and T17 O4. It seems plausible that a similar interaction could occur between the β-hydroxyl of the BDT moiety and G17 O6 in the S,S-BDT-A‚G oligodeoxynucleotide, although it was not possible to detect spectroscopic evidence for such an interaction. Structure-Activity Relationships. The low level of mutations induced by the ras61 S,S-BDT-(61,2) adduct in E. coli has been presumed to reflect its minor perturbation of the DNA duplex (59), such that the major groove orientation of the BDT moiety does not interfere with translesion replication. When ligated into 85-mer templates and examined as to translesion replication in vitro, the S,S-BDT-(61,2) adduct did not block translesion replication by the E. coli polymerases Klenow fragment exo-, polymerase II, or polymerase III (62). All of these polymerases successfully bypassed this adduct with little or no loss of efficiency or fidelity. The ras61 S,S-BDT(61,2) adduct did not result in a significant block to the replication of site specifically modified bacteriophage in vivo, as evidenced by no decrease in plaque-forming ability of phage-infected bacteria. Presumably, the low level of AfC mutations observed for the ras61 S,S-BDT(61,2) adduct results from occasional misincorporation of dGTP opposite the lesion. The present structural studies reveal that misincorporation of dGTP opposite the ras61 S,S-BDT-(61,2) adduct could allow formation of an A‚G mismatch in which both mismatched bases are stacked into the DNA duplex. Biological Implications. The recognition and repair of A‚G mismatches play an important role in maintaining the integrity of the genome, both in prokaryotes and in eukaryotes. Oxidative damage to DNA results in formation of the mutagenic 8-oxo-dG, which, if not successfully removed by Fpg glycosylase (92, 93), miscodes for the insertion of dATP during translesion synthesis (94-96). Oxidative damage to dG not repaired by Fpg represents a significant source of A‚G mismatches in vivo. In E. coli, the Mut Y glycosylase excises adenine from A‚G mispairs (97, 98). In humans, the homologous MYH glycosylase


performs the same function (99). It is perhaps significant that the major conformation of the S,S-BDT-A‚G mismatch maintains a structure similar to the corresponding A‚G mismatch in the unmodified ras61 oligodeoxynucleotide. This suggests that it might also be targeted by enzymes that act upon A‚G mismatches. The mismatch repair recognition protein Mut S was examined bound to DNA containing a G‚T mismatch (100) or an abasic site opposite dG (101). These revealed an intrahelical orientation for the mismatched bases, minor groove binding of domain 1 of Mut S allowing recognition of the mismatch by conserved PheX-Glu motif (100, 101), and DNA binding contacts with the phosphodiester backbone. Each of these structural features was consistent with the fact that Mut S recognizes a variety of mismatched bases and in a variety of DNA sequence contexts. The present structural studies suggest that the S,S-BDT-A‚G mismatch would not be expected to perturb these protein-DNA interactions. In this regard, the observation of a second minor conformation is intriguing. The low level genotoxicity associated with this adduct (62) might result from inhibition of mismatch repair by a minor conformation of the adduct. Summary. Incorporation of dG opposite the ras61 S,SBDT-(61,2) adduct allowed formation of an X6‚G17 mismatch in which both mismatched purines were intrahelical and in the anti conformation about the glycosyl bond. They exhibited face-to-face base pairing in which X6 N1 was within hydrogen bonding distance of G17 N1H and X6 N6H was within hydrogen bonding distance of G17 O6. The BDT moiety was oriented in the major groove. If not repaired, the resulting mismatch would generate the observed AfC mutations associated with this adenine N6 BDT adduct (62).

Acknowledgment. Markus Voehler and Dr. Jaison Jacob assisted with NMR spectroscopy. Dr. Jarrod Smith assisted with structural refinement. This work was supported by NIH Grant ES-05509 (M.P.S.). Funding for the NMR spectrometers was supplied by Vanderbilt University, NIH Grant RR-05805, and by the Vanderbilt Center in Molecular Toxicology, ES-00267. The Vanderbilt-Ingram Cancer Center is supported by NIH Grant CA-68485.


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Mispairing of a Site Specific Major Groove (2S,3S)-N6-(2,3,4

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