Fluorescence Characteristics of Site-Specific and Stereochemically

metabolite anti-BPDE (the diol epoxide r7,t8-dihydroxy-t9,10epoxy-7,8,9,10-tetrahydrobenz-. [a]pyrene) to the exocyclic amino groups of guanine ([BP]-...
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Chem. Res. Toxicol. 2002, 15, 118-126

Fluorescence Characteristics of Site-Specific and Stereochemically Distinct Benzo[a]pyrene Diol Epoxide-DNA Adducts as Probes of Adduct Conformation Weidong Huang, Shantu Amin,† and Nicholas E. Geacintov*,‡ Chemistry Department, New York University, New York, New York 10003-5180, and American Health Foundation, Valhalla, New York 10595 Received August 14, 2001

Spectroscopic fluorescence quenching techniques are described for distinguishing the conformational characteristics of adducts derived from the binding of the benzo[a]pyrene metabolite anti-BPDE (the diol epoxide r7,t8-dihydroxy-t9,10epoxy-7,8,9,10-tetrahydrobenz[a]pyrene) to the exocyclic amino groups of guanine ([BP]-N 2-dG) and adenine ([BP]-N 6-dA) in double stranded oligonucleotides. These methods are calibrated by comparing the fluorescence quenching and UV absorbance characteristics of different, stereoisomeric anti-[BP]-N 2-dG adducts of known adduct conformations, previously established by high-resolution NMR techniques. It is shown that intercalative adduct conformations can be distinguished from solvent-exposed adduct conformations, e.g., adducts in which the pyrenyl residues are positioned in the minor groove. These low resolution fluorescence methods are at least 4 orders of magnitude more sensitive than the high-resolution NMR techniques; the fluorescence methods are useful for distinguishing adduct conformations when either small amounts of material are available or the NMR signals are of such poor quality that high-resolution structures cannot be determined. This methodology is illustrated using a variety of anti-BPDE-modified oligonucleotides of varying adduct conformations. It is shown that the 10S (+)-trans-anti-[BP]N 6-dA adduct in an oligonucleotide duplex containing an N-ras protooncogene sequence, believed to be conformationally heterogeneous and disordered, is significantly more exposed to the solvent environment than the stereoisomeric, intercalated 10R adduct [Zegar et al. (1996) Biochemistry 35, 6212]. These differences suggest an explanation for the greater efficiencies of excision of the 10S adduct (relative to the 10R adduct) by human nucleotide excision repair enzymes [Buterin et al. (2000) Cancer Res. 60, 1849].

Introduction Benzo[a]pyrene, a typical polycylcic aromatic hydrocarbon (PAH), is metabolized in mammalian cells to the chemically reactive, stereoisomeric diol epoxides 7,8dihydroxy-9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (BPDE), that form covalent adducts with native DNA (1). Both diastereomeric forms of BPDE, anti and syn-BPDE, can be resolved into (+) and (-) enantiomers, and each enantiomer can react via trans or cis addition at its C10 position with the exocyclic amino groups of guanine and adenine residues to form a set of stereoisomeric adducts (2). The conformations of many of these stereoisomeric [BP]-N 2-dG (3-6) and [BP]-N 6-dA covalent adducts (7-11) (Figure 1) in double stranded DNA in various sequence contexts are known, and some examples relevant to this work are depicted in Figure 2. The influence of [BP]-DNA adduct conformation on the susceptibilities of these lesions to DNA repair is beginning to provide insights into the factors that govern the recognition and excision of these bulky lesions by prokaryotic (12, 13) and human (14-16) nucleotide excision * To whom correspondence should be addressed. Phone: (212) 9988407. Fax: (212) 998-8421. E-mail: [email protected]. † American Health Foundation. ‡ New York University.

repair (NER) enzymes. However, the detailed mechanisms governing the removal of PAH-DNA adducts of different conformations by NER remain to be established. Furthermore, [BP]-DNA adducts of different conformations are known to persist on mouse skin for different lengths of time (17), an effect that is probably associated with differential, adduct conformation-dependent DNA repair. While NMR methods provide the most detailed molecular views of the conformations of adducts in DNA, milligram quantities of samples are needed for such studies. Other techniques for distinguishing overall adduct conformations that require smaller amounts of stereochemically well-defined PAH-DNA adduct samples would be highly useful. We describe here a simple fluorescence quenching method that can distinguish intercalated from solvent-exposed [BP]-DNA adduct conformations. It is shown that the fluorescence quenching efficiencies are correlated with UV absorption properties of the [BP]-DNA adducts selected for study. The UV absorbance (18-20) and induced circular dichroism spectra (21, 22) of BPDE-modified, site-specific oligonucleotides have been previously utilized to gain information about the conformations of the pyrene-like aromatic residues. Fluorescence methods are more sensitive and have also been employed to study the charac-

10.1021/tx010135y CCC: $22.00 © 2002 American Chemical Society Published on Web 01/30/2002

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Figure 1. Stereochemical properties of BPDE adducts of 2′-deoxyguanosine and 2′-deoxyadenosine.

Figure 2. Conformations of anti-[BP]-N 2-dG lesions (A, B, and C). (A) (+)-trans- in duplex I (3), (B) (+)-cis- in duplex I (5), and (C) (+)-trans-adduct in the deletion duplex I-Del (6). (D) Typical conformation of the 10R anti-[BP]-N 6-dA adduct in double stranded DNA (from ref 11). The BP residues are shown in red, while the modified purines and Watson-Crick partner bases (except in panel C) are shown in green.

teristics of [BP]-DNA adducts (17, 23-25). However, the relationships between adduct conformation and fluorescence either were not reliably established (18, 25) or required liquid helium temperatures for distinguishing adducts of different conformations from one another (17). Site-specific, stereoisomeric [BP]-N 2-dG and -N 6-dA ad-

ducts in double stranded oligonucleotides with conformations that have been fully characterized by NMR methods in certain base sequence contexts are now available (10, 26, 27). However, the conformational properties of some [BP]-N 2-dG and [BP]-N 6-dA lesions are difficult or impossible to determine by NMR techniques because of

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conformational heterogeneity and interconversion between different conformers, effects that may depend on base sequence context (26, 28, 29). The objectives of this study are to correlate the fluorescence and UV absorbance characteristics of selected PAH-DNA adducts with their conformations (Figure 2) to provide a benchmark for future studies of [BP]DNA adducts of unknown conformations, e.g., in different sequence contexts. A second goal is to characterize differences in the physical characteristics of the 10R (-)trans-anti-[BP]-N 6-dA and the isomeric 10S (+)-transanti-[BP]-N 6-dA lesions in an identical DNA sequence context. The conformational properties of these two stereoisomeric lesions in double stranded DNA are of great interest because they are excised with different efficiencies by human DNA repair proteins (16).

Materials and Methods The racemic (()-anti-BPDE was purchased from the National Cancer Institute Chemical Carcinogen Reference Standard Repository. The BPDE-modified 11-mer oligonucleotides 5′-d (CCATC[G*]CTACC) were generated by employing a direct synthesis method, purified, and identified, as previously described (18, 30). The BPDE-modified 5′-CGGAC[A*]AGAAG oligonucleotides were synthesized using an automated DNA synthesizer and the trans-anti-[BP]-N 6-dA phosphoramidite derivatives as described in detail elsewhere (31). The BPDEmodified oligonucleotides were dissolved in 20 mM phosphate buffer solution, pH 7, 100 mM NaCl and annealed with complementary, or partially complementary strands by heating at 70 °C for 10 min, followed by cooling to room-temperature overnight. Electrophoresis-grade acrylamide was obtained from Bio-Rad (Hercules, CA), and was used as received. The 2′deoxyuridine was purchased from Sigma Chemical Co. (St. Louis, MO) and was purified by G-10 Sephadex chromatography column before use. The concentrations of 2′-deoxyuridine were determined by absorption spectroscopy utilizing an extinction coefficient of 8500 M-1 cm-1 at 260 nm. All UV absorption spectra were determined using an AVIV 140S UV-Vis spectrophotometer (Lakewood, NJ). The melting curves of the duplexes were obtained by monitoring the absorbance at 260 nm and at 348 nm as the temperature was increased from 15 °C to 70 °C at a rate of 1 °C/min. The temperature of the optical cell was controlled by an Endocal refrigerated circulating bath. The fluorescence quenching studies were performed utilizing a modified, computer-controlled MPF-2A Hitachi-Perkin-Elmer fluorescence spectrophotometer. All fluorescence emission spectra were scanned from 360 to 500 nm in air-saturated solutions in 0.4 × 1.0 cm cells (spectral excitation and emission bandwidths: 4 nm) at temperatures of 13 ( 1 °C. All of the reported fluorescence intensities represent values integrated from 360 to 460 nm. Since the fluorescence lifetimes of these [BP]-DNA adducts are of the order of nanoseconds, or less (32-34), the amount of O2 in air-equilibrated aqueous solutions is insufficient to affect the fluorescence intensities of the adducts (32). In the acrylamide fluorescence quenching experiments, small aliquots of concentrated acrylamide solutions (5.6 M) were added into the BPDE-modified duplex solution at room temperature. Small aliquots of concentrated acrylamide or 2′-deoxyuridine aqueous solutions were added to solutions containing the BPDE-modified duplexes, and appropriate amounts of buffer solutions were also added in order to maintain the adduct concentrations at constant values at the different fluorescence quencher concentrations. The [BP]-DNA adducts can decompose under prolonged irradiation with 300-360 nm light (35) to yield the highly fluorescent tetraols BPT. These tetraols, even when present in amounts of a few percent, can lead to incorrect analysis of the fluorescence quenching data (32, 36). Therefore, the exposure of the samples to near-UV light was minimized. Furthermore,

Huang et al. after each series of experiments, the samples were reanalyzed by reversed-phase HPLC methods (30) to determine if substantial quantities of BPT had been formed during the experiments. The DNA adduct and BPT levels were monitored in-line by their UV absorbance and fluorescence excited at 343 nm (the fluorescence excitation maximum of BPT) (37).

Results BPDE-Modified Sequences and Adduct Conformations. The pyrene-like fluorescence of [BP]-DNA adducts, occurring in the 360-500 nm region of the spectrum, is strongly quenched by the DNA bases (32, 34, 38) by an electron-transfer mechanism (38) and is highly sensitive to the microenvironment, particularly to fluorescence-quenching species in the aqueous phase (32, 39). We have selected for study a set of stereoisomeric (+)trans, (-)-trans, and (+)-cis-[BP]-N 2-dG adducts, [G*], incorporated in the 11mer oligonucleotide duplex I:

The conformations of each of these adducts in this sequence have been characterized by NMR methods (3, 4, 40), allowing for a correlation between their conformations (Figure 2) and fluorescence quenching efficiencies. The aromatic [BP] residues in the (+)-trans- and (-)trans-lesions are positioned in the minor groove, pointing either into the 5′-direction (Figure 2A) or the 3′-direction relative to the modified guanine residue. In the 10R (+)cis-anti-[BP]-N 2-dG adduct (Figure 2B), the BP residue is intercalated and the modified guanine and partner cytidine in the complementary strand are displaced into the minor and major groove, respectively. Removal of the cytidine in the partner strand opposite the lesion results in a dramatic change in the conformation of the (+)-trans adduct from a minor groove to an intercalative conformation (Figure 2C) (6). The fluorescence characteristics of the 10R (-)-transand 10S (+)-trans-[BP]-N 6-dA adducts were studied in the sequence context of oligonucleotide duplex II with

[A*] representing the lesion. In this N ras codon 61 (CAA) sequence context, the 10R (-)-trans adduct is known to be intercalated from the major groove, without base displacement, on the 5′-side of the modified adenine residue (8). The conformations of the 10R adducts in other sequences (7, 8, 41) have been established and are similar. The conformation of a typical 10R adduct, a (+)cis-anti-[BP]-N 6-dA adduct (11), is depicted in Figure 2D. The NMR signals of the stereoisomeric 10S adducts are of insufficient quality for a detailed NMR structural analysis due to line broadening and the existence of multiple, interconverting conformers (10, 27). The structure of the 10S adduct is therefore unknown and is believed to be disordered (42). Nevertheless, there are a number of experimental (10, 27) and theoretical considerations (26, 43) that suggest that in the 10S adducts the [BP] residue should be positioned on the 3′-side of the modified adenine. Absorption Spectra. In normal duplexes I with all of the bases complementary to one another, the (+)-trans-

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Figure 4. Typical Stern-Volmer fluorescence quenching curves (plots of the quenching factors F(0)/F(Acr) as a function of acrylamide quencher concentration). F(0) and F(Acr) are the fluorescence yields in the absence and presence respectively of acrylamide. (b) (+)--anti-[BP]-N 2-dG single base (mononucleoside) adduct; ([) same (+)-cis adduct in duplex I; (0) (-)-trans adduct in duplex I. Excitation wavelength: 350 nm.

Figure 3. Absorption spectra of [BP]-dG and [BP]-dA adducts in duplexes I, I-DEL, I-A, or II. For comparison, the absorption spectrum of BPT is shown in panel C.

and (-)-trans-anti-[BP]-N 2-dG adducts exhibit absorption maxima near 346-347 nm (Figure 3, panels A and B), slightly red-shifted with respect to the absorption maximum of free BPT1 at 343 nm (panel C). In contrast, the absorption maximum of the stereoisomeric (+)-cis adduct in the same duplex is shifted to 352 nm (Figure 3, panel C). The absorption spectra of the trans-anti-[BP]-N 2-dG adducts (19) undergo a significant red shift if the dC residue opposite G* in the partner strand is deleted as in the duplex I-DEL (Figure 3, panels A and B):

A similar red-shift is observed when the partner strand dC opposite G* is replaced by a dA residue (Figure 3, panels A and B):

These shifts in the absorption maxima are correlated with conformational changes of the lesions as discussed below. The absorption characteristics of the (+)-trans- and (-)trans-anti-[BP]-N 6-dA duplexes II also differ from one another as shown in Figure 3, panel D. The 10S (+)-trans adduct exhibits somewhat sharper, blue-shifted absorption bands than the 10R (-)-trans-adduct. Thermodynamic Stabilities of Duplexes. Typical melting curves of some of the BPDE-modified duplexes studied in this work are shown in Supporting Information. Duplexes with [BP]-N 2-dG (19, 20) or [BP]-N 6-dA (7, 31, 44) adducts are known to be destabilized by the 1 Abbreviations: BPDE, 7,8-dihydroxy-9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene; BPT, 7,8,9,10-tetrahydroxytetrahydrobenzo[a]pyrene; PAHs, polycyclic aromatic hydrocarbons; NER, nucleotide excision repair; [BP]-N 6-dA, covalent adducts derived from the binding of BPDE to the exocyclic amino group of adenine in DNA; [BP]-N 2dG, covalent adducts derived from the binding of BPDE to the exocyclic amino group of guanine in DNA; [BP]-DNA, stereochemically unspecified adducts derived from the binding of BPDE to DNA.

lesions. The melting points, Tm, depend on the type of adduct and the base sequence context and are routinely lower than the Tm of the unmodified sequences and range from a lowest value of 25.8 °C for the 10S (+)-trans-anti[BP]-N 6-dA adduct in duplex II (31) to a high of 46.5 °C for the (+)-cis-anti-[BP]-N 2-dG adduct in sequence I (19). All fluorescence quenching experiments were thus conducted at temperatures of 12-14 °C to ensure that the DNA was in the double-stranded form. Fluorescence Quenching by Acrylamide. Acrylamide is a well-known fluorescence quencher that is commonly used in biochemical applications (45) and is known to quench the fluorescence of adducts derived from the heterogeneous binding of anti-BPDE to native DNA (32). The acrylamide Stern-Volmer fluorescence quenching curves, corresponding to plots of F(0)/F(acr) as a function of quencher concentration, are shown in Figure 4; F(0) and F(acr) are the steady-state fluorescence intensities in the absence and presence of acrylamide, respectively. There is a clear difference for the duplexes with the (-)trans-anti-[BP]-N 2-dG stereochemistry (external, minor groove conformation) and those with (+)-cis-anti-[BP]N 2-dG stereochemistry (base displaced intercalation, Figure 2B), though both have R absolute configuration at the [BP]-N 2-dG linkage site. The fluorescence of the (-)-trans adducts is quenched by acrylamide, while the fluorescence of the (+)-cis adducts is not, indicating that the pyrenyl residues are not solvent-accessible in the (+)cis adducts. The F(acr)/F(0) quenching curve for the single-base (+)-cis-anti-[BP]-N 2-dG adduct is shown for comparison in Figure 4. This single nucleoside adduct, in contrast to the same adduct in the duplex, is rather strongly quenched by acrylamide, as expected for a solvent or quencher-accessible fluorophore. Quenching of Fluorescence by 2′-Deoxyuridine (dU). Since the mechanism of fluorescence quenching of fluorophores by acrylamide is not well understood, and we desired to test the generality of the above results with another fluorescence quencher, we selected the nucleoside dU as the quencher. Nucleosides such as dU quench the fluorescence of pyrenyl residues by an electron-transfer mechanism by both dynamic and static (complex-forming) mechanisms (46, 47). The quenching is favored by a close contact between the fluorophore and the nucleoside quenchers. Because dU is a more bulky molecule than

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Figure 5. Comparisons of fluorescence quenching factors F(0)/ F(dU) of anti-[BP]-N 2-dG adducts with trans- and cis-adduct stereochemistry in normal duplex I as a function of 2′-deoxyuridine quencher concentration. (0) (-)-trans adduct in duplex I; (b) (+)-trans adduct in duplex I; ([) (+)-cis adduct in duplex I. Excitation wavelength: 350 nm.

acrylamide, our hypothesis is that the quenching of the fluorescence of pyrenyl residues in [BP]-N 2-dG adducts by dU would be particularly sensitive to the conformation of these lesions in DNA duplexes. The F(0)/F(dU) factors vs concentration of dU in different duplexes are presented in Figure 5. These quenching factors for the (+) and (-)-trans-anti-[BP]-N 2dG adducts in the 11-mer duplexes I (minor groove adduct conformation) exhibit a rapid initial rise, followed by a slower rise as the quencher concentration is increased above [dU] ) 0.05 M. As in the case of acrylamide, dU does not quench the fluorescence of the stereoisomeric, intercalated (+)-cis-[BP]-N 2-dG adducts, and the F(0)/F(dU) ratios are ∼1.0, within experimental error, at all dU concentrations studied (Figure 5). The fluorescence quenching curves are similar at different excitation wavelengths corresponding to the longest wavelength absorption band (e.g., at 350, 352 and 354 nm for the (+)-cis adduct in duplex I, data not shown). To ascertain that significant amounts of the tetraol BPT were not formed by a photoinduced dissociation of the covalent adducts due to the unavoidable UV-irradiation of the samples during the collection of the fluorescence quenching data, the irradiated samples were subjected to HPLC analysis. Tetraols were not detected, within experimental error, by fluorescence-detected HPLC elution profiles (data not shown). We conclude that the observed fluorescence is associated with the covalent [BP]-N 2-dG adducts, rather than with BPT photodissociation products that can form during the photoirradiation of the adducts (35). Fluorescence Quenching Curves with [BP]-N 2dG in Duplexes I-DEL and I-A. The rather dramatic changes in the absorption spectra of the (+)-trans- and (-)-trans-anti-[BP]-N 2-dG adducts that occur when the partner strand dC residue opposite G* is deleted, or replaced by dA, are accompanied by significant decreases in fluorescence quenching efficiencies as shown in Figure 6 (panels A and B, respectively). The (+)-trans- and (-)trans-anti-[BP]-N 2-dG adducts are quenched by dU in the full duplexes I but not in the deletion duplexes I-DEL (Figure 6, panel A). Similar absorption and fluorescence quenching characteristics are exhibited in the I-A “mismatch” duplexes (Figure 6, panel B). Fluorescence Quenching Curves Determined for [BP]-N 6-dA Lesions in Duplexes II. The fluorescence quenching curves with dU as the quencher obtained with

Figure 6. Comparisons of fluorescence quenching factors F(0)/ F(dU) of anti-[BP]-N 2-dG adducts in normal duplexes I, in deletion duplexes I-DEL, and in “mismatched” duplex I-A as a function of 2′-deoxyuridine concentration. (0) (-)-trans adduct in duplex I; (b) (+)-trans adduct in duplex I; (A) (3) (+)-trans adduct in the I-DEL duplex; (/) (-)-trans adduct in the I-DEL duplex; (B) (4) (+)-trans adduct in the I-A duplex; (×) (-)-trans[BP]-N 2-I-A. Excitation wavelength: 350 nm.

Figure 7. Fluorescence quenching factors F(0)/F(dU) of anti[BP]-N 6-dA adducts in normal duplex II as a function of 2′-deoxyuridine concentration. (0) 10R (-)-trans adduct; (9) 10S (+)-trans adduct. Excitation wavelength: 352 nm.

duplexes with [A*] ) 10R (-)-trans- or the 10S (+)-transanti-[BP]-N 6-dA adducts in duplex II are depicted in Figure 7. The fluorescence of the 10S adduct is more strongly quenched than that of the 10R adduct.

Discussion Previous fluorescence quenching studies on native [BP]-DNA adducts obtained by treating DNA with BPDE suffered from the drawbacks that the adducts were structurally and conformationally heterogeneous, thus greatly complicating the interpretation of the results (32). In the case of randomly modified [BP]-DNA adducts, the interpretation of the data is complicated because mixtures of cis and trans adducts are formed, different DNA bases (dG or dA) are modified, and it is not known exactly how stereoisomeric DNA adduct conformations influence the fluorescence quenching parameters.

Fluorescence of BPDE-DNA Adducts

Comparison of the Results from the Fluorescence Quenching Experiments and UV Absorption Spectroscopy with NMR Solution Structures. Highresolution NMR structural studies show that the BP residues in (+) and (-)-trans-anti- [BP]-N 2-dG adducts in duplexes I are positioned in the minor grooves, oriented on the 5′-side or the 3′-side, respectively, of the modified guanine residue (3, 4). The BP residues are thus partially exposed to the aqueous solvent environment, which is consistent with a small, ∼3 nm red shift in the absorption maximum of the pyrenyl residues from 343 nm in BPT to 346 nm in the two trans-anti-[BP]-N 2-dG adducts in duplex I (Figure 3, panels A and B). The fluorescence of the pyrenyl residues in duplexes I is thus at least partially accessible to the fluorescence quenchers acrylamide and dU (Figures 4 and 5). The plots of F(0)/ F(Q) vs quencher concentrations, Q, are nearly identical for the (+)-trans and (-)-trans adducts in duplex I, as expected since both exhibit minor groove adduct conformations. However, it should be noted that there is a difference in adduct conformations at low temperatures. Even though both the (-)-trans and (+)-trans adducts in duplex I are predominantly minor groove structures, detailed low-temperature fluorescence studies reveal that the (+)-trans adduct also exists as a base-stacked conformer (∼ 25%), while the (-)-trans adduct is almost exclusively of the minor groove type (23). Thus, there are differences in the heterogeneities of adduct conformations between these two stereoisomeric trans adducts, but this difference does not appear to be extensive at roomtemperature judging from the fluorescence quenching data (Figure 5). The (+)-cis adducts are characterized by base-displaced intercalative conformations (5). Consistent with the extensive pyrenyl-base stacking interactions in these conformations, a large 9-11 nm red shift in the absorption maximum to 352-354 nm is observed in the (+)-cis adduct (Figure 3, panel C). The intercalated pyrenyl residues are expected to be poorly accessible to the aqueous solvent, and thus to water-soluble fluorescence quenchers. This hypothesis is indeed confirmed by the inefficient quenching of the fluorescence of the (+)-cisanti-[BP]-N 2-dG adducts (Figures 4 and 5). Overall, the differences in the fluorescence quenching curves obtained with stereoisomeric minor groove transand intercalated cis-anti-[BP]-N 2-dG adducts, suggests that fluorescence quenching techniques can be used to distinguish intercalative from other conformations in which the BP residues are more exposed to the aqueous solvent environment. This concept was tested employing other adducts of known conformation. Conformations of Adducts in Duplexes with a Deleted Base Opposite the Lesion. The pyrenyl residue of the (+)- and (-)-trans-anti-[BP]-N 2-dG adducts, positioned in the minor groove, undergo dramatic changes in conformation when the dC residue opposite the modified guanine residue is removed from duplex I to form the duplex I-DEL. In the deletion duplex I-DEL the pyrenyl residue in both the (+)- and (-)-trans adducts is intercalated as shown by NMR methods (6, 48). Consistent with this model (Figure 2C), shifts in the absorption maxima from 346 nm in duplexes I to ∼354 nm in duplexes I-DEL are observed (Figure 3, panels A and B). The values of the fluorescence quenching factors F(0)/F(dU) dramatically decrease from a maximum value of ∼2 in the minor groove structures in duplexes I, to

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∼1.1 in the deletion duplexes I-DEL (Figure 6, panel A). Thus, the pyrenyl ring system of the BP residues becomes less accessible to the dU quencher molecules in the deletion duplexes I-DEL than in the full duplex I. Conformations of Duplexes with a Mismatched Adenine Opposite the Lesion. The conformation of duplex I-A is of interest because the most frequently observed base substitution mutations in a variety of cell systems treated with (+)-anti-BPDE are G f T transversions (49, 50). Similar results have been obtained in site-directed mutagenesis studies in different cell systems (51-53). Thus, during error-prone translesion synthesis, a dA is inserted opposite the modified guanine and, upon further replication, a mismatched duplex results. It is therefore of interest to examine the conformations of such “mismatched” duplexes. The NMR solution structure of (+)-trans-anti-[BP]-N 2-dG with the mismatched base dA opposite the adduct instead of the normal dC has not yet been studied. However, the red-shifted absorption maxima of both the (+)-trans and the (-)-trans mismatched duplexes (Figure 3, panels A and B, respectively) clearly suggests that the duplexes I-A are both of the intercalation type (19, 20) since the absorption maximum is shifted from 346 nm in duplexes I to 352 nm in duplexes I-A. Thus, substituting the C opposite the BPDE-modified guanine in the normal duplex by a single A, induces a dramatic change in the adduct conformation from a minor groove to an intercalated conformation. This conclusion is fully supported by the fluorescence quenching data shown in Figure 6B; the fluorescence quenching by dU is abolished in the mismatched duplexes I-A. Differences in the Characteristics of Stereoisomeric trans-anti-[BP]-N 6-dA Lesions. An interesting example of a striking difference in adduct conformations associated with stereoisomeric PAH-DNA adducts is the 10R (-)-trans- and 10S (+)-trans-anti-[BP]-N 6-dA pair of adducts in the CA*A sequence context of ras codon 61 of duplex II. The NMR solution structure of the 10R, but not that of the 10S duplex, has been established (8, 42). This structure is similar to the one established by Schurter et al. for the stereochemically identical 10R lesion in another sequence context (54). In this adduct, the BP residue is intercalated from the major groove on the 5′-side of the modified adenine residue. All base pairs, including the modified A*-T base pair, are intact and of the Watson-Crick type. However, the conformation of the 10S (+)-trans-adduct with a T in the complementary strand opposite A* has not been reported. Yeh et al. found that the NMR characteristics of these adducts, in contrast to the characteristics of the 10R (-)-trans-anti-BPDEN 6-dA adducts (8, 9), are poorly defined. Several considerations suggest that the 10S (+)-trans adduct should be generally positioned on the 3′-side of the modified adenine residue (9, 10, 27, 43). It is therefore of interest to examine the fluorescence quenching properties of these two adducts to determine if both are intercalated and if there are any differences in adduct conformations that can be discerned by molecular spectroscopic methods. The fluorescence quenching curves depicted in Figure 7 show that the F(0)/F(dU) factors reach a value of about 1.45 for the intercalated 10R (-)-trans-anti-[BP]-N 6-dA adduct in duplex II. This factor is higher than observed in the case of the base-displaced intercalated 10R (+)cis-anti-[BP]-N 2-dG adducts in duplex I (Figure 5). Thus, the accessibility to the solvent-borne fluorescence quencher dU of the 10R (-)-trans-anti-[BP]-N 6-dA adducts is

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somewhat greater than in the case of the 10R (+)-cisanti-[BP]-N 2-dG adducts. The fluorescence quenching characteristics of the 10S (+)-trans-anti-[BP]-N 6-dA adduct in sequence II are, however, quite different than those expected for an intercalation complex (Figure 7), as are the UV absorption spectra (Figure 3, panel D). The fluorescence quenching factor F(0)/F(dU) rises with increasing quencher concentration and reaches values as high as 2.2. This result suggests that the BP residue is much more exposed to the aqueous solvent environment in the 10S (+)-transadduct than in the stereoisomeric 10R (-)-trans-anti[BP]-N 6-dA adduct in the same duplex. This difference in exposure to the aqueous solvent environment was predicted by a recent computational analysis (55). These differences are consistent with the available NMR data for these BPDE-modified adenine adducts in oligonucleotide duplexes, and with the UV absorption spectra (Figure 3, panel D) that suggest a significant degree of solvent exposure since the absorption bands are more narrow and sharply defined in the case of the 10S (+)trans than in the intercalated 10R (-)-trans-adduct. These remarkable differences in the fluorescence quenching characteristics are correlated with the generally lower Tm values of [BP]-N 6-dA lesions in double stranded oligonucleotides (7, 31, 44). In the case of duplex II, the higher solvent-accessibility of the 10S adduct is correlated not only with a higher exposure to solvent, a lower Tm value (31), but to a greater heterogeneity of adduct conformations, probably involving an anti-syn glycosidic bond angle equilibrium (10, 27). These differences suggest that the 10S adducts are more disordered than the 10R (-)-trans-anti-[BP]-N 6-dA adducts. These factors might account for the greater susceptibilities of these 10S adducts to removal by nucleotide excision repair enzymes (16).

Conclusions Fluorescence quenching methods combined with UV absorption spectroscopy, constitute a rapid and sensitive approach for distinguishing between intercalative and solvent-exposed conformations of [BP]-DNA adducts. The UV absorbance measurements in the 300-360 nm region can be conducted with samples of 1 µM concentrations (in BP residues), and thus the amount of sample needed is more than a ∼1000 times smaller than required for detailed NMR studies. The fluorescence methods are even more sensitive, depending on the particular fluorimeter and associated signal/noise characteristics. Low-resolution fluorescence and UV absorption characteristics can be employed to distinguish intercalative adduct conformations from solvent-exposed ones using microgram or, in some cases, submicrogram quantities of BPDE-modified oligonucleotide adduct samples.

Acknowledgment. This work was supported by the National Institutes of Health, National Cancer Institute, Grant CA 76660. The (()-anti-BPDE was purchased from the National Cancer Institute Chemical Carcinogen Reference Standard Repository. We are grateful to Shixiang Yan and Professor Suse Broyde for generating Figure 2. Supporting Information Available: Thermal stabilities of duplexes and a figure of the melting curves of (+)-trans-IDEL and (+)-trans-I-A duplexes. This material is available free of charge via the Internet at http://pubs.acs.org.

Huang et al.

References (1) Harvey, R. G. (1991) Polycyclic Aromatic Hydrocarbons: Chemistry and Carcinogenicity, Cambridge University Press. (2) Szeliga, J., and Dipple, A. (1998) DNA adduct formation by polycyclic aromatic hydrocarbon dihydrodiol epoxides. Chem. Res. Toxicol. 11, 1-11. (3) Cosman, M., de los Santos, C., Fiala, R., Hingerty, B. E., Singh, S. B., Ibanez, V., Margulis, L. A., Live, D., Geacintov, N. E., and Broyde, S., et al. (1992) Solution conformation of the major adduct between the carcinogen (+)-anti-benzo[a]pyrene diol epoxide and DNA. Proc. Natl. Acad. Sci. U.S.A. 89, 1914-8. (4) de los Santos, C., Cosman, M., Hingerty, B. E., Ibanez, V., Margulis, L. A., Geacintov, N. E., Broyde, S., and Patel, D. J. (1992) Influence of benzo[a]pyrene diol epoxide chirality on solution conformations of DNA covalent adducts: the (-)-transanti-[BP]G.C adduct structure and comparison with the (+)-transanti-[BP]G.C enantiomer. Biochemistry 31, 5245-52. (5) Cosman, M., de los Santos, C., Fiala, R., Hingerty, B. E., Ibanez, V., Luna, E., Harvey, R., Geacintov, N. E., Broyde, S., and Patel, D. J. (1993) Solution conformation of the (+)-cis-anti-[BP]dG adduct in a DNA duplex: intercalation of the covalently attached benzo[a]pyrenyl ring into the helix and displacement of the modified deoxyguanosine. Biochemistry 32, 4145-55. (6) Cosman, M., Fiala, R., Hingerty, B. E., Amin, S., Geacintov, N. E., Broyde, S., and Patel, D. J. (1994) Solution conformation of the (+)-trans-anti-[BP]dG adduct opposite a deletion site in a DNA duplex: intercalation of the covalently attached benzo[a]pyrene into the helix with base displacement of the modified deoxyguanosine into the major groove. Biochemistry 33, 11507-17. (7) Schurter, E. J., Yeh, H. J. C., Sayer, J. M., Lakshman, M. K., Yagi, H., Jerina, D. M., and Gorenstein, D. G. (1995) NMR solution structure of a nonanucleotide duplex with a dG mismatch opposite a 10R adduct derived from trans addition of a deoxyadenosine N 6-amino group to (-)-(7S,8R,9R,10S)-7,8-dihydroxy9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene. Biochemistry 34, 1364-75. (8) Zegar, I. S., Kim, S. J., Johansen, T. N., Horton, P. J., Harris, C. M., Harris, T. M., and Stone, M. P. (1996) Adduction of the human N-ras codon 61 sequence with (-)-(7S,8R,9R,10S)-7,8-dihydroxy9,10-epoxy-7,8,9,10-tetrahydrobenzo[a] pyrene: structural refinement of the intercalated SRSR(61,2) (-)-(7S,8R,9S,10R)-N 6-[10(7,8,9,10-tetrahydrobenzo[a]pyrenyl)]-2′-deoxyadenosyl adduct from 1H NMR. Biochemistry 35, 6212-24. (9) Yeh, H. J. C., Sayer, J. M., Liu, X., Altieri, A. S., Byrd, R. A., Lakshman, M. K., Yagi, H., Schurter, E. J., Gorenstein, D. G., and Jerina, D. M. (1995) NMR solution structure of a nonnucleotide duplex with a dG mismatch opposite a 10S adduct derived from trans addition of a deoxyadenosine N 6-amino group to (+)(7R,8S,9S,10R)-7,8-dihydroxy-9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene: an unusual syn glycosidic torsion angle at the modified dA. Biochemistry 34, 13570-13581. (10) Volk, D. E., Rice, J. S., Luxon, B. A., Yeh, H. J., Liang, C., Xie, G., Sayer, J. M., Jerina, D. M., and Gorenstein, D. G. (2000) NMR evidence for syn-anti interconversion of a trans opened (10R)-dA adduct of benzo[a]pyrene (7S,8R)-diol (9R,10S)-epoxide in a DNA duplex. Biochemistry 39, 14040-53. (11) Mao, B., Gu, Z., Gorin, A., Chen, J., Hingerty, B. E., Amin, S., Broyde, S., Geacintov, N. E., and Patel, D. J. (1999) Solution structure of the (+)-cis-anti-benzo[a]pyrene-dA ([BP]dA) adduct opposite dT in a DNA duplex. Biochemistry 38, 10831-42. (12) Zou, Y., Liu, T.-M., Geacintov, N. E., and Van Houten, B. (1995) Interaction of the UvrABC Nuclease System with a DNA Duplex Containing a Single Stereoisomer of dG-(+)- or dG-(-)-anti-BPDE, Biochemistry 34, 13582-93. (13) Hoare, S., Zou, Y., Purohit, V., Krishnasamy, R., Skorvaga, M., Van Houten, B., Geacintov, N. E., and Basu, A. K. (2000) Differential incision of bulky carcinogen-DNA adducts by the UvrABC nuclease: comparison of incision rates and the interactions of Uvr subunits with lesions of different structures. Biochemistry 39, 12252-61. (14) Hess, M. T., Gunz, D., Luneva, N., Geacintov, N. E., and Naegeli, H. (1997) Base pair conformation-dependent excision of benzo[a]pyrene diol epoxide-guanine adducts by human nucleotide excision repair enzymes, Mol. Cell. Biol. 17, 7069-76. (15) Custer, L., Zajc, B., Sayer, J. M., Cullinane, C., Phillips, D. R., Cheh, A. M., Jerina, D. M., Bohr, V. A., and Mazur, S. J. (1999) Stereospecific differences in repair by human cell extracts of synthesized oligonucleotides containing trans-opened 7,8,9,10tetrahydrobenzo[a]pyrene 7,8-diol 9,10-epoxide N 2-dG adduct stereoisomers located within the human K-ras codon 12 sequence. Biochemistry 38, 569-81.

Fluorescence of BPDE-DNA Adducts (16) Buterin, T., Hess, M. T., Luneva, N., Geacintov, N. E., Amin, S., Kroth, H., Seidel, A., and Naegeli, H. (2000) Unrepaired fjord region polycyclic aromatic hydrocarbon-DNA adducts in ras codon 61 mutational hot spots. Cancer Res. 60, 1849-56. (17) Suh, M., Ariese, F., Small, G. J., Jankowiak, R., Hewer, A., and Phillips, D. H. (1995) Formation and persistence of benzo[a]pyrene-DNA adducts in mouse epidermis in vivo: importance of adduct conformation, Carcinogenesis 16, 2561-9. (18) Geacintov, N. E., Cosman, M., Mao, B., Alfano, A., Ibanez, V., and Harvey, R. G. (1991) Spectroscopic characteristics and site I/site II classification of cis and trans benzo[a]pyrene diolepoxide enantiomer-guanosine adducts in oligonucleotides and polynucleotides. Carcinogenesis 12, 2099-108. (19) Ya, N.-Q., Smirnov, S., Cosman, M., Bhanot, S., Ibanez, V. and Geacintov, N. E. (1994) Thermal stabilities of benzo[a]pyrene diol epoxide-modified oligonucleotide duplexes. Effects of mismatched complementary strands and bulges. In Structural Biology: The State of the Art. Proceedings of the 8th Converstaion (Sarma, R. H. S.a. M. H., Ed.) Vol. 2, pp 349-66, Adenine Press, Schenectady, NY. (20) Arghavani, M. B., SantaLucia, J., Jr., and Romano, L. J. (1998) Effect of mismatched complementary strands and 5′-change in sequence context on the thermodynamics and structure of benzo[a]pyrene-modified oligonucleotides. Biochemistry 37, 8575-83. (21) Pradhan, P., Jernstrom, B., Seidel, A., Norden, B., and Graslund, A. (1998) Induced circular dichroism of benzo[a]pyrene-7,8dihydrodiol 9,10-epoxide stereoisomers covalently bound to deoxyribooligonucleotides used to probe equilibrium distribution between groove binding and intercalative adduct conformations. Biochemistry 37, 4664-73. (22) Pradhan, P., Jernstrom, B., Seidel, A., Norden, B., and Graslund, A. (1999) Studies on the adduct heterogeneity of benzo[a]pyrene 7,8-dihydrodiol 9,10-epoxide stereoisomers covalently bound to deoxyribooligonucleotides by induced circular dichroism and light absorption spectroscopy. Chem. Res. Toxicol. 12, 403-11. (23) Suh, M., Ariese, F., Small, G. J., Jankowiak, R., Liu, T.-M., and Geacintov, N. E. (1995) Conformational studies of the (+)-trans, (-)-trans, (+)-cis, and (-)-cis adducts of anti-benzo[a]pyrene diolepoxide to N 2-dG in duplex oligonucleotides using polyacrylamide gel electrophoresis and low-temperature fluorescence spectroscopy. Biophys. Chem. 56, 281-296. (24) Jankowiak, R., Ariese, F., Hewer, A., Luch, A., Zamzow, D., Hughes, N. C., Phillips, D., Seidel, A., Platt, K. L., Oesch, F., and Small, G. J. (1998) Structure, conformations, and repair of DNA adducts from dibenzo[a, l]pyrene: 32P-postlabeling and fluorescence studies. Chem. Res. Toxicol. 11, 674-85. (25) Zhao, R., Liu, T. M., Kim, S. K., MacLeod, M. C., and Geacintov, N. E. (1992) Identification and quantitative detection of isomeric benzo[a]pyrene diolepoxide-DNA adducts by low-temperature conventional fluorescence methods. Carcinogenesis 13, 1817-24. (26) Geacintov, N. E., Cosman, M., Hingerty, B. E., Amin, S., Broyde S., and Patel, D. J. (1997) NMR Solution Structures of Stereoisomeric Polycyclic Aromatic Carcinogen-DNA Adducts: Principles, Patterns, and Diversity, Chem. Res. Toxicol. 10, 111-146. (27) Pradhan, P., Tirumala, S., Liu, X., Sayer, J. M., Jerina, D. M., and Yeh, H. J. (2001) Solution structure of a trans-opened (10S)dA adduct of (+)-(7S,8R,9S,10R)-7,8-dihydroxy-9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene in a fully complementary DNA duplex: evidence for a major syn conformation. Biochemistry 40, 5870-81. (28) Xu, R., Mao, B., Amin, S., and Geacintov, N. E. (1998) Bending and circularization of site-specific and stereoisomeric carcinogenDNA adducts. Biochemistry 37, 769-78. (29) Fountain, M. A., and Krugh, T. R. (1995) Structural characterization of a (+)-trans-anti-benzo[a]pyrene-DNA adduct using NMR, restrained energy minimization, and molecular dynamics. Biochemistry 34, 3152-61. (30) Pirogov, N., Shafirovich, V., Kolbanovskiy, A., Solntsev, K., Courtney, S. A., Amin, S., and Geacintov, N. E. (1998) Role of hydrophobic effects in the reaction of a polynuclear aromatic diol epoxide with oligodeoxynucleotides in aqueous solutions. Chem. Res. Toxicol. 11, 381-8. (31) Krzeminski, J., Ni, J., Zhuang, P., Luneva N., Amin, S., and Geacintov, N. E. (1999) Total synthesis, mass spectrometric sequencing, and stabilities of oligonucleotide duplexes with single trans-anti-BPDE-N6-dA lesions in the N-ras codon 61 and other sequence contexts. Polycyclic Aromat. Compd. 17, 1-10. (32) Geacintov, N. E., Zinger D., Ibanez V., Santella R., Grunberger D., and Harvey, R. G. (1987) Properties of covalent benzo[a]pyrene diol epoxide-DNA adducts investigated by fluorescence techniques. Carcinogenesis 8, 925-35. (33) Ya, N. (1994) Studies of base sequence effects of interactions in covalent benzo[a]pyrene-7,8-diol-9,10-epoxide-oligodeoxynucleotide

Chem. Res. Toxicol., Vol. 15, No. 2, 2002 125

(34)

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(37)

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(41)

(42)

(43)

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(45) (46)

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(50)

adducts by UV spectroscopy (benzo(a)pyrene), Ph.D. Dissertation, New York University. LeBreton, P. R., Huang, C.-R., Fernando, H., Zajc B., Lakshman, M. K., Sayer, J. M., and Jerina, D. M. (1995) Multiple fluorescence lifetimes for oligonucleotides containing single, site-specific modifications at guanine and adenine corresponding to trans addition of exocyclic amino groups to (+)-(7R,8S,9S,10R)- and (-)(7S,8R,9R,10S)-7,8-dihydroxy-9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene. Chem. Res. Toxicol. 8, 338-48. Zinger, D., Geacintov, N. E., and Harvey, R. G. (1987) Conformations and selective photodissociation of heterogeneous benzo[a]pyrene diol epoxide enantiomer-DNA adducts. Biophys. Chem. 27, 131-8. Geacintov, N. E. (1987) Principles and applications of fluorescence techniques in biophysical chemistry. Photochem. Photobiol. 45, 547-53. Ibanez, V., Geacintov, N. E., Gagliano, A. G., Brandimarte, S., and Harvey, R. G. (1980) Physical binding of tetraols derived from 7,8-dihydroxy-9,10-epoxybenzo[a]pyrene to DNA, J. Am. Chem. Soc. 102, 5661-6. Geacintov, N. E., Zhao, R., Kuzmin, V. A., Kim, S. K., and Pecora, L. J. (1993) Mechanisms of quenching of the fluorescence of a benzo[a]pyrene tetraol metabolite model compound by 2′-deoxynucleosides. Photochem. Photobiol. 58, 185-94. Undeman, O., Lycksell, P. O., Graslund, A., Astlind, T., Ehrenberg A., Jernstrom, B., Tjerneld, F., and Norden, B. (1983) Covalent complexes of DNA and two stereoisomers of benzo(a)pyrene 7,8dihydrodiol-9,10-epoxide studied by fluorescence and linear dichroism. Cancer Res. 43, 1851-60. Cosman, M., Hingerty, B. E., Luneva, N., Amin, S., Geacintov, N. E., Broyde, S., and Patel, D. J. (1996) Solution conformation of the (-)-cis-anti-benzo[a]pyrenyl-dG adduct opposite dC in a DNA duplex: intercalation of the covalently attached BP ring into the helix with base displacement of the modified deoxyguanosine into the major groove. Biochemistry 35, 9850-63. Zegar, I. S., Chary P., Jabil, R. J., Tamura, P. J., Johansen, T. N., Lloyd, R. S., Harris, C. M., Harris, T. M., and Stone, M. P. (1998) Multiple conformations of an intercalated (-)-(7S,8R,9S,10R)-N 6-[10-(7,8,9,10-tetrahydrobenzo[a]pyrenyl)]-2′-deoxyadenosyl adduct in the N-ras codon 61 sequence. Biochemistry 37, 1651628. Le, P. T., Harris, C. M., Harris, T. M., and Stone, M. P. (2000) Altered electrophoretic migration of polycyclic aromatic hydrocarbon and styrene oxide adducts at adenine N(6) correlates with adduct-induced structural disorder. Chem. Res. Toxicol. 13, 6371. Tan, J., Geacintov, N. E., and Broyde, S. (2000) Principles governing conformations in stereoisomeric adducts of bay region benzo[a]pyrene diol epoxides to adenine in DNA: Steric and hydrophobic effects are dominant. J. Am. Chem. Soc. 122, 302132. Chatuverdi, S., and Lakshman, M. K. (1996) Site-specific modification of the human N-ras proto-oncogene with each diol epoxide metabolite of benzo[a]pyrene and thermal denaturation studies of the adducted duplexes. Carcinogenesis 17, 2747-2752. Lakowicz, J. R. (1999) Principles of fluorescence spectroscopy, 2nd ed., Kluwer Academic/Plenum, New York. Netzel, T. L., Nafisi, K., Headrick, J., and Eaton, B. E. (1995) Direct observation of photoinduced electron transfer in pyrenelabeled dU nucleosides and evidence for protonated 2′-deoxyuridine anion, dU(H), as a primary electron-transfer product. J. Phys. Chem. 99, 17948-55. Shafirovich, V. Y., Courtney, S. H., Ya, N., and Geacintov, N. E. (1995) Proton-coupled photoinduced electron transfer, deuterium isotope effects, and fluorescence quenching in noncovalent benzo[a]pyrenetetraol-nucleoside complexes in aqueous solutions. J. Am. Chem. Soc. 117, 4920-9. Feng, B., Gorin, A., Kolbanovskiy, A., Hingerty, B. E., Geacintov, N. E., Broyde, S., and Patel, D. J. (1997) Solution conformation of the (-)-trans-anti-[BP]dG adduct opposite a deletion site in a DNA duplex: intercalation of the covalently attached benzo[a]pyrene into the helix with base displacement of the modified deoxyguanosine into the minor groove. Biochemistry 36, 1378090. Rodriguez, H., and Loechler, E. L. (1993) Mutational specificity of the (+)-anti-diol epoxide of benzo[a]pyrene in a supF gene of an Escherichia coli plasmid: DNA sequence context influences hotspots, mutagenic specificity and the extent of SOS enhancement of mutagenesis. Carcinogenesis 14, 373-83. Wei, S. J., Chang, R. L., Hennig, E., Cui, X. X., Merkler, K. A., Wong, C. Q., Yagi, H., Jerina, D. M., and Conney, A. H. (1994) Mutagenic selectivity at the HPRT locus in V-79 cells: comparison of mutations caused by bay-region benzo[a]pyrene 7,8-diol-9,-10-

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Chem. Res. Toxicol., Vol. 15, No. 2, 2002

epoxide enantiomers with high and low carcinogenic activity. Carcinogenesis 15, 1729-35. (51) Moriya, M., Spiegel, S., Fernandes, A., Amin, S., Liu, T., Geacintov, N. and Grollman, A. P. (1996) Fidelity of translesional synthesis past benzo[a]pyrene diol epoxide-2′-deoxyguanosine DNA adducts: marked effects of host cell, sequence context, and chirality. Biochemistry 35, 16646-51. (52) Page, J. E., Zajc, B., Oh-hara, T., Lakshman, M. K., Sayer, J. M., Jerina, D. M., and Dipple, A. (1998) Sequence context profoundly influences the mutagenic potency of trans-opened benzo[a]pyrene 7,8-diol 9,10-epoxide-purine nucleoside adducts in site-specific mutation studies. Biochemistry 37, 9127-37. (53) Shukla, R., Jelinsky, S., Liu, T., Geacintov, N. E., and Loechler, E. L. (1997) How stereochemistry affects mutagenesis by N 2deoxyguanosine adducts of 7,8-dihydroxy-9,10-epoxy-7,8,9,10tetrahydrobenzo[a]pyrene: Configuration of the adduct bond is

Huang et al. more important than those of the hydroxyl groups. Biochemistry 36, 13263-13269. (54) Schurter, E. J., Sayer, J. M., Oh-hara, T., Yeh, H. J., Yagi, H., Luxon, B. A., Jerina, D. M., and Gorenstein, D. G. (1995) Nuclear magnetic resonance solution structure of an undecanucleotide duplex with a complementary thymidine base opposite a 10R adduct derived from trans addition of a deoxyadenosine N 6-amino group to (-)-(7R,8S,9R,10S)-7,8-dihydroxy-9,10-epoxy-7,8,9,10tetrahydrobenzo[a]pyrene. Biochemistry 34, 9009-20. (55) Yan, S., Shapiro, R., Geacintov, N. E., and Broyde, S. (2001) Stereochemical, structural, and thermodynamic origins of stability differences between stereoisomeric benzo[a]pyrene diol epoxide deoxyadenosine adducts in a DNA mutational hot spot sequence. J. Am. Chem. Soc. 123, 7054-66.

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