Conformational Determinants of Structures in Stereoisomeric Cis

The Nonmutagenic (R)- and (S)-β-(N-Adenyl)styrene Oxide Adducts Are Oriented in the Major Groove and Show Little Perturbation to DNA Structure. Chris...
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Chem. Res. Toxicol. 2000, 13, 811-822

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Conformational Determinants of Structures in Stereoisomeric Cis-Opened anti-Benzo[a]pyrene Diol Epoxide Adducts to Adenine in DNA Jian Tan,†,‡ Nicholas E. Geacintov,*,† and Suse Broyde*,‡ Chemistry and Biology Departments, New York University, New York, New York 10003 Received April 18, 2000

As part of a comprehensive effort to understand the origins of the variety of structural motifs adopted by (+)- and (-)-cis- and trans-anti-[BP]-N2-dG and -N6-dA adducts, with the goal of contributing to the elucidation of the structure-function relationship, we present results of our comprehensive computational investigation of the C10R (+)-cis- and C10S (-)-cis-anti[BP]-N6-dA adducts on the nucleoside level. We have surveyed the potential energy surface of these two adducts by varying systematically, at 5° intervals in combination, the three key torsion angle determinants of conformational flexibility (χ, R′, and β′) in each adduct, creating 373 248 structures, and evaluating each of their energies. This has permitted us to map the entire potential energy surface of each adduct and to delineate the low-energy regions. The energy maps possess a symmetric relationship in the (+)/(-) adduct pair. This symmetry in the maps stems from the mirror image configuration of the benzylic rings in the two adducts, which produces opposite orientations of the BP residues in the C10R and C10S adducts on the nucleoside level. These opposite orientations result from primary steric hindrance between the base and the BP moiety which ensues when a (+) stereoisomer is rotated to the conformation favored by the (-) stereoisomer, and vice versa. Moreover, this steric hindrance manifested on the nucleoside level governs the structure on the duplex DNA level, accounting for observed opposite orientations in high-resolution NMR studies of C10R/C10S adduct pairs.

Introduction Metabolic activation of the environmental precarcinogen benzo[a]pyrene (BP)1 produces among other metabolites the enantiomer pair (+)- and (-)-anti-benzo[a]pyrene diol epoxide (BPDE) (1). Each of these BPDE isomers can react with purines of DNA by cis- and transepoxide opening, leading to C10R (+)-cis/(-)-trans and C10S (-)-cis/(+)-trans-anti adducts to guanine and adenine at their exocyclic amino groups (2, 3). The guanine adducts are more prevalent (2-6). However, minor adducts may well play an important role in initiating carcinogenesis, and the adenine adducts display intriguing structural (7-12) and biological (13-21) properties. To summarize briefly, the conformational themes that have been delineated in the cis and trans-anti adducts (reviewed in ref 22) are as follows: (1) opposite orientations with respect to the DNA helix in a (+)/(-) adduct pair, irrespective of the specific position of the pyrenyl moiety with respect to the B-DNA duplex, or the modified base; (2) a minor groove (23-25) or a base-displaced intercalated conformation in guanine adducts (26-31); and (3) quasi-classical intercalation in adenine adducts (7-12). The interesting finding of a dose-dependent enhancement of mutations targeted to adenines, relative * To whom correspondence should be addressed. N.E.G.: phone, (212) 998-8407; fax, (212) 998-8421; e-mail, [email protected]. S.B.: phone, (212) 998-8231; fax, (212) 995-4015; e-mail, [email protected]. † Chemistry Department. ‡ Biology Department. 1 Abbreviations: BP, benzo[a]pyrene; (+)- and (-)-anti-BPDE, 7(R),8(S)-dihydroxy-9(S),10(R)-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene and the corresponding 7(S),8(R),9(R),10(S) enantiomer, respectively.

to mutations targeted to guanines, with low (nontoxic) versus high concentrations of (+)-anti-BPDE (13-15) has highlighted the possible importance of adenine adducts. Recent site specific mutagenicity studies on the (+)and (-)-cis-anti-[BP]-N6-dA adducts introduced into human and SOS-induced Escherichia coli cell systems reveal distinctly different mutagenic outcomes, governed by the stereochemical nature of the adduct, the cell system, and the neighboring sequence context (16, 17). These studies underscore the complexity governing mutagenesis, while confirming the distinct effect of stereochemistry as one key element governing biological effects. Earlier studies had also revealed differential mutagenic outcomes as well as efficiencies of DNA synthesis past these two adducts in in vivo (18) and in vitro systems (18-20). In addition, in vitro nuclease digestion studies (21) have revealed differential processing of the two lesions. As part of a comprehensive effort to understand the origins of the variety of structural motifs adopted by (+)and (-)-cis- and trans-anti-[BP]-N2-dG and -N6-dA adducts, with the goal of contributing to the elucidation of structure-function relationships, we present results of our comprehensive computational investigation of the C10R (+)-cis- and C10S (-)-cis-anti-[BP]-N6-dA adducts (Figure 1) on the nucleoside level. We surveyed the potential energy surface of these two adducts by varying systematically, at 5° intervals in combination, the three key determinants of conformational flexibility (χ, R′, and β′) of these two adducts, creating 373 248 structures, and evaluating each of their energies. This permitted us to map the entire potential energy surface of each adduct

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Figure 1. Structures of (+)- and (-)-cis-anti-[BP]-N6-dA nucleoside adducts. Torsion angles χ, R′, and β′ are defined as follows: χ, O4′-C1′-N9-C4; R′, N1-C6-N6-C10(BP); and β′, C6-N6-C10(BP)-C9(BP).

and delineate the low-energy regions. The energy maps possess a symmetric relationship in the (+)/(-) adduct pair. This symmetry in the maps stems from the mirror image configuration of the benzylic rings in the two adducts, which produces opposite orientations of the BP residues on the nucleoside level. In turn, these opposite orientations result from primary steric hindrance (22) between the base and the BP moiety which ensues when a (+) stereoisomer is rotated to the conformation favored by the (-) stereoisomer, and vice versa. Moreover, this steric hindrance manifested on the nucleoside level also governs the structure on the duplex DNA adduct level. An adenine cis adduct NMR solution structure exists only for the C10R (+)-cis-anti-[BP]-N6-dA adduct (12). This adduct adopts a quasi-classically intercalated conformation with the BP moiety inserted on the 5′-side of modified dA while maintaining Watson-Crick hydrogen bonding at the modified A. It is plausible that the C10S (-)-cis-anti-[BP]-N6-dA adduct would adopt a similar type of conformation, but with the BP moiety intercalated to the 3′-side of the modified dA.

Experimental Procedures Creating Starting Conformations. We employed the structure of the 10S (+)-trans-anti-[BP]-N6-dA adduct (32) to create the 10S (-)-cis-anti-[BP]-N6-dA adduct by interchanging the position of the Hs and OHs on C7, C8, and C9 of the benzylic ring, using INSIGHTII from MSI, Inc., which was also used to visualize the structures. The resulting structure was energy minimized with AMBER 5.0 (33) using the force field from Cornell et al. (34) with the PARM96.DAT parameter set. This produced the geometry of the 10S (-)-cis-anti-[BP]-N6-dA adduct. The same procedure was employed to produce the geometry of the 10R (+)-cis-anti-[BP]-N6-dA adduct from the structure of the 10R (-)-trans-anti-[BP]-N6-dA adduct (32). Table S1 (Supporting Information) gives conformational features of these structures. Starting structures for the energy calculations were then created with a torsion driver, a program which rotates the

Tan et al. torsion angles χ, R′, and β′ (Figure 1) to chosen values and then computes the coordinates of the resulting structures. The starting structures uniformly surveyed the potential energy surface of the molecule with conformers in which χ, R′, and β′ sampled their 360° conformation space at 5° intervals, in combination, giving a total of 373 248 [(360°/5° ) 72)3] conformers for each adduct. Energy Computation. Energies of each of the 373 248 structures for each adduct were computed with the molecular mechanics program AMBER 5.0 (33), using the force field from Cornell et al. (34) with the PARM96.DAT parameter set. Since there are no negatively charged phosphates in the nucleoside, Na+ counterions were not needed. A sigmoidal distance-dependent dielectric function (35) which is a suitable treatment for the dielectric constant (36) was employed in the Coulombic term of the force field, to model the dielectric effects of solvent water. Parameters added to the AMBER 5.0 force field for the [BP]N6-dA adducts are the same as those given previously (32) (Table S2, Supporting Information), except for the partial charges for the modified nucleosides; these were computed with Gaussian 94 (37) at the 6-31G* basis set level which is compatible with the rest of the AMBER 5.0 force field, and then the least-squares charges fitting algorithm RESP (38) provided with AMBER 5.0 was used to fit the charge to each atomic center. Partial charges, atom types, and topology information are given in Table S3 (Supporting Information). Computations were carried out at the Department of Energy’s National Energy Research Supercomputer Center (Berkeley, CA), at the National Science Foundation National Partnership for Advanced Computational Infrastructure (San Diego, CA), and on our own SGI workstations. Statistical Weights and Thermodynamic Quantities. The computed energies were used to construct both threedimensional energy surfaces as a function of the χ, R′, and β′ torsion angles and two-dimensional energy contour slices through them; slices were made at 5° intervals of χ as a function of R′ and β′, and also at selected β′ values as a function of χ and R′. The program TECPLOT was used for this purpose. These plots revealed five low-energy wells or domains (Figure 2). This permitted grouping the full data set into five regions, each encompassing one of the five low-energy domains: region 1, χ ) 0-120° and 305-355°, R′ ) 270-355° and 0-85°, β′ for the (+) adduct ) 0-175°, β′ for the (-) adduct ) 180-355°; region 2, χ ) 125-300°, R′ ) 270-355° and 0-85°, β′ ) 0-355°; region 3, χ ) 125-300°, R′ ) 90-265°, β′ ) 0-355°; region 4, χ ) 0-120° and 305-355°, R′ ) 90-265°, β′ ) 0-355°; region 5, χ ) 0-120° and 305-355°, R′ ) 270-355° and 0-85°, β′ for the (+) adduct ) 180-355°; β′ for the (-) adduct ) 0-175°. With this grouping, we computed fractional statistical weights as well as conformational free energies, enthalpies, and entropies as described in detail previously (32, 39, 40).

Results Near Mirror Image Symmetry of Adducts Derived from (+)- and (-)-cis-anti-BPDE Enantiomers. Figure 2 depicts the three-dimensional AMBER energy topographies as a function of the torsion angles χ, R′, and β′, up to 8.5 kcal/mol above the lowest energy, for the 10R (+)- and 10S (-)-cis-anti-[BP]-N6-dA adducts. Five low-energy domains (potential energy wells) are found for each of the two adducts. Domains I, IV, and V have syn glycosidic torsion angles χ, and domains II and III are in the anti region. Table 1 summarizes the ranges in the values of χ, R′, and β′ that characterize each domain. Table 2 gives the number of conformers in each 1 kcal/ mol energy shell from 0 to 5 kcal/mol. The energy landscapes of the 10R (+)- and 10S (-)cis-anti adducts were studied further as a function of the torsion angles R′ and β′ that determine the relative orientation of the BP with respect to the nucleoside

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Figure 2. Three-dimensional χ, R′, and β′ energy topographies to 8.5 kcal/mol. I-V represent the five low-energy domains. Torsion angles cycle over 360°, and 359° is actually contiguous with 0°: (A) the (+)-cis-anti-[BP]-N6-dA adduct and (B) the (-)-cis-anti-[BP]N6-dA adduct. Table 1. Domains of Low-Energy Wells in 10R (+)- and 10S (-)-cis-anti-[BP]-N6-dA Adductsa R′ domain (deg) domain

χ domain (deg)

I II III IV V

35 + 55/-85 215 + 95/-50 215 + 70/-40 35 + 10/-10 35 + 20/-40

(+) adduct

(-) adduct

-5 ( 50 5 ( 50 -5 ( 40 5 ( 40 165 ( 30 195 ( 30 165 ( 45 195 ( 45 35 ( 20 325 ( 30

β′ domain (deg) (+) adduct

(-) adduct

100 ( 40 100 ( 30 100 ( 25 100 ( 40 240 ( 15

-100 ( 40 -100 ( 30 -100 ( 25 -100 ( 40 120 ( 20

a Domains of the 10 kcal/mol surface were approximated to (5°, from the approximate well center. χ and R′ domains are the same for the (+) and (-) adducts. For R′, this is the case because 0° and 180° are the same as their sign-inverted values.

residues (Figure 1). Specifically, we investigated slices through the approximate centers of the wells in the syn (35°) and anti (215°) domains of χ, from the energy contour maps in the R′, β′ plane (Figure 3). In addition, Figures S1 and S2 (Supporting Information) show the full set of R′, β′ energy contour maps at 5° intervals of χ for the (+)- and (-)-cis-anti-[BP]-N6-dG adducts. The twodimensional 10R (+)- and 10S (-)-cis-anti adduct maps show a clear symmetry between them. In particular, the map for the (+) adduct can be essentially transformed into that of the (-) adduct by inverting the sign of the torsion angles R′ and β′. This corresponds to a 180° rotation about a central symmetry axis positioned perpendicular to the map plane at R′ and β′ ) 180°. Consequently, a given energy feature in a specific R′, β′ region of the (+) adduct map has a corresponding feature in the (-) adduct map in the -R′, -β′ region. This torsion angle symmetry is a hallmark of mirror image pairs of molecules, although the symmetry is not exact in the nucleoside adduct pair. Transitions between Domains. Possible pathways between conformational domains are suggested from our results. The three-dimensional χ, R′, β′ energy topography of Figure 2 indicates that a path may exist between syn domain I and anti domain II along the χ ) 310° trajectory, above 8.5 kcal/mol. To elucidate this possible

connectivity further, a two-dimensional energy contour slice was generated through the approximate centers of domains I and II, at β′ ) 105° for the (+)-cis-anti adduct and 255° (-105°) for the (-)-cis-anti adduct. This map, shown in Figure S3 (Supporting Information), reveals the connecting path at ∼9-10 kcal/mol. Figure S3 also reveals a path between syn domain IV and anti domain III at χ ) 310° at ∼11-12 kcal/mol. These paths represent upper limits in the energy pathways, since our computations could only survey the key sources of conformational flexibility, the torsion angles χ, R′, and β′. Low-Energy Conformations. Representative structures of each of the five low-energy domains of the (+)and (-)-cis-anti adducts are shown in Figures 4-6. These figures show that for each domain, the (+)-cis-anti adduct structure is nearly a mirror image of the (-)-cis-anti structure, with the mirror image symmetry broken by the sugar residues, whose O4′ atoms point in the same rather than the opposite directions in the (+)- and (-)cis-anti adducts in each domain. The five structural types are approximately related to one another by torsional rotations as follows: χ + ∼180°

domain I 98 domain II R′ + ∼180°

domain II 98 domain III χ + ∼180°

domain III 98 domain IV β′ ( ∼140°

domain I 98 domain V Syn domain I differs from anti domain II in the orientation of the sugar relative to the adenine residue. A ∼180° rotation of R′, which converts domain II to domain III, turns the BP long axis in the opposite direction. A ∼180° rotation of χ then produces domain IV. Syn domain V is produced from syn domain I by a ∼140° rotation of β′. Steric Hindrance Produces Opposite Orientations. The steric origin of the opposite orientations was

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Table 2. Number of Conformers in 1 kcal/mol Shells in 10R (+)- and 10S (-)-cis-anti-[BP]-N6-dA Adducts, to 5 kcal/mol 0-1 kcal/mol

1-2 kcal/mol

2-3 kcal/mol

3-4 kcal/mol

4-5 kcal/mol

total

domain

(+)

(-)

(+)

(-)

(+)

(-)

(+)

(-)

(+)

(-)

(+)

(-)

I II III IV V total

99 0 0 0 0 99

95 0 0 0 0 95

172 0 0 0 0 172

178 0 0 0 0 178

244 0 0 0 0 244

251 0 0 0 0 251

270 39 0 29 0 338

271 27 0 0 0 298

287 345 0 125 0 757

310 328 0 57 0 695

1072 384 0 154 0 1610

1105 355 0 57 0 1517

Figure 3. R′, β′ energy contour maps to 25 kcal/mol. I-V represent the five low-energy domains. Torsion angles cycle over 360°, and 359° is actually contiguous with 0°: (A) the (+)-cis-anti-[BP]-N6-dA adduct, with χ ) 35° (syn); (B) the (-)-cis-anti-[BP]-N6-dA adduct, with χ ) 35° (syn); (C) the (+)-cis-anti-[BP]-N6-dA adduct, with χ ) 215° (anti); and (D) the (-)-cis-anti-[BP]-N6-dA adduct, with χ ) 215° (anti).

elucidated with contour maps which computed just the van der Waals component of the total energy (Figure 7). These van der Waals energy maps reveal the same overall regions below 25 kcal/mol, with the same symmetries as those employing the full energy potential (Figure 3). The energy boundaries above 25 kcal/mol are determined by repulsive steric components of the Lennard-Jones potential employed to compute van der Waals energies (41). Therefore, steric repulsions are predominantly responsible for the starkly different energy landscapes in the (+)- and (-)-cis adducts and for the symmetries between them. The energy regions below 25 kcal/mol contain differing details in the full energy maps of Figure 3 and the van der Waals component maps of Figure 7, due to modulating effects from electrostatic, torsional, and other energy terms included in the total energy maps. In addition, small differences in detail stem

from the non-mirror image nature of the sugars and attached C4′ and C5′. To further illustrate the relationship between the steric repulsions and the energy landscape, each structure shown in Figure 4 was rotated into that preferred by its stereoisomer, by a 180° rotation around β′ (Table 1). The transformed structures are shown in Figures 8 and 9. For domains I and II of both (+) and (-) adducts, after rotation, there is crowding in the region between N1 of adenine and the C6A-C10A region of the BP moiety; for domains III and IV, the rotated structures are disfavored because of crowding between N7 of adenine and C6A of the BP moiety. The rotated structures of domain V, however, show no serious crowding because rotated domain V is rather similar to the normal domain I (see Table 1). Statistical Weights and Thermodynamic Parameters. We computed the fractional statistical weights

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Figure 4. Color views of representative structures for (+)- and (-)-cis-anti-[BP]-N6-dA adducts. I-V represent the five low-energy domains. In each pair, the left structure is the (+)-cis-anti-[BP]-N6-dA adduct and the right structure is the (-) adduct. χ, R′, and β′ values and energies, ∆E, relative to the global minimum are as follows. Domain I: (+) adduct, 35°, 355°, 105°; (-) adduct, 35°, 5°, 225°; ∆E, (+) 0.00, (-) 0.02 kcal/mol. Domain II: (+) adduct, 215°, 355°, 105°; (-) adduct, 215°, 5°, 255°; ∆E, (+) 3.77 kcal/mol, (-) 3.88 kcal/mol. Domain III: (+) adduct, 215°, 165°, 105°; (-) adduct, 215°, 195°, 255°; ∆E, (+) 7.29 kcal/mol, (-) 8.07 kcal/mol. Domain IV: (+) adduct, 35°, 165°, 105°; (-) adduct, 35°, 195°, 255°; ∆E, (+) 3.51 kcal/mol, (-) 4.25 kcal/mol. Domain V: (+) adduct, 40°, 35°, 240°; (-) adduct, 40°, 325°, 120°; ∆E, (+) 7.22 kcal/mol, (-) 7.00 kcal/mol. The view is edge-on along the adenine with C8H directed toward the viewer. Table 3. Statistical Weights and Thermodynamic Parameters of Low-Energy Domains in 10R (+)- and 10S (-)-cis-anti-[BP]-N6-dA Adductsa W (%) domain (+) I II III IV V

(-)

G (kcal/mol) (+)

(-)

H (kcal/mol) TS (kcal/mol) (+)

(-)

(+)

(-)

99.3 99.5 -2.449 -2.437 0.865 0.892 3.314 3.329 0.5 0.4 0.712 0.772 4.503 4.559 3.791 3.787 0.0 0.0 4.267 5.035 8.053 8.726 3.786 3.692 0.2 0.1 1.157 1.865 4.477 5.117 3.319 3.252 0.0 0.0 4.856 4.574 7.924 7.660 3.068 3.086

a Statistical weights, W, are given as a percentage of the population. G, H, and S are conformational free energy, enthalpy, and entropy, respectively. T ) 300 K.

together with conformational free energies, enthalpies, and entropies for each domain of the (+)- and (-)-cisanti-[BP]-N6-dA adduct, as described in earlier work (32, 40, 41). Table 3 gives thermodynamic parameters and statistical weights. Computation of these thermodynamic parameters for each domain treats these as individual species, which seems justifiable on the basis of the apparent barriers between the domains (Figures 2, 3, and S3). Differences in population between the (+) and the (-) adduct stem from the symmetry-breaking effect of the sugar residues and the C4′ and C5′ atoms. We note that domains III and V, with ∆G values ∼7 kcal/mol above that of the most favored domain (I), have essentially no statistical weights on the nucleoside level

(∼0.001% or lower). However, domain III becomes important on the duplex DNA level, and domain V could provide one possible avenue for overcoming the opposite orientation phenomenon, as will be discussed below.

Discussion Mirror Image Benzylic Rings Cause Opposite Orientations. Five pairs of low-energy conformational domains for the (+)- and (-)-cis-anti-[BP]-N6-dA adducts have been computed. In each domain, the pyrenyl moieties adopt a near mirror image symmetry as viewed along the HN8-containing adenine edge in the (+)-cis/ (-)-cis adduct pair, resulting in opposite orientations of the BP residue (Figure 4). The energy landscape of the five adducts reveals this near mirror image symmetry; they can nearly be transformed into one another by sign inversion of R′ and β′. Thus, a given feature in the (+) adduct map at a certain R′, β′ region is approximately found in the -R′, -β′ region of the (-) adduct map (Figure 3). This is rooted in the near mirror image symmetry of the BP benzylic ring in the two adducts. The low-energy regions of R′ are at ∼0° and ∼180°, which are the same when their signs are inverted (0° ) 0°; 180° ) -180°). Consequently, the low-energy domains of the 10S (-)and 10R (+)-cis-anti adducts actually differ from one another only by a ∼180° rotation in the key torsion angle β′ (Table 1). Rotation about β′, the bond closest to the

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Figure 5. Stereoviews of the (+)-cis-anti-[BP]-N6-dA adduct. Representative structures of Figure 4. I-V represent the five low-energy domains.

Figure 6. Stereoviews of the (-)-cis-anti-[BP]-N6-dA adduct. Representative structures of Figure 4. I-V represent the five low-energy domains.

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bulky BP, from the region preferred by one stereoisomer into the region preferred by the other isomer causes crowding between atoms on the adenine and the benzylic ring of the BP residues (Figures 8 and 9), termed “primary” steric hindrance (22). Thus, primary steric hindrance plays an important part in keeping β′ out of high-energy regions (Figure 3) and near the region that is favored for each isomer. This phenomenon is even more pronounced in the case of the more crowded cis adducts than in the trans adducts because the C9-OH and C8-OH are on the same side of the benzylic ring as guanine and sugar in cis adducts and on the opposite side in the trans adducts (Figures 1 and 4-6). Structural Domain Preferences. (1) Syn and Anti Conformations. Domains I (syn) and II (anti) are essentially the same except for the difference in glycosidic torsion angles. The stabilization of domain I compared to domain II is enthalpic in origin as can be seen from the data in Table 3. The enthalpic stabilization probably stems mainly from the fact that the sugar residue is in contact with both aromatic rings of the adenine in the syn conformation; when the adenine residue is in the anti orientation, the sugar contacts only its five-membered ring. In addition, a small favorable electrostatic interaction between the deoxyadenosine N3 and the sugar H(O5′) [N3-H(O5′) distance ) 4.04 Å] may contribute somewhat to the enthalpic stabilization; H(O5′) and N3 often form a hydrogen bond when the adenine glycosidic bond adopts the syn orientation (42), although they are not hydrogen bonded in this case. The stabilization characteristic of the syn domain is therefore intrinsic to the deoxyadenosine moiety and is not significantly influenced by the BP residue. Syn domain IV is favored over anti domain III for the same reason. (2) r′ and β′ Regions. Torsion angle R′ is centered at 0° or 180° in each of the low-energy domains (Table 1). These two R′ regions are favored because of the maximal overlap of the adenine aromatic ring π orbitals with the N6 lone pair p orbital when R′ ∼ 0° or 180°, since the N6 p and the adenine ring π orbitals are maximally parallel in these cases. With these R′ values, the preferred β′ values are in the 90° and 270° (-90°) regions. Steric crowding between BP and adenine occurs when other combinations of R′ and β′ are adopted. The R′ ) 0° region is preferred over the 180° region in these adenine mononucleoside adducts (see Tables 1 and 3). This is the case for mononucleosides, because the adenine five-membered ring lies over the benzylic ring when R′ is in the 180° region, while it is positioned farther away from the benzylic ring when R′ is near 0° (see Figures 5 and 6). This leads to a larger, more entropically favored R′ ) 0° region (Table 2). However, in normal duplexes, the R′ ∼ 180° region is required to form the Watson-Crick hydrogen bond that involves the free hydrogen atom on N6-dA. Consequently, the R′ ) 180° region is more favored in duplexes. In contrast, in the 10S (-)- and 10R (+)-cis-anti-N2dG adducts, the five-membered ring of guanine is on the edge of the base opposite the linkage site to BP, far from the benzylic ring, and does not influence the preferred R′ region. In the guanine adducts, the R′ ) 0° region is somewhat disfavored because of proximity between H1(N1) and the benzylic ring, which causes the conformational space to be more constrained than when R′ is in the 180° region (40).

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Figure 7. R′, β′ van der Waals energy component maps to 25 kcal/mol. I-V represent the five low-energy domains. V and VI are higher-energy domains that do not contribute to the statistical weights. Torsion angles cycle over 360°, and 359° is actually contiguous with 0°: (A) the (+)-cis-anti-[BP]-N6-dA adduct, with χ ) 35° (syn); (B) the (-)-cis-anti-[BP]-N6-dA adduct, with χ ) 35° (syn); (C) the (+)-cis-anti-[BP]-N6-dA adduct, with χ ) 215° (anti); and (D) the (-)-cis-anti-[BP]-N6-dA adduct, with χ ) 215° (anti).

cis and trans-N6-dA Adduct Structures. Role of the Absolute Configuration about C10, C9, and C8. (1) C10 Configuration. The C10R (-)-trans-anti-[BP]N6-dA adduct has the same absolute configuration at the benzylic ring C10 as the C10R (+)-cis-anti-[BP]-N6-dA adduct, and similarly, the C10S (+)-trans-anti adduct has the same absolute configuration as the C10S (-)-cis-anti adduct. The absolute configuration at C10 plays the key role in governing the opposite orientations of the BP moiety in 10R and 10S adduct pairs (40), due to the steric crowding engendered when a C10R adduct adopts the β′ domain of its C10S isomer and vice versa. This is manifested in the NMR solution structures of the C10R (-)-trans-anti-[BP]-N6-dA duplexes (10, 11) when compared to the C10R (+)-cis-anti-[BP]-N6-dA duplex (12). In both cases, the benzo[a]pyrenyl ring systems are on the 5′-side of the modified adenine, as shown in Figure 10. Moreover, on the nucleoside level, the structures in each domain of the C10R adducts [(+)-cis/(-)-trans] and of the C10S adducts [(-)-cis/(+)-trans] are essentially the same (Figure 4) (32), and the similarity on the DNA level originates with the nucleosides. (2) C9 and C8 Configurations. In the cis adducts, the C9-OH and C8-OH are on the same side of the benzylic ring as the C10-N6 linkage, while they are on the opposite side of the benzylic ring in the case of the trans adducts. Therefore, in the cis configuration, the

O9-OH and O8-OH are in a comparatively more crowded situation with respect to the N7 edge of adenine. This character of cis-dA adducts has an impact on its lowenergy regions, making the energy contour maps in the R′, β′ plane (Figure 3) of cis-dA adducts different from those of trans-dA adducts (32). Specifically, the R′, β′ ranges at low energy are narrower in the cis adducts than in the trans adducts, especially in the torsion angle β′. Both the area (Figure 3) and the number of conformers in low-energy domains (Table 2) are lower in cis adducts than in trans adducts (32). This makes the conformations in domain III and IV less favorable than in the trans adducts. As a result, the statistical weight of the less favored domain III becomes essentially zero on the nucleoside level. In addition, there are no paths linking domains I and IV, or II and III, as in the trans adducts. A similar phenomenon was observed in a comparison of cis- and trans-anti-[BP]-N2-dG adducts (39, 40). Preferred Domains and Relevance to DNA Duplexes. For the case of the cis-anti-[BP]-N6-dA adducts, only one high-resolution NMR solution structure in a DNA duplex is available to date, namely, the one with (+)-cis-anti adduct stereochemistry (12). Table 4 gives χ, R′, and β′ values in that NMR structure, which correspond to our computed domain III. Preference for domain III stems from the fact that in a duplex where Watson-Crick base pairing is maintained, χ must be anti

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Figure 8. Stereoviews of (+) adduct structures rotated to wells of the (-) adduct as detailed in Figure 6. I-V represent the five low-energy domains.

Figure 9. Stereoviews of (-) adduct structures rotated to wells of the (+) adduct as detailed in Figure 5. I-V represent the five low-energy domains.

and R′ must be in the ∼180° region, as in domain III. Domain I, which is most preferred in the nucleoside, has

Tan et al.

the glycosidic bond χ syn and R′ near 0°. Thus, since a modified nucleoside is more flexible than a modified duplex, a conformational domain that is higher energy in the nucleoside can predominate in the duplex. However, in modified single strands or in a duplex containing a mismatch at the lesion site, domains with χ syn and/or R′ near 0° would be feasible. Adenine versus Guanine Adducts. N6 versus N2 Linkage Governs Low-Energy Domains in Nucleosides and BP Orientation in Duplexes. (1) Nucleosides. In guanine, the N2 position and the N9 linkage to sugar are on the same edge of the purine ring. Therefore, the interaction between the sugar ring and the bulky BP moiety linked to N2 is a primary factor in determining the preferred low-energy domains. This effect is especially important when the glycosidic linkage adopts the syn conformation, since N2 is closer to the sugar in the syn than in the anti orientation. In the adenine adducts, the BP moiety at N6 is separated from the sugar by the purine rings, and the interaction between the sugar and the BP moiety becomes less important. In cis-anti-[BP]N6-dA adducts, the most favored domain (with a statistical weight of >99.0%) is domain I with a syn conformation of the glycosidic linkage. This is mainly due to an inherent preference for the syn conformation of dA, which stems primarily from favorable electrostatic and van der Waals interactions between sugar and both aromatic adenine rings in the syn conformation, while only the five-membered adenine ring is contacted by the sugar in the anti conformation. However, in the cis-anti-[BP]-N2dG adducts, crowding between the sugar and the BP moiety caused by the syn conformation (40) dominates, and hence, these adducts favor anti domain III. In [BP]-N6-dA adducts, a different crowding pattern becomes possible because the N6-BP linkage site is next to the adenine N7 edge of the five-membered ring. This produces steric constraints between the N7 edge and the benzylic ring on the BP moiety when R′ = 180°. Therefore, domains III and IV with R′ centered around 180° are less favored in both trans and cis adenine adducts than in the guanine adduct analogues, due to the crowding between the C10-C9 edge in the benzylic ring and the adenine N7 edge. This trend is accentuated in the cisanti-[BP]-N6-dA adducts, with the C9-OH and C8-OH on the same side as the C10-N6 linkage of the benzylic ring, and therefore closer to the N7 edge than in the transanti-[BP]-N6-dA adducts. For this reason, domain III has essentially no statistical weight in the cis-anti-[BP]-N6dA adducts. Interestingly, we find an analogy in [BP]N2-dG adduct domain preferences, where the NH1 next to the N2 linkage site causes domain III with R′ = 180° to be favored over domain II, where R′ = 0° due to steric constraints in domain II (39, 40). (2) Duplexes. In normal B-DNA duplexes, the N6 linkage site of [BP]-N6-dA adducts is in the major groove. However, a major groove position for the BP moiety conformation is less preferred due to the entropic hydrophobic energy penalty associated with exposing the aromatic pyrene rings to aqueous solvent in the major groove (32). This results in the quasi-classical intercalation of the BP moiety in the adenine adducts (7-12), in which the DNA duplex stretches and unwinds to accommodate the inserted BP residue, without rupturing the Watson-Crick base pair when a normal partner T is present. In [BP]-N2-dG adducts, the N2 linkage site in normal B-DNA is in the minor groove. In the cis-anti-

Conformational Determinants in cis-[BP]-dA Isomers

Chem. Res. Toxicol., Vol. 13, No. 9, 2000 819

Figure 10. (A) NMR solution structure of the (+)-cis-anti-[BP]-N6-dA adduct with normal partner T (12). Central-modified duplex 5-mer is shown in stereo. (B) NMR solution structures of the (-)-trans-anti-[BP]-N6-dA adduct with normal partner T (10). Centralmodified duplex 5-mer is shown in stereo. Table 4. χ, r′, β′ Orientation in NMR Solution Structure of the 10R (+)-cis-anti-[BP]-N6-dA Adduct

a

duplex type

χ, R′, β′ (deg)

normal partnera

258 ( 6°, 160 ( 10°, 107 ( 14°

From ref 12.

[BP]-N2-dG adducts, the minor groove conformation is disfavored because O9H and O8H would be directed into the groove (40). Moreover, classical intercalation is less favored in the BP guanine adducts, possibly due to greater difficulty in insertion of the covalently linked BP, without displacement of the modified G, from the narrow minor groove side. Consequently, base-displaced intercalation is adopted (26, 28-30). Relationship between 3′ and 5′-Directionality, Opposite Orientations, and the Torsion Angle β′. We note from Table 1 that β′ is always rotated ∼180° oppositely in the (+)- and (-)-cis-anti-[BP]-N6-dA adduct pair. In domains I-IV, β′ is in the ∼90° region in the 10R (+)-cis adduct and in the ∼-90° (270°) region in the 10S (-)-cis adduct. (However, in domain V, which has essentially no statistical weight, the favored β′ regions are reversed.) Thus, the opposite orientations are governed by a favored β′ of ∼90° in the 10R adduct and ∼-90° in the 10S adduct, and steric hindrance disfavors rotation into the wrong β′ domain. In the observed domain III for the C10R (+)-cis-anti-[BP]-N6-dA adduct, the BP moiety is placed on the 5′-side of the modified dA (12), and it is likely that the (-)-cis-anti adduct is intercalated on the 3′-side, by analogy to the C10R (-)trans-anti and C10S (+)-trans-anti adducts (8-11). In a normal partner context, domain III would again be anticipated, but in a mismatched context, domains I or V (χ is syn, R′ = 0°), II (R′ = 0′), or IV (R′ = 0°) could be possible.

Our computed domains II and IV, which have not yet been observed, place the BP on the 5′-side of the modified dA residue in the 10S isomer, and on the 3′-side in the 10R isomer. The key point is the opposite orientations in a given domain adopted by the 10S/10R pair, which is determined by the favored β′ region, rather than the 3′or 5′-direction. Domains I, II, and IV could be particularly relevant to cases where Watson-Crick pairing is not possible, such as in single strands or with a mismatched base opposite the modification site; then domain II when R′ ) 0° or domains I and IV with χ in the abnormal syn region could become more favored. Another point, which can be discerned from Figure 4, is that it is possible for a (+) stereoisomer to adopt one domain and a (-) stereoisomer to adopt another (domains I and IV, or I and III), such that the BP moiety is pointing in the same direction relative to the 3′- or 5′-sugar direction in both isomers, while β′ remains in its preferred region in each case. In addition, the following question arises. Could the wrong β′ for a given stereoisomer domain be accommodated? Our results indicate that is possible through the least favored domain V, which brings the BP moiety of the (+) adduct near domain I of the (-) adduct, and vice versa (see Table 1).

Summary and Significance This work completes a series of comprehensive computational studies on the nucleoside level of C10S (+)and C10R (-)-trans-anti-[BP]-N2-dG adducts (39), C10R (+)- and C10S (-)-cis-anti-[BP]-N2-dG adducts (40), C10S (+)- and C10R (-)-trans-anti-[BP]-N6-dA adducts (32), and C10R (+)- and C10S (-)-cis-anti-[BP]-N6-dA adducts. In these efforts, we have elucidated fundamental chemical origins governing the variety of conformational motifs

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adopted by these differing stereoisomeric lesions in DNA duplexes. Accordingly, we summarize here the principles that we have deduced from these fruitful nucleoside adduct studies. (1) Opposite orientations of the BP moiety in a given (+)/(-) adduct pair are rooted in the exact mirror image nature of the benzylic ring in the (+)/(-) pair. The torsion angle β′ plays a key role in determining the preferred domains for each isomer, which therefore favor the 90° and -90° regions. The absolute configuration at the C10 atom determines the favored β′ domains (since rotation about the C10-N bond is β′) and hence the preferred orientation, and the absolute configurations of the OH groups at C9 and C8 do not play a role. Primary steric hindrance (22) between the benzylic ring and atoms on the guanine or adenine occurs when a stereoisomer is rotated into the β′ domain favored by its partner in the (+)/(-) pair. Structures of the adducts in DNA duplexes are governed by the favored β′ domains on the nucleoside level, which are found in the duplexes and produce the opposite orientations observed in all (+)/(-) adduct pairs. Opportunities for an adduct pair to be oriented in the same direction exist, but only by exacting a price in energy, either involving occupancy of structural domains that are up to 7 kcal/mol higher in conformational free energy than the lowest-energy domain in nucleosides (without violating steric hindrance) or necessitating the energetically more costly rotations into the wrong β′ domain of a given isomer, with the attendant steric hindrance. It is therefore plausible that preservation of the correct β′ domain may well be maintained in a biological setting, since the primary steric hindrance, engendered in the wrong β′ domain, is inherent in the BP to base covalent linkage and cannot be avoided. (2) Cis adducts are more sterically constrained than trans adducts because the linkage between BP and the base is on the same side of the benzylic ring as O9H and O8H in cis adducts, while the BP-base linkage is on the side of the benzylic ring opposite O9H and O8H in the trans adducts. This explains why cis-anti-[BP]-N2-dG adducts do not occupy the B-DNA minor groove, while trans adducts can reside in the minor groove; when the cis adducts are positioned in the minor groove, O9H and O8H point into the narrow groove, causing crowding, while they point outward in the trans adducts. Therefore, the cis-anti-[BP]-N2-dG adducts adopt base-displaced intercalated conformations, with the modified G and its attached benzylic ring displaced into the major groove in the case of the C10R (+) adduct, and to the opposite minor groove side in the C10S (-) adduct (26, 28, 29). Thus, the absolute configuration of the C9-OH and C8-OH groups governs the structures of cis and trans adducts in N2-dG adducts. (3) C10S (+)- and C10R (-)-trans-anti-[BP]-N2-dG adducts adopt a minor groove position in full duplexes (23-25) [5′-directed in C10S (+) adducts and 3′-directed in C10R (-) adducts] because this orientation retains Watson-Crick base pairing, normal base-base stacking, and exacts the price of exposing only one face of the aromatic pyrenyl ring system to aqueous solvent. However, when the partner residue to the modified dG is missing (in a deletion duplex), a base-displaced intercalated conformation is adopted with opposite orientations, so the modified guanine and its attached benzylic ring are displaced into the minor groove side of the helix cylinder in the C10S (+) adduct, and the opposite major

Tan et al.

groove side in the C10R (-) adduct (27, 31). This conformation is preferred in the deletion duplexes because the opportunity for normal Watson-Crick base pairing no longer exists since the partner here is missing; hence, the cost of unstacking the modified guanine and displacing it into a groove is compensated by the removal of the aromatic pyrene rings from the aqueous milieu, avoiding the entropic hydrophobic penalty, as well as by providing enthalpic stabilization via new stacking interactions between the BP and adjacent bases. It is noteworthy that the structural motifs of the C10R (+)-cisanti-[BP]-N2-dG adducts [a normal full duplex (26) and a deletion duplex (28)] are the same as the motif of C10R (-)-trans-anti-[BP]-N2-dG adduct in the deletion duplex (31). Also, the C10S (-)-cis-anti-[BP]-N2-dG adduct conformations in a normal, full duplex (29) are the same as the C10S (+)-trans-anti-[BP]-N2-dG adduct conformations in a deletion duplex (27). The same absolute configuration at C10 in (+)-trans/(-)-cis and (-)-trans/ (+)-cis adducts governs these structures. (4) The [BP]-N6-dA adducts adopt classical intercalation conformations (7-12) with the BP moieties inserted into the double helix because of the following favorable factors: Watson-Crick base pairing is maintained in duplexes with the normal partner; additional favorable stacking interactions between the BP ring system and adjacent base pairs are achieved; and the aromatic pyrene ring system avoids the entropic hydrophobic penalty, being sequestered from aqueous solvent. The energetic price for this conformation is stretching and unwinding the B-DNA duplex. Both cis and trans adducts can adopt this conformation because the absolute configuration at C9-OH and C8-OH, with the OHs on the same side of the benzylic ring as the adenine N6-C10 linkage in cis adducts, does not interfere with this structure. However, the C10R adducts [(-)-trans/(+)-cis] and the C10S adduct for which data are available [(+)trans] are oppositely oriented, to the 5′-side of the modified A in the (-)-trans/(+)-cis adducts and to the 3′side of the modified A in the (+)-trans adduct, adopting β′ domains favored on the nucleoside level. High-resolution NMR structures are not yet available at this writing for the C10S (-)-cis adduct, but it is plausible that the BP moiety would be intercalated on the 3′-side of the modified adenine residue. (5) The [BP]-N2-dG adducts appear less likely to adopt the classical intercalation position of the [BP]-N6-dA adducts because the guanine amino group to which the BP is tethered is on the narrow minor groove side of the B-DNA helix, providing less maneuverability for insertion into the duplex above or below the modified base, without rupturing the Watson-Crick hydrogen bonds. However, classical intercalation can be more easily achieved by the [BP]-N6-dA adducts because the adenine amino group to which the BP is covalently linked is in the roomy B-DNA major groove, more readily permitting the tethered BP to insert into the helix without base displacement. Furthermore, the entropic hydrophobic penalty for remaining in the groove is less in the minor groove where only one face of the aromatic pyrene ring is exposed to solvent (23-25) than in the major groove where both faces can be exposed (32). The stereochemical origins of the differing conformational themes adopted by stereoisomeric benzo[a]pyrenyl moieties bound to N2-dG or N6-dA elucidated from our computational studies govern the conformations adopted

Conformational Determinants in cis-[BP]-dA Isomers

in DNA by these stereoisomeric lesions, and are likely to play an important role in producing their differing biological properties during replication, transcription, and repair.

Acknowledgment. This research is supported by NIH Grants CA 28038 and RR-06458, DOE Grant DE-FG0290ER60931 to S.B., and NIH Grant CA-20851 to N.E.G. Supporting Information Available: Table S1 listing conformational features of energy-minimized modified nucleosides in the starting structures, Table S2 giving additional force field parameters, Table S3 giving partial charges, topology, and atom type assignments for the 10R (+)- and 10S (-)-cis-anti[BP]-N6-dA nucleoside adducts, Figures S1 and S2 showing the full set of R′, β′ energy contour maps at 5° intervals of χ for the 10R (+)- and 10S (-)-cis-anti-[BP]-N6-dA adducts, respectively, and Figure S3 showing χ, R′ energy contour maps to 25 kcal/ mol with β′ ) 105° for the (+) adduct and 255° (-105°) for the (-) adduct. This material is available free of charge via the Internet at http://pubs.acs.org.

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References (1) Conney, A. H. (1982) Induction of microsomal enzymes by foreign chemicals and carcinogenesis by polycyclic aromatic hydrocarbons. Cancer Res. 42, 4875-4917. (2) Meehan, T., and Straub, K. (1979) Double stranded DNA stereoselectively binds benzo[a]pyrene diol epoxides. Nature 277, 410412. (3) Cheng, S. C., Hilton, B. D., Roman, J. M., and Dipple, A. (1989) DNA adducts from carcinogenic and noncarcinogenic enantiomers of benzo[a]pyrene dihydrodiol epoxide. Chem. Res. Toxicol. 2, 334-340. (4) Weinstein, I. B., Jeffrey, A. M., Jennette, K. W., Blobstein, S. H., Harvey, R. G., Harris, C., Autrup, H., Kasai, H., and Nakanishi, K. (1976) Benzo[a]pyrene diol epoxides as intermediates in nucleic acid binding in vitro and in vivo. Science 193, 592-594. (5) Jeffrey, A. M., Jennette, K. W., Blobstein, S. H., Weinstein, I. B., Beland, F. A., Harvey, R. G., Kasai, H., Miura, I., and Nakanishi, K. (1976) Benzo[a]pyrene-nucleic acid derivative found in vivo: structure of benzo[a]pyrenetetrahydrodiol epoxide-guanine adduct. J. Am. Chem. Soc. 98, 5714-5715. (6) Osborne, M. R., Beland, F. A., Harvey, R. G., and Brookes, P. (1976) The reaction of (+)-7R,8β-dihydroxy-9β,10β-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene with DNA. Int. J. Cancer 18, 362368. (7) Schurter, E. J., Sayer, J. M., Oh-hara, T., Yeh, H. J. C., 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 N6-amino group to (-)-(7R,8S,9R,10S)-7,8-dihydroxy-9,10-epoxy-7,8,9,10tetrahydrobenzo[a]pyrene. Biochemistry 34, 9009-9020. (8) Yeh, H. J., 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 nonanucleotide duplex with a dG mismatch opposite a 10S adduct derived from trans addition of a deoxyadenosine N6-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. (9) Schwartz, J. L., Rice, J. S., Luxon, B. A., Sayer, J. M., Xie, G., Yeh, H. J., Liu, X., Jerina, D. M., and Gorenstein, D. G. (1997) Solution structure of the minor conformer of a DNA duplex containing a dG mismatch opposite a benzo[a]pyrene diol epoxide/ dA adduct: glycosidic rotation syn to anti at modified deoxyadenosine. Biochemistry 36, 11069-11076. (10) 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)-N6-[10(7,8,9,10-tetrahydrobenzo[a]pyrenyl)]-2′-deoxyadenosyl adduct from 1H NMR. Biochemistry 35, 6212-6224. (11) Schurter, E. J., Yeh, H. J., 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

(17)

(18)

(19)

(20)

(21)

(22)

(23)

(24)

(25)

(26)

(27)

a 10R adduct derived from trans addition of a deoxyadenosine N6-amino group to (-)-(7S,8R,9R,10S)-7,8-dihydroxy-9,10-epoxy7,8,9,10-tetrahydrobenzo[a]pyrene. Biochemistry 34, 1364-1375. 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, 10831-10842. Wei, S. J., Chang, R. L., Wong, C. Q., Bhachech, N., Cui, X. X., Hennig, E., Yagi, H., Sayer, J. M., Jerina, D. M., and Preston, B. D. (1991) Dose-dependent differences in the profile of mutations induced by an ultimate carcinogen from benzo[a]pyrene. Proc. Natl. Acad. Sci. U.S.A. 88, 11227-11230. Wei, S. J., Chang, R. L., Bhachech, N., Cui, X. X., Merkler, K. A., Wong, C. Q., Hennig, E., Yagi, H., Jerina, D. M., and Conney, A. H. (1993) Dose-dependent differences in the profile of mutations induced by (+)-7R,8S-dihydroxy-9S,10R-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene in the coding region of the hypoxanthine (guanine) phosphoribosyltransferase gene in Chinese hamster V-79 cells. Cancer Res. 53, 3294-3301. Wei, S. J., Chang, R. L., Wong, C. Q., Cui, X. X., Dandamudi, N., Lu, Y. P., Merkler, K. A., Sayer, J. M., Conney, A. H., and Jerina, D. M. (1999) The ratio of deoxyadenosine to deoxyguanosine adducts formed by (+)-(7R,8S,9S,10R)-7,8-dihydroxy-9,10-epoxy7,8,9,10-tetrahydrobenzo[a]pyrene in purified calf thymus DNA and DNA in V-79 cells is independent of dose. Int. J. Oncol. 14, 509-513. Lavrukhin, O. V., and Lloyd, R. S. (1998) Mutagenic replication in a human cell extract of DNAs containing site-specific and stereospecific benzo[a]pyrene-7,8-diol-9,10-epoxide DNA adducts placed on the leading and lagging strands. Cancer Res. 58, 887891. Page, J. E., Pilcher, A. S., Yagi, H., Sayer, J. M., Jerina, D. M., and Dipple, A. (1999) Mutational consequences of replication of M13mp7L2 constructs containing cis-opened benzo[a]pyrene 7,8diol 9,10-epoxide-deoxyadenosine adducts. Chem. Res. Toxicol. 12, 258-263. Chary, P., Latham, G. J., Robberson, D. L., Kim, S. J., Han, S., Harris, C. M., Harris, T. M., and Lloyd, R. S. (1995) In vivo and in vitro replication consequences of stereoisomeric benzo[a]pyrene7,8-dihydrodiol 9,10-epoxide adducts on adenine N6 at the second position of N-ras codon 61. J. Biol. Chem. 270, 4990-5000. Christner, D. F., Lakshman, M. K., Sayer, J. M., Jerina, D. M., and Dipple, A. (1994) Primer extension by various polymerases using oligonucleotide templates containing stereoisomeric benzo[a]pyrene-deoxyadenosine adducts. Biochemistry 33, 1429714305. Chary, P., and Lloyd, R. S. (1995) In vitro replication by prokaryotic and eukaryotic polymerases on DNA templates containing site-specific and stereospecific benzo[a]pyrene-7,8-dihydrodiol-9,10-epoxide adducts. Nucleic Acids Res. 23, 1398-1405. Chary, P., and Lloyd, R. S. (1996) Impact of the stereochemistry of benzo[a]pyrene 7,8-dihydrodiol 9,10-epoxide-deoxyadenosine adducts on resistance to digestion by phosphodiesterases I and II and translesion synthesis with HIV-1 reverse transcriptase. Chem. Res. Toxicol. 9, 409-417. Geacintov, N. E., Cosman, M., Hingerty, B. E., Amin, S., Broyde, S., and Patel, D. J. (1997) NMR solution structures of stereoisomeric covalent polycyclic aromatic carcinogen-DNA adduct: principles, patterns, and diversity. Chem. Res. Toxicol. 10, 111-146. 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. (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, 1914-1918. 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: (-)-trans-anti[BP]G‚C adduct structure and comparison with (+)-trans-anti[BP]G‚C enantiomer. Biochemistry 31, 5245-5252. 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, 3152-3161. 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 covalently attached benzo[a]pyrenyl ring into helix and displacement of modified deoxyguanosine. Biochemistry 32, 4145-4155. 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

822

(28)

(29)

(30)

(31)

(32)

(33)

(34)

Chem. Res. Toxicol., Vol. 13, No. 9, 2000 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, 11507-11517. Cosman, M., Fiala, R., Hingerty, B. E., Amin, S., Geacintov, N. E., Broyde, S., and Patel, D. J. (1994) Solution conformation of the (+)-cis-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 33, 11518-11527. 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 covalently attached BP ring into the helix with base displacement of modified deoxyguanosine into major groove. Biochemistry 35, 9850-9863. 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 covalently attached BP ring into the helix with base displacement of modified deoxyguanosine into major groove. Biochemistry 35, 9850-9863. Feng, B., Gorin, A., Kobanovskiy, 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, 1378013790. 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, 30213032. Case, D., Pearlman, D., Caldwell, J., Cheatham, T., Ross, W., Simmerling, C., Darden, T., Merz, K., Stanton, R., Cheng, A., Vincent, J., Crowley, M., Ferguson, D., Radner, R., Seibel, G., Singh, U. C., Weiner, P., and Kollman, P. (1997) AMBER 5.0 Documentation, University of California, Berkeley, CA. Cornell, W., Cieplak, P., Bayly, C. I., Gould, I. R., Merz, K. M., Ferguson, D. M., Spellmeyer, D. C., Fox, T., and Kollman, P. A.

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(37)

(38)

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(1995) A second generation force field for the simulation of proteins and nucleic acids. J. Am. Chem. Soc. 117, 5179-5197. Hingerty, B. E., Ritchie, R. H., Ferrell, T. L., and Turner, J. E. (1985) Dielectric effects in biopolymers: the theory of ionic saturation revisited. Biopolymers 24, 427-439. Friedman, R. A., and Honig, B. (1992) The electrostatic contribution to DNA base-stacking interactions. Biopolymers 32, 145159. Frisch, M. J., Trucks, G. W., Schlegel, H. B., Gill, P., Johnsone, B. G., Robb, M. A., Cheeseman, J. R., Keith, T. A., Petersson, G. A., Montgomery, J. A., Raghavachari, K., Al-Laham, M. A., Zakrzewski, V. G., Ortiz, J. V., Foresman, J. B., Cioslowski, J., Stefanov, B. B., Nanayakkara, A., Challacombe, M., Peng, C. Y., Ayala, P. Y., Chen, W., Wong, M. W., Andres, J. L., Reploge, E. S., Gomperts, R., Martin, R. L., Fox, D. J., Binkley, J. S., Defrees, D. J., Baker, J., Stewart, J. P., Head-Gordon, M., Gonzalez, C., and Pople, J. A. (1995) Gaussian 94, revision A.1, Gaussian, Pittsburgh, PA. Bayly, C. I., Cieplak, P., Cornell, W. D., and Kollman, P. A. (1993) A well-behaved electrostatic potential based method using charge restraints for deriving atomic charges: the RESP model. J. Phys. Chem. 97, 10269-10280. Xie, X. M., Geacintov, N. E., and Broyde, S. (1999) Stereochemical origin of opposite orientations in DNA adducts derived from enantiomeric anti-benzo[a]pyrene diol epoxides with different tumorigenic potentials. Biochemistry 38, 2956-2968. Xie, X. M., Geacintov, N. E., and Broyde, S. (1999) Origins of conformation differences between cis and trans DNA adducts derived from enantiomeric anti-benzo[a]pyrene diol epoxides. Chem. Res. Toxicol. 12, 597-609. Atkins, P. W. (1990) Physical Chemistry, 4th ed., W. H. Freeman Co., New York. Sundaralingam, M. (1973) The Concept of a Conformationally “Rigid” Nucleotide and Its Significance in Polynucleotide Conformational Analysis, pp 417-456, Israel Academy of Science & Humanities, Jerusalem.

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