Molecular Modeling of Four Stereoisomers of the Major B[a

Chem. Res. Toxicol. 2002, 15, 1429-1444

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Molecular Modeling of Four Stereoisomers of the Major B[a]PDE Adduct (at N2-dG) in Five Cases Where the Structure Is Known from NMR Studies: Molecular Modeling Is Consistent with NMR Results Chiu Hong Lee, Sushil Chandani, and Edward L. Loechler* Biology Department, Boston University, Boston, Massachusetts 02215 Received April 5, 2002

The potent mutagen/carcinogen benzo[a]pyrene (B[a]P) is metabolically activated to (+)anti-B[a]PDE, which is known to induce a variety of mutations (e.g., GC f TA, GC f AT, etc.). One hypothesis for this complexity is that different mutations are induced by different conformations of its major adduct [+ta]-B[a]P-N2-dG when bypassed during DNA replication (perhaps by different DNA polymerases). Our previous molecular modeling studies have suggested that conformational complexity might be extensive in that B[a]P-N2-dG adducts appeared capable of adopting at least sixteen potential conformational classes in ds-DNA [e.g., Kozack and Loechler (1999) Carcinogenesis 21, 1953], although only eight seemed likely to be relevant to base substitution mutagenesis. Such molecular modeling studies are only likely to be valuable for the interpretation of mutagenesis results if global minimum energy conformations for adducts are found and if the differences in the energies of these different conformations can be computed reasonably accurately. One approach to assessing the reliability of our molecular modeling techniques is considered herein. Using a five-step molecular modeling protocol, which importantly included a molecular dynamics version of simulated annealing, eight conformations are studied in each of five cases. (The five cases are listed below, and were chosen because in each case the preferred solution conformation is known from a NMR study.) Of the eight conformations studied, the one computed to be lowest in energy is the same conformation as the one observed by NMR in four of the five cases: 5′-CGC sequence with [+ta]-, [-ta]-, and [+ca]-B[a]P-N2-dG, and 5′-TGC sequence with [+ta]-B[a]P-N2-dG. In the fifth case (5′-CGC sequence with [-ca]-B[a]P-N2-dG), the known NMR conformation is computed to be second lowest in energy, but it is within ∼1.7 kcal of the computed lowest energy conformation. These results suggest that molecular modeling is surprisingly accurate in computing lowest energy conformations and that it should be useful in assessing the relative energies of different conformations. This is especially important given that currently molecular modeling is the only means available to study the energetics of minor conformations of DNA adducts.

Introduction (B[a]P)1

Benzo[a]pyrene is a potent mutagen/carcinogen and an example of a polycyclic aromatic hydrocarbon (PAH), which is a class of substances produced by incomplete combustion and found ubiquitously in the environment (1-7). B[a]P is metabolized in cells to the ultimate mutagen/carcinogen (+)-anti-B[a]PDE, which gives one predominant DNA adduct [+ta]-B[a]P-N2-dG (Figure 1) (8, 9). * To whom correspondence should be addressed. Phone: (617) 3539259. Fax: (617) 353-6340. E-mail: [email protected]. 1 Abbreviations: B[a]P, benzo[a]pyrene; (+)-anti-B[a]PDE, 7R,8Sdihydroxy-9S,10R-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene; [+ta]B[a]P-N2-dG, the major adduct of (+)-anti-B[a]PDE, formed by trans addition of N2-dG to (+)-anti-B[a]PDE; [-ta]-, [+ca]-, and [-ca]-B[a]PN2-dG, other stereoisomers of B[a]P-N2-dG (see Figure 1); PAH, polycyclic aromatic hydrocarbon; NMR, nuclear magnetic resonance; t, annealing time; T0, initial annealing temperature; τ, molecular dynamics time step; Κ1, constraint force applied to the hydrogen bonds in a base pair during initial conjugate gradient minimization; Κ2, constraint force applied to the hydrogen bonds in a base pair during simulated annealing; BPmi5, BPmi3, BPma5, BPma3, Gmi5, Gmi3, Gma5, and Gma3, as well as eight other conformations are represented pictorially in Figure 1.

Early mutagenesis studies were done with a racemic mixture of (+)- and (-)-anti-B[a]PDE (10-14), but mutational spectra for pure (+)-anti-B[a]PDE have been generated in Escherichia coli (15, 16) and in mammalian (CHO) cells (17-20). In E. coli, base substitutions, frameshifts, insertions, and deletions were all induced by (+)-anti-B[a]PDE (15, 16). For mutations at G:C base pairs alone, G:C f T:A (57%), G:C f A:T (23%), and G:C f C:G (20%) mutations were each significant (3, 4), and the patterns were influenced by the DNA sequence context. Our adduct site-specific mutational studies with the major adduct, [+ta]-B[a]P-N2-dG, reinforced this conclusion (21-25), in that G f T mutations dominated in a 5′-TGC-3′ sequence context [>95% (21)], and G f A mutations dominated in a 5′-AGA-3′ sequence context [∼95% (24)], while a mixture of G f T, A and C mutations were all prevalent in a 5′-CGG-3′ sequence context (25). Others have also shown that sequence context can influence both the qualitative and quantitative pattern of mutagenesis by B[a]P-N2-dG adducts (2630).

10.1021/tx0200257 CCC: $22.00 © 2002 American Chemical Society Published on Web 10/12/2002

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Lee et al.

Figure 1. Structures for the four stereoisomers [+ta]-, [-ta]-, [+ca]-, and [-ca]-B[a]P-N2-dG, and a pictorial representation of the 16 classes of conformations that B[a]P-N2-dG adducts appear capable of adopting in ds-DNA (58). The B[a]P moiety of [+ta]-B[a]PN2-dG is depicted as a rectangle (half-open/half-solid) with the solid portion representing the “a-face”, which is defined in the [+ta]B[a]P-N2-dG structure in the upper center of Figure 1. The dG moiety of the adduct is represented as a rectangle with a “G” toward its center. The base pairs on the 3′- and 5′-side are located above and below the adducted base pair, respectively. The major and minor grooves are oriented to the left and right of the adduct and DNA, respectively. The abbreviation representing each conformation is based on the designation of the moiety in the groove. For example, “BPmi5” indicates that the B[a]P moiety of the adduct is in the minor groove and pointing toward the base on the 5′-side of the adduct. In the case of base displaced structures, using “Gmi5” as an example, the dG moiety is in the minor groove, while the “a-face” of the B[a]P moiety of the adduct is pointing toward the base on the 5′-side the adduct bond. (See text for additional discussion.) The base paired and base displaced conformations retain the same number of steps in the DNA helix as unadducted DNA. Four double displaced conformations are also shown (G/BPma5, G/BPma3, G/BPmi5, and G/BPmi3), and in each case they have both the B[a]P and the dG moiety of the adduct in a groove. Such structures might be expected to be precursors to a -1 frameshift mutation when at a ds/ss-DNA junction. Four intercalated conformations are also indicated (Ima5, Ima3, Imi5, and Imi3), and in this case, the “5” and “3” indicates the direction of the a-face, and not on which side the B[a]P moiety is intercalated. Such structures might be expected to be precursors to a +1 frameshift mutation when at a ds/ss-DNA junction.

These results raised the question: how can a single adduct induce different kinds of mutations and why does the pattern change in different sequence contexts? Our working hypothesis for this mutational complexity is that an adduct can adopt multiple conformations in DNA, where each conformation can cause a different kind of mutation, and adduct conformation can be controlled by factors such as DNA sequence context (16, 31, 32). The notion that adduct mutagenesis is controlled by adduct conformation (a chemical effect) has experimental support (reviewed in ref 32). The outcome of adduct bypass is also affected by the properties of the DNA polymerase involved (32). Thus, it is likely that an understanding of B[a]P mutagenic mechanism will only emerge once more is known about the patterns of mutations induced by [+ta]-B[a]P-N2-dG, the relationship of these patterns to the conformations that [+ta]-B[a]P-N2-dG can adopt in

DNA, and how these conformations are processed by relevant lesion-bypass (i.e., Y-family) DNA polymerases. The notion that an adduct can adopt multiple conformations has direct experimental support. By NMR, the pyrene moiety of a [+ta]-B[a]P-N2-dG adduct in a 5′-TGC3′ sequence context is in the minor groove and pointing toward the base on the 5′-side (a conformation we refer to as “BPmi5”, see discussion of Figure 1 below); however, there is a second, minor conformation as well, whose structure could not be determined (33). [+ta]-B[a]P-N2dG also adopted the BPmi5 conformation in a 5′-CGC-3′ sequence context in fully duplex DNA (34); however, when the base complementary to the adducted dG was removed, the conformation became base displaced (discussed in ref 35). Fluorescence studies also suggest that [+ta]-B[a]P-N2-dG can adopt multiple conformations in duplex DNA, although the conformations are not deter-

Validating Simulated Annealing Modeling

minable by this technique (36-38). Furthermore, four stereoisomers of the B[a]P-N2-dG adduct were studied and each was in a different conformation as determined by NMR, and in several cases minor species were also observed (34, 39-41; reviewed in 35; see below). Molecular modeling and computational chemistry have also been used to study conformational complexity of B[a]P-N2-dG adducts. Early studies on [+ta]-B[a]P-N2dG based on model building, suggested that the B[a]P moiety could be placed in the minor groove (42), the major groove (43), or be intercalated (44, 45). Energy minimized versions of these structures were later offered by a variety of groups (46-53), including us (54). The most revealing studies on B[a]P-N2-dG adducts have been done by Broyde and colleagues, both to refine NMR structures (34, 35, 39-41), and in purely theoretical work, notable achievements being the prediction of the lowest energy conformations for both [+ta]- and [-ta]-B[a]P-N2-dG (55), and studies to probe the fundamental reason for the conformational preference for various stereoisomers of B[a]P-N2-dG adducts (56; addressed more extensively in Results and Discussion). On the basis of our own molecular modeling work we suggested (57-60) that B[a]P-N2-dG adducts can adopt at least 16 classes of conformations in ds-DNA when considering the same number of bases in each strand and with a dC moiety in the strand opposite the adduct (pictorially represented in Figure 1). Eight of these conformations seemed most likely to be relevant to base substitution mutagenesis (two leftmost columns in Figure 1), since they retain the same number of steps in the DNA helix, while the other eight conformations seemed more likely to be associated with frameshift mutagenesis. Regarding Figure 1, the [+ta]-B[a]P-N2-dG adduct is in the middle, and the base pair on the 5′- and 3′-sides are below and above, respectively, while the major and minor groove are left and right, respectively. We proposed a set of abbreviations to designate these conformations (58, 59), based on the moiety in the groove. For example, “BPmi5” indicates that the B[a]P moiety of the adduct is in the minor groove and pointing toward the base on the 5′-side of the adduct. Four conformations have the B[a]P moiety in a groove (Figure 1, first column). BPmi5 and BPmi3 have the dG of the adduct in a Watson-Crick base pair with dC and have the B[a]P moiety in the minor groove pointing toward the base on the 5′- or 3′-sides, respectively. BPma5 and BPma3 result from an anti f syn rotation about the glycosylic bond of the dG moiety of the adduct giving the Hoogstein base orientation, which moves the B[a]P moiety into the major groove, where it can point toward the base on the 5′- or 3′-sides, respectively. There are no other ways to accommodate the B[a]P moiety in a groove without significantly distorting DNA. The four conformations in column two of Figure 1 have the B[a]P moiety of the adduct stacked with the surrounding base pairs and the dG moiety is in a groove; these are “base-displaced” structures. Regarding nomenclature, for example “Gmi5” indicates that the dG moiety is in the minor groove; in this case, the ‘5’ designates that the “a-face” (see Figure 1) of the B[a]P moiety of the adduct is pointing toward the base on the 5′-side. Previously, we studied the eight conformations for [+ta]-B[a]P-N2-dG (i.e., BPmi5, BPmi3, BPma5, BPma3, Gmi5, Gmi3, Gma5, and Gma3) using a four-step protocol (57-60): a canonical structure was developed (step 1),

Chem. Res. Toxicol., Vol. 15, No. 11, 2002 1431 Table 1. Parameters Used in the “24-Parameters” Protocola 1. t ) 40 2. t ) 50 3. t ) 60 4. t ) 80 5. t ) 100 6. t ) 120 7. t ) 140 8. k1 ) k2 ) 30 9. τ ) 1.5 10. κ1 ) κ2 ) 3 11. κ2 ) 30 12. κ2 ) 3

13. κ1 ) κ2 ) 80b 14. κ2 ) 80b 15. T0 ) 500 16. T0 ) 625 17. T0 ) 875 18. T0 ) 500; t ) 60 19. T0 ) 500; t ) 80 20. T0 ) 625; κ2 ) 30 21. t ) 50; τ ) 1.5 22. t ) 60; κ2 ) 3 23. T0 ) 625; t ) 60; κ2 ) 30 24. T0 ) 500; τ ) 1.5; κ2 ) 3

a The values in Table 1 are the parameters in a run that varied from the canonical parameters: T0 ) 750 K, t ) 40 ps, τ ) 1.0 fs, κ1 ) 30 kcal/mol/Å, κ2 ) 30 kcal/mol/Å. Abbreviations: T0, initial annealing temperature (in step 3); t, annealing time (in step 3); τ, molecular dynamics time step (in step 3); κ1, harmonic potential force constant applied during the initial conjugate gradient minimization (in step 2); κ2, harmonic potential force constant applied during simulated annealing (in step 3). Note: κ1 and κ2 constraints were placed on all base pairs in BPmi5, and on all base pairs but the adducted dG and its complementary dC in Gma5, because this base pair does not have Watson-Crick-type hydrogen bonds. b In runs 13 and 14 for BPmi5, the κ1 and/or κ2 constraints were 80 kcal/mol/Å for all base pairs except the adducted base pair, which makes these runs more analogous to runs with Gma5, which also do not have constraints on the adducted base pair.

after which the structure was subjected to an initial conjugate gradient minimization with constraints (step 2), followed by a molecular dynamics version of simulated annealing (step 3), then a final conjugate gradient minimization without constraints (step 4). To explore conformational space more fully, parameters in the simulated annealing step were varied (e.g., the initial annealing temperature). In total, 24 unique sets of simulated annealing parameters (Table 1) were used to evaluate eight conformations in five different DNA sequence contexts for which we had the most information about [+ta]-B[a]P-N2-dG base substitution mutagenesis. We noted a correlation between our molecular modeling results and mutagenesis results that suggested the hypothesis that Gma5 is the G f T conformation and Gmi3 is the G f A conformation (59). The significance of the correlation noted in the previous paragraph depends on whether our computational approach reliably finds low energy structures and correctly estimates the relative energy of different conformations. To improve our chances of finding global energy minimum structures, herein we describe the addition of a fifth step to our protocol, involving iteration of the simulated annealing step (step 3). We find that after several iterations, we have converged on a best structure that cannot be improved with further rounds of iteration. In terms of assessing the reliability of this five step molecular modeling protocol, we could conceive of only one approach. The dominant conformations for [+ta](32), [-ta]- (39), [+ca]-, (40) and [-ca]-B[a]P-N2-dG (41) in a 5′-CGC sequence context are known from NMR studies to be BPmi5, BPmi3, Gmi3, and Gma5, respectively, while the dominant conformation for [+ta]-B[a]PN2-dG in a 5′-TGC sequence context is BPmi5 (33). Using molecular modeling, we have studied the eight conformations BPmi5, BPmi3, BPma5, BPma3, Gmi5, Gmi3, Gma5, and Gma3 for each of these stereoisomers in the relevant DNA sequence contexts and show that our approach appears to be surprisingly accurate in com-

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parison to what is known in the NMR studies. In the Results and Discussion, we also address why we think that modeling BP-N2-dG adducts in ds-DNA might be relevant to our mutagenesis results, although this is currently unknown and the actual situation is likely to be very complex.

Experimental Procedures Software and Force Field. B[a]P-N2-dG-containing structures were constructed, viewed, manipulated and analyzed using Insight II [Molecular Simulations, Inc., version 98.0 (61)]. A CHARMm force field (62) was used to calculate energies, and direct all minimizations and simulated annealings of structures, although we optimized parameters for DNA (63) and B[a]P (57). Hydrogens bound to oxygen or nitrogen were included explicitly, while a united atom force field was used for hydrogens bound to carbon, except that all hydrogens were included on B[a]P moieties. As done previously, the total charge on each phosphate group was reduced from -1.00 to -0.32 to simulate charge dampening due to counterions and solvent. Long-range interactions were truncated by a switching function centered at 10.5 Å with a width of 1.0 Å (see below). Electrostatic interactions were modified by a dielectric constant ) R, where R is the interatomic distance. The force field and settings have had been used in our previous molecular modeling studies of B[a]P-N2dG DNA (57-60). Most calculations were conducted on either an SGI O2 or an Origin 200. Conformational Search Protocol. To search for low energy conformations, we used a previously developed four-step protocol (57), to which a fifth step was added, as outlined below. Input, starting structures for each of eight conformations were constructed (step 1) and were minimized with constraints to retain Watson-Crick hydrogen bonding in complementary base pairs (step 2), after which simulated annealing was conducted (step 3) followed by a final minimization without constraints (step 4). A new, fifth step was added: iteration of steps 3-4 (using low-energy structures) until a lowest energy structure was located. Step 1: Construction of Initial, Input Conformations of the B[a]P-N2-dG Adducts in DNA. Four stereoisomers [+ta]-, [-ta]-, [+ca]-, and [-ca]-B[a]-N2-dG were studied in a 5′-CGC sequence context. In the case of each stereoisomer, eight classes of adduct conformations (BPmi5, BPmi3, BPma5, BPma3, Gmi5, Gmi3, Gma5, and Gma3: described in the Introduction) were constructed in a duplex heptamer that was identical in sequence to the seven innermost base pairs (5′-ATCGCTA) of an 11-mer used in several NMR studies (5′-CCATCGCTACC). Previous work (57) indicated that heptamers were a reasonable compromise in that they are big enough to minimize end effects, yet small enough to allow the calculation to maximally explore conformational space in the vicinity of the adduct rather than in peripheral regions of the DNA molecule. Methods to construct input structures were described previously (57, 58), but are outlined here. BPmi5 and BPmi3 were constructed using coordinates for canonical fiber diffraction B-DNA (64) and superimposing a [+ta]-B[a]P-N2-dG moiety over the central dG. BPma5 was constructed by rotating the dG moiety in BPmi3 from anti to syn to place the B[a]P moiety in the major groove in the Hoogstein orientation. BPma3 was constructed from BPma5 by rotation of the B[a]P moiety. Gmi5, Gmi3, Gma5, and Gma3 were built by trial and error from BPmi5 following rotations of appropriate bonds to stack the B[a]P moiety with surrounding base pairs and to place the dG moiety in the appropriate groove. In all cases, these initial structures were tweaked to minimize van der Waals contacts and to find starting coordinates that ultimately led to relatively low energy conformations. The conformations BPmi5, BPmi3, Gmi3, and Gma5 were observed by NMR with [+ta]-B[a]P-N2dG (33, 34), [-ta]-B[a]P-N2-dG (39), [+ca]-B[a]P-N2-dG (40), and [-ca]-B[a]P-N2-dG (41), respectively, so our starting structures

Lee et al. for these conformations were built to be consistent with (although not identical to) these NMR structures. The initial structures with [+ta]-B[a]P-N2-dG were used to generate the structures for [-ta]-B[a]P-N2-dG, [+ca]-B[a]P-N2dG, and [-ca]-B[a]P-N2-dG by simply changing the stereochemistry of the hydroxyl groups. The initial structures for [+ta]B[a]P-N2-dG in the 5′-CGC sequence was used to generate structures for the 5′-TGC sequence (5′-TGTGCAC), a sequence derived from an NMR study (33). As noted previously (58), Gma3 is a particularly bad structure, so we tried many variations for it, but none was particularly successful. Interestingly, Gma5, as well as Gma3, have the most latitude in terms of potential input structures, so many versions of it were also tried (see below). In addition to the 40 conformers that we constructed, we also used the four published NMR structures for [+ta]-, [-ta]-, [+ca]-, and [-ca]-B[a]P-N2-dG in the 5′-CGC sequence (34, 39-41) as input structures for our calculations (coordinates graciously provided by Dr. Suse Broyde). Only the seven innermost base pairs of each 11-mer NMR structure were used. Step 2: Initial Minimization. Each initial input structure was minimized using the conjugate gradient algorithm until the tolerance gradient was less than 0.01. A distance constraint (κ1 ) 30.0 kcal/mol) was added during the minimization to retain the lengths of the Watson-Crick hydrogen bonds between base pairs [1.80 Å for O‚‚‚H bonds and 1.87 Å for N‚‚‚H bonds in G:C base pairs; 1.81 Å for O‚‚‚H bonds and 1.86 Å for N‚‚‚H bonds in A:T base pairs (65)]. Constraints were not placed on the B[a]P-containing base pair in the BPma3, BPma5, Gma3, Gma5, Gmi3, and Gmi5 conformations, since they cannot form Watson-Crick hydrogen bonds. Steps 3 and 4: Simulated Annealing and Final Minimization. Following minimization (step 2), a structure was subjected to a molecular dynamics version of simulated annealing (step 3), involving instantaneous heating (“heat-shock”) followed by cooling to 0 K with time (in 5 K steps). Heat-shock provided the structures sufficient energy to surmount energy barriers around their local minimum, while the cooling phase allowed the structure to explore conformational space before settling into a lower energy local minimum. At the end of each simulated annealing, the structure was minimized (conjugate gradient, tolerance gradient 0.01) without hydrogen bonding constraints (step 4). Thereafter, the twooutermost base pairs were removed (yielding a pentamer), and the point energy was calculated. The energy of the innermost five residues was shown to be a better reflection of the true energy relevant to the adduct than the heptamer itself (57). Because we did not know in advance what annealing conditions would provide lowest energy structures, different conditions were used (Table 1), which differed in four variables: initial temperature (T0), time step (τ), the strength of the constraint holding the hydrogen bonds between base pairs (κ1 and κ2), and the time for cooling the structures from the initial temperature to absolute zero (t). We call this protocol “24parametes”, and it was described previously (57, although it was not named as such). The term “24-parameters” arises because step 3 was done on each input structure 24 times using different parameter sets (Table 1). Step 5: Iterative Simulated Annealing. For a particular stereoisomer and conformation of B[a]P-N2-dG, 24 structures were generated (i.e., following the completion of step 4 for each of the 24 parameters in Table 1). Each conformation was inspected and those with distorted structures were disregarded (see Results and Discussion). The lowest energy, nondistorted structure in a set of 24 structures (R1 for “round 1”) was subjected to a second round (R2) of simulated annealing and final minimization (i.e., steps 3 and 4 were repeated), then analyzed. This iterative procedure was continued (i.e., R3, R4, etc.) until the energy of the lowest energy conformation increased between rounds of iteration. More modeling attention (i.e., more simulated annealing runs) was focused on Gma5 than other conformations for the following

Validating Simulated Annealing Modeling reason. The B[a]P or dG moiety projects into the minor groove in the case of the BPmi5, BPmi3, Gmi5, and Gmi3 conformations. These conformations give low energy structures with minimal structural heterogeneity, because the minor groove is relatively narrow and confining. The B[a]P or dG moiety projects into the major groove in the case of the BPma5, BPma3, Gma5, and Gma3 conformations. These conformations tend to show greater structural heterogeneity, because the major groove is more spacious. As noted previously and as reinforced by the findings reported herein, BPma3 and BPma5 tend to be highenergy structures as does Gma3, which is very distorted (58). Thus, of the conformations with a moiety projecting into the major groove, Gma5 is most likely to be quantitatively important. Because of this and because of its relative flexibility (i.e., with the dG moiety projecting into the major groove), more iterative runs were required to find a lowest minimum-energy structure. In addition, more input structures for Gma5 were also tried, e.g., four different orientations of the dC moiety opposite the adducted base were tried; in each case, the best output structures were of similar energy. Other Considerations. The results obtained herein were generated with a 10.5 Å cutoff for nonbonded parameters; however, we investigated whether a change in this value (in the range of 7.5 to 11.5 Å) affected the relative energies of the BPmi5 vs Gma5 conformations for [+ta]-B[a]P-N2-dG in the 5′CGC sequence. BPmi5 was always lower in energy than Gma5, although the relative energy differed using different cutoff distances. We were unable to identify a pattern that allowed us to make a rational decision about what cutoff made the most sense. We decided to use a 10.5 Å cutoff, because this value is the default in CHARMm, it is often recommended, we have used it in the past (57-60), and (ultimately) it gave results for B[a]PN2-dG adducts that conformed to what has been obtained by NMR (as discussed herein), implying that it is a sensible cutoff from a practical point of view. In the course of a simulated annealing run, some atoms will oscillate around the nonbonded cutoff value, but CHARMm must avoid dropping these atoms in and out of the calculation in order to maintain consistent energetic comparisons. CHARMm offers several procedures to cope with this issue for both electrostatic and van der Waals interactions. To investigate whether the choice of these two functions affected the relative energies of different conformations, the energy of the BPmi5 and Gma5 conformers for [+ta]-B[a]P-N2-dG in the 5′-CGC sequence (after minimization) were compared using four combinations: switch-vswitch, switch-vshift, shift-vswitch, and shift-vshift, where “shift” and “switch” were the smoothing functions for electrostatic interactions, while “vshift” and “vswitch” were the functions for van der Waals interactions. In all cases, BPmi5 was lower in energy, and there was no obvious pattern that allowed us to chose a particular combination, so we used switch-vswitch, because it is the default in CHARMm, it is frequently used and we have used it previously. We wished to determine whether the lowest energy structure we obtained after multiple rounds of iteration was dependent on the structure chosen after R1 of simulated annealing, where in all cases we chose the lowest energy structure. A higher energy structure from R1 (∼4 kcal/mol higher than the lowest energy structure) was chosen for BPmi5, BPmi3, BPma5, BPma3, Gma5, and Gmi3 of [+ta]-B[a]P-N2-dG in the 5′-CGC sequence and iterative rounds of simulated annealing was conducted. (Gma3 and Gmi5 were not studied because all of their structures were distorted after R1.) BPma3 and Gma5 gave slightly higher energy structures (2.2 and 2.3 kcal/mol lower, respectively), but the other four conformers gave rise to virtually identical structures with energy difference less than 1 kcal/mol. We conclude that the use of a higher energy conformation from R1 of simulated annealing does not give rise to a lower energy structure in R2.

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Results and Discussion Input Structures. The four stereoisomers [+ta]-, [-ta]-, [+ca]-, and [-ca]-B[a]P-N2-dG were studies by molecular modeling in a sequence context that corresponds to the seven innermost base pairs of the 11-mer sequence (5′-CCATCGCTACC) used in a series of NMR studies (34, 39-41). In principle, B[a]P-N2-dG adducts can adopt at least 16 classes of conformations [Figure 1 (58)], of which eight (BPmi5, BPmi3, BPma5, BPma3, Gmi5, Gmi3, Gma5, and Gma3) are most likely to be relevant to base substitution mutagenesis. Canonical, input (starting) structures for each of these eight conformations were constructed (58; Experimental Procedures). An NMR study on [+ta]-B[a]P-N2-dG in a 5′-TGC sequence has also been published (33), and we followed the identical protocol to generate eight, initial, input structures in the sequence (5′-TGTGCAC). In addition, Dr. Suse Broyde graciously provided coordinates that she generated by molecular modeling using the NMR NOE distance constraints for [+ta]-, [-ta]-, [+ca]-, and [-ca]-B[a]P-N2-dG in the BPmi5, BPmi3, Gmi3, and Gma5 conformations (34, 39-41), respectively, and they were used as input structures. [In the case of [+ta]-B[a]P-N2-dG in the 5′-TGC sequence, the coordinates based on the NMR study (33) were not available.] An Example of the Experimental Approach: BPmi5 for [+ta]-B[a]P-N2-dG in a 5′-CGC Sequence. Consider for the moment the generation of output (lower energy) structures for one example: the BPmi5 conformation of [+ta]-B[a]P-N2-dG in the 5′-CGC sequence. Initially, a four-step protocol was followed: construction of the input conformation (step 1; see Experimental Procedures), initial energy minimization with constraints (step 2), simulated annealing (step 3), and final energy minimization without constraints (step 4). Twenty-four output structures were generated from the single input structure via 24 independent runs, which differed in their annealing parameters in step 3 [Table 1; conditions established in our previous work (57)]. The following annealing parameters were varied: initial temperature (T), length of the cooling period (t), length of the molecular dynamics time step (τ), and constraints between the two strands of the DNA molecule (see Table 1 and Experimental Procedures). The final minimization (step 4) was performed on each structure after each of the 24 runs. Figure 2 shows the results for [+ta]-B[a]P-N2-dG in the 5′-CGC sequence context starting from our canonical BPmi5 input conformation. The solid squares indicate the energies following steps 1-4. The plot simply shows the energy (ordinate) of the 24 output structures when ranked from lowest to highest energy (numbered 1-24 on the abscissa) and equally spaced apart. [Only 18 solid square data points are present in Figure 2 even though 24 runs were conducted, because distorted structures emerged that were excluded (addressed in the next section)]. We note that neither the slope nor the intercept has any obvious meaning. The lowest energy structure after the first round of analysis (i.e., the structure corresponding to the leftmost solid square in Figure 2) was subjected to a second round of the calculations using the same 24 parameters, and the results are plotted as the open squares in Figure 2. Similarly, a third and fourth round of analysis were

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Table 2. Calculated Energy for the Lowest Energy Conformations of [+ta]-, [-ta]-, [+ca]- and [-ca]-B[a]P-N2-dG in a 5′-ATCGCTA Duplex, and of [+ta]-B[a]P-N2-dG in a 5′-TGTGCAC Duplex [+ta]-a 5′-CGCb canonical input structuresd BPmi5 (00.00)e BPmi3 (4.42) Gmi3 (6.21) BPma3 (9.12) Gma5 (10.84) Gmi5 (15.64) Gma3 (21.29) BPma5 (30.62) NMR input structuresd BPmi5 (+1.32)

[-ta]5′-CGCb

[+ca]5′-CGCb

[-ca]5′-CGCb

[+ta]5′-TGCc

BPmi3 (00.00) Gmi3 (1.20) BPmi5 (2.58) Gmi5 (6.17) Gma5 (9.62) BPma5 (10.51) Gma3 (13.36) BPma3 (17.99)

Gmi3 (00.00) BPmi5 (3.55) BPmi3 (6.39) Gma5 (10.67) BPma3 (15.43) BPma5 (17.19) Gmi5 (22.43) Gma3 (33.23)

BPmi5 (00.00) Gma5 (1.66) BPmi3 (3.72) BPma3 (10.63) Gmi3 (11.75) Gma3 (13.69) BPma5 (16.23) Gmi5 (21.03)

BPmi5 (00.00) Gma5 (1.40) BPmi3 (2.57) BPma3 (17.99) Gmi3 (19.83) Gmi5 (22.79) BPma5 (25.31) Gma3 (29.36)

BPmi3 (-2.39)

Gmi3 (-0.22)

Gma5 (+0.62)

NA

a

The designations [+ta]-, [-ta]-, [+ca]-, and [-ca]- indicate the stereoisomers of the B[a]P-N2-dG adduct (see Figure 1). b 5′-CGC indicates a 5′-ATCGCTA sequence. c 5′-TGC indicates a 5′-TGTGCAC sequence. d Canonical input structures were developed de novo as described in the Experimental Procedures. In the case of [+ta]-, [-ta]-, [+ca]-, and [-ca]-B[a]P-N2-dG in a 5′-CGC sequence, NMR studies established that BPmi5 (34), BPmi3 (39), Gmi3 (40), and Gma5 (41), respectively, were observed in solution, and these NMR structures were also used as input structures for our studies. e Ranking (lowest on top) of different conformations with their relative energies in kcal/mol in parentheses. For ease of comparison, the lowest energy conformation is arbitrarily assigned a relative energy value of 00.00.

Figure 2. Relative energies for [+ta]-B[a]P-N2-dG in the canonical input conformation for BPmi5 in a 5′-CGC sequence. The plot shows the energy (ordinate) following final energy minimization (step 4) for each of the structures generated by the 24 runs using the “24-parameters” protocol (numbered 1-24 on the abscissa), when equally spaced apart. The solid squares show the results after the first round (R1) of the 24-parameters protocol. (As discussed in the Experimental Procedures, some final structures were disregarded because they were distorted, which accounts for why there are fewer than 24 points.) The lowest energy structure from R1 (square at position 1) was used as an input structure for a second round (R2) of the 24parameters protocol, and the results are shown as open squares. Iterations using the lowest energy structure from a round were continued until no improvement in the lowest energy structure was obtained, which in this case was reached after three rounds of iteration (R3, solid triangles; R4, open triangles).

also conducted starting from the lowest energy structure in the previous round (closed and open triangles, respectively). We refer to the iterative step in our protocol as “step 5”, and iteration was continued until the lowest energy structure in round [n + 1] was higher than the lowest energy structure in round [n]. In this case the lowest energy structure was obtained in round three. We have named this five-step procedure: “iterative 24 parameters”. The lowest energy BPmi5 structure is shown in Figure 5. The iterative 24 parameters protocol was repeated for each of the other eight input structures for [+ta]-B[a]PN2-dG in the 5′-CGC DNA sequence context, and the results are given in the eight panels in Figure 3 [(A) BPmi5 [NMR input]; (B) BPmi3; (C) BPma3; (D) BPma5; (E) Gmi3; (F) Gmi5; (G) Gma3; and (H) Gma5]. The energy of the lowest energy structure for each of these conformations is listed in Table 2.

Energies for BPma5, BPma3, Gma3, and Gmi5 are relatively high (Table 2). As noted previously (58), BPma5 and BPma3 consistently give high-energy structures, and Gma3 gave high energy structures that are significantly perturbed. In cases when B[a]P-N2-dG is surrounded by G:C base pairs in the minor groove Gmi5 was also high in energy and markedly perturbed (Figure 4); in fact, only one acceptable structure for Gmi5 emerged in the first round of the 24-parameters protocol (see Figure 4 and its legend). Distorted Structures. This section addresses the distorted structures that were ignored. We use the word “distorted” to describe significantly deformed and unprecedented structures, and the word “perturbed” to describe moderately deformed but acceptable structures. The number of data points for the BPmi5 conformation of [+ta]-B[a]P-N2-dG in Figure 2 are 18, 18, 10, and 6 for rounds 1, 2, 3, and 4 respectively, even though 24 runs were conducted in each round, because distorted structures emerged and were ignored. To illustrate this issue, consider R1 (solid squares in Figure 2). Six structures were judged to be distorted, of which two (R1d1 and R1d2) were lower in energy (Table 3) than the structure that was chosen for the second round of the 24parameters protocol (i.e., that gave the open squares in Figure 2). The lowest energy distorted structure R1d1 is shown in Figure 6A. At the bottom, one base is bent ∼90°, while near the top the penultimate T:A base pair has only a single hydrogen bond due to a severe propeller twist in the adenine resulting in it interacting electrostatically with the C:G base pair below it. R1d1 and R1d2 were subjected to the iterative 24parameters protocol, and a portion of that analysis is provided in Table 3. After two rounds of the iterative 24parameters protocol, the lowest energy structure derived from R1d1 was R3d111 (Figure 6B; Table 3). Base pairing has resolved at the bottom of the structure, but the adenine in the penultimate T:A base pair has collapsed into the major groove, and the structure shows an ungainly kink. To the best of our knowledge there is no precedent for structures such as R3d111, so we believe that it is an artifact of our calculations (see below). Another low-energy structure derived from R1d1 is also shown (R2d12; Figure 6C), and it is also distorted. R3d221 is the lowest energy structure derived from R1d2 after two rounds of the 24-parameters protocol; R3d221



Figure 3. Relative energies for [+ta]-B[a]P-N2-dG in eight conformations in a 5′-CGC sequence. Each plot is arranged as described in the legend to Figure 2. The eight panels are (A) BPmi5 [NMR input]; (B) BPmi3; (C) BPma3; (D) BPma5; (E) Gmi3; (F) Gmi5; (G) Gma3; and (H) Gma5.

is distorted (Figures 6D). While Figure 6 shows only four structures, all structures in R2 and R3 from R1d1 and R1d2 are dramatically distorted. Consider another example, the Gma5 conformation of [+ta]-B[a]P-N2-dG. After R1, two low energy structures (R1d3 and R1d4) were ignored because they were distorted. R1d3 (Figure 7A) shows a severe tilt/kink to the right and its upper end is becoming unraveled. The lowest energy structure in the second round is R2d31 (Figure 7B), which is significantly distorted as were structures derived from it (data not shown). R3d321 (Figure 7C) is the lowest energy structure derived from R1d32. The tilt is more exaggerated in R3d321 compared to R1d3, and the topmost adenine in the A:T base pair has slid down, collapsing the minor groove and causing a severe kink in the sugar-phosphate backbone. To the best of our knowledge there is no precedent for the formation of such a structure. R1d3, R2d31, and R3d321 can be compared with the lowest energy structure that

was ultimately accepted for the Gma5 conformation of [+ta]-B[a]P-N2-dG (Figure 7D), which is a reasonable structure, whose character is fully consistent with NMR structures for Gma5 for [-ca]-B[a]P-N2-dG (41). One final example of a rejected/distorted structure can be found at the website http://www.bu.edu/biology/ Faculty_Staff/loechler.html (last accessed Oct 2002) which directly compares one of our low-energy, distorted Gmi3 conformations to a known Gmi3 NMR counterpart. While only BPmi5, Gma5, and Gmi3 examples are discussed herein, these results typify our overall findings vis-a`-vis distorted structures. Several points emerge. (1) Low-energy, distorted structures in R1 were pursued into subsequent rounds of the iterative 24-parameters protocol, and the structures that emerged were invariably more distorted. (2) Some low-energy distorted structures after R1 might be considered “acceptable” (e.g., Figure 6A for R1d1 with BPmi5 of [+ta]-B[a]P-N2-dG); however, these structures are never lower in energy than the

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Figure 4. Stereoview of the lowest energy version of the Gmi5 conformation for [+ta]-B[a]P-N2-dG in duplex DNA of sequence 5′-ATCGCTA-3′. The view is into the minor groove with the adduct-containing strand running 5′-to-3′ from bottom to top. The structure is distorted, e.g., the cramped minor groove forces the dG moiety of the adduct to be tilted outward (toward the viewer), leaving a wedge above into which the complementary dC inserts. As a result the helix is elongated, e.g., in comparison to the helix for Gmi3 structure in Figure 6. Table 3. Calculated Energies for Selected Distorted Structures in Rounds 1-3 of the Iterative 24-Parameters Protocol for the BPmi5 and Gma5 Conformations of [+ta]-B[a]P-N2-dG in a 5′-ATCGCTA Duplex R1a

R2 BPmi5

nondistortedb R1 (best) distortedd R1d1e (F6A)f

R1d2

R3

[+ta]-B[a]P-N2-dG

-159.96c -165.38

-160.23

R2d11

-171.50

R2d12 (F6C) R2d13

-166.95 -166.05

R2d21

-166.98

R2d22

-166.64

(best overall)

-165.99

R3d111 (F6B) R3d112 R3d121 R3d131 R3d132 R3d211 R3d212 R3d221 (F6D) R3d222

-179.77 -176.32 -173.94 -170.10 -169.64 -165.97 -164.88 -171.75 -171.51

(best overall) (F7D)

-155.15

R3d111 R3d321 (F7C) R3d322

-171.27 -158.01 -156.03

Gma5 [+ta]-B[a]P-N2-dG nondistortedb R1 (best) distortedd R1d3(F7A) R1d4

-149.66 -151.01

R2d31 (F7B) R2d32

-151.90 -151.89

-150.15

R2d21

-163.08

a

R1, R2, and R3 indicate the first, second and third round of the 24-parameters protocol. b The lowest energy nondistorted structures from R1 (“best”) and following multiple rounds of the 24-parameters protocol (“best overall”) for comparison to the lowest energy distorted structures. c Energy in kcal/mol. d The best (lowest energy) distorted structure from R1, R2, and R3. e “R1d1” indicates a structure from the first round (“R1”) that is distorted (“d”) and lowest in energy (“1”). “R3d112”, for example, indicates a structure from the third round (“R3”) that is distorted (“d”) and derived from the lowest energy structure in the first and second round, but the second lowest energy structure in the third round (“112”). f “F6A” indicates that this structure can be viewed in Figure 6A.

nondistorted structure that eventually emerge via the iterative 24-parameters protocol (e.g., the BPmi5 conformation for [+ta]-B[a]P-N2-dG in Figure 5). Thus structures in R1 (e.g., R1d1), which only generate distorted structures in R2 or R3 (e.g., R3d111), cannot be used to find the best acceptable structures, and must be disregarded. (3) As apparent from Figures 6 and 7, only structures with major deformations were rejected. Minor deformations (e.g., extreme propeller twists) were not

used to reject a structure. (4) Decisions about the rejection of structures were made prior to the determination of the energies of those structures. This raises the question: what is the likely source of the generation of these distorted, artifactual structures? We believe the answer is that our calculations do not include water. The bases in DNA have hydrogen-bonding donors and acceptors that would be interacting with water if it were implicitly included. Without water, these



Figure 5. Stereoview of the lowest energy version of the BPmi5 conformation for [+ta]-B[a]P-N2-dG in duplex DNA of sequence 5′-ATCGCTA-3′. The view is into the minor groove with the adduct-containing strand running 5′-to-3′ from bottom to top.

donors and acceptors can form Coulombic interactions with other atoms in the structure, and this can start the structure down an unrealistic path toward a distorted structure. Perhaps the path from R1d3 (Figure 7A) to R3d321 (Figure 7B) illustrates this best; if the water molecules in the spine of hydration of the minor groove had been present, then the minor groove probably would not have collapsed. A similar analysis also applies for R1d1 (Figure 6A) and R1d4 (Figure 7C). Regarding the inclusion of water, preliminary studies were conducted. The B[a]P-N2-dG adduct was located at the center of a rectangular cell with dimensions of 30 Å × 30 Å × 35 Å (the minimum to fit the adduct-containing heptamers), the rest of the cell was filled with water molecules, a periodic boundary condition was applied to the rectangular cell, and then our calculations were performed. In the output structures, the energy of the water molecules themselves and their interaction with the adduct dominated the energetics (>90%), such that gratuitous differences/uncertainties in water structure overwhelmed the overall energy. We concluded that the inclusion of explicit water was likely to cause more artifacts than it eliminated. We are currently exploring studies with implicit solvent. Additional Results for B[a]P-N2-dG Adducts in Both 5′-CGC and 5′-TGC Sequences. The iterative 24parameter protocol was repeated for all eight conformations (BPmi5, BPmi3, BPma5, BPma3, Gmi5, Gmi3, Gma5, and Gma3) for each of the other three stereoisomers [-ta]-, [+ca]-, and [-ca]-B[a]P-N2-dG in the 5′-CGC sequence context, as well as for [+ta]-B[a]P-N2-dG in the 5′-TGC sequence context. The energy of the lowest energy structure for each conformation is given in Table 2. We note that in two cases ([+ta]- and [-ca]-B[a]PN2-dG) our canonical input, starting structure gave a lower energy final, output structure than did the NMR input starting structure, while in two other cases ([-ta]-and [+ca]-B[a]P-N2-dG) the NMR input starting structure gave lower energy structures. In all cases, however, the final output structure was similar in structure and energy for both our canonical starting structure and the NMR starting structure. These results suggest that neither was a significantly superior starting structure.

Lowest Energy Structures for [+ta]-, [-ta]-, and [+ca]-B[a]P-N2-dG. Of the eight conformations studied, BPmi5 was calculated to be lowest in energy for [+ta]B[a]P-N2-dG in the 5′-CGC sequence (Table 2, Figure 5). BPmi5 was also the conformation observed in an NMR study (34). BPmi5 is low in energy (Table 2) whether we started with our canonical structure for BPmi5 or with the coordinates generated in the NMR study (33). The next lowest energy structure is BPmi3, which is significantly higher in energy (∼4.4 kcal). BPmi3 was calculated to be the lowest energy conformation for [-ta]-B[a]P-N2-dG in the 5′-CGC sequence (Table 2, Figure 8), and it was the conformation observed in an NMR study (39). BPmi3 is low in energy whether we started with our canonical structure for BPmi3 or with the NMR coordinates. However, Gmi3 is close in energy to BPmi3 (∼1.2 kcal higher); BPmi3 and Gmi3 are sufficiently close in energy that it would be difficult to distinguish between them based on computed values alone. Gmi3 was calculated to be the lowest energy conformation for [+ca]-B[a]P-N2-dG in the 5′-CGC sequence (Table 2, Figure 9), and it was the conformation observed in an NMR study (40). Gmi3 is low energy whether we started with our canonical structure for Gmi3 or with the NMR coordinates. The next lowest energy structure is BPmi5, which was higher in energy (∼3.6 kcal). NMR studies showed a minor conformation for [+ca]-B[a]P-N2-dG in the 5′-CGC sequence (40); BPmi5 is a good candidate, although the true solution energy difference would have to be less than what we have estimated. BPmi5 was calculated to be the lowest energy conformation for [+ta]-B[a]P-N2-dG in the 5′-TGC sequence (Table 2; the structure is virtually identical to the one in Figure 5), and it was the conformation observed in an NMR study (33). However, Gma5 is close in energy to BPmi5 (∼1.4 kcal higher); these two are sufficiently close that it is difficult to distinguish between them based on computed values alone. It is of interest to note that in the NMR studies, a second conformation was observed (33). The authors speculated that it appeared to be some kind of base-displaced conformation, and our calculations suggest that Gma5 is a good candidate for it.

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Figure 6. Stereoview of four low energy, but distorted versions of BPmi5 conformations for [+ta]-B[a]P-N2-dG in duplex DNA of sequence 5′-ATCGCTA-3′. The view is into the minor groove with the adduct-containing strand running 5′-to-3′ from bottom to top in each case. Panels A, B, C, and D show R1d1, R3d111, R2d12, and R3d221, respectively, and are described in the text and in Table 3.



Figure 7. Stereoview of three low energy, but distorted, versions of Gma5 conformations for [+ta]-B[a]P-N2-dG in duplex DNA of sequence 5′-ATCGCTA-3′, as well as the lowest energy nondistorted Gma5 conformation. The view is into the minor groove with the adduct-containing strand running 5′-to-3′ from bottom to top in each case. Panels A, B, and C show R1d1, R3d121, and R1d2 respectively, and are described in the text and in Table 3. For comparison, panel D shows the overall lowest energy nondistorted Gma5 structure.

Low Energy Structures for [-ca]-B[a]P-N2-dG. By NMR, Gma5 was the conformation observed for [-ca]B[a]P-N2-dG in a 5′-CGC sequence (41). Using the itera-

tive 24-parameters procedure, BPmi5 is calculated to be the lowest energy structure, while Gma5 (Figure 10) is second lowest, independent of whether we initiated the

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Figure 8. Stereoview of the lowest energy version of the BPmi3 conformation for [-ta]-B[a]P-N2-dG in duplex DNA of sequence 5′-ATCGCTA-3′. The view is into the minor groove with the adduct-containing strand running 5′-to-3′ from bottom to top.

Figure 9. Stereoview of the lowest energy version of the Gmi3 conformation for [+ca]-B[a]P-N2-dG in duplex DNA of sequence 5′-ATCGCTA-3′. The view is into the minor groove with the adduct-containing strand running 5′-to-3′ from bottom to top.

Figure 10. Stereoview of the lowest energy version of the Gma5 conformation for [-ca]-B[a]P-N2-dG in duplex DNA of sequence 5′-ATCGCTA-3′. The view is into the major groove with the adduct-containing strand running 5′-to-3′ from bottom to top.

calculation from our canonical Gma5 starting structure (∼2.7 kcal higher than BPmi5, Table 2) or from the Gma5 NMR structure (∼0.6 kcal higher than BPmi5, Table 2).

This discrepancy led us to evaluate other Gma5 input structures for [-ca]-B[a]P-N2-dG. The NMR structure for [-ca]-B[a]P-N2-dG has the complementary dC moiety


oriented with the C2-carbonyl tilted into the major groove, and we used that orientation when we built our canonical version of Gma5 for [-ca]-B[a]P-N2-dG (and all other Gma5 starting structures). We rotated this dC about the glycosylic bond, trying three other orientations, but none ultimately gave lower energy conformations. Several other Gma5 input structures for [-ca]-B[a]P-N2dG were also tried, but none gave lower energy conformations. Finally, previous work suggested that B[a]Pcontaining 7-mers were preferable to 9-mers or 11-mers; however, this decision was based on work for [+ta]-B[a]PN2-dG in the BPmi5 conformation in a 5′-CGC sequence. We explored the possibility that an 11-mer, which corresponds exactly to the sequence studied by NMR, might be preferable for [-ca]-B[a]P-N2-dG in the 5′-CGC sequence context, but this did not appear to be the case (data not shown). In summary, we find that BPmi5 is calculated to be lower in energy than Gma5 for [-ca]-B[a]P-N2-dG even though the latter is observed by NMR. However, the calculated difference in energy between BPmi5 and Gma5 can be minimal (e.g., as low as 0.6 kcal/mol, Table 2), so this discrepancy is not great. NMR studies showed a minor conformation for [-ca]-B[a]P-N2-dG in the 5′-CGC sequence (41), and BPmi5 is certainly a good candidate for it. Basis for Conformational Preferences. Dr. Suse Broyde and colleagues have studied nucleosides adducts of different stereoisomers of BP-N2-dG (56) to probe the fundamental basis for their conformational preferences. They pointed out that the cis adducts [+ca]- and [-ca]BP-N2-dG have two, adjacent hydroxyl groups pointing in the same direction as their adduct bonds (Figure 1), which thrusts these two hydroxyl groups into the helix axis when a groove-like orientation is adopted (i.e., with BPmi5, BPmi3, BPma5, or BPma3). The resulting steric crowding makes the groove-like orientations relatively higher in energy, and probably is the major reason base displaced conformations are favored for both cis adducts. The position of adduction (C10 on the saturated benzylic ring) places the greatest bulk of BP-N2-dG adducts below the adduct bond as the structures are drawn in Figure 1. Furthermore, [+ta]- and [-ca]-BP-N2-dG have S-stereochemistry about the adduct bond, while [-ta]and [+ca]-BP-N2-dG have R-stereochemistry. Given both the asymmetry in bulk and the adduct bond stereochemistry, there is a natural tendency for adducts with S- vs R-stereochemistry to prefer mirror image orientations in the case of the nucleoside adducts (56). This fundamental tendency is also apparent in DNA (66, 67), although we find some exceptions when considering all four stereochemical examples in the 5′-CGC sequence context (Table 2). BPmi5 is lower in energy than BPmi3 for [+ta]- and [-ca]-BP-N2-dG (both S-configurations), while BPmi3 is lower in energy for [-ta]-BP-N2-dG (an R-configuration). [+ca]-BP-N2-dG is the exception, where BPmi5 is preferred to BPmi3, because of steric clashes involving the hydroxyl groups in the case of the BPmi3 conformation (data not shown). Considering base displaced conformations, Gmi3 is lower in energy than Gma5 for [-ta]- and [+ca]-BP-N2-dG (both R-configurations), while Gma5 is lower in energy for [+ta]-BP-N2-dG (an S-configuration). [+ta]-BP-N2-dG is the exception in this case, where Gmi3 is preferred to Gma5. It is less obvious what underlying principle dictates this exception, since both Gma5 and Gmi3 appear to fit nicely in DNA without major steric


clashes. In this regard, we note that (1) the calculated energy difference between Gma5 and Gmi3 is less in the case of [+ta]- (4.63 kcal/mol) than [-ta]- (8.42 kcal/mol) or [+ca]- (10.67 kcal/mol) or [-ca]-BP-N2-dG (10.09 kcal/ mol); and (2) Gma5 is lower in energy than Gmi3 in the case of [+ta]-BP-N2-dG in the 5′-TGC sequence (Table 2). Finally, we note that this same pattern (i.e., one exception to the rule that mirror image conformers show opposite conformational preferences) also exists for the relatively higher energy conformational pairs BPma5 vs BPma3 (exception: [+ca]-BP-N2-dG), and Gmi5 vs Gma3 (exception: [+ta]-BP-N2-dG) (Table 2). In summary, two simple principles derived from modeling studies of nucleoside adducts of BP-N2-dG stereoisomers seem to influence strongly the underlying conformational preferences (56), although exceptions emerge when considering these adducts in duplex DNA, since the situation is complex. A Comparison of Our Methods to Those of Others. The work reported herein is the only example (of which we are aware), where each of the reasonable conformational possibilities for BP-N2-dG adducts in duplex DNA (i.e., the eight conformations in the two leftmost columns in Figure 1) have been studied explicitly, and the best structures for each conformation have been compared to each other, as well as to what is known from NMR studies. On the basis of probabilities, there was only an ∼50% chance that one of the five structures would be predicted correctly, so our record of 4 of 5 correct (plus coming close on the fifth) suggests that our methods are better than random and seem likely to have some validity. Since no other group has conducted studies similar to our own, we cannot compare the success of our method to the methods of others. As noted above, Dr. Broyde and colleagues have laid the foundation for our understanding of the principles of conformational preferences based on their studies of B[a]P-N2-dG nucleoside adducts (56). They also correctly predicted that BPmi5 and BPmi3 would be the preferred conformations for [+ta]- and [-ta]-B[a]P-N2-dG, respectively (55), and have extended this work more recently (66, 67). Despite our apparent successes, it is important to point out that our methods might be improved. An extensive study of duplex DNA was recently conducted in which the coordinates of modeled structures were compared to NMR and X-ray crystal structures (68). Agreement was improved when the energy barriers were reduced between the C2′- and C3′-endo conformations of the deoxyribose, when the Lennard-Jones nonbonded energy terms were shifted to a distance ∼0.4 Å greater than the sum of the van der Waals’ radii and when the dielectric function assumed a steep distance-dependence. Furthermore, advances in the use of free energy calculations combined with implicit solvent (or “continuum” models) have been developed for nucleic acids. In the future, we must consider each of these approaches and techniques (69). Modeling in ds-DNA. It is important to address why we think that modeling BP-N2-dG adducts in ds-DNA might be relevant to our mutagenesis studies. Certainly, the “mutagenic mechanism point”, for example, for dA insertion (i.e., the G f T pathway) vs dT insertion (i.e., the G f A pathway) involves a transition state between an incoming dNTP and a ss/ds-DNA junction in the active site of a relevant DNA polymerase; this state does not

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resemble ds-DNA. However, our mutagenesis results concern, for example, the relative probability of following the G f T vs G f A pathway (principally as a function of DNA sequence context), which is the “mutagenic decision point”. It is not axiomatic that the “mutagenic mechanism point” and the “mutagenic decision point” are the same. We have argued that, if adducts cannot undergo conformational rearrangements after being bound in a DNA polymerase active site, then the probability that the DNA polymerase will follow, for example, the G f T vs the G f A pathway could be dictated by the probability that the polymerase encounters the adduct in the conformation responsible for the G f T vs the G f A mutation (59, 60). In fact, there are at least five lines of evidence in support of this view (60), including several suggesting that the G f T vs G f A mutational conformations for [+ta]-BP-N2-dG do not interconvert rapidly, at least in some circumstances. If true, then the mutagenic decision point is defined by the relative fraction of the G f T vs G f A conformation in ds-DNA prior to the arrival of DNA polymerase, which is why it makes sense to model BP-N2-dG adducts in ds-DNA. Figure 1 shows 16 conformations, and it seems likely that some of these will interconvert rapidly, while others will not. Furthermore, interconversion will be influenced by factors such as DNA sequence context, the timing between DNA strand separation and DNA replication, the properties of the relevant DNA polymerase(s), etc. This complexity will make it exceedingly difficult to understand the relationship between adduct conformation and adduct mutagenesis, and we do not mean to imply that we know what is correct, However, currently our best “guess” is that modeling in ds-DNA makes sense, given our interest in understanding the G f T vs G f A mutational pathway for BP-N2-dG adducts.

Conclusions Our five-step 24-parameters molecular modeling procedure correctly predicts the lowest energy conformation determined by NMR in four of five cases (i.e., for [+ta]-, [-ta]-, and [+ca]-B[a]P-N2-dG in 5′-CGC and [+ta]-B[a]PN2-dG in 5′-TGC), while in the fifth case ([-ca]-B[a]PN2-dG in 5′-CGC) the known NMR conformation (Gma5) is computed to be second lowest in energy and close to the lowest computed energy conformation (BPmi5). This level of agreement is surprisingly high given the fact that eight different conformations were considered in our modeling studies and the fact that B[a]P-N2-dG adducts adopted four different conformations as determined by NMR studies in these five cases. Thus, we are encouraged that our five-step protocol (“iterative 24 parameters”) may provide useful insight into the energetics of B[a]P-N2dG adducts.

Acknowledgment. This work was supported by United States Public Health Services Grant R01CA50432. We thank Dr. Suse Broyde for graciously making coordinates available for B[a]P-N2-dG adducts. We also thank the Scientific Computing and Visualization group at Boston University for allocations of computational resources.

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