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Effect of Ring Size on Conformations of Aromatic Amine-DNA Adducts: The Aniline-C8 Guanine Adduct Resides in the B-DNA Major Groove Robert Shapiro,*,† Stephen Ellis,† Brian E. Hingerty,‡ and Suse Broyde*,§ Chemistry and Biology Departments, New York University, New York, New York 10003, and Life Sciences Division, Oak Ridge National Laboratory, Oak Ridge, Tennessee 37831 Received November 25, 1997
While the one-ring amine aniline (AN) has only slight genetic activity, the polycyclic aromatic amines 2-aminofluorene (AF) and 1-aminopyrene (AP) are significant mutagens and carcinogens. Moreover, the bulkier AP is more mutagenic per adduct than AF in the tetracyclineresistance gene of plasmid pBR322 [Melchior et al. (1994) Carcinogenesis 15, 889]. To elucidate possible conformational origins of the differing mutagenic effects of these three adducts, which may stem from their differing ring sizes, we have examined their conformations in two mutation-susceptible sequences from the above gene: TTGAG*GCCG (sequence I) and GAATG*GTGC (sequence II), where G* ) C8-modified guanine. No experimental highresolution NMR data are yet available for the aniline adduct in a DNA duplex. Minimized potential energy calculations were carried out, using the molecular mechanics program DUPLEX to explore the conformation space of these adducts. In the case of AN, a relatively unperturbed B-DNA helix with the amine in the major groove was strongly favored in both sequences. In the case of AF- and AP-modified DNA, however, several differing conformations were competitive in energy. They included major groove structures, as well as conformations with syn-modified guanine and the polycyclic amine in the minor groove, or the amine rings intercalated into the helix with displacement of the modified guanine, in overall harmony with high-resolution NMR solution structures. Thus, aniline distorts DNA structure to a lesser extent than larger aromatic amine ring systems, since a number of different conformations are energetically feasible and have been observed for the larger systems. This result may be relevant to their enhanced mutagenicity and their repair propensity, in contrast to aniline’s low mutagenic effect.
Introduction The aromatic amines have been noteworthy carcinogens because of their historical importance and wide human exposure (1, 2). The reactivity and ease of preparation of the amines have made them central to the chemical industry for more than a century. It was recognized as early as 1895 that workers in the dye industry developed bladder cancer as a consequence of their long exposure to aromatic amines (3). According to one review: “The aromatic amines are one of the few classes of chemical carcinogens for which there is convincing evidence that some members induce cancer in humans” (3). The risk from aromatic amines is not confined to industrial workers but also extends to the general public. They are components of cigarette smoke and synthetic fossil fuels derived from coal (4). Further, a threat of undetermined magnitude exists from the potent aromatic amine mutagens and carcinogens that are formed during the cooking of meat and other protein-rich foods (reviewed in refs 5 and 6). * Corresponding authors. † Chemistry Department, New York University. ‡ Oak Ridge National Laboratory. § Biology Department, New York University.
The biological potency of the aromatic amines is strongly affected by their structure. In a survey of 34 diverse amines, it was found that their potency covered 9 orders of magnitude for mutagenesis and 4 orders for carcinogenesis (7). In an attempt to correlate structural features with potency, only the number of rings and the presence of C-methyl substituents were significant, with the number of rings having the greater weight. It is widely believed that most chemical mutagens exert their effect through reaction of their activated forms with DNA. The structures of such damaged DNAs very likely contribute to their mutagenic potential, and it is hoped that an understanding of the details of structure will ultimately enable us to predict many of the genetic effects of chemical mutagens. We are using molecular mechanics calculations with energy minimization to elucidate the feasible conformations of DNA segments modified by mutagenic aromatic amines. Although more than one product may result from the reaction of activated aromatic amines with DNA, the principal product in most instances arises from attachment of the amine nitrogen to C8 of guanine (1). To gain further understanding of the effect of the number of rings on DNA conformation, we have explored the conforma-
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Figure 1. Structures of adducts and definitions of linkage site torsion angles (A-B-C-D). The angle A-B-C-D is measured by a clockwise rotation of D with respect to A, looking down the B-C bond. A eclipsing D is 0°. (A) AN: R′ ) N9-C8N(AN)-C1(AN); β′ ) C8-N(AN)-C1(AN)-C2(AN). (B) AF: R′ ) N9-C8-N(AF)-C2(AF); β′ ) C8-N(AF)-C2(AF)-C1(AF). (C) AP: R′ ) N9-C8-N(AP)-C1(AP); β′ ) C8-N(AP)-C1(AP)C1OA(AP). The torsion angle designations for the DNA sugar backbone and glycosidic torsion angles are R, O3′-P-O5′-C5′; β, P-O5′-C5′-C4′; γ, O5′-C5′-C4′-C3′; , C4′-C3′-O3′-P; ζ, C3′-O3′-P-O5′; χ (pyrimidines), O4′-C1′-N1-C2; χ (purines), O4′-C1′-N9-C4. Sugar pucker is defined by the pseudorotation parameter P (49). The thymine methyl torsion angle, Me, is defined as C6-C5-C-H.
tions of DNA duplex nonamers in two sequences with adducts of three aromatic amines of differing number of rings: aniline (AN),1 2-aminofluorene (AF), and 1-aminopyrene (AP). Figure 1 shows structures of these adducts. These three amines have been selected for study because of considerable interest in them over the years and as paradigms for the effects of ring size. The sequences selected for the present work (only modified strand shown) are sequence I, TTGAG*GCCG, and sequence II, GAATG*GTGC. The modified G* has a normal partner, C. Both sequences were reported as sites of point and/or frameshift mutations by AF, AP, and other polycyclic aromatic amines in the tetracyclineresistance gene of plasmid pBR322 in Escherichia coli (8). Aniline. Although the name “aniline cancer” was first applied to the disease contracted by dye workers (3), this substance is at most a borderline carcinogen (reviewed in ref 9; see also refs 10 and 11). Although the lack of potency of aniline may be due to the lesser reactivity of its activated derivatives with DNA (9), we thought it important to compare the conformational effects of a system containing a single unsubstituted aromatic ring with those of larger ones and have therefore studied aniline. 2-Aminofluorene. The aromatic amines most studied in terms of their genetic effects and mechanism of action are 2-aminofluorene and its acetylated derivative, 2-(acetylamino)fluorene. They were prepared synthetically and tested for use as insecticides by the U.S. Department of Agriculture more than 50 years ago (12). However, they were found to produce tumors in the liver 1 Abbreviations: AN, aniline; AF, 2-aminofluorene; AP, 1-aminopyrene.
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and other organs of rats and many other species and, hence, were never used as pesticides. Instead, AF and AAF have found a career as models for the carcinogenic process. The pair has been termed “superb tools for the exploration of the mechanism of carcinogenesis” (13), and it has been judged in a recent review that they are “among the most intensively studied of all chemical mutagens and carcinogens” (14). The depth of the literature on AAF and AF can be judged from the length (over 100 pages) of that review. Additional discussion targeted toward mutagenic mechanisms is provided in recent reviews by Hoffmann and Fuchs (15) and Shibutani and Grollman (16). We have included AF with its two in-plane methylene-bridged aromatic rings in this study as a representative amine of intermediate bulk. 1-Aminopyrene. Aminopyrene residues are bound to DNA in a process that starts with exposure of an organism to the widespread environmental carcinogen 1-nitropyrene. The latter substance occurs in diesel engine exhaust, coal fly ash, urban air particulates, some foods, and a number of other sources (17). 1-Nitroreduction processes in vivo then afford activated derivatives that react with DNA to produce an aminopyrene adduct to C8 of guanine. This process can initiate tumors in the mammary glands, livers, and lungs of rodents (reviewed in ref 1). In the mutagenicity study of Melchior et al. (8), 1-aminopyrene was found to be more mutagenic (per adduct) than 2-aminofluorene. Mutagenesis data on 1-aminopyrene in other systems are summarized in Malia et al. (18) and Gu et al. (19). 1-Aminopyrene, with its four aromatic rings, has been selected as an example of a larger system.
Methods Energy Minimization. Minimized potential energy calculations were carried out with DUPLEX, our molecular mechanics program for carcinogen-modified nucleic acids (20). DUPLEX performs potential energy minimizations in the reduced variable domain of torsion angle space. It employs a force field for nucleic acids that was devised in the laboratory of Olson (21, 22). We employ a distance-dependent dielectric function (r), which mimics the interpenetration of aqueous solvent as the distance, r, between an atom pair increases: (r) ) ′/e-βr. A value for the Debye screening parameter β of 0.1 is used. This simulates the screening effect of a 0.1 M monovalent salt concentration (23). In the trimer trials the variable ′ was assigned a value of 4 as in earlier work (20). In the nonamer trials, ′ was assigned a value of 1, producing a less steep distance dependence of the dielectric function, which gave results more consistent with experiment in studies on unmodified nonamer duplexes in our laboratory (unpublished data). Appropriate choices for distance-dependent dielectric functions are currently under investigation in our laboratory (24). Geometries and force-field parameters for the AN, AF, and AP adducts are the same as those employed in earlier work (9, 25, 26). Search Strategy. The search strategy for all but the basedisplaced-intercalated structures started with modified duplex trimers whose sequence was the same as that of the central trimers in the modified nonamers of sequences I and II. A total of 80 trials were carried out for each modified trimer sequence in each adduct, using the search strategy described previously in detail (26-28). Thus, 480 trials were performed at the trimer level for three adducts in two sequences. Trimers representative of the various types of computed conformations for each adduct were then selected for building to the nonamer level. The nonamers were generated by embedding the trimer in a B-DNA nonamer of the desired sequence and minimizing the energy in stages, according to our previously described procedure (26, 27).
Major Groove Aniline Adduct
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Figure 2. AN-modified structure (stereoview). Sequence I: major groove, ∆E ) 0.0 kcal/mol. Color code: DNA, blue; modified guanine, yellow; AN, red. The starting torsion angles were those of the selected trimer, flanked by two B-DNA trimers, to constitute a nonamer. Torsion angles for the flanking DNA starting conformation were those of an ideal B-DNA fiber diffraction model (29), except that syn-purines at the lesion site were oriented with glycosidic torsion angles of 60°. Since the systematic trimer searches had not located low-energy base-displaced-intercalated structures, torsion angles to search for such structures directly on the nonamer level were obtained from representative structures determined by NMR studies and earlier computations (26, 30). Furthermore, in most cases additional trials were run for a particular structural type and sequence using the angles of a favored minimum that had already been located in another sequence with the same amine, or in the same sequence with another amine, employing the procedure described previously (26, 27). This was done to maximize the possibility that the lowest-energy variant of a given structural type had been located, in view of the multiple minimum problem. Torsion angle definitions are given in Figure 1.
Table 1. Representative Low-Energy Conformations of Modified DNA Nonamersa carcinogen/sequence
χ
R′
β′
∆E
class
AN/sequence I
237
162
42
0.0
major
AN/sequence II
230
188
39
0.0
major
AF/sequence I
67 67 236 229 340
261 249 165 197 220
316 141 227 39 42
0.0 0.8 2.3 2.4 4.0
minor minor major major BD
AF/sequence II
67 228 232 68 341 235
248 203 191 259 218 160
137 212 45 314 40 233
0.0 0.1 0.7 1.2 3.2 3.5
minor major major minor BD major*
AP/sequence I
236 336 64
161 218 243
228 220 139
0.0 0.5 4.6
major BD minor
AP/sequence II
230 231 340 66 235
191 151 214 247 164
223 73 222 137 232
0.0 1.7 2.8 3.1 3.7
major major BD minor major*
Results The structures considered were double-stranded nonamers of sequences I and II, modified at the central G by AN, AF, and AP (Figure 1). Bases and sugars in nonamers are referred to numerically according to the following scheme: 1 – 2 – 3 – 4 – 5 – 6 – 7 – 8 – 9 18 – 17 – 16 – 15 – 14 – 13 – 12 – 11 – 10
1 and 10 are the 5′ residues of the two chains, and 5 is the modified base. “Base pair 4” in this notation refers to the 4-15 pair. The full torsion angle sets for the central trimers (base pairs 4-6) for the best minima in each class of structure up to 5 kcal/mol above the lowest-energy conformation (∆E ) 0.0 kcal/mol) are presented in Supporting Information Tables S1-S6. Table 1 gives the key χ, R′, and β′ torsion angles that govern the orientation of the aromatic amine and relative energies for these minima. Selected structures are shown in Figures 2-4. In general, the structures of a particular class were quite similar in appearance, irrespective of the amine or sequence. However, energies do vary with the sequence or amine.
a Torsion angles in degrees; ∆E in kcal/mol; major, major groove; major*, shielded major groove; minor, minor groove; BD, basedisplaced-intercalated. Sequence I (modified strand) ) TTGAG*GCCG. Sequence II (modified strand) ) GAATG*GTGC.
Major Groove Structures (Figures 2, 3C, 4A,C). In this class of structure, DNA exists in a normal double helix with base pairing generally intact and the modified guanine anti. The aromatic residue projects into the major groove, but least for the one-ring aniline. The most favorable structures of this type for the three amines all have the aromatic system oriented in a 5′-direction on the modified strand, with the ring bearing the amino group in contact with one or both protons of C2′ of deoxyribose-4. A stretched hydrogen bond generally exists between the amine NH and the 5′-OH of deoxyribose-5. In the case of AF and AP (but not symmetrical AN), two positions of the fluorene or pyrene residue can be generated by a 180° rotation around the N to hydro-
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carbon bond (torsion angle β′). These are designated low and high β′, corresponding to the relative magnitudes of their values. In Table 1, for example, in the case of AF sequence I, 227° is the high β′ value and 39° is the low β′ value for the two major groove structures. The conformers produced by this rotation are usually competitive in energy. The most favored major groove structures of sequence I are generally almost unbent (Figures 2 and 4A), while those of sequence II are quite bent (Figures 3C and 4C). Shielded Major Groove Structures (Figure 3E). In this group, the aromatic ring system lies flat against the walls of the major groove in a 3′-direction on the modified strand, with contacts to base pairs 6 and 7. The opposite face of the ring system is exposed, except for contacts to the 2′-CH2 protons of deoxyribose-4. The antimodified guanine and opposite cytosine are coplanar but do not base-pair. Their closest approach involves their carbonyl oxygens. Other base pairs are intact. This structural type was previously noted for AP in a different sequence, as detailed in Nolan et al. (26). Minor Groove Structures (Figures 3A,B, 4D). The modified guanine is rotated to a syn conformation, which places the aromatic amine residue in the minor groove. Watson-Crick pairing at the modified G is disrupted, though one Hoogsteen bond from O6 of guanine to the amino group of cytosine-14 exists in some cases. The modified G and its partner C remain stacked within the helix. The other base pairs of the helix are intact. The aromatic rings may lie in the minor groove in a way that has most of their bulk oriented in either a 3′- or 5′-direction along the modified strand. However, the 5′oriented structures are considerably higher in energy (at ∼15 kcal/mol). The conformers in which the aromatic amine has a 3′ orientation still present two alternatives generated by rotation around torsion angle β′, as in the case of the major groove structures. Base-Displaced-Intercalated Structures (Figures 3D, 4B). The modified guanine is displaced to the minor groove in the vicinity of base pairs 3 and 4. Its glycosidic torsion angle is syn but in an unusual domain, near 340° (-20°), while the angle range centered about 60° is the prevalent region in unmodified DNAs where syn-guanines occur (31). The amine residue occupies, approximately, the position of the displaced guanine in the helix. Base pairing in the helix is intact, except at the modified guanine and its partner. The helix is somewhat bent. The aromatic rings stack with base pair 6 and partly with base 4. Cytosine-14 is mostly exposed on the side of base pair 4 and has loose contacts to deoxyribose-13 hydrogens on its other side. This structural type had also furnished the global minimum in our calculations for the duplex nonamer with the sequence TCATGAPATTC on the modified strand (26).
Discussion Comparison of AN, AF, and AP: Effect of Ring Size. In the two DNA nonamers with a central base pair
Figure 3. AF-modified structures (stereoviews). (A) Sequence I: minor groove 3′, high β′, ∆E ) 0.0 kcal/mol. (B) Sequence II: minor groove 3′, low β′, ∆E ) 0.0 kcal/mol. (C) Sequence II: major groove, high β′, ∆E ) 0.1 kcal/mol. (D) Sequence II, basedisplaced-intercalated, ∆E ) 3.2 kcal/mol. (E) Sequence II: shielded major groove, ∆E ) 3.5 kcal/mol.
Major Groove Aniline Adduct
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modified by AN, an unperturbed major groove structure is favored (Figure 2), and other conformers are much poorer in energy. For AF and AP, on the other hand, alternative conformers are closely spaced in energy, making it difficult to be sure of their relative importance because of the multiple minimum problem and uncertainties in the force field. However, our data do show a greater tendency toward minor groove structures for AF and to base displacement with intercalation by AP, in our two sequences. Major groove structures are also competitive in energy in both AF and AP. The larger the ring size the greater the advantage to the hydrophobic moiety in avoiding solvent and in stacking with base. Both minor groove and base-displacedintercalated structures shield the aromatic hydrophobic surface, while the base-displaced one also has the added advantage of stacking the aromatic system with bases of the helix. However, the carcinogen-base stacking in base-displaced intercalation comes at the expense of greater helix distortion than occurs in minor groove structures: the modified base and possibly its partner must sacrifice their stacked-in position to accommodate the carcinogen in the case of base-displaced intercalation, while these bases remain stacked when the carcinogen is in the minor groove. On the other hand, both these types of structures exact the price of ruptured WatsonCrick base pairing with rotation of the modified guanine to the abnormal syn domain, in contrast with the major groove structures which retain the normal B-DNA form. Clearly, these factors are in a delicate balance, which is modulated also by base sequence. The present study indicates that in the case of the one-ringed aniline adduct the energy balance strongly favors the undistorted major groove conformation, since exposure of the small hydrophobic surface is minimal. The advantages of burial in the minor groove or base-displaced intercalation are not worth the energetic cost. Sequence Effects and Comparison with Experiment. Our results are in overall harmony with experimental observations on related structural types in solution. Base-displaced-intercalated conformers with synguanine have been shown to predominate in solution for AP and AF modification in an 11-mer duplex with modified strand sequence CCATCG*CTACC (30, 32). However, the AP adduct was essentially 100% basedisplaced-intercalated, while the AF adduct was only ∼70% in this conformation; the balance of the population contained the AF in the major groove. Our computed greater predilection for base-displaced intercalation in the AP adduct compared to the AF one in the present study is in line with these solution studies. A major groove structure for AF modification predominates (∼90%) in solution in a duplex with modified strand sequence CTCGG*CGCCATC (33), with the balance of the population base-displaced-intercalated. An approximately equal proportion of base-displaced-intercalated conformers and major groove conformers has been observed in solution by Eckel and Krugh (34, 35) for the AF-modified sequence CACCAG*GAAC. Cho et al. (36) also observed a predominant major groove conformer (∼60-70%) for AF Figure 4. AP-modified structures (stereoviews). (A) Sequence I: major groove, high β′, ∆E ) 0.0 kcal/mol. (B) Sequence I: base-displaced-intercalated, ∆E ) 0.5 kcal/mol. (C) Sequence II: major groove, high β′, ∆E ) 0.0 kcal/mol. (D) Sequence II, minor groove 3′, low β′, ∆E ) 3.1 kcal/mol.
340 Chem. Res. Toxicol., Vol. 11, No. 4, 1998
modification, in the sequence TACTCTTCTTG*ACCT, as did Zhou et al. (37) (∼55%) in a truncated version of this sequence. In addition, minor groove conformers have been observed for AF modification in the sequence CCATCG*CTACC when an A (38), G, or I (39) is placed opposite the modified guanine. It is noteworthy that this is the same sequence in which AF was ∼70% basedisplaced-intercalated and ∼30% in the major groove when the normal partner C was opposite the lesion (32), as mentioned above. However, when this sequence is modified by AP with A opposite the lesion, a basedisplaced-intercalated conformer is observed (19). In the sequence CTCATG*ATTCC with AP modification, on the other hand, a more heterogeneous conformational mix precluded detailed NMR characterization, but spectroscopic data and calculations suggested the presence of both intercalative and external types of structures (26). Thus, experimental studies show that base sequence effects can clearly modulate the adopted conformations with exquisite precision; this is also suggested from the present results in which similar structural types have differing energy rankings for a given adduct in the two sequences investigated. One interesting sequence effect for AP modification in the current work is the much greater stabilization of the shielded major groove structure in sequence II than in sequence I, although this structure, which addresses the hydrophobicity in a novel way, has not yet been observed experimentally. The greater stabilization in sequence II likely stems from the fact that base pair 7, which is contacted by the aromatic ring system, is a T‚A pair in sequence II, while it is a C‚G pair in sequence I: the more flexible T‚A pair can permit closer stabilizing interactions between the major groove walls and the aromatic rings. Our search strategy employed duplex trimers to locate without experimental information major and minor groove type low-energy structures similar to observed ones. These were then built to the nonamer level by embedding them into B-form DNA. However, low-energy base-displaced-intercalated structures were not found by this strategy. Our accumulated experience has suggested that duplex trimers may be too short to favor structures of this type, probably because the stabilizing effect of additional residues is needed to counterbalance the local distortion. In the present work base-displaced-intercalated structures were sought in duplex nonamers by employing experimental and previously computed structures of this type (for different carcinogens and/or different base sequences) as starting models. Previously, in the absence of such models, we have computed basedisplaced-intercalated structures in duplex dodecamers de novo, by embedding low-energy single-stranded dimer and trimer conformers with carcinogen-base stacking into B-DNA duplex dodecamers (40). These proved to be in agreement with subsequent independent NMR determinations of such structural types (41). Relevance to Replication and Repair. The larger amines, AF and AP, show a greater tendency to disturb duplex DNA structure than does AN. If this tendency is also expressed during stages of the replication process, then it is consistent with the marked activity of AP and AF as mutagens and carcinogens, and also the borderline status of AN. The various conformations displayed by AP and AF in double-stranded DNA are especially relevant to questions of repair of the damaged duplexes. It has been suggested
Shapiro et al.
that differential rates of repair of the same adduct in different sequences may be one factor that determines mutagenic hotspots (42-44). DNA repair systems have been shown to be sensitive to DNA conformation governed by adduct type and base sequence context (41, 4547). The rate of repair of intercalative conformations appears to exceed that of other structures that distort DNA to a lesser extent (45, 46). Gunz et al. (48) suggested that mammalian excision repair is primarily targeted to structural defects that destabilize the doublehelical conformation of DNA. By this criterion, adducts that produce unfavorable changes in free energy would be primary targets for repair. While experimental information on the effects of DNA sequence on both adduct conformation and adduct repair rates is still very limited, as more data become available and correlations become apparent, predictions of repair susceptibility on the basis of structure may become feasible.
Acknowledgment. This work was supported by NIH CA 28038 and 75449, NIH RR 06458, and U.S. DOE DEFG02-90ER 60931 (S. Broyde and R. Shapiro) and also by U.S. DOE Contract DE-AC05-84OR21400 with MartinMarietta Energy Systems (B. E. Hingerty). Computations were carried out at the Department of Energy’s National Energy Research Supercomputer Center and the National Science Foundation’s San Diego Supercomputer Center. Supporting Information Available: Full torsion angle sets for the central trimers (base pairs 4-6) for the best minima in each class of structure to 5 kcal/mol above the lowest-energy conformation (∆E ) 0.0 kcal/mol) (6 pages). Ordering information can be found on any current masthead page.
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