Nuclear Magnetic Resonance Solution Structures ... - ACS Publications

MAY 1998 VOLUME 11, NUMBER 5 © Copyright 1998 by the American Chemical Society

Invited Review Nuclear Magnetic Resonance Solution Structures of Covalent Aromatic Amine-DNA Adducts and Their Mutagenic Relevance Dinshaw J. Patel,*,† Bing Mao,† Zhengtian Gu,† Brian E. Hingerty,‡ Andrey Gorin,† Ashis K. Basu,§ and Suse Broyde*,∇ Cellular Biochemistry and Biophysics Program, Memorial Sloan-Kettering Cancer Center, New York, New York 10021, Life Sciences Research Division, Oak Ridge National Laboratory, Oak Ridge, Tennessee 37831, Chemistry Department, University of Connecticut, Storrs, Connecticut 06269, and Department of Biology, New York University, New York, New York 10003 Received December 2, 1997

1. Introduction and Background 2. Normal DNA Duplexes with Equal Numbers of Residues on the Two Strands and Standard Watson-Crick Complement Opposite the Lesion Site: A View Prior to Replication 2.1. AAF 2.2. AF 2.3. ABP 2.4. AP 3. DNA Single-Strand-Double-Strand Junctions: Models of an Arm of a Replication Fork 3.1. The Lesion Site as Next Base To Be Replicated 3.2. The Lesion Site with a Normal Partner Opposite the Adduct * Corresponding authors. Dinshaw J. Patel: phone, 212-639-7207; fax, 212-717-3066; e-mail, [email protected]. Suse Broyde: phone, 212998-8231; fax, 212-995-4015; e-mail, [email protected]. † Memorial Sloan-Kettering Cancer Center. ‡ Oak Ridge National Laboratory. § University of Connecticut. ∇ New York University.

3.3. The Lesion Site with a Mismatch Opposite the Adduct 4. DNA Duplexes with Equal Numbers of Residues on the Two Strands Containing a Mismatch Opposite the Lesion Site: A View Following Replication and Extension Past a Base Substitution Mutation 4.1. AF 4.2. AP 5. DNA Duplexes Containing Unpartnered Bases on the Parent Strand: A View Following Replication and Extension Past a Frameshifting Deletion Mutation 5.1. AF 5.2. AAF 5.3. Thermal Stabilization of Bulged AF- and AAF-Modified Duplexes 6. Conformational Themes and Mutagenic Relevance 6.1. Normal Duplexes and Mismatched Full Duplexes

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392 Chem. Res. Toxicol., Vol. 11, No. 5, 1998

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6.2. AF at Single-Strand-Double-Strand Junctions 6.3. Bulged Duplexes 6.4. Conformation and Biological Effects of AF and AAF Adducts 6.5. Base Sequence Effects on Conformational Population Balance 7. Conformational Themes Common to PAH and Aromatic Amine Adducts 8. Conclusion and Future Directions

1. Introduction and Background The aromatic amines 2-aminofluorene (AF)1 and 2-(acetylamino)fluorene (AAF) have been the subject of intense interest for over 50 years. The very extensive literature has been reviewed by Beland and Kadlubar (1), Kriek (2), Heflich and Neft (3), and Hoffman and Fuchs (4). Research summarized very briefly in this Introduction and Background is detailed with comprehensive literature citations of the many hundreds of published articles in these reviews. AF and AAF were originally synthesized as insecticides but fortunately were never used for their intended purpose, since they were determined to be powerful mammalian carcinogens. Their highly potent carcinogenicity became the focus of a very large research effort to uncover the molecular origin of their tumorigenic effect. As aromatic amines they are representative of a class of substances that are known carcinogens and that are present in the environment in tobacco smoke, in automobile exhaust, in broiled meats and fish, and as byproducts of various industrial processes (1, 5-8). Hence, molecular understanding in the cases of AF and AAF, the prototypical aromatic amines for which the most biological data is available, can have broad applicability to a category of harmful substances to which humans are widely exposed. The earlier work determined that AF and AAF are metabolically activated to reactive derivatives that form covalent reaction products with DNA, which are termed adducts (1). The major in vivo adducts are to C8 of guanine (Figure 1). A minor AAF adduct to guanine N2 is also known. The major AAF adduct can also be enzymatically deacetylated in vivo; in many systems the major adduct observed is, in fact, the deacetylated AF one. The simple difference between the AF and the AAF guanine C8 adduct, the presence or absence of an acetyl group, has spurred much research effort to elucidate the biological and conformational distinctions that result. These two adducts are now readily accessible to synthesis of site-specifically modified oligomers in vitro in quantities sufficient for biological and high-resolution NMR studies; however, synthesis of the minor guanine N2 AAF adduct is more difficult (9), and only biological in vitro studies have been reported as yet (10). Consequently, we focus here on the guanine C8 adducts, although the minor AAF guanine N2 adduct may well be important because of its in vivo persistence (1, 11). Extensive mutagenicity studies (reviewed in ref 3), both in vivo and in vitro, have determined that the AF 1Abbreviations: AF, 2-(amino)fluorene; AAF, 2-(acetylamino)fluorene; ABP, 4-aminobiphenyl; AP, 1-aminopyrene; anti-BPDE, 7r,8tdihydroxy-t9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene; PAH, polycyclic aromatic hydrocarbon.

Figure 1. Schematics and numbering schemes of (a) [AF]G (1), (b) [AAF]G (2), (c) [ABP]G (3), and (d) [AP]G (4) adducts.

and AAF guanine C8 adducts can have miscoding consequences, but the predominant types of mutations produced differ: the AAF adduct causes largely frameshift mutations, while the AF adduct produces mainly mismatches. However, both types of mutations have been observed for each adduct in one system or another. In addition, the AAF adduct is much more disposed to act as a block to polymerases than the AF adduct, in general, but specific base sequence contexts foster blockage by AF as well (4, 12, 13). AF adducts are also in general more persistent than AAF adducts (reviewed in ref 1). The G1-G2-C-G3-C-C NarI mutational hotspot sequence of Escherichia coli is an important one first discovered by Fuchs and co-workers (4, 14, 15). This sequence has the remarkable property of inducing -2 frameshift mutations, leading to the sequence G-G-C-C only when G3 is modified (16). Moreover, AAF has a much greater propensity to induce frameshifts at this site than does AF (16-20). However, the intriguing observation of AF’s low but very interesting capacity for frameshift mutations at the NarI sequence (18) highlights the issue of the relationship between the structural distinctions of these two carcinogen-bound DNAs and their functional distinctions. Belguise-Valladier and Fuchs (12) began to address this question with chemical-probing experiments which revealed that “some AF adducts (as at position G3) behave almost as AAF adducts in terms of structural distortions induced”. These workers also noted that the AF adduct is largely bypassed by polymerase at G1 or G2 but acts as a block at G3, while AAF is a blocking lesion at all three positions. Moreover, even next nearest neighbors modulate the mutagenicity of AAF at G3 (21). The similarity between AF and AAF modification at G3 uncovered by Belguise-Valladier and Fuchs (12) suggests that structural studies of AF-modified G3 in the NarI sequence may also shed light on AAF modification. Recent perspectives (4, 22) discuss genetic and other molecular data in relation to mechanisms of frameshift mutagenesis in AF and AAF adducts, highlighting also other frameshift-prone sequences. Mutagenicity is now known to be an underlying cause of tumorigenesis, whose origin resides in structural damage to DNA in the case of chemical carcinogens such as the aromatic amines and hydrocarbons (23). Mutations may activate oncogenes (24, 25) or inactivate tumor suppressors such as the p53 gene, which has been found

Invited Review

mutated in about one-half of all cancer cases (26, 27). Understanding the nature of this damage in atomic resolution detail is a necessary first step to understanding why these substances cause mutations that can lead to cancer. The present review focuses on the molecular structures of AF guanine C8 adducts in solution, determined by a combination of high-resolution NMR studies and molecular mechanics/molecular dynamics computations in our laboratories, as well as related studies on these and other aromatic amine adducts [AAF, ABP (4-aminobiphenyl), and AP (1-aminopyrene)] to guanine C8 (Figure 1) investigated by ourselves and other groups. Both 4-aminobiphenyl and 1-nitropyrene are ubiquitous environmental pollutants that are known mutagens and carcinogens (1). They are the only other such aromatic compounds, besides AF and AAF, whose metabolic activation products form adducts to guanine C8, which have been studied by high-resolution NMR to date, as far as we are aware. These structures have provoked intriguing insights into possible mechanistic origins to mutagenicity, some of which had been previously hypothesized from molecular mechanics computations. Moreover, general structural principles that bridge adducts of activated polycyclic aromatic amines and hydrocarbons have been uncovered, and these unifying principles may underlie the mutagenicity of both types of adducts. A recent review has described structures of the activated polycyclic aromatic hydrocarbon adducts (28). This review is organized to present structures as they model some stages of the replication cycle: (1) Normal DNA duplexes with equal numbers of residues on the two strands and the standard Watson-Crick complement opposite the lesion site; this models a view prior to a round of replication. (2) Duplexes containing a singlestrand-double-strand junction with (a) the damaged base unpartnered at the junction, modeling a view during replication in which the replication machinery has proceeded up to but not including the lesion, and (b) the damaged base partnered normally or mismatched with adenine, modeling replication that has proceeded to the lesion site. (3) Full duplexes containing a mismatched base opposite the modified one; this models a view following mutagenic replication in which the replication machinery has placed a noncomplementary base opposite the lesion and has proceeded beyond it to yield a duplex containing a mismatch mutation. (4) Duplexes in which the modified residue, or the modified residue and its 3′ or 5′ neighbor on the same strand, are unpartnered, so that there are one or two fewer residues on the partner strand than on the damaged one; this models a view following mutagenic replication in which the replication machinery has skipped the modified base, or the modified base and one of its neighbors, to produce a duplex containing a -1 or -2 deletion, a frameshift mutation if in a coding region. We then elucidate unifying principles emerging from these and related studies on DNAs modified by activated polycyclic aromatic hydrocarbons to connect them to the mutagenic outcome at this stage of the work. Of course, much remains to be done, and this will be mentioned at the end. Full details of methods are given in the original works and in the review by Geacintov et al. (28) on polycyclic aromatic hydrocarbon adducts and are thus not treated here.

Chem. Res. Toxicol., Vol. 11, No. 5, 1998 393 Chart 1. Aromatic Amine-C8-Guanine Adduct-Containing Sequencesa

a Adducted G designated by *G. *G4, *G5, and *G7 adducts were incorporated one at a time into sequence V.

2. Normal DNA Duplexes with Equal Numbers of Residues on the Two Strands and Standard Watson-Crick Complement Opposite the Lesion Site: A View Prior to Replication 2.1. AAF. The first and, to date, only solution structure of an AAF-modified DNA oligomer was presented by O’Handley et al. (29) for sequence I, with G5 modified (Chart 1). In this work the structure of the major conformer, whose population was of the order of about 70%, was elucidated. The overall features of the structure were in remarkable agreement with the base displacement model first proposed by Grunberger and coworkers (30), also termed insertion-denaturation by Fuchs and Daune (31). This insightful model has now been observed in a variety of different contexts, elaborated in subsequent sections, and may perhaps play a causative role in frameshift mutagenesis. In the solution structure of this AAF adduct the modified guanine adopts the syn conformation and is displaced from its normal position stacked within the helix. The modified base pair is consequently ruptured, and the aromatic fluorenyl moiety is inserted in its place. The modified G5 base is displaced into the major groove, while the C14 partner remains stacked within the helix. The methylene bridge (C9 edge) of the fluorenyl moiety is directed toward the major groove side of the helix cylinder, and the acetyl group is positioned so that it protrudes into the major groove with its carbonyl oxygen directed so that it is trans to guanine C8 in the models of the structure. While conformational heterogeneity led to uncertainty in details of the model, the main features of syn-guanine with displacement of the modified base and insertion of the


Patel et al.

Figure 2. (a) Stereopair of NMR distance-refined solution structure of the central pentamer duplex segment of the [AF]G basedisplaced, intercalated conformer opposite C in sequence IV with AF modification at G6 (42). This view looks into the major groove and shows the rotamer with the C9-containing edge of AF facing the major groove. The [AF]G6 and C17 residues are shown in darkened bonds. (b) Stereopair of NMR distance-refined solution structure of the central pentamer duplex segment of the [AF]G external, major groove conformer opposite C in sequence V with AF modification at G5 (43). This view looks into the major groove and shows the rotamer with the C9-containing edge of AF oriented toward the 3′ direction of the modified strand. The [AF]G5 and C20 residues are shown in darkened bonds.

fluorenyl moiety were established. Moreover, the observed syn-guanine is in line with computations that have indicated that the syn domain is strongly favored over the anti domain for this adduct, even in small subunits including nucleosides and deoxydinucleoside monophosphates, due to steric interference between the bulky acetyl group and the sugar attached to the modified base when the normal B-DNA anti conformation is adopted (32-34). As we will discuss below, this strong preference for the syn region distinguishes the acetylated AAF derivative from the unacetylated AF one, which can adopt either conformation. 2.2. AF. Eckel and Krugh (35, 36), Cho et al. (37), and Zhou et al. (38) have investigated AF-modified duplexes in sequences from c-Ha-ras protooncogenes with modification within the critical codon 61, a site where a G to T transversion mutation is observed to induce protooncogene activation to oncogene. This mutation has been observed to follow treatment of this gene with activated AAF derivatives (39, 40). Eckel and Krugh (35, 36) studied sequence II, with AF modification at G6, in which C3-C4-A5-G6-G7-A8 are from the coding strand of the human c-H-rasI protooncogene and C4-A5-G6 are codon 61. Cho et al. (37) studied sequence III, and Zhou et al. (38) studied a 12-mer segment of sequence III, spanning a portion of the mouse c-Ha-ras protooncogene, with AF modification at G11, the first base of codon 61 in the noncoding strand. These groups noted conformational heterogeneity and were able to characterize one conformer, representing about 50% (35, 36), about 6070% (37), and 55% (38) of the population mix, as a structure with the AF positioned externally in the major groove of a minimally perturbed B-DNA helix with all base pairs essentially intact and the modified guanine in the anti conformation. In the model presented by Eckel and Krugh (35, 36), the fluorenyl moiety has its

C9-containing edge directed 5′ along the modified strand, while the work of Cho et al. (37) and Zhou et al. (38) did not include molecular models. The second conformer delineated by Eckel and Krugh (35, 36) was of the modified base-displaced, fluorenyl ring-intercalated type. Zhou et al. (38) proposed that their second conformer was also a stacked one. While Eckel and Krugh (35, 36) were unable to uncover the glycosidic torsion angle of the modified nucleoside in this structure from the interpretation of their NMR data, their favored model contained a modified guanine anti glycosidic conformation with the AF inserted so that its C9-containing edge is directed toward the major groove. The displaced guanine was situated in the minor groove, and its non-hydrogenbonded partner C was displaced into the major groove. Their basis for favoring the anti domain resided in the belief that the interconversion between the anti and the syn domain, as observed by them at 30 °C, with a 10-ms lifetime, was less likely to be possible in the presence of the bulky AF at the modified guanine. A syn inserted structure that satisfied their data was also shown in Supporting Information. In the work of Cho et al. (37), it was noted that the minor conformer(s) manifested collective shielding of their protons with chemical shifts that closely matched those of syn-modified guanine structures previously observed in an AF-modified duplex containing a mismatch (41) in which the AF is located in the minor groove and in the AAF-modified duplex with base-displaced syn-modified guanine (29). These workers suggested, therefore, that such syn-type structures represented possible structural types for their minor conformer(s). Mao et al. (42) have obtained the solution structure of the AF guanine C8 adduct in sequence IV, with AF modification at G6. There was also a conformational equilibrium between external major groove and base-

Invited Review

Chem. Res. Toxicol., Vol. 11, No. 5, 1998 395

Figure 3. (a) Stereopair of NMR distance-refined solution structure of the central trimer duplex segment of the [AF]G base-displaced, intercalated conformer opposite C in sequence IV with AF modification at G6 (42). This view looks down the helix axis and shows the rotamer with the C9-containing edge of AF facing the major groove. The [AF]G6 and C17 residues are shown in darkened bonds. (b) Stereopair of NMR distance-refined solution structure of the central trimer duplex segment of the [AF]G external, major groove conformer opposite C in sequence V with AF modification at G5 (43). This view looks down the helix axis and shows the rotamer with the C9-containing edge of AF oriented toward the 3′ direction of the modified strand. The [AF]G5 and C20 residues are shown in darkened bonds.

displaced, intercalated structures in slow exchange in this sequence context, but the population is about 70% of the base-displaced, intercalation type, which permitted a clearer delineation of its structure (Figures 2a and 3a). A major feature is that the modified guanine adopts the syn conformation, is displaced into the major groove, and is tilted slightly in the 3′ direction. The fluorenyl moiety is intercalated in its place between intact neighboring base pairs. The cytidine partner to the AF-modified guanine is also displaced into the major groove. In addition, there are indications of two rapidly interconverting rotameric orientations of the intercalated fluorenyl moiety, namely, with its C9-containing edge facing the minor groove or the major groove. The pair of rotamers is related by an approximately 180° rotation about the torsion angle β′ {[AF]G(C8)-[AF](N)-[AF](C2)-[AF](C1)} (see Table 1 for definitions of torsion angles governing the carcinogen-base linkage conformation), while the R′ torsion angle {[AF]G(N9)-[AF]G(C8)[AF](N)-[AF](C2)} is about the same in the two rotamers (see Table 1). Each of these rotamers is in harmony with the set of NMR data, although broadened line widths of aminofluorene H1, H3, and H4 proton resonances and overlapped aminofluorene H6 and H7 proton resonances precluded unambiguous characterization of the interconversion of the two conformers. Mao et al. (42) adopt the view that the high barrier to interconversion between the syn and anti alignment in the AF-intercalated and AFexternal conformers accounts for the observed slow exchange between these two conformations. (Fast and slow exchange between conformations on the NMR time scale reflects the interconversion rates between states relative to the chemical shift separation; separate resonances are observed under slow exchange conditions, while an averaged resonance is observed under fast exchange conditions. Broadening of the resonances occurs under intermediate exchange conditions when the interconversion rates are on the order of the chemical shift separation between states.) As will be discussed

further below, the syn conformation is a unifying feature of the modified guanine in all base-displaced, intercalated conformers of guanine C8 adducts except for the proposed anti orientation in the model of Eckel and Krugh (36). In addition, the observed shift in equilibrium from about 50% base-displaced intercalated in the A-[AF]G-G sequence of Eckel and Krugh (36) to about 70% in the C-[AF]G-C sequence of Mao et al. (42) and about 3045% in the T-[AF]G-A sequence of Cho et al. (37) and Zhou et al. (38) (assuming their minor conformer is also of this type) reveals the strong influence of sequence context on the equilibrium balance, a theme that has also emerged in studies of adducts of activated benzo[a]pyrene (28), and that is highlighted further below. Mao et al. (43) studied three AF adducts in sequence V, with site-specific modification at G4, G5, or G7, which contains the NarI mutational hotspot. Mao et al. (43) found that there was a slowly exchanging equilibrium between an external major groove structure with modified guanine anti and a base-displaced, AF-intercalated structure with modified guanine syn, when the modification was at any one of the three sites. However, the proportion of the two states differed markedly in the three sequence contexts. The C3-[AF]G4-G5 and G4-[AF]G5-C6 sequence contexts favor the external major groove conformer by approximately 70% and 90%, respectively, while the C6-[AF]G7-C8 hotspot position contains an approximately equal population of syn-base-displaced, AF-intercalated and anti-AF-external major groove conformers. The AF-external major groove conformer was characterized in detail directly from the high-resolution NMR data together with the molecular mechanics calculations for the [AF]G5-modified duplex of sequence V (Figures 2b and 3b). The experimental restraints were only satisfied for a pair of rapidly interchanging rotamers in this external major groove conformer. These differed by an approximate 180° rotation about the aminofluorene long axis, defined by an approximately 180° difference in the torsion angle β′ (see Table 1). In one rotamer the


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Table 1. Summary of Structures and Torsion Angles in AF- and AP-Modified Duplexesa,b adduct/sequence/modification site AF IV G6

base-displaced, intercalated, G-syn, normal duplex

AF V G5

major groove, G-anti, normal duplex

AP IV G6 AF VI-A G4 AF VI-B G4

AF VII G6 AP VII G6 AF VIII G6

base-displaced, intercalated, G-syn, normal duplex base-displaced, intercalated, G-syn, single-strand-double-strand junction base-displaced, intercalated, G-syn, single-strand-double-strand junction, normal partner C opposite [AF[G base-displaced, intercalated, G-syn, single-strand-double-strand junction, mismatched A opposite [AF[G minor groove, 3′-directed, G-syn, [AF]G‚A mismatch base-displaced, intercalated, G-syn, [AP]G‚A mismatch base-displaced, intercalated, G-syn, -1 deletion

AF IX G6 AF X G7

base-displaced, intercalated, G-syn, -2 deletion base-displaced, intercalated, G-syn, -2 deletion

AF VI-C G4

a

adduct conformation

χ

R′

β′

ref

65° 58° 223° 230° 62° 20° 59°

212° 197° 200° 191° 209° 183° 183°

138° 322° 38° 213° 141° 54° 41°

42

49 50 51

72°

207°

327°

52

71° 64° 64° 65° 59° 47°

208° 150° 90° 92° 87° 227°

317° 225° 109° 283° 80° 186°

41 58 59

43

60 61

Torsion angle definitions A-B-C-D: χ R′(AF) β′(AF) R′(AP) β′(AP)

O4′-C1′-N9-C4 G(N9)-G(C8)-AF(N)-AF(C2) G(C8)-AF(N)-AF(C2)-AF(C1) G(N9)-G(C8)-AP(N)-AP(C1) G(C8)-AP(N)-AP(C1)-AP(C10A)

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°. b Torsion angles given in the table are molecular mechanics distance-refined values. Values following second-stage relaxation matrix refinement against NOE intensities, where available, are AP VII G6 AF VI-A G4 AF VI-B G4 AF VI-C G4 AF X G7

χ 76° ( 6° 31° ( 21° 105° ( 8° 98° ( 10° 68° ( 16°

C9-containing edge of the AF moiety was oriented in the 5′ direction of the modified strand, while it was 3′directed in the second rotamer. One of the two rotamers was very similar to a previously computed major groove structure for an AF-modified duplex, characterized by torsion angles R′ and β′ of 181° and 42°, respectively (44), compared to 200° and 38° (Table 1) in the solution structure. The NMR data indicated that the external major groove structure in all three modified duplexes was similar. The base-displaced, intercalated conformer in slow exchange with the major groove structure was found, from the NMR data, to be virtually identical to the one characterized in detail in sequence IV by Mao et al. (42), described above. Consequently, the central trimer of this structure was employed to represent the central C6-[AF]G7-C8 trimer structure of the same type, since the neighboring bases are the same as in the [AF]G7 hotspot. The major groove structure at the [AF]G7 hotspot sequence was obtained from the G4-[AF]G5-C6 sequence structure by removing the AF from G5, docking it to G7 in the same orientation and reminimizing. Thus, the structures of the NarI sequence hotspot have been delineated in detail in this normal duplex modified by AF, and the profound influence of nearest neighbors on the population mix in slowly interchanging conformers became clear. Moreover, a strong influence of even next nearest neighbors on the population mix emerged: the AF base-displaced, intercalated and external major groove structures are of equal population in the G5-C6-[AF]G7C8-C9 duplex of sequence V (43), but the base-displaced, intercalated one is predominant (about 70%) in the T4C5-[AF]G6-C7-T8 sequence IV (42), although the nearest neighbors are the same.

R′ 149° ( 9° 231° ( 28° 229° ( 10° 210° ( 8° 210° ( 8°

β 214° ( 9° 11° ( 23° 10° ( 4° 307° ( 9° 188° ( 14°

2.3. ABP. Cho et al. (45) studied the guanine C8 major adduct of ABP (Figure 1c) by high-resolution NMR, albeit without molecular modeling, in the same sequence III of the mouse c-Ha-ras protooncogene as they employed in their AF study (37). ABP is of special structural interest because it resembles AF except that the C9 methylene bridge of AF is missing, and this imparts flexibility between the two phenyl rings. Computations have indicated that the two rings are twisted in relation to one another by about 30-40° and that the ABP can reside in the major groove of a normal B-DNA helix (46, 47). The computed twist is in line with observed orientations in model biphenyls (48). Mutagenicity studies indicate a lower capacity of ABP to induce frameshift mutations compared to AF (19). In line with the computations, Cho et al. (45) found a major conformer in which the ABP is situated in a minimally perturbed B-DNA helix, in the major groove, together with one or more minor conformers (5-10% of the population mix) which could not be well-characterized, but upfield shifts of the ABP protons in the minor conformer(s) suggested the possibility of stacking with bases. The higher proportion of external major groove conformation for the ABP adduct compared to the AF adduct observed by Cho et al. (37, 45) in the same sequence context could be related to the twisted nature of the biphenyl moiety in contrast with the planar AF, which would make insertion into the helix more difficult (the two phenyls would need to be flattened into plane at some energetic cost, or only one ring could be inserted). However, Cho et al. (45) did not obtain data regarding the twist, but they did note that the ABP ring system was rapidly rotating. 2.4. AP. Mao et al. (49) have obtained the solution structure of the guanine C8 adduct of AP (Figure 1d) in

Invited Review


Figure 4. (a) Stereopair of NMR distance-refined solution structure of the central pentamer duplex segment of the [AP]G basedisplaced, intercalated conformer opposite C in sequence IV with AP modification at G6 (49). This view looks into the major groove with [AP]G6 and C17 highlighted in darkened bonds. (b) Stereopair of NMR intensity-refined solution structure of the central pentamer duplex segment of the [AP]G base-displaced, intercalated conformer opposite A in sequence VII with AP modification at G6 (58). This view looks into the major groove with [AP]G6 and A17 highlighted in darkened bonds.

sequence IV, with modification at G6. This is the same sequence context and modification site studied for the AF adduct (42). The AP adduct is of special interest because of its larger bulk compared to the AF one. This AP structure strongly resembles the major AF conformer in the same sequence context. The AP moiety is inserted into the helix with displacement of the syn-modified guanine and its partner cytidine into the major groove (Figure 4a). The AP-modified G is tilted in the 3′ direction of the modified strand, the C5-C6-C7-containing edge of the AP is directed toward the G16-C17-G18 segment of the unmodified strand, the C8-C9-containing edge of AP is directed toward the minor groove, and the C3-C4-containing edge is directed toward the major groove. One significant difference between the AF adduct and the AP adduct in the same sequence context is the population mix. In the AP adduct the base-displaced, intercalated conformer represents essentially 100% of the population mix in this sequence while it is only 70% of the mix, in equilibrium with the external major groove conformer, in the AF adduct. The greater aromatic surface of the AP compared to AF causes AP to more greatly favor stacking within the helix.

3. DNA Single-Strand-Double-Strand Junctions: Models of an Arm of a Replication Fork 3.1. The Lesion Site as Next Base To Be Replicated. Mao et al. (50) obtained the solution structure of sequence VI-A, with AF modification at G4, as a model for an arm of a replication fork in which replication of the AF-modified strand has proceeded up to but not including the [AF]G4. In this structure (Figure 5a) the

syn-modified guanine is displaced into the major groove and the AF ring is inserted, stacking over the C5‚G22 base pair. The other face of the AF also stacks with the purine ring of the unpaired, nonadjacent A2, while the C3 neighbor is unstacked and positioned outside the helix. The NMR data revealed the existence of one rotamer with the C9-containing edge directed toward the major groove, although the existence of a second rotamer flipped ≈180° about the torsion angle β′ (Table 1) so that the C9-containing edge is directed toward the minor groove could not be entirely ruled out. 3.2. The Lesion Site with a Normal Partner Opposite the Adduct. A solution structure has also been obtained for sequence VI-B with AF modification at G4 by Gu et al. (51). In this structure replication has proceeded up to the [AF]G4 and a normal partner C23 has been placed opposite the lesion (Figure 5b). Interestingly, this structure is strikingly similar to the one in which the [AF]G4 has no partner in that the modified G4 is syn and displaced to the major groove side, while the AF is inserted in its place and stacked with the C5‚G22 pair, and its C9-containing edge is directed toward the major groove. Again, the other face of the AF is stacked with A2, while C3 is unstacked and looped out to the minor groove side. The striking aspect of this structure is the looped out position of the C23 opposite the syn-[AF]G4. The C23 is unstacked and positioned flexibly on the minor groove side of A2 and G22. 3.3. The Lesion Site with a Mismatch Opposite the Adduct. Gorin et al. (52) have also obtained the solution structure of sequence VI-C with AF modification at G4, in which replication has again proceeded up to the lesion but a mismatched A23 has been placed


Patel et al.

Figure 5. (a) Stereopair of the entire NMR distance-refined solution structure of [AF]G in sequence VI-A, modeling an arm of a replication fork with replication up to but not including the AF-modified G4 (50). This view looks into the major groove with the [AF]G4 residue highlighted in darkened bonds. (b) Stereopair of a segment of the NMR intensity-refined solution structure of [AF]G in sequence VI-B, modeling an arm of a replication fork with replication up to the AF-modified G4 and incorporation of normal partner C23 (51). This view looks into the major groove and spans the (A2-C3-[AF]G4-C5-T6)‚(A21-G22-C23) segment with [AF]G4 and C23 highlighted in darkened bonds.

opposite (Figure 6a and 7a). The features of this structure are very similar to those in sequence VI-B with C23 opposite [AF]G4 except for the markedly different position of the mismatched A23. In contrast to the flexible, looped out position of the C23 on the minor groove side, A23 is well-defined among the refined structures with its purine ring positioned over the van der Waals surface of the floor of the major groove and its amino group directed toward the double-helical segment. The Hoogsteen edge of the modified guanine of [AF]G4 and the Watson-Crick edge of A23 are approximately coplanar and directed toward each other (Figure 6a) but are separated by twice the hydrogen-bonding distance required for pairing (Figure 6b).

4. DNA Duplexes with Equal Numbers of Residues on the Two Strands Containing a Mismatch Opposite the Lesion Site: A View Following Replication and Extension Past a Base Substitution Mutation 4.1. AF. Norman et al. (41) studied sequence VII, in which the modified [AF]G6 is opposed by the mismatched A17, by high-resolution NMR and molecular mechanics

computations. An [AF]G‚A mismatch was chosen for investigation in this work because this is the predominant mutation induced by AF (3, 18, 53, 54). In this structure (Figures 6b and 7b) the modified guanine adopts the syn conformation, but it remains stacked within the B-DNA helix as does its opposite adenine, which adopts the normal anti conformation. With this optimal stacking of the modified base and its opposite A, the AF is sandwiched within the walls of the minor groove which it spans so that its distal ring is directed toward the G16-A17-G18 sugar-phosphate backbone of the partner strand. The C9-containing edge of the fluorenyl ring system is directed toward the interior of the helix, and the C4-C5-containing edge is directed toward solvent. Both faces of the AF ring are sandwiched between the two strands that constitute the walls of the minor groove with only the AF edge exposed, and the AF is tilted in the 3′ direction of the modified strand. This orientation is very reminiscent of the observed situation of noncovalently minor groove-bound drugs such as a netroposin-DNA oligomer complex which had been observed earlier (55, 56). The [AF]G and its mismatched A are not hydrogen-bonded at neutral pH, although all

Invited Review


Figure 6. (a) Stereopair of a segment of the NMR intensity-refined solution structure of [AF]G in sequence VI-C, modeling an arm of a replication fork with replication up to the AF-modified G4 and misincorporation of A23 (52). This view looks into the major groove and spans the (A2-C3-[AF]G4-C5-T6)‚(A21-G22-A23) segment with [AF]G4 and A23 highlighted in darkened bonds. (b) Stereopair of the central pentamer duplex segment of the NMR distance-refined solution structure of [AF]G opposite A in sequence VII with AF modification at G6 (41). This view looks into the major groove with [AF]G6 and A17 highlighted in darkened bonds.

Figure 7. (a) Stereopair of the dimer segment of the NMR intensity-refined solution structure of [AF]G in sequence VI-C, modeling an arm of a replication fork with replication up to the AF-modified G4 and misincorporation of A23 (52). This view looks at base pair overlaps down the helix axis and spans the ([AF]G4-C5)‚(G22-A23) segment with [AF]G4 and A23 highlighted in darkened bonds. Note that the aminofluorene ring is stacked over the C5‚G22 pair, while the modified guanine of [AF]G4 and A23 are displaced into the major groove. (b) Stereopair of the central dimer duplex segment of the NMR distance-refined solution structure of [AF]G opposite A in sequence VII with AF modification at G6 (41). This view looks at base pair overlaps down the helix axis and spans the ([AF]G6-C7)‚(G16-A17) segment with [AF]G6 and A17 highlighted in darkened bonds. Note that the modified guanine of [AF]G6 and A17 are stacked over the C7‚G16 pair, while the aminofluorene ring is positioned in the minor groove.

normal Watson-Crick hydrogen bonds are intact at the other residues (41). Thus, the neutral pH conformation is stabilized by hydrophobic interactions between the faces of the AF moiety and the walls of the minor groove and by the maintenance of a stacked-within-the helix position of the syn-[AF]G and the opposite A. At acidic pH the NMR data indicates that the AF ring is displaced away from the helix axis relative to the neutral pH conformation (41). A single hydrogen bond between N1protonated A17 and O6 of [AF]G6, proposed for the acidic structure, could produce this small realignment of the

AF under the acidic conditions that permit N1 protonation. This structure of [AF]G opposite A in a fully paired duplex (Figures 6b and 7b) (41) can be compared with the corresponding structure of [AF]G opposite A at a model template-primer junctional site (Figures 6a and 7a) (52). The alignment of the Hoogsteen edge of syn[AF]G positioned opposite the Watson-Crick edge of anti-A has been observed for both systems with the separation greater in the case of the junctional alignment in the model template-primer system (Figure 7a) relative


to the duplex (Figure 7b). However, the aminofluorene ring is positioned in the minor groove in the fully paired duplex (Figure 7b), while it stacks over the junctional base pair in the template-primer system (Figure 7a). Abuaf et al. (57) reported the NMR/molecular mechanics solution structure of [AF]G in the same sequence context as the [AF]G structure of Norman et al. (41), except that the mismatch opposite the lesion was either a G or an I (base hypoxanthine, nucleoside deoxyinosine, abbreviated I). G was chosen as the mismatched base because mutational data (54) had indicated that [AF]G‚G mismatches are also observed, and the I derivative (G without the amino group) was studied as an aid to modeling the G‚G structure. Interestingly, the features of these mismatched structures were essentially the same as those of the [AF]G‚A mismatch, with the syn-modified guanine and its mismatched opposite base stacked into the helix and the AF sandwiched into the minor groove with its C9 edge buried and its C4-C5 edge exposed, directed toward the partner and tilted in the 3′ direction of the modified strand (57). 4.2. AP. Gu et al. (58) obtained the solution structure of sequence VII with AP modification at G6, which is opposed by the mismatched A17. This sequence is the same as sequence IV except that the base opposite the AP-modified G6 is a mismatched adenine instead of the normal partner cytosine. The AP structures in the two sequences are similar in that the modified guanine is syn and displaced into the major groove, as is the opposing base, while the AP moiety is intercalated into the helix between the adjacent base pairs (Figure 4b). However, the mismatched structure differs from the normally paired one in the position of the base opposing the lesion. While C17 is looped out and not stacked with AP (Figure 4a), the A17 residue is stretched so that it is approximately coplanar with G18 and partially stacked with AP as well, in several of the refined structures (Figure 4b). No hydrogen bonding exists between the APmodified G6 and the mismatched A17. Another difference is the apparent predominant orientation of the G6 in the normal and mismatched duplexes, which is 3′directed in the former case (Figure 4a) and 5′-directed in the latter (Figure 4b). The modified base may be able to assume different orientations in different basedisplaced, intercalated structures because of its external displaced position, which could be somewhat mobile and therefore oriented differently in different contexts. The proton of the N-H at the carcinogen-base linkage site is a marker for the orientation, but it was not always identifiable in the NMR spectra. The alignment in the models is the one produced following computations and selection of the structure with the best overall goodnessof-fit to the NMR data. Of considerable interest is the distinction between the structure of the AP-modified duplex in sequence VII (58) and the structure of the AF-modified duplex in the same mismatched sequence (41), as this affords an assessment of the influence of the greater bulk of AP. While the AP adopts a base-displaced, intercalated structure (Figure 4b), the AF resides in the minor groove (Figure 6b). The smaller AF can be neatly sandwiched into the minor groove with only its edge exposed, but the larger AP would protrude such that the aromatic surface would be solvent-exposed if it were situated in the minor groove. By intercalating, this exposure is avoided and stacking is optimized by interaction of the AP with adjacent base

Patel et al.

pairs and with A17 in this sequence. In the AF case, optimal stacking is achieved by retaining the modified G6 and its mismatched A in an intrahelical position.

5. DNA Duplexes Containing Unpartnered Bases on the Parent Strand: A View Following Replication and Extension Past a Frameshifting Deletion Mutation 5.1. AF. Mao et al. (59) obtained a structure for an [AF]G lesion by high-resolution NMR/molecular mechanics calculations in sequence VIII, in which the modified strand has 11 residues, the unmodified partner has 10, and the modified G6 base has no partner. This models the situation following mutagenic replication in which the replication machinery has skipped the modified base and extended beyond it, producing a -1 deletion in the partner strand. In a coding region this results in a highly deleterious shift in the codon-reading frame, yielding a protein whose amino acid residues are all aberrant beyond the frameshift. While frameshift mutations are unusual for AF modification, they have been observed (3, 18-20) and are of interest because of the profound harm they could cause in encoded proteins, although sequence VIII is not one in which frameshift mutations are common. In this -1 deletion sequence context, the [AF]G adopts a syn conformation and is displaced into the major groove (Figure 8a). The displacement of the guanine makes room for the intercalation of the AF ring in its place between the C5‚G17 and C7‚G16 neighboring base pairs (59). The intercalation site is wedge-shaped with the C5 and C7 bases on the modified strand separated by a greater distance than the G16 and G17 bases on the partner strand. All complemented bases are Watson-Crick hydrogen-bonded, but the C5‚G17 base pair is buckled and propeller-twisted significantly, in agreement with NMR data indicating selective perturbation of this base pair. Moreover, the [AF]G6 is tilted in the 5′ direction of the modified strand so that it stacks with C5. The AF moiety undergoes rapid 180° flips about its long axis on the NMR time scale, so that its C9containing edge is directed toward either the minor groove or the major groove, and these two different orientations are reflected in approximately 180° differences in the torsion angle β′ (Table 1) in the two rotamers. Mao et al. (60) also studied the solution structure of [AF]G in sequence IX, in which the [AF]G6 and its 3′ neighboring A7 have no partner. This models the situation following mutagenic replication in which the replication machinery has skipped both the modified base and its 3′ neighbor and has extended beyond it, producing a -2 deletion in the daughter strand. Here, again, the AF-modified guanine is in a syn alignment displaced into the major groove and the fluorenyl ring is inserted into the helix in its place, intercalated between flanking G‚C base pairs (Figure 8b). The AF moiety is oriented with its long axis parallel to the long axis of these flanking base pairs as in the -1 deletion case. The unpartnered adenine in the -2 deletion duplex is also displaced into the major groove and is stacked with the unpartnered AF-modified guanine, to form a stacked two-base bulge. The C9-containing edge of the AF was in the major groove in the energetically favored conformers, but conformers with this edge in the minor groove, resulting from 180° flips about the AF long axis, could not be ruled out from the NMR data. The stacking patterns of the AF ring with

Invited Review


Figure 8. (a) Stereopair of NMR distance-refined solution structure of the central 5/4-mer segment of [AF]G in sequence VIII in which the AF-modified G6 contains no partner residue (59). The aminofluorene ring intercalates into the helix with base displacement of the modified guanine into the major groove. This view looks into the major groove and shows the rotamer with the C9-containing edge facing the major groove. The [AF]G6 residue is shown by darkened bonds. (b) Stereopair of NMR distance-refined solution structure of the central 6/4-mer segment of [AF]G in sequence IX in which the AF-modified G6 and its 3′ neighbor A7 have no partner residues (60). The aminofluorene ring intercalates into the helix with base displacement of the modified guanine and its adjacent, mutually stacked A7 residue into the major groove. This view looks into the major groove and shows the rotamer with the C9-containing edge positioned in the major groove. The [AF]G6 and adjacent A7 residues are shown by darkened bonds.

Figure 9. Stereopair of NMR distance-refined solution structure of the central 6/4-mer segment of AF-modified G7 in sequence X, the NarI hotspot, in which the [AF]G7 and its 5′ neighbor C6 have no partner residues (61). The aminofluorene ring intercalates into the helix with base displacement of the modified guanine and its adjacent C6 residue into the major groove. This view looks into the major groove with [AF]G7 and adjacent C6 residues shown by darkened bonds.

respect to flanking base pairs differed in the -1 and -2 deletion duplexes: in the -2 deletion case the AF ring is displaced toward the minor groove (Figure 8b), while it is displaced toward the major groove in the -1 deletion case (Figure 8a). Mao et al. (61) have obtained a solution structure of an AF-modified duplex in sequence X, which contains the NarI hotspot sequence C-G1-G2-C-G3-C-C with modification at G3. [AF]G7 corresponds to the G3 hotspot for -2 deletions. In the modified duplex, which contains 12 residues on the AF-containing strand and 10 on the partner, two different pairing alignments are possible: C6 and [AF]G7 may be unpartnered, or [AF]G7 and C8 may be unpartnered. The biological frameshift mutation data cannot distinguish between these two possibilities in the NarI hotspot as either of them could lead to the observed outcome of a -2 deletion from CGGCGCC to

CGGCC. The NMR data revealed unequivocally that C6 and [AF]G7 were unpartnered in this case. In this structure (Figure 9) the modified guanine is again in the syn orientation and displaced into the major groove, as is the adjacent dC6 residue. The AF ring is intercalated between the adjacent G5‚C18 and C8‚G17 base pairs. The [AF]G7 base plane stacks over the minor groove sugar face of the dC6 and is tilted somewhat toward the 5′ direction of the modified strand. The C9-containing edge of the AF is directed toward the minor groove in this structure, and only this one rotamer is observed. A comparison of the structure of the -2 deletion duplex in the NarI sequence context d(G-C-[AF]G-C)‚d(G-C) (sequence X) (61) versus the d(C-[AF]G-A-C)‚d(G-G) context (sequence IX) (60) reveals the common features of syn[AF]G displaced into the major groove with intercalation of the AF and displacement of the neighboring unpaired


Patel et al.

Table 2. Summary of Melting Temperatures in AF- and AAF-Modified Bulged Duplexes and Their Unmodified Counterparts

Table 3. Base Sequence Dependence of Conformational Mix in AF-Modified Normal Duplexesa

melting temperature, °C

adduct/sequence/ modification site

unmodified

modified

ref

AF VIII G6 AF IX G6 AF X G7 AAF XI G6 AAF XII G7

24 22 33 31 34

38 33 47 46 49

59 60 61 62 63

A into the major groove as well. However, the extra adenine is 3′ to the modification site in sequence IX, while the extra cytidine is 5′ in sequence X. Moreover, the displaced [AF]G is tilted 5′ and stacked with its bulged out 3′ neighbor A in sequence IX (Figure 8b), while the [AF]G is also tilted in the 5′ direction but not stacked with the bulged out 5′ neighbor C in the NarI sequence X (Figure 9). 5.2. AAF. Milhe´ et al. (62) obtained NMR data for sequence XI, in which the [AAF]G6 is unpartnered opposite a -1 deletion site, but the conformational heterogeneity did not allow for the definition of interproton distance bounds for molecular modeling. Their interpretation of the data suggested an equilibrium between a conformer in which the AAF is inserted into the helix and a second conformer in which the AAF is external, with 70% of the population in the latter state. In this external conformer the authors considered the NMR data to be consistent with the AAF pointing in the 5′ direction and the whole [AAF]G moiety extruding from the helix. Base pairs surrounding the bulge were intact in this structure, as is the case for all the [AF]G bulges described above. However, this is the only example of a bulged structure in which the fluorenyl moiety is judged to be externally situated. No information on the syn versus anti orientation of the [AAF]G could be deduced from the data. Milhe´ et al. (63) have also carried out NMR studies of an AAF-modified -2 deletion duplex in the NarI sequence XII, with AAF modification at G7. This sequence again has the option of two different possible unpartnered residues: C6 and [AAF]G7 or [AAF]G7 and C8. Conformational heterogeneity again prevented the definition of interproton distances for molecular modeling. The data were interpreted to be consistent with intercalation of the AAF moiety with displacement of the modified guanine which was deemed to be in the syn conformation, in the major conformer (about 80% of the population mix). Moreover, Milhe´ et al. (63) believed that all the conformers in the equilibrium are similar in that the AAF is inserted, based on the observed chemical shift ranges. Milhe´ et al. (63) interpreted their NMR evidence to conclude that [AAF]G7 and C8 were unpartnered in this case, rather than C6 and [AAF]G7, as observed by Mao et al. (61) in the [AF]G-modified NarI sequence described above. However, difficulties in the interpretation of the NMR data due to the conformational heterogeneity made it difficult to make this assessment with certainty. 5.3. Thermal Stabilization of Bulged AF- and AAF-Modified Duplexes. Table 2 gives melting temperatures of modified and unmodified duplexes containing -1 and -2 deletion sites with AF and AAF modification. In every case the modified duplex is more stable than the unmodified one, and this is the case also for deletion duplexes containing modification by anti-benzo-

adduct/sequence/ modification site

% major groove

AF II G6 AF III G11 AF III G11 AF IV G6 AF V G4 AF V G5 AF V G7

50 60-70 55 30 70 90 50

adduct-containing sequence C-A-[AF]G-G-A T-T-[AF]G-A-C T-T-[AF]G-A-C T-C-[AF]G-C-T T-C-[AF]G-G-C C-G-[AF]G-C-G G-C-[AF]G-C-C

ref 35, 36 37 38b 42 43 43 43

a Balance of population: base-displaced, intercalated conformer. Sequence studied was a 12-mer segment of sequence III. AFstacked conformer proposed for balance of population.

b

[a]pyrenediol epoxide (BPDE) (28, 64). Moreover, all the deletion duplexes investigated by NMR methods, including modification by AF, AAF [except for the major conformer in the -1 deletion duplex of Milhe´ et al. (62)], and BPDE, share the common structural feature of basedisplaced intercalation: the modified guanine is displaced from its normal position stacked within the helix, while the aromatic moiety is inserted. This shared conformational theme is the likely explanation for the surprising stabilization induced by the carcinogen: insertion into the helix offers sufficient stabilizing stacking interactions to make the modified duplex more stable than its unmodified counterpart.

6. Conformational Themes and Mutagenic Relevance 6.1. Normal Duplexes and Mismatched Full Duplexes. The structures of the modified duplexes with normal Watson-Crick partner and equal numbers of residues on the two strands are most relevant to the issue of lesion repair versus persistence, and the full duplexes containing mismatches are also relevant to this issue. DNA repair enzymes are believed to seek out distorting lesions as substrates. The nature of such distortions is currently not known, but kinking, unwinding, and local denaturation are among the features that the enzyme might well-recognize. The double-stranded structures of AF-modified DNA all manifest an equilibrium between structures in which the double helix is essentially normal and the AF is external and located in the major groove (Figures 2b and 3b) and structures in which the AF is intercalated into the helix with local unwinding and with displacement of the modified guanine (Figures 2a and 3a) so that the lesion site is denatured. Moreover, this guanine is in the abnormal syn conformation in all structures where the glycosidic orientation could be elucidated. The population balance between the two conformers is highly sequence-dependent with even next nearest neighbors exerting a contributing influence (Table 3). It is plausible to consider the base-displaced, intercalated conformer as a likely repair enzyme substrate, while the nondistorting, external major groove conformer could be envisioned to be less repair-prone. Moreover, the sequence dependence of the population balance is in harmony with the observation of sequencedependent incision rates of AF adducts by the UvrABC nuclease, the nucleotide excision repair complex of E. coli (65). We can also compare the AF structures with those of ABP and AP. We note that in the same sequence (sequence III) ABP (45) has a greater propensity to

Invited Review

remain in the normal, essentially undistorted B-form than AF (37). The AP adduct (49) favors the intercalated conformer essentially 100% (Figure 4a), both in sequence IV, where the AF adduct (42) favors this conformer by only 70% (Figure 2a), and in the mismatched sequence VII (Figure 4b) (58), where the AF is positioned in the minor groove (Figure 6b) (41). The AAF adduct has not been investigated in the same sequence as an AF adduct, but its major conformer (about 70%) in the sequence studied (29) is also of the syn-guanine, base-displaced, intercalated type, and the remainder of the population also contains syn-modified guanine structures that are denatured at the lesion site (66). Taken together, these results would suggest that the AAF and AP adducts would be more repair-prone than the AF adduct and that the ABP adduct would be least likely to be repaired. The observed faster incision rates of AAF adducts compared to AF adducts by the UvrABC nuclease are consistent with this concept (65). It would be interesting to obtain repair data in a single system for all these adducts to test this conformational interpretation. 6.2. AF at Single-Strand-Double-Strand Junctions. The observed structures with AF modification at a single-strand-double-strand junction [sequence VI-A, Mao et al. (50); sequence VI-B, Gu et al. (51); sequence VI-C, Gorin et al. (52)] all share the features of synmodified [AF]G displaced into the major groove with AF inserted in its place, whether there is a normal partner C, a mismatched A, or no partner opposite the [AF]G. These structures are relevant to mutagenic replication since accurate translesion synthesis presumably requires the [AF]G to be in the normal anti conformation. The similarities between the structures of sequences VI-A (Figure 5a) and VI-B (Figure 5b), with no partner opposite the lesion in VI-A and cytosine opposite in VIB, are striking. The looped out position of the additional C opposite [AF]G suggests how an incoming dCTP might fail to position itself properly for faithful extension opposite an [AF]G in which the G is syn. Furthermore, if the dCTP were incorporated, a structure of this type could plausibly be responsible for polymerase blockage, with the opportunity for slippage mispairing which can introduce frameshift mutations, in a suitable sequence context such as the NarI sequence (10, 67-71). Indeed, the mechanism proposed by Schaaper et al. (69) for -2 deletions at the NarI hotspot requires incorporation of C opposite the lesion prior to slippage. On the other hand, the relative alignment between the [AF]G and the mismatched A in sequence VI-C suggests that A can be misincorporated because the [AF]G can adopt the abnormal syn conformation, and when this does happen, a mismatched A can be stably accommodated opposite the lesion (Figure 6a). A small movement within the polymerase could possibly bring the A into hydrogenbonding range of the modified G, utilizing O6 of G and the amino group hydrogen of A. One can also draw correlations between [AF]G aligned opposite A at junctional sites (52) and within fully paired duplexes (41). The R′ and β′ linkage torsion angles are similar for the syn-[AF]G residue in the fully paired (sequence VII, [AF]G‚A alignment) and junctional (sequence VI-C) sites (Table 1). Further, the Hoogsteen edge of [AF]G is directed toward the Watson-Crick edge of the opposing A in both fully paired (Figure 7b) and junctional (Figure 7a) systems. This suggests that the [AF]G opposite A junctional alignment (Figures 6a and 7a) could be readily


incorporated within a duplex (Figures 6b and 7b) by bringing the opposing bases toward each other and through a translation of this entity toward the minor groove. Thus, there would be a switch from a stabilizing stacking interaction involving the AF ring and flanking bases/base pairs associated with the junctional alignment (Fiugre 7a) to a stabilizing stacking interaction involving the modified guanine and opposing adenine and flanking base pairs associated with a fully paired duplex (Figure 7b). Similarly, there would be a switch from a stabilizing van der Waals interaction involving the modified guanine and opposing adenine aligned face down within the walls of the major groove in the junctional alignment (Figure 6a) to a stabilizing van der Waals interaction involving the aminofluorene ring sandwiched between the walls of the minor groove in the fully paired duplex (Figure 6b). Such an analysis suggests how an incoming dATP might be misincorporated and even extended, since the primer terminus 3′-OH of the mismatched adenine is positioned near normally. However, the vast majority of replication events observed past AF lesions result in incorporation of the normal partner C (reviewed in refs 3 and 4). This is in line with AF’s capacity to easily permit the modified guanine to adopt the anti domain as well. The neighboring sequence might well-determine whether an anti or a syn structure occurs at a replication fork, as it governs the balance between them in the duplex, and an equilibrium between them might occur at a replication fork also. Of course, the polymerase would also play an important role here. 6.3. Bulged Duplexes. All the bulged duplexes in which there are fewer residues on the partner than on the AF-modified strand share a common structure of synmodified guanine with base-displaced intercalation (Figures 8 and 9) (59-61). Moreover, one of the two AAFmodified bulged structures has a similar structure (63). These modified bulged structures are all stabilized relative to their unmodified counterparts (Table 2) due to favorable stacking interactions by the intercalated fluorene. A structure of this type in a nonextended bulge, where replication has proceeded only one residue past the unpartnered base, could also be similarly stabilizing, encouraging the bulge rather than rearrangement back to an unbulged structure (sequence permitting). Thus, the deletion or insertion would become permanent during the replication process. Of course, a bulged out duplex is a natural substrate for repair, but failing this, a deletion mutation would be the outcome. The mismatch repair system has the capacity to correct bulged duplexes (72, 73). There is no structural information yet available on bulged duplexes for other aromatic amines. However, the results associated with the propensity of normal duplexes containing AF, ABP, and AP adducts (section 2) to form syn-guanine, base-displaced, intercalated conformers suggest the order AP > AF > ABP. Furthermore, AAF is also highly disposed toward structures of this type (29, 63). It is interesting that this propensity for forming syn, base-displaced, intercalated conformations parallels ring size. This trend has also been noted in computations which include the single-ringed aniline adduct by Shapiro et al. (74). They note that the greater proclivity for the larger-ringed aromatics to intercalate with base displacement stems from the advantage gained by them, compared to smaller ones, in avoiding solvent and in achiev-


Patel et al.

Figure 10. Structures of the central trimer duplex segments of [AF]G in sequence V with modification at G7, the NarI mutation hotspot, in space-filling color models. Top: The AF-external, major groove conformer (43). This view shows the rotamer with the C9-containing edge oriented toward the 3′ direction of the modified strand. Bottom: The base-displaced, AF-intercalated conformer (42). This view shows the rotamer with the C9-containing edge oriented toward the major groove. Color scheme: AF, cyan; modified G, yellow; partner C, gray.

ing favorable stacking interactions with DNA bases, at the expense of a lost Watson-Crick pair. Moreover, the propensity for inducing frameshift mutations in bacteria (3, 19-20, 75) also parallels increasing ring size. This would suggest that stabilization of bulges by basedisplaced structures with intercalated aromatic rings is governed by ring size. A greater disposition of the larger ringed structures to stabilize bulges could account for their greater manifestation of frameshift mutations. 6.4. Conformation and Biological Effects of AF and AAF Adducts. The observation by Belguise-Valladier and Fuchs (12) that AF adducts are similar to AAF adducts at the NarI G3 hotspot, but not at G1 and G2, in terms of structure and ability to permit polymerase bypass could well-be related to the fact that the AF resides mainly in the major groove in a normal B-DNA duplex when bound at G1 and G2, but at the G3 hotspot there are equal proportions of normal anti and abnormal syn-modified guanines, as noted by Mao et al. (43) in their studies of sequence V with modification at G4, G5, and G7: sequence V contains the NarI sequence, and G4, G5, and G7 correspond to G1, G2, and the G3 hotspot, respectively. The distorted syn structures could account for the polymerase blockage at the hotspot site. Furthermore, syn structures are preferred to a much greater extent for the AAF adduct than the AF one (34), which could explain why AAF has a much greater propensity

for inducing polymerase blockage than AF (12, 13). These features could be responsible for AAF’s greater propensity than AF to produce frameshift mutations in sequences where bulged out, rearranged, slipped intermediates can form (67-71). Moreover, the greater susceptibility of the AAF adduct to repair (11) could also be correlated with its syn conformation, in contrast with the AF adduct’s ability to adopt both domains. 6.5. Base Sequence Effects on Conformational Population Balance. The finding, summarized in Table 3, of profound base sequence effects in AF-modified duplexes on the equilibrium between base-displaced, intercalated conformations and major groove, external ones is striking. As mentioned above, in the NarI sequence context it is a plausible explanation for differences in polymerase blockage and responses to chemicalprobing experiments observed by Belguise-Valladier and Fuchs (12) upon AF modification of each of the guanines; greater blockage and distortion could be associated with a higher proportion of syn-guanine, base-displaced, intercalated conformers. A quantitative mapping of the conformational populations in the solution studies with the polymerase blockage data is, of course, not possible since the polymerase could certainly affect the population balance. Figure 10 shows color views of the two conformers present in about equal proportion at the NarI hotspot (see section 2.2). More broadly, such observed exquisite

Invited Review

sequence dependence of the conformational balance, if operative at replication forks and in other systems, could well-relate to the puzzling phenomenon of mutational hotspots in carcinogen-modified DNA. Rodriguez and Loechler (76) have proposed conformational heterogeneity to underlie mutational heterogeneity in a hotspot sequence modified by (+)-anti-BPDE. In the case of frameshifts such as -2 deletions at the NarI G3 hotspot, a primary sequence permitting slippage is the first prerequisite, and only G3 contains such a sequence context. However, the fact that different bulky adducts, notably AAF and AF, have quite different proclivities for inducing such mutations at the hotspot, suggests an important adduct-specific conformational component that may wellbe modulated by sequence context.

7. Conformational Themes Common to PAH and Aromatic Amine Adducts A striking parallel between the aromatic amines and the PAH BPDE adducts is the capability for forming both external structures and base-displaced, intercalated ones via an equilibrium whose balance is governed by base sequence context. The key forces at play in this equilibrium are carcinogen-base stacking in the intercalated conformers versus maintenance of Watson-Crick base pairing in the external ones. The details for the PAHs have been reviewed in Geacintov et al. (28). In the case of the PAHs, where modification on guanine is at N2, the external conformer with base pairs intact naturally positions the PAH in the minor groove, rather than the major groove as in the guanine C8 modification by aromatic amines. Moreover, the intercalated conformer adopts the anti domain rather than the syn domain at the modified guanine (except at the single-stranddouble-strand junction). However, the overall parallel concerning a sequence context-governed equilibrium is notable (28, 77). It is in harmony with the overarching concept that nondistorting external structures might represent structures that escape repair while distorting intercalated ones that are accessible could be mutagenic, as was first suggested more than a decade ago by Broyde and Hingerty (33, 44, 78) and advanced more recently in relation to mutagenicity for AF by Eckel and Krugh (35, 36).

8. Conclusion and Future Directions Of course, all of the above studies are just a beginning. To elucidate in greater detail the effects of base sequence context, so intriguingly beginning to emerge, is clearly an important direction to investigate, in normal and bulged duplexes and in replication intermediates. And, most certainly, it will be necessary to consider the role of the enzyme(s), since biological outcome is governed by interactions with them and can vary specifically with the specific enzyme system (79-81).

Acknowledgment. This research is supported by NIH Grant CA-49982 to D.J.P.; NIH Grant CA-75449, Grant CA-28038, and Grant RR-06458 and DOE Grant DE-FG02-90ER60931 to S.B.; NIH Grant ES-07946 and Grant ES-09127 to A.K.B.; DOE Contract DE-AC05960R22464 with Lockheed Martin Energy Research to B.E.H. We thank Professors Nicholas E. Geacintov and Robert Shapiro, Department of Chemistry, New York University, for ongoing stimulating discussions. Compu-


tations were carried out at the Department of Energy’s National Energy Research Supercomputer Center and the National Science Foundation’s San Diego Supercomputer Center. Coordinate Deposition: Coordinates of the distancerefined structures of the adducts solved by these authors are available from Suse Broyde, e-mail address [email protected], while their intensity-refined counterparts have been deposited in the Brookhaven Data Bank and can be retrieved on request to this depository.

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