NMR Solution Structures of Stereoisomeric Covalent Polycyclic

FEBRUARY 1997 VOLUME 10, NUMBER 2 © Copyright 1997 by the American Chemical Society

Invited Review NMR Solution Structures of Stereoisomeric Covalent Polycyclic Aromatic Carcinogen-DNA Adducts: Principles, Patterns, and Diversity Nicholas E. Geacintov,*,† Monique Cosman,‡,§ Brian E. Hingerty,| Shantu Amin,⊥ Suse Broyde,*,# and Dinshaw J. Patel*,‡ Chemistry and Biology Departments, New York University, New York, New York 10003, Cellular Biochemistry and Biophysics Program, Memorial Sloan Kettering Cancer Center, New York, New York 10021, Health Sciences Research Division, Oak Ridge National Laboratory, Oak Ridge, Tennessee 37831, and American Health Foundation, Valhalla, New York 10595 Received August 7, 1996

1. Introduction 2. Background 2.1. Structural Characteristics of Precarcinogenic PAH Compounds 2.2. Biological Activities and Absolute Configurations of PAH Diol Epoxide Stereoisomers 2.3. DNA Binding Specificity 2.4. PAH-DNA Adduct Characteristics 2.4.1. Low-Resolution Spectroscopic Studies of BPDE-DNA Adduct Conformations 2.4.2. Computed Adduct Conformations 3. NMR Solution Structures of Stereoisomeric PAH Diol Epoxide-DNA Adducts 3.1. NMR Characteristics of PAH-DNA Adduct Conformations

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* Corresponding authors. † Chemistry Department, New York University. ‡ Memorial Sloan Kettering Cancer Center. § Present address: Biology and Biotechnology Research Program, Lawrence Livermore National Laboratory, Livermore, CA 94550. | Oak Ridge National Laboratory. ⊥ American Health Foundation. # Biology Department, New York University.

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3.2. From the NMR Data to the Molecular Views 3.3. Conformations of Benzo[a]pyrene Diol Epoxide-N2-Deoxyguanosine Adducts 3.3.1. Overview 3.3.2. Solution Structures of anti-[BP]-N2-dG Lesions in 11-mer/ 11-mer Duplexes I 3.3.3. Conformations of the 10(S)-(+)-trans-, 10(R)-(-)-trans-, and 10(R)-(+)-cis-anti-[BP]-N2-dG Lesions in the 11-mer/10-mer Deletion Duplexes II 3.3.4. trans-anti-[BP]-N2-dG Lesions at Template/Primer Junctions (Sequence III) 3.3.5. Diversity of Stereochemistry- and Sequence Context-Dependent Conformational Motifs 3.3.6. Effects of Flanking Bases 3.4. Conformations of 5-Methylchrysene Diol Epoxide-N2-Deoxyguanosine Adducts in Duplex I 3.5. Minor Groove Styrenyl Oxide-N2-dG Adducts © 1997 American Chemical Society

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3.6. Conformations of PAH Diol Epoxide -N6-Deoxyadenosine Adducts 3.6.1. Benzo[c]phenanthrene Diol Epoxide-N6-Deoxyadenosine Adducts 3.6.2. Benzo[a]pyrene Diol Epoxide-N6-Deoxyadenosine Adducts 3.6.3. Styrene Oxide-N6-Deoxyadenosine Adducts 4. Principles, Patterns, and Diversity of Adduct Conformations 4.1. Comparisons with Other Types of Polynuclear Aromatic DNA Adducts 4.2. Stereochemical Effects and Relationships between Different Types of Adduct Conformations 4.3. Adduct Conformations at a Single Strand-Double Strand Junction 4.4. Effects of Bases Flanking the anti-[BP]-N2-dG Lesions on the Modified Strand 4.5. Structural and Steric Hindrance Factors That Determine anti-[BP]-N2-dG Adduct Conformations 4.5.1. The Torsion Angles That Define the Orientation of the BP Residues 4.5.2. The Torsion Angle β′ and the 5′f3′ and 3′f5′ Minor Groove Orientation Motifs 4.5.3. Classically Intercalated trans-anti-[BP]-N2-dG Adducts? 4.6. Structural Relationships between Minor Groove and Base-Displaced anti-[BP]-N2-dG Adduct Conformations 4.6.1. Computed trans-anti-[BP]-N2-dG Adduct Conformations: Structural Characteristics 4.6.2. Similarity of Torsion Angles R′ and β′ in Minor Groove and Base-Displaced Intercalative Conformers 4.6.3. Equilibrium between Minor Groove and Base-Displaced Intercalative Conformers 4.6.4. The Torsion Angles in the 11-mer/ 10-mer Deletion Duplexes 4.7. Base Stacking and Hydrogen Bonding in the Classically Intercalated trans-anti-[BPh]-N6-dA Adducts 4.8. The Importance of Secondary Steric Hindrance Effects 5. Adduct Conformations: Biological and Biochemical Implications 5.1. Detection and Identification of [BP] -DNA Adducts in Vitro and in Vivo 5.2. Stereoselective Digestion of [BP] -DNA Adducts by Exonucleases 5.3. Transcription 5.4. DNA Replication and Influence of Adduct Stereochemistry

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5.5. Stereochemical Effects on Excision of anti-[BP]-N2-dG Lesions by the UvrABC Nuclease System 5.6. Slipped Frameshift Intermediates during DNA Replication 6. Summary and Future Outlook

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1. Introduction Exogenous chemicals have been implicated in causing mutations that are responsible for the initiation and progression of tumors (1-5). Mutations in the p53 gene, a tumor suppressor gene in its nonmutated form, are common genetic alterations in human cancers, including breast cancer (3). Mutations of ras genes give rise to oncogenes that are believed to endow cells with a selective growth advantage resulting in tumors upon clonal expansion. There is accumulating evidence that a successive series of mutagenic events is fundamental to cancer causation (1, 5). Polycyclic aromatic hydrocarbons (PAH)1 are a wellknown class of environmental pollutants that, upon metabolic activation to highly reactive diol epoxides (6), bind chemically to cellular DNA and cause mutations. The genotoxic effects of PAH diol epoxide derivatives, especially their mutagenic and tumorigenic activities, have been well documented (for reviews, see refs 6-8). Of particular interest for elucidating chemical structurebiological activity relationships are the observations that the various PAH diol epoxide stereoisomers are characterized by strikingly different mutagenic and tumorigenic activities. While these remarkable stereochemical effects have long been of interest to chemists and molecular biologists, the influence of steric factors in determining the biological activities of PAH diol epoxides is still poorly understood. Detailed studies of these correlations on a molecular level may provide new insights into the still obscure mechanisms of mutations induced by chemicals and, ultimately, the mechanisms of induction of tumorigenesis by this class of compounds. The structural characteristics of the bulky adducts derived from the binding of PAH diol epoxides to cellular DNA are likely to play a critical role in a number of important events in the cell that determine its ultimate fate. For example, the recognition and rates of excision of these lesions by repair enzyme systems may be conformation-dependent. If the lesions escape repair, they can block DNA replication and transcription, thus leading to cell death. On the other hand, translesional bypass by polymerases may lead to mutations. The large-scale direct synthesis of various stereochemically defined and precisely positioned PAH diol epoxide lesions in various oligodeoxyribonucleotides of defined sequences developed in our laboratories (9-11) has allowed for breakthroughs in determining their solution 1Abbreviations: PAH: polycyclic aromatic hydrocarbons; 5-MeC: 5-methylchrysene; BP: benzo[a]pyrene; BPh: benzo[c]phenanthrene; anti-BPDE: 7r,8t-dihydroxy-t9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene; syn-BPDE: 7r,8t-dihydroxy-c9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene; (+)-anti-BPDE: 7(R),8(S),9(S),10(R) absolute configuration; (-)-anti-BPDE: 7(S),8(R),9(R),10(S) absolute configuration; (+)anti-5-MeCDE: 1(R),2(S)-dihydroxy-3(S),4(R)-epoxy-1,2,3,4-tetrahydro5-methylchrysene; (-)-anti-5-MeCDE: 1(S),2(R)-dihydroxy-3(R),4(S)epoxy-1,2,3,4-tetrahydro-5-methylchrysene; (+)-anti-BcPhDE: 3(R),4(S)dihydroxy-1(S),2(R)-epoxy-1,2,3,4-tetrahydrobenzo[c]phenanthrene; (-)anti-BcPhDE: 3(S),4(R)-dihydroxy-1(R),2(S)-epoxy-1,2,3,4-tetrahydrobenzo[c]phenanthrene; AF: 2-aminofluorene; AAF: N-acetyl-2-aminofluorene.

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Figure 1. Structures and numbering schemes of the polycyclic aromatic hydrocarbon compounds benzo[a]pyrene (BP), benzo[c]phenanthrene (BcPh), and 5-methylchrysene (5-MeC). The heavy lines define the bay regions (BP and 5-MeC) or the fjord region (BcPh).

structures by NMR techniques (12-23). Total synthesis and post-oligomerization approaches have been employed by other groups to synthesize site-specifically modified oligonucleotide adducts (24-27); NMR structural data on adducts synthesized by these methods are emerging from other laboratories at an increasing pace (28-35). These combined efforts are beginning to provide new insights into the relationships between adduct stereochemistry, adduct conformations, and the biological end points associated with these lesions. New results on the structures of DNA adducts derived from the binding of aflatoxin (36, 37), a structurally related sterigmatocystin derivative (38), and aromatic amines2 (39-46) to oligodeoxyribonucleotides have recently appeared as well. In this article, however, we limit ourselves to reviewing and discussing the effects of stereochemical factors on the conformations of adducts derived from the binding of PAH diol epoxides (12-23, 28-32), and a related aromatic epoxide (33-35), to oligonucleotides of defined sequence. The relationships between the experimental NMR data and the structural features of the adducts are illustrated using examples mainly from our own laboratories. We examine the diverse conformational features of PAH diol epoxideDNA lesions and discuss the structural principles that have emerged from these high-resolution NMR studies. We begin by recalling the basic characteristics of some of the relevant PAH diol epoxides, their biological activities, and their modes of covalent binding to DNA.

2. Background 2.1. Structural Characteristics of Precarcinogenic PAH Compounds. Our initial selection of compounds focused on sets of diol epoxide stereoisomers derived from the metabolic activation of three typical PAH compounds, benzo[a]pyrene (BP), benzo[c]phenanthrene (BPh), and 5-methylchrysene (5-MeC), each representing an important structural motif (Figure 1). The biologically important metabolites of BP are the diol epoxides 7,8-dihydroxy-9,10-epoxybenzo[a]pyrene in which the epoxide group is situated in the bay region of this molecule (the region encompassed by the C10-C11 carbon atoms denoted by heavy lines in Figure 1). BPh repre2B. Mao, B. E. Hingerty, S. Broyde, and D. J. Patel, Biochemistry, submitted.

Figure 2. Structures of PAH diol epoxide enantiomers that were used to prepare modified oligonucleotide sequences for NMR studies.

sents another structural motif, that of a sterically crowded region, called the fjord region (the region encompassed by the C1-C12 carbon atoms, Figure 1). In this compound, the steric hindrance between hydrogen atoms at C1 and C12 causes a deviation from planarity of the normally flat aromatic ring system (47). The methylsubstituted PAH compound 5-MeC represents still another motif of steric crowding since the methyl group at the 5-position is tilted out of the plane of the aromatic ring system and causes an in-plane widening of the bay region (48). The position of the methyl group is critical to the biological activity of the methylchrysene derivatives: only the 5-MeC derivative is highly tumorigenic, while the other single methyl group-substituted positional isomers are either inactive or much less active in rodents (49). 2.2. Biological Activities and Absolute Configurations of PAH Diol Epoxide Stereoisomers. The bay or fjord region diol epoxides of BP, 5-MeC, and BPh are the biologically most active metabolites of these three compounds. There are two diastereomeric forms of each diol epoxide that are distinguished from one another because the most distant hydroxyl group can either be anti or syn relative to the orientation of the epoxide group. In the case of the bay region BP diol epoxides, each of the two diastereomers, 7r,8t-dihydroxy-t9,10epoxy- and 7r,8t-dihydroxy-c9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (called anti- and syn-BPDE, respectively), can be resolved into two optically active (+)- and (-)-enantiomers. In the case of anti-BPDE, the absolute configurations of the two enantiomers are (+)-7(R),8(S),9(S),10(R) and (-)-7(S),8(R),9(R),10(S) ((+)- and (-)-antiBPDE, respectively). In our own NMR structural studies, we have concentrated on adducts derived from the covalent binding of the biologically highly active antidiol epoxides of BP and 5-MeC to guanine, and those of BPh to adenine residues (Figure 2). Other researchers


have focused on adducts derived from the binding of antiBPDE (28, 30-32) and syn-BPDE (29) to adenine, and styrene oxide to adenine (33, 34) and guanine (35) residues. The biological activities of racemic anti-BPDE have been investigated more extensively than those of the syn diastereomer. Racemic anti-BPDE is known to cause mutations in the critical codons 12 and 61 of the human c-Ha-ras1 protooncogene (50). Chirality exerts a profound influence on the biological activities of antiBPDE: the (+)-anti-BPDE enantiomer is highly tumorigenic while the (-)-anti-BPDE isomer is not (51, 52). Moreover, the (+)-enantiomer is more mutagenic in mammalian cell systems (53); however, in bacterial cell systems, the mutagenic efficiencies of the different stereoisomers are strain-dependent (54, 55). Like BP, 5-MeC is metabolically activated to highly reactive and biologically active bay region diol epoxide derivatives of varying biological activities (56, 57). Among these are the enantiomers 1(R),2(S)-dihydroxy-3(S),4(R)epoxy-1,2,3,4-tetrahydro-5-methylchrysene ((+)-anti-5MeCDE) and 1(S),2(R)-dihydroxy-3(R),4(S)-epoxy-1,2,3,4tetrahydro-5-methylchrysene ((-)-anti-5-MeCDE), depicted in Figure 2. Paralleling the characteristics of the two anti-BPDE enantiomers, the (+)-enantiomer is more tumorigenic in the lung of newborn mice and on mouse skin (56, 57) than the (-)-enantiomer and is also more mutagenic in Salmonella typhimurium strain TA 100 (57). The (+)-anti-5-MeCDE isomer has been implicated in causing mutations in the K-ras gene isolated from 5-MeC-induced lung tumors in mice (58). The biologically active metabolites of BPh are the fjord region diol epoxides anti- and syn-3,4-dihydroxy-1,2epoxy-1,2,3,4-tetrahydrobenzo[c]phenanthrene (anti- and syn-BPhDE, respectively). All four stereoisomers are mutagenic (59), and all of them, except for the (-)-syn isomer, are tumorigenic (60). The (-)-anti-BPhDE isomer is one of the most tumorigenic PAH diol epoxide derivatives known (60). The structures of the antiBPhDE enantiomers (+)-1(S),2(R),3(R),4(S) and (-)1(R),2(S),3(S),4(R) enantiomers are also depicted in Figure 2. Recent bioassays have shown that (()-anti-BPhDE is a potent mammary gland carcinogen in female rats, while (()-anti-BPDE is, at best, weakly active (61). Both of these diol epoxide derivatives (51, 52, 60-62), as well as 5-MeCDE (56), are strong carcinogens on mouse skin and in the lung of newborn mice. The exceptionally high biological activities of other fjord region and other sterically hindered diol epoxides have also been documented (63-71). The molecular origins of these differences in biological activities of fjord region and bay region PAH diol epoxides are presently not understood. 2.3. DNA Binding Specificity. The exocyclic amino groups of purine residues in native DNA are the primary targets of the covalent binding reactions of PAH diol epoxides. The attachment of PAH residues to the N2 groups of guanine or N6 of adenine residues can occur by either trans or cis addition as defined in Figure 3 for anti-BPDE-N2-dG adducts (see, for example, refs 72-76). Thus, each PAH diol epoxide enantiomer can give rise to two adducts with different absolute configurations about the PAH-exocyclic amino group linkage site. The spectrum of adducts formed when (+)- or (-)-anti-BPDE reacts with nucleic acids in an aqueous environment depends on the nucleic acid composition, secondary structure of the DNA, solution composition, etc. Typical

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Figure 3. Structures of the four stereoisomeric adduct moieties, anti-[BP]-N2-dG, derived from the trans- or cis-covalent binding of (+)-anti-BPDE or (-)-anti-BPDE to deoxyguanosine residues in DNA. The torsion angles R′ and β′ for the (+)-transadduct are indicated; the values of these torsion angles for the different strereoisomeric adducts are summarized in Table 1.

results on the binding of these two enantiomers to native double-stranded DNA were reported by Cheng et al. (73); in the case of the (+)-anti-BPDE enantiomer, the dominant adduct (>90%) is the (+)-trans-anti-BPDE-N2-dG adduct, while in the case of (-)-anti-BPDE, a smaller proportion of (-)-trans-adducts (63%) but greater proportions of (-)-cis-anti-BPDE-N2-dG (22%) and (-)-transanti-BPDE-N6-dA adducts (15%) were found. The (+)and (-)-anti-5-MeCDE isomers also bind predominantly to the exocyclic amino group of guanine residues by trans addition (75); smaller amounts (by factors of ∼10 and ∼4 in the case of (+)- and (-)-anti-5-MeCDE, respectively) of adenine adducts are also formed (57). In contrast to BPDE and 5-MeCDE, BPhDE binds more extensively to adenine than to guanine residues (76). Significant differences in the reactivity patterns of fjord and bay region PAH diol epoxides with DNA have also been noted for other sterically hindered PAH diol epoxides derived from 5,6-dimethylchrysene (77) and benzo[g]chrysene (70); these PAH diol epoxides also bind extensively to the exocyclic amino group of adenine residues in DNA. There has been some speculation that PAH diol epoxideadenine adducts may be highly mutagenic and therefore of importance in the initiation stages of tumorigenesis (76, 77). In general, minor DNA adducts could be quite important because their mutagenic potentials might be greater than those of the major adducts; however, specific comparative data relevant to this issue are still lacking at this time. 2.4. PAH-DNA Adduct Characteristics 2.4.1. Low-Resolution Spectroscopic Studies of BPDE-DNA Adduct Conformations. In the decade or so before the first NMR solution structure was published (12), extensive optical spectroscopic studies provided some insights into the different types of BPDEDNA adduct conformations (for reviews, see refs 78-82). Based on linear dichroism studies of covalent (()-antiBPDE-DNA adducts, it was suggested that the pyrenyl residues are predominantly situated at external binding sites, most likely in the minor groove (83). However, the notion that the bulky hydrophobic pyrenyl ring system

Invited Review

is situated at a water-exposed DNA site seemed unattractive; ligands with hydrophobic flat aromatic ring systems generally prefer to intercalate between adjacent base pairs in DNA, thus minimizing their contacts with the aqueous solvent environment. Hogan et al. (84) proposed a wedge-shaped intercalation model in which the pyrenyl ring system is partially buried between adjacent base pairs at a distorted native DNA binding site. Linear dichroism and other spectroscopic data on the noncovalent and covalent binding of different PAH diol epoxides to DNA indicated that two types of binding sites can be distinguished, one with intercalative characteristics (site I) and the other with external binding site attributes (site II) (85). The noncovalent, prereaction complexes formed between anti- or syn-BPDE and native DNA are of the site I type (86, 87); these conclusions are consistent with other studies that suggest that noncovalent anti-BPDE-DNA complexes are intercalative in nature (88-90). As the anti-BPDE molecules react with DNA to form covalent adducts, site I and site II adducts are formed in different proportions, depending on the anti-BPDE enantiomer (91, 92). The linear dichroism (91, 92) and fluorescence characteristics (9396) of covalent BPDE-DNA adducts are consistent with the heterogeneity of adduct conformations (97, 98). Based on comparisons of the UV optical characteristics of anti-BPDE-polynucleotide and stereochemically defined, site-specific oligonucleotide adducts, it was shown that trans-anti-BPDE-N2-dG adducts have site II conformations, while the isomeric cis-adducts have site I adduct conformations (99). 2.4.2. Computed Adduct Conformations. Computational methods to study the conformations of anti-[BP]N2-dG lesions positioned in double-stranded DNA were the only means for obtaining detailed structural views of these adducts before high-resolution NMR experimental studies became feasible. Singh et al. (100) used the program DUPLEX (see below) to carry out wide-scale conformational searches of the structures of the (+)- or (-)-trans-anti-BPDE-N2-dG lesions in an alternating selfcomplementary dG-dC dodecamer duplex. Three types of lower energy conformations were found for each of these adducts. Two were of the site II type and one was of the site I type. In the site II type conformers, the pyrenyl moiety was placed either in the B-DNA minor groove with all base pairs essentially intact, or in the major groove with the modified deoxyguanosine glycosidic torsion angles in the syn domain. In the site I type conformation, the pyrenyl moiety was inserted into the helix by displacement of the modified guanine, so that the base pair at the modification site was ruptured. The site II type conformer with the pyrenyl moiety in the minor groove was lowest in energy for the (+)-enantiomer adduct, while the minor and major groove conformers were of about equal energy for the (-)-adduct in this sequence. The computations predicted that, for each type of conformation, the pyrenyl moiety in the (+)- and (-)adducts would be directed either toward the 5′-end or toward the 3′-end of the modified strand for minor groove site II conformers, respectively. For the base-displaced intercalative site I conformers, structures with the aromatic pyrenyl residues pointing either toward or away from the minor groove were found for the (+)- and (-)adducts, respectively. Overall, these conformational themes proved to be remarkably close to the experimentally measured ones.


Several other groups have modeled the properties of trans-anti-[BP]-N2-dG lesions (101-106). None of the earlier modeling efforts had uncovered the opposite orientations of the (+)- and (-)-adducts, although Weston et al. (102) had explored the orientation issue for a (+)adduct minor groove structure and had correctly concluded that the 5′-orientation was favored.

3. NMR Solution Structures of Stereoisomeric PAH Diol Epoxide-DNA Adducts 3.1. NMR Characteristics of PAH-DNA Adduct Conformations. The solution conformations of PAH carcinogen-modified duplexes in H2O and D2O buffer solutions can be studied by multidimensional and heteronuclear NMR techniques. We briefly outline some of the more important principles of these methods, particularly those that are used routinely in our own research. Exchangeable Proton Spectra. The 1D NMR spectra of the PAH-modified duplexes in H2O buffer solution provide information on duplex formation and whether single or multiple conformations are present. Upfield chemical shifts of sharp and narrow imino protons may indicate the presence of significant ring current effects due to stacking interactions between the DNA bases and an intercalated PAH aromatic ring system. On the other hand, upfield chemical shifts of broad, rapidly exchanging imino protons may be due to the disruption of the base pair hydrogen bonding. The presence of NOEs between guanosine iminos and cytidine amino protons is indicative of Watson-Crick G:C base pair alignment, while NOEs between thymidine imino and adenosine H2 protons are characteristic of Watson-Crick A:T base pairs. The imino to imino connectivity in a normal B-DNA helix can be followed from one base pair to the base pairs stacked above and below it. A disruption of this connectivity between two or more DNA base pairs in the modified duplex may be due to the presence of an intercalated PAH residue. Nonexchangeable proton spectra measured in D2O are used to assign all of the base and sugar protons (except for the superimposed H5′ and H5′′ resonances) using known principles (107). Expanded NOESY contour plots are used to determine connectivities between purine H8 (or pyrimidine H6) protons and H1′ protons on their own sugar rings or those of 5′-flanking bases. These directional connectivities are traced from base to adjacent base to establish a right-handed alignment of base pairs. The absence of some of these connectivities at or near the lesion site may signal the presence of intercalated PAH ring systems. Unusual chemical shifts due to ring current effects of some of the proton resonances on the bases, or the aromatic rings, can provide further evidence for the insertion of the aromatic ring system into the helix. In contrast, for minor groove adduct conformations, chemical shifts in some of the sugar protons located in the minor groove near the sites of the lesion can be used to establish the orientation of the PAH aromatic ring system. The coupling constant patterns are determined by phase-sensitive COSY methods and are compared with patterns calculated from modified Karplus relationships, thus providing information on the conformation of the benzylic ring. The NOE cross-peaks (50 ms mixing time) between base protons and sugar H1′ protons are used to differentiate between syn and anti glycosidic torsion angles, since NOEs are either strong


or weak, respectively, for these different base-sugar conformations. Phase-sensitive COSY and DQF-COSY spectra are used to determine the sugar proton-proton vicinal coupling constants and thus to distinguish between different families of sugar pucker and A- and B-DNA forms (107). Analysis of NOEs. The analysis of NOESY data sets measured in H2O and D2O buffer solutions provides information about through-space connectivities for protons that are located within less than ∼5.5 Å of one another. NOEs between the covalently attached PAH moieties and nearby DNA protons provide a set of interproton distances with upper and lower bounds that are used as distance restraints in the molecular mechanics computations of the adduct conformations. 3.2. From the NMR Data to the Molecular Views. Different approaches can be used to generate threedimensional adduct structures from the NMR data (12, 23, 28, 32). In our approach, the set of interproton distances with lower and upper bounds are used to determine the structure of the PAH diol epoxide-modified oligonucleotides using the torsion angle space molecular mechanics program DUPLEX (108). In torsion angle space energy minimizations, intramolecular motions are limited to rotations about single bonds (and sugar pucker flexing), the major factors that govern the shape of the DNA adducts. Bond lengths and bond angles are fixed to observed equilibrium values, and aromatic moieties are constrained to their planar equilibrium positions, unless suggested otherwise by the data. This greatly diminishes the number of variables that must be optimized, from 3N - 6 to 9 (where N is the number of atoms), in a nucleotide covalently linked to a PAH residue. The ability of the minimizer to locate low energy minima on the potential energy surface is thus markedly enhanced, with the added advantage that structures with anomalous geometries cannot occur. The potential energy surface is searched with a series of energy minimization trials that employ arbitrary, unbiased orientations of the carcinogen as initial positions; the trials include the available NMR-derived distance restraints as penalty functions which guide the minimization algorithm in the direction of the observed solution structure. At this stage, small subunits, usually duplex trimers to pentamers, are investigated. The structure that best fits the data, which is also among the lowest energy forms, is built up to the oligomer level, and all restraints are then released. If the NMR-derived distances are not retained at this point, distance restraints can be re-evaluated, especially when conformational heterogeneity is a concern, and further searches are performed until a final structure is achieved that is an unrestrained minimum energy conformation in agreement with the data. These final structures are also fully consistent with NMR data other than the NOESY proton-proton distance data sets employed as restraints; for example, chemical shifts due to ring current and other effects provide independent evidence for the orientation and positioning of the bulky polycyclic aromatic residues at their binding sites; interproton distances that have not been restrained also serve as cross-checks. Other adduct structures (23, 28-35) have been successfully solved by restrained energy minimization or molecular dynamics calculations with programs such as Discover from Biosym Technologies, Inc., X-Plor (109), and Amber 4.0 (110).

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Figure 4. Oligodeoxyribonucleotide sequences discussed in the text. The starred deoxyguanosine (G*) or deoxyadenosine (A*) residues denote the modified bases. I: 11-mer/11-mer duplex, II: 11-mer/10-mer deletion duplex (complementary base C opposite the modified G* is missing), III: 13-mer/9-mer template/ primer junction. Sequences IV-VI are normal duplexes in which all of the bases in the partner strands are complementary to those in the modified strands.

3.3. Conformations of Benzo[a]pyrene Diol Epoxide-N2-Deoxyguanosine Adducts 3.3.1. Overview. The most abundant reaction products of the (+)- and (-)-anti-BPDE enantiomers with native DNA are adducts with a covalent linkage between the C10 position of BPDE and the exocyclic amino group of deoxyguanosine residues (designated here as the anti[BP]-N2-dG lesions). These lesions have been selected for our initial detailed conformational NMR studies. We have explored a number of structural motifs using the four possible stereochemically distinct anti-[BP]-N2-dG lesions (Figure 3) embedded in several different sequences (Figure 4). These include 11-mer/11-mer duplexes with their normal complementary strands (I), 11mer/10-mer duplexes with a missing partner base on the complementary strand opposite the modified guanine residue (“deletion” duplexes II), and anti-[BP]-N2-dG lesions situated at double strand-single strand junctions as in a DNA replication fork, but with one arm of the fork missing (template/primer 13-mer/9-mer sequence III). 3.3.2. Solution Structures of anti-[BP]-N2-dG Lesions in 11-mer/11-mer Duplexes I. The adduct conformations for the stereoisomeric, but chemically identical (+)-trans-, (-)-trans-, (+)-cis-, and (-)-cis-anti-[BP]N2-dG lesions (G6*) embedded in duplex I (Figure 4) were investigated in detail (12, 13, 20, 21). The (+) and (-) designations denote mirror image relationships between the absolute configurations of the -OH groups at the C7, C8, and C9 chiral carbon centers, while the cis and trans notations designate the relative configurations of the adducted deoxyguanosyl moiety at C10 of the BP residue. The absolute configuration of the adducts at the C10 carbon atom is 10(S) for the (+)-trans- and (-)-cisadducts, and 10(R) for the (-)-trans- and (+)-cis-lesions (Figure 3).

Invited Review

The 10(S)-(+)- and 10(R)-(-)-trans-anti-BPDE-N2-dG Adducts. All of the imino proton resonances were assigned (12, 21) and are situated in the region of 12.014.0 ppm in both the (+)-trans- and (-)-trans-duplexes I. The NOEs between deoxyguanosine iminos and the hydrogen-bonded cytidine amino protons, and between thymidine imino and adenosine H2 protons, are characteristic of Watson-Crick hydrogen bonding for all 11 base pairs. The B-DNA-type base stacking conformations at all base pairs, as well as the lack of upfield-shifted imino proton resonances, establishes that the pyrenyl residues are not intercalatively inserted between any of the adjacent base pairs. In contrast, unusual chemical shifts are seen in some of the sugar protons of the bases in the vicinity of the modified G6* residue. In the case of the (+)-trans-adduct, the dramatic upfield chemical shifts of the H1′ sugar protons of G18, and to a smaller extent the H1′ proton of A19, suggest that the aromatic pyrenyl residue stacks primarily over the sugar ring of G18 in the minor groove, and to a lesser extent over that of A19, and is thus oriented toward the 5′-end of the modified strand. In the case of the (-)-trans-duplex I, pronounced upfield chemical shifts of the H1′ protons of the C17 and G16 sugars clearly indicate that the aromatic pyrenyl residue stacks primarily over the sugar ring of C17 on the partner strand in the minor groove and is thus oriented toward the 3′-end of the modified strand. Thus, the most striking difference between the conformations of the (+)-trans- and (-)-trans-adduct duplexes I is the orientation of the pyrenyl aromatic ring system and the benzylic ring of the BP residue relative to the modified deoxyguanosyl residue G6* (Figures 5A and 5B). These opposite orientations of the BP residues are manifestations of the chiral relationships between the (+)-antiBPDE and (-)-anti-BPDE enantiomers. The 10(R)-(+)-cis- and 10(S)-(-)-cis-anti-[BP]-N2-dG Lesions. The predominant adduct conformations of the (+)-cis- and (-)-cis-[BP]-N2-dG lesions in duplexes I (Figures 6A and 6B) are characterized by base-displaced intercalative conformations (13, 20) and are thus remarkably different from those of the stereoisomeric transadducts (Figures 5A and 5B). In the major (+)-cis-adduct structure, the pyrenyl ring system is intercalated between adjacent base pairs and displaces the modified guanine residue G6* into the minor groove, and the partner base C17 into the major groove. Thus, the intercalation pocket is created by displacing the modified base pair from its normal intrahelical position, rather than by stretching and unwinding the helix as in classical intercalation. The structural differences between the two trans- and the two cis-adducts manifest themselves in terms of markedly dissimilar NMR characteristics. In the two trans-adducts, all of the NOE connectivities between the purine H8 (or pyrimidine H6) protons and H1′ protons, on their own sugar rings or those of 5′-flanking bases, could be traced from the first to the 11th base pair of duplexes I. In contrast, in the cis-adducts in the C5G6*-C7 duplex segment, these NOEs are weak or very weak, suggesting that the normal connectivity (as in double-stranded DNA) is disrupted around the lesion site. The C17 H1′ proton is downfield-shifted, suggesting a loss of base stacking interactions and thus a displacement of this cytidine residue from the double helix. The BP aromatic protons (though not the benzylic ring protons) are significantly more upfield-shifted in the cis- than in the trans-adducts (Figure 7). Furthermore, the BP


proton-G16 and -G18 imino proton NOEs pinpoint the intercalative alignment of the pyrenyl ring system between the intact hydrogen-bonded base pairs C5:G18 and C7:G16, with the nonplanar benzylic ring in the minor groove. The H2O exchangeable proton spectrum supports this interpretation: for example, three well-resolved and upfield-shifted G16, G6*, and G18 imino protons are observed in the case of the (+)-cis-adduct at 12.1, 11.2, and 10.8 ppm, respectively. The dramatic upfield shift of the G18 imino proton resonance to 10.8 ppm, as well as the more modest upfield shift of the G16 imino proton to 12.1 ppm, are consistent with stacking interactions with the aromatic pyrenyl ring system These unusual upfield shifts of the G6* imino proton occur because of a loss of hydrogen bonding to the normal partner base C17; this conclusion is supported by the loss of an NOE signal between the imino proton of G6* and the amino proton of C17, the absence of an NOE between the imino proton of G6* and the imino proton of the adjacent G16, and only a weak NOE between the imino protons of G6* and G18. However, an NOE is observed between the imino proton of G6* and the H1′ proton of the C5 sugar residue. This suggests that the G6* residue is displaced from the helix and is situated in the minor groove and is oriented toward the 5′-end of the modified strand. The G16 and G18 imino protons exhibit strong NOEs to the respective amino protons of their partner C7 and C5 bases, and both base pairs flanking the lesion are thus hydrogen-bonded. The overall conformational features of the (-)-cisadducts (20) are similar to those of the (+)-cis-adducts (13), but with some important differences in the positioning of the pyrenyl moiety in the intercalation pocket generated by the displaced modified deoxyguanosyl residue (Figures 6A and 6B). The benzylic ring is in the minor groove in the (+)-cis-adduct, while in the (-)-cisadduct it is situated oppositely, rotated 180° about the helix axis, in the major groove. The pyrenyl ring system points toward the major groove in the (+)-cis-, and toward the minor groove in the (-)-cis-adduct. Furthermore, the covalently modified guanine residues are displaced into the minor groove in the case of the (+)-cis-adduct, and into the major groove in the case of the (-)-cis-adduct. The partner base C17, which normally is hydrogenbonded with the modified base G6* in duplex I, is displaced into the major groove in both cases. In the (-)cis-adduct, the modified guanine residue is also directed toward the 5′-end of the modified strand and stacks over the major groove edge of the 5′-flanking cytosine residue C5. More Than One Conformation in the 10(R)-(+)-cis- and (-)-cis-Adducts. In the (+)-cis-adduct, the existence of imino proton exchange cross-peaks at 1 °C establishes the presence of a minor conformation (∼15%) in which the G6*:C17 bases are hydrogen-bonded and the pyrenyl ring system is not intercalated (13). The 1D and 2D NMR spectra of the major, intercalated form were thus measured at 10 °C or less, since the spectral resolution deteriorated as the temperature was raised to ambient values. Exchange cross-peaks in the NOESY spectra of the 10(S)-(-)-cis-adduct indicate that there is also a slow equilibrium between the major intercalative base-displaced conformer and a minor conformer in this temperature range (1-10 °C); however, as in the (+)-cis-adduct case, the conformation of the minor conformer could not be established (20). In contrast to the two cis-adducts, the NMR resonances of the two trans-adducts are well-


Geacintov et al.

Figure 5. NMR solution structures (stereoviews) of the two stereoisomeric trans-anti-[BP]-N2-dG lesions in the 11-mer/11-mer duplexes I; the 5′-ends of the modified strands are on the top in all cases, and the views are into the minor groove and normal to the helix axis. Only the central five-base-pair segments are shown. (A) 10(S)-(+)-trans-anti-[BP]-N2-dG adduct, and (B) 10(R)-(-)-transanti-[BP]-N2-dG adduct. These views have been prepared for viewing with a stereoviewer; for viewing with crossed eyes, the left and right images should be interchanged.

Figure 6. NMR solution structures (stereoviews) of the two stereoisomeric cis-anti-[BP]-N2-dG lesions in the 11-mer/11-mer duplexes I; the 5′-ends of the modified strands are on the top in all cases, and the views are into the minor groove and normal to the helix axis. Only the central five-base-pair segments are shown. (A) 10(R)-(+)-cis-anti-[BP]-N2-dG adduct, and (B) 10(S)-(-)-cis-anti-[BP]N2-dG adduct.

Invited Review


Figure 7. Chemical shifts of H1-H12 [BP] proton resonances in the four stereoisomeric anti-[BP]-N2-dG adducts in the 11mer/11-mer duplexes I (from ref 20).

resolved even at the higher temperatures of 25 °C (12, 21). Adduct Conformations and Thermal Stabilities of Duplexes. The covalently bound BP residues significantly lower the melting points of the 11-mer/11-mer duplexes I (Table 1). In the case of the two trans-adducts, the characteristic duplex melting points Tm are lowered by 8-10 °C relative to the Tm (51 °C) of the unmodified 11mer/11-mer duplex. In the (+)-cis- and (-)-cis-11-mer/ 11-mer duplexes, the Tm values are only ∼4-5 °C lower

than that of the unmodified duplex I (111). Therefore, the stacking of the pyrenyl ring system with the adjacent bases in the (+)-cis- and (-)-cis-adduct duplexes I probably contributes significantly to the stability of the duplexes, and partially compensates for the loss of hydrogen bonding in the base displaced structures. 3.3.3. Conformations of the 10(S)-(+)-trans-, 10(R)-(-)-trans-, and 10(R)-(+)-cis-anti-[BP]-N2-dG Lesions in the 11-mer/10-mer Deletion Duplexes II. Bulky PAH-DNA lesions are known to stall DNA polymerases at or near the sites of the adducts, which could allow for the formation of bulged intermediates in which the modified nucleic acid residues are no longer paired with their partner bases on the complementary strands. Such slipped, misaligned frameshift intermediates are believed to give rise to deletion and point mutations (112-114). The detailed structural features of these intermediates are therefore of interest (see section 5.6). As a model system for the bulged intermediates, we investigated the high-resolution NMR characteristics of sequence II, which is identical to sequence I except that the base (C17) opposite the anti-[BP]-N2-dG lesion is deleted in the 11-mer/10-mer duplex II (Figure 4). In the H2O imino proton spectrum of the (+)-trans-anti[BP]-N2-dG deletion duplex II, dramatic upfield shifts are observed in the imino proton resonances of G6*, G17, and G16, as well as in the amino protons of the neighboring C5 and C7 on the modified strand, and there are no unusual upfield chemical shifts in the H1′ sugar proton of the bases flanking the modified G6* site on the 5′-side (15). The large upfield chemical shifts are due to ring current effects arising from the intercalation of the pyrenyl ring system between the C7:G16 and C5:G17 base pairs. NOEs are observed between the C7 and C5 amino protons and the G16 and G17 imino protons, which establishes the existence of Watson-Crick base pairing around the flanking BPDE-modified G6*. Base pairing is observed at all other positions as well. However, the G6* imino proton appears to be shifted upfield primarily because of a loss of hydrogen bonding; the broadness of this resonance (15) and the lack of connectivities to other protons indicate that the G6* guanine ring is solventexposed and that its imino proton is rapidly exchanging with water. Indeed, nonexchangeable proton-proton NOEs indicate that the guanine ring is looped out into the major groove and stacks over the base portion of the 5′-flanking C5. A detailed analysis of the chemical shifts

Table 1. Summary of Conformations of anti-[BP]-N2-dG Oligonucleotide Adducts, and the Torsion Angles A-B-C-D at the Modified dG Residuesa [BP]-N2-dG adductb stereochemistry (-)-trans-11/11-mer duplex I (+)-trans-11/11-mer duplex I (+)-trans-11/10-mer deletion duplex II (+)-trans-13/9-mer template/ primer sequence IIIe (+)-cis-11/11-mer duplex I (-)-cis-11/11-mer duplex I (+)-cis-11/10-mer deletion duplex II (+)-trans-11/11-mer duplex IVg (-)-trans-[MC]-11/11-mer duplex I a

base opposite the lesion dC dC none none dC dC none dC dC

adduct conformation

χ, deg

R′, deg

β′, deg

Tm, °Cc

minor groove, 3′-orientation minor groove, 5′-orientation base-displaced intercalationd stacked with 3′-base pair, base-displaced base-displaced intercalationf base-displaced intercalationd base-displaced intercalationf minor groove, 5′-orientation minor groove, 5′-orientation

260 257 301 71

152 137 138 272

78 258 263 201

43 (51) 41 (51) 30 (24) 42 (41)

189 285 168

160 135 169

136 268 91

282

176

63

47 (51) 46 (51) 49 (24) 42 (55) 40 (51)

The base to sugar glycosidic angle χ at the purine-carcinogen linkage site is defined as the O4′-C1′-N9-C4 angle. The torsion angles defining the relative orientations of the PAH and purine residues are R′ (N1-C2-N2-(BP)C10) and β′ (C2-N2-(BP)C10-C9) for the [BP]-N2-dG adducts, and R′ (N1-C2-N2-(MC)C4) and β′ (C2-N2-(MC)C4-C3) for the [MC]-N2-dG adducts. See also Figure 19. b Unless mentioned otherwise. c From Ya et al., ref 111 (except for the 13/9-mer Tm value); all melting points measured at duplex concentrations of 20 µM. Values for the unmodified duplexes are in parentheses. d Orientation of benzylic ring f distal aromatic ring on pyrenyl residue: major to minor groove. e Torsion angles for the 13-mer/9-mer have not been published previously. f Orientation of benzylic ring f distal aromatic ring on pyrenyl residue: minor to major groove. g Fountain and Krugh (23).


Geacintov et al.

Figure 8. Stereoview of the 10(S)-(+)-trans-anti-[BP]-N2-dG adduct in the 11-mer/10-mer deletion duplex II. The 5′-end of the modified strand is on the top, and the view is into the minor groove and normal to the helix axis (top), or down the helix axis (bottom). The central five-base-pair segment is shown on the top, and only the d(C5-G6*-C7)‚d(G16-G17) segment is shown in the bottom view.

Figure 9. Stereoview of the 10(R)-(+)-cis-anti-[BP]-N2-dG adduct in the 11-mer/10-mer deletion duplex II. The 5′-end of the modified strand is on the top, and the view is into the minor groove normal to the helix axis (top), or down the helix axis (bottom). The central five-base-pair segment is shown on the top, and only the d(C5-G6*-C7)‚d(G16-G17) segment is shown in the bottom view.

and NOEs shows that the benzylic ring of BP is situated in the major groove, while the most distant aromatic ring (the [BP]C1-C2-C3 edge) protrudes into the minor groove. The long axis of the pyrenyl ring is oriented perpendicular to the long axis of the flanking C5:G17 and

C7:G16 base pairs (Figure 8). In summary, the BP residue displaces the modified guanine ring into the major groove and intercalates into a wedge-shaped intercalation site (the G16-G17 bases form the narrow side of the wedge) with the benzylic ring directed toward

Invited Review

the major groove and the pyrenyl ring system toward the minor groove. Interestingly, recent experiments with the stereoisomeric (-)-trans-anti-[BP]-N2-dG-11/10-mer deletion duplex suggest a similar conformation, but with the benzylic ring situated in the minor groove and the pyrenyl ring system thus oriented in the opposite direction, toward the major groove.3 The difference in the conformations of the BP residues in the minor groove (+)-trans-11-mer/11-mer duplex I (Figure 5A) and the base-displaced intercalative (+)trans-11-mer/10-mer deletion duplex II (Figure 8) is especially striking. However, the torsion angles R′ and β′, defining the carcinogen-base linkage conformation (defined in Figure 3 and Table 1, and discussed in greater detail below), are nearly identical in these two duplexes (Table 1); this suggests that relatively small, energetically feasible concerted movements in the glycosidic and nearby DNA backbone torsion angles can effect this rearrangement. By removing the C17 partner base of G6*, the loss of hydrogen bonding imposes a change from a minor groove conformation of the BP residue in the 11mer/11-mer duplex I, to an intercalative base-displaced conformation in the deletion 11-mer/10-mer duplex II. In contrast, there is little change in the conformation of the major (+)-cis-anti-[BP]-N2-dG lesion upon removing the C17 base from the complementary strand of duplex I to generate the deletion duplex II (16). However, in the (+)-cis-11-mer/10-mer deletion duplex II, only one major conformation was evident, while in the full (+)cis-11-mer/11-mer duplex I the major form was in slow equilibrium with a minor conformer at 1-10 °C (13). The (+)-cis-anti-[BP]-N2-dG deletion duplex II shares many of the same structural features with the base-displaced intercalative (+)-trans-anti-[BP]-N2-dG deletion duplex II. As in the latter, the imino protons of G6*, G17, and G16 in the (+)-cis-anti-[BP]-N2-dG deletion duplex II are strongly shifted upfield, reflecting ring current shifts due to the intercalated BP aromatic ring system. However, in the (+)-trans-anti-[BP]-N2-dG deletion duplex II, the benzylic ring is situated in the major groove (Figure 8), while in the (+)-cis-anti-[BP]-N2-dG deletion duplex II it is positioned in the minor groove (Figure 9). The long axis of the pyrenyl ring system is aligned orthogonal to the axis joining the adjacent C5:G17 and C7:G16 base pairs in both adducts. In the normal (+)-cis-anti-[BP]N2-dG duplex I, the G16 and G17 bases are not stacked on top of one another, although they are stacked in the (+)-cis-anti-[BP]-N2-dG deletion duplex II (Figure 9). This factor may account for the surprisingly higher melting point Tm (49 °C) of the (+)-cis deletion 11-mer/10-mer duplex II than the Tm (47 °C) of the normal (+)-cis-11mer/11-mer duplex I (Table 1), even though the former has one less intact base pair. 3.3.4. trans-anti-[BP]-N2-dG Lesions at Template/ Primer Junctions (Sequence III). During DNA replication, one DNA strand serves as the template while the other strand is extended by the incorporation of 2′deoxynucleotide 5′-triphosphates catalyzed by DNA polymerases. As a model system, we have selected for study the 13-mer/9-mer sequence III (Figure 4) with either a (+)- or a (-)-trans-anti-[BP]-N2-dG lesion at the single strand-double strand junction. This sequence was selected because it was neither too long for a detailed NMR structural study, nor too short for comparisons with 3B. Feng, B. E. Hingerty, N. E. Geacintov, S. Broyde, and D. J. Patel, Biochemistry, submitted.


ongoing parallel in vitro polymerase-catalyzed DNA synthesis studies (115). The mechanisms of polymerase-catalyzed DNA replication past bulky BPDE-DNA lesions, especially the effects of stereochemistry, have long been of interest for understanding the influence of adduct conformation on mutagenic specificities (116, 117). In the ternary DNA polymerase-primer/template complexes, the kinetics of addition of the deoxynucleoside triphosphates to the nascent primer strands are expected to be particularly affected near the BPDE-modified guanine on the template strand (117). In sequence III, the primer strand extends up to the base C5 flanking the anti-[BP]-N2-dG lesion on the 3′-side. In view of the fact that in the (+)trans- and (-)-trans-[BP]-N2-dG adduct duplexes I the aromatic portions of the BP residues point toward the 5′- or toward the 3′-end of the modified strand, respectively, it is of interest to determine if this preferred orientation manifests itself as well at primer-template junctions. The Unmodified Control Sequence III. The imino proton resonances in the G22:C5 f G14:C13 segment are well resolved, and the connectivities between imino protons on adjacent base pairs can be traced from one end of this segment to the other. Watson-Crick base pairing is indicated in this segment by the presence of characteristic NOEs between the imino protons and the base and amino protons. However, the NOE cross-peak between the imino protons of T6 and G22 is very weak, suggesting that the terminal junction base pair G22:C5 is subject to fraying. The nonexchangeable protons are also well resolved, and sequential NOEs between the base protons and their own and 5′-flanking sugar H1′ protons can be traced from one end of the double-stranded region to the other end of the same strand. All of the resonances in the single-stranded region d(A1-A2-C3-G4-...) were also assigned (19). The (+)-trans-anti-[BP]-N2-dG 13-mer/9-mer Sequence III. Analysis of the 1D and 2D NMR characteristics of the (+)-trans-anti-[BP]-N2-dG 13-mer/9-mer duplex III shows that Watson-Crick base pairing is intact in the G22:C5 f G14:C13 segment, as it is in the control unmodified 13-mer/9-mer sequence. However, the presence of the BP moiety causes a marked deterioration of the quality of the resonances due to line broadening in the d(A1-A2-C3-G4*-C5)‚d(G22) portion of III. This suggests that the single-stranded region is more mobile than in the control sequence III. The G22 imino proton and the two amino protons of the partner base C5 are shifted upfield by ∼1.1 ppm; smaller upfield shifts are also observed for the imino proton of T6 (0.14 ppm) and the H2 base proton of A21 (0.41 ppm). An analysis of the relevant NOEs of the nonexchangeable proton resonances of the BP moiety indicates that it is stacked with the G22:C5 base pairs at the junction; the magnitudes of these upfield shifts are consistent with the exposure of one of its faces to the aqueous solvent environment. The C9-C10-C11-C12 edge of BP is oriented toward the modified strand, while the opposite C2-C3-C4 edge is positioned over the C5:G22 base pair. Most interestingly, a strong H1′ to H8 NOE cross-peak at the BP-modified base G4* indicates that the glycosidic torsion angle for this modified deoxyguanosine residue is in the syn rather than in the usual anti orientation. The conformation of the modified deoxyguanosyl residue G4* is different from the base-base stacked conformation of G4 in the unmodified 13-mer/9-mer complex. Using the NMR data


Geacintov et al.

Figure 10. Stereoview of the 10(S)-(+)-trans-anti-[BP]-N2-dG adduct in the 13-mer/9-mer template/primer sequence III. The 5′end of the modified strand is on the top, and the view is into the minor groove, normal to the helix axis of the duplex segment.

to guide the computational searches, a number of restrained structures of a 9-mer/5-mer segment from the (+)-trans-anti-[BP]-N2-dG 13-mer/9-mer sequence III were generated. These were closely related structures, each consistent with the NMR input data. A representative structure from this ensemble was built up to the 13mer/9-mer level, and all restraints were released. This structure, shown in Figure 10, is consistent with the NMR data. The (-)-trans-anti-[BP]-N2-dG Adduct in the 13-mer/ 9-mer Sequence III. The NMR characteristics of this stereoisomeric template/primer junction sequence are quite different from those of the (+)-trans-anti-[BP]-N2dG 13/9-mer sequence III. There is a striking loss of resolution at and around the (-)-trans-anti-[BP]-N2-dG lesion site. Only the imino protons of the last six base pairs C8:G19 f C13:G14, i.e., those furthest removed from the BP-modified G4* residue, can be resolved. Thus, the presence of the (-)-trans-anti-[BP]-N2-dG moiety causes a significant structural disturbance that propagates over the three-base-pair d(C5-T6-A7):d(G22-A21T20) duplex region flanking the modified G4* on the 3′side. Much of the NMR data were characterized by very broad, overlapping cross-peaks, especially at, and in the vicinity of, the lesion site. The presence of exchange cross-peaks in both the NOESY and TOCSY data sets establishes that there is more than one conformation of the (-)-trans-anti-[BP]-N2-dG moiety, and the two or three residues flanking the lesion on either side. Thus, the exact orientation of the BPDE residue could not be ascertained from the NMR data. 3.3.5. Diversity of Stereochemistry- and Sequence Context-Dependent Conformational Motifs. The ef-

fects of stereochemistry are strikingly represented in the family of four chemically identical but stereochemically distinct anti-[BP]-N2-dG lesions embedded in the fully complementary 11-mer/11-mer duplexes I. In the full 11mer/11-mer duplexes, the two cis-anti-[BP]-N2-dG adducts assume base-displaced intercalative conformations of the site I type, while the two trans-adducts are characterized by external, site II type, minor groove binding sites. The chirality of the two anti-BPDE enantiomers manifests itself in opposite orientations relative to the modified guanine sites either along the minor groove (trans-adducts) or perpendicular to the helix axis (cis-adducts). In the (+)-cis- and (-)-cis-adducts, the stereochemical difference manifests itself by opposite orientations via ∼180° rotations in a plane perpendicular to the helix axis, rather than along the minor groove as in the two trans-adducts. Space-filling (CPK) models of these four structures are depicted in Figures 11 and 12. All views are into the minor grooves of the duplexes. The exposure of the pyrenyl residues to the aqueous solvent environment is clearly evident in the two minor groove structures (Figure 11). The opposite orientations of the pyrenyl residues relative to the minor grooves are also evident in the two cis-adduct structures (Figure 12). The (+)-trans- (Figure 8), (-)-trans-, and (+)-cis-anti[BP]-N2-dG (Figure 9) adduct conformations in the 11mer/10-mer deletion duplexes II are also of the basedisplaced intercalation type; the structural features of the (+)- and (-)-trans deletion duplexes II are like those of the (-)-cis and (+)-cis full duplexes I, respectively. In the case of the (+)-trans-anti-[BP]-N2-dG 13-mer/9-mer primer/template complex IV, the conformation of the pyrenyl ring system is closer to that of the base-displaced

Invited Review


Figure 11. Space-filling models of the (A) 10(S)-(+)-trans-anti-[BP]-N2-dG and (B) 10(R)-(-)-trans-anti-[BP]-N2-dG adducts in the 11-mer/11-mer duplex I. The views are into the minor groove, normal to the helix axis. The 5′-ends of the modified strands are on the top left. Carcinogen: cyan; modified base and partner: yellow.

Figure 12. Space-filling models of the (A) 10(R)-(+)-cis-anti-[BP]-N2-dG and (B) 10(S)-(-)-cis-anti-[BP]-N2-dG adducts in the 11mer/11-mer duplex I. The views are into the minor grooves, normal to the helix axis. The 5′-ends of the modified strands are on the top left. Carcinogen: cyan; modified base and partner: yellow.

intercalation adducts, although the glycosidic angle χ is in the syn rather than in the anti domain (Table 1). However, when the primer strand is elongated by one base so that a normal cytidine residue is now positioned across from the modified G4* residue (13-mer/10-mer primer/template complex), the pyrenyl residue is again oriented in the 5′-direction of the modified strand3 in

duplex III. It is thus evident that the base sequence context in the complementary strand can exert a powerful effect on the conformations of the anti-[BP]-N2-dG lesions in DNA. The localization of the hydrophobic BP residues in the minor grooves in the trans-anti-[BP]-N2-dG 11-mer/11mer duplexes I comes at the expense of the exposure of


one face of the aromatic pyrenyl residue to the aqueous solvent environment (12, 21). This unfavorable environment for the pyrenyl residue is apparently compensated by the maintenance of the G6:C17 Watson-Crick base paired structure in duplex I. These findings suggest that the maintenance of Watson-Crick base pairing and the stacking of adjacent base pairs are important factors in determining the positioning of the pyrenyl residues in the minor groove in duplexes I and reflect the effects of competing forces. In the two cis-adducts in duplexes I, on the other hand, the pyrenyl residue is localized in solvent-protected hydrophobic intercalation sites where it can participate in base-stacking interactions with neighboring bases on the modified or complementary strands. This is achieved, however, by the disruption of a single base pair and the displacement of the modified guanine residue to allow for insertion of the carcinogen in its place. The different conformations adopted by the stereoisomeric anti-[BP]-N2-dG lesions in the 11-mer/11mer duplexes I appear to result from an optimization of free energies in which these competing effects are balanced against one another. As we discuss in the next section, the sequence context of the modified duplex also plays a key role in determining adduct conformations. 3.3.6. Effects of Flanking Bases. The effects of base sequence are of critical importance in hotspot phenomena observed in mutagenesis experiments with (+)- and (-)anti-BPDE (116, 118-120). The bases flanking the modified base on either the 5′- or the 3′-side can have a profound influence on the conformations of the anti-[BP]N2-dG lesions as suggested by low-resolution spectroscopic data (95, 98, 121, 122). The apparent conformations of anti-[BP]-N2-dG adducts depend significantly on the nature of the flanking base pairs. Flow linear dichroism measurements have shown that synthetic polynucleotides with dA:dT base pairs flanking the BPDE-N2-dG lesions are associated with a significantly greater flexibility than those in which dG:dC base pairs flank the lesion sites (98). Furthermore, the presence of dA:dT base pairs adjacent to the BP-modified guanines gives rise to a local destabilization of the duplexes as indicated by a lowering of the duplex melting points (111, 123). The effect of flanking dA:dT base pairs on increasing the local mobilities of trans-anti-[BP]-N2-dG lesions has been demonstrated by examining the NMR characteristics of these lesions in sequences4 IV (23) and V. In the BP-modified sequence IV studied by Fountain and Krugh (23), the (+)-trans-anti-[BP]-N2-dG lesion is flanked by a dT on the 5′-side and a dC on the 3′-side of the modified strand. As in duplex I with dC’s flanking the modified guanine residue on both sides, in duplex IV the pyrenyl ring system is positioned in the minor groove and is tilted toward the 5′-end of the modified strand; however, line broadening due to chemical exchange effects of protons located in the 5′-d(...-T7-G8*-C9-...)‚d(...-G14-C15-A16-...) portion of duplex IV indicates that a second, minor conformation is also present. Fountain and Krugh (23) concluded that the major and minor forms are in conformational equilibrium with one another, but could not characterize the structure of the minor conformer, although they hypothesized that it might be an intercalative structure. Since the stereochemical characteristics of the (+)-trans-anti-[BP]-N2-dG 4B.

Mao, unpublished results.

Geacintov et al.

lesion are the same in sequences I and IV, the results of Fountain and Krugh provide an important clue to the effects of base sequence on adduct conformations. The conformational equilibrium in IV, since it is not observed in duplex I (12), is attributed to a higher local flexibility associated with the presence of the less stable dA:dT base pair on the 5′-side of the lesion in IV (23). This hypothesis is reasonable in view of the previous observations of the lowering of the apparent persistence lengths of synthetic polynucleotides containing BP-modified guanine lesions flanked by dA:dT base pairs (98). Mao4 studied the effects of flanking dA:dT base pairs, one on each side of the (+)-trans-anti-[BP]-N2-dG lesion, on the adduct conformations in sequence V. Mao was unable to observe sufficiently well resolved NMR resonances for a detailed structural analysis both in water and in D2O, especially on the 5′-side of the lesion site. This observation can also be attributed to the lower stabilities of the flanking dA:dT base pairs in V as compared to those of the flanking dG:dC base pairs in I. According to this hypothesis, in sequence V the flanking dA:dT base pairs give rise to a higher flexibility, and therefore to a broadening of the NMR resonances. The NMR proton resonances for the central d(...T5-G6*T7...)‚d(...A16-C17-A18...) segment in sequence V indicate that there is more flexibility on the 5′-side than on the 3′-side of the lesion; this effect is consistent with an orientation of the pyrenyl residue on the 5′-side of the modified deoxyguanosine residue G6*, as in duplex I. The NMR characteristics of V indicate that several conformers may be in slow equilibrium with one another on the NMR time scale; these results are consistent with the observed formation of small circular molecules of different sizes (77, 88, 99, 110, 121, and 132 base pairs in size) when the 11-mer sequence V with a (+)-transanti-[BP]-N2-dG lesion is ligated using T4 ligase (124). 3.4. Conformations of 5-Methylchrysene Diol Epoxide-N2-Deoxyguanosine Adducts in Duplex I. The effects of methyl substituents on the conformations of PAH diol epoxide-DNA adducts and the possible structural deformations caused by these hydrophobic groups are of great interest. We have studied the conformations of adducts derived from the covalent binding of (-)-anti-5-MeCDE to the exocyclic amino group of the deoxyguanosine residue G6* in duplex I (G6* ) 4(R)-(-)-trans-anti-[MC]-N2-dG). Preliminary experiments with the stereoisomeric 4(S)-(+)-trans-anti-[MC]N2-dG lesion in the 11-mer/11-mer duplex I show that the NMR signals are less well resolved.5 The results on the adduct derived from the (-)-enantiomer have been published (18). The 4(R)-(-)-trans-anti-[MC]-N2-dG adduct (Figure 13) and the 10(R)-(-)-trans-anti-[BP]-N2-dG (Figure 3) are stereochemically similar; these two adducts differ from one another only by one aromatic ring and by one methyl group. It is therefore interesting to compare their structural characteristics in the identical sequence context of duplex I with the lesions situated at G6* in both cases. Both the 10(R)-(-)-trans-anti-[BP]-N2-dG 11-mer/11mer duplex (21) and the 4(R)-(-)-trans-anti-[MC]-N2-dG 11-mer/11-mer duplex (18) manifest single conformations in solution. Like the BP pyrenyl residue, the 5-methylchrysenyl polycyclic ring system is aligned in the minor 5M.

Cosman, unpublished results.

Invited Review

Figure 13. Structures of the two stereoisomeric trans-anti[MC]-N2-dG adducts. The torsion angles R′ and β′ are indicated for the (+)-trans-adduct and are defined in Table 1.

groove of a B-DNA duplex with intact Watson-Crick base pairing (Figure 14). The aromatic chrysenyl residue is tilted toward the 3′-end of the modified strand; again, this orientation is similar to the tilt of the aromatic pyrenyl residue toward the 3′-end of the modified strand in the (-)-trans-anti-[BP]-N2-dG 11-mer/11-mer duplex I. A number of interesting features emerge from an analysis of the structural model shown in Figure 14. The -CH3 group in the (-)-trans-anti-[MC]-N2-dG 11-mer/11mer duplex gives rise to structural perturbations that are not evident in the (-)-trans-anti-[BP]-N2-dG 11-mer/11mer duplex. The methyl group is wedged into the helix between the base pairs G6*:C17 and C7:G16 and is positioned toward the floor of the minor groove, thus minimizing its exposure to the polar solvent (Figure 14). In order to accommodate this alignment, the chrysenyl residue is oriented more edgewise in the minor groove than the pyrenyl residue in the (-)-trans-anti-[BP]-N2dG 11-mer/11-mer duplex. The three methyl protons are characterized by a single resonance (2.45 ppm), which indicates that the methyl group rotates freely. In order to permit the chrysenyl methyl group to rotate freely, the aromatic ring is slightly distorted from planarity (the C4A-C5A-C5B-C11 dihedral angle is -5.7°) and the 5-methyl group is positioned somewhat out of the plane of the chrysenyl ring (the C7-C6-C5-C4 dihedral angle is -9.2°). A similar effect is observed in the crystallographic structure of 5-MeC in which the methyl group is positioned in a slightly widened bay region and is forced out of the plane of the aromatic ring, which itself is approximately planar (48). Because the methyl group is wedged between adjacent base pairs in the (-)-transanti-[MC]-N2-dG 11-mer/11-mer duplex, its proton reso-


nances appear to be upfield-shifted due to ring current effects arising from neighboring base pairs; this conclusion is supported by the observed upfield shift of 1.5 ppm of the MC(H6) proton relative to the chemical shift value of the BP(H12) proton at the analogous position of the MC and BP aromatic ring systems (18). In the energy-minimized structure that is consistent with all of the NOE restraints (Figure 14), the wedgeshaped alignment of the chrysenyl residue is accompanied by (1) a 22° buckling of the modified G6*:C17 base pair, (2) a local widening of the minor groove, (3) a compression of the major groove, and (4) a small unwinding of the helix, manifested by a twist angle of 29.5° between the G6*:C17 and C7:G16 base pairs (in B-DNA in solution the average value of the twist angle is about 35°). These structural distortions are associated with a bend of about 47°, which is more pronounced than in the (-)-trans-anti-[BP]-N2-dG 11-mer/11-mer duplex. We note that bends in DNA duplexes are generally difficult to characterize by NMR methods due to the short-range (45°) from R′ ) 180° are not favored because one Watson-Crick hyrogen bond is lost. Furthermore, adopting a value of β′ in the wrong domain may entail other unfavorable effects such as severe bending or major changes in the shape of the minor groove to alleviate crowding. 4.5.3. Classically Intercalated trans-anti-[BP]-N2dG Adducts? We distinguish two possible types of intercalated trans-anti-[BP]-N2-dG adduct conformations: (1) intercalation without base displacement, and (2) intercalation with base displacement. In (1), an ideal intercalation complex would have the plane of the pyrenyl ring system as close as possible to a parallel orientation relative to the planes of the neighboring DNA bases and the modified guanine base itself. The latter should still be hydrogen-bonded to its partner base in the unmodified strand. It is useful to recall the results of several previous studies that modeled the structures of intercalative [BP]-DNA adducts. Zakrzewska and Pullman (104) used energy minimization with computer graphics modeling to theoretically examine the possible conformations of intercalated structures of the (+)-trans-anti-[BP]-N2-

Invited Review

dG lesions embedded in a DNA duplex. Intercalative (+)anti-[BP]-DNA adduct conformations were generated with the BP inserted into the helix either from the 3′- or the 5′-side, with the latter mode being favored energetically over the former. However, there were large local distortions of the normal DNA structure, because the modified guanine residues were significantly displaced from their normal positions. In another modeling effort, Taylor et al. (105) concluded that the intercalative insertion of the pyrenyl residue on either the 5′- or the 3′-side of the modified guanine residue in duplex DNA required a large local kink of at least 39°. Thus, classically intercalative conformations without significant structural perturbations of the B-DNA duplex structure appear to be unlikely for trans-anti-[BP]-N2-dG adducts, and indeed have not been observed. The experimentally determined intercalation-type structures are all basedisplaced. 4.6. Structural Relationships between Minor Groove and Base-Displaced anti-[BP]-N2-dG Adduct Conformations 4.6.1. Computed trans-anti-[BP]-N2-dG Adduct Conformations: Structural Characteristics. Singh et al. (100) made an extensive search of the torsion angle conformation space with both 10(S)-(+)- and 10(R)-(-)trans-anti-[BP]-N2-dG lesions in the center of the selfcomplementary dodecamer (dG-dC)6‚(dG-dC)6 duplex, studying first small subunits followed by building to the dodecamer level. Three types of minimum energy structures were found for these two trans-anti-[BP]-N2-dG adducts, and two of these are especially relevant to our experimentally determined anti-[BP]-N2-dG 11-mer/11mer duplex I adduct conformations. In order of increasing energy, these two structural types had the following conformational characteristics: Conformers (A): Minor groove conformations with the pyrenyl residues pointing in the 5′-direction of the modified strand for the computed 10(S)-(+)-trans-adduct, and in the 3′-direction for the computed 10(R)-(-)-transadduct conformers, as observed experimentally (12, 21). Conformers (B): Base-displaced intercalated structures in which the modified guanine is rotated out of the duplex and the pyrenyl moiety is inserted, so that the distal aromatic rings are directed toward the minor or major groove in the computed 10(S)-(+)- and 10(R)-(-)-transanti-[BP]-N2-dG adduct conformers, respectively. No classical intercalation structures were found in this conformational search by Singh et al. (100). In the family of experimentally observed adduct conformations of anti[BP]-N2-dG lesions, only the 10(S)-(+)-trans and the 10(R)-(-)-trans stereoisomeric adducts in the full 11-mer/ 11-mer duplexes I prefer the minor groove conformer (A) structure (Table 1). Type (B), base-displaced intercalative structures, have been observed for the two stereoisomeric cis-adducts, and for the same two trans-adducts in the somewhat different sequence context of deletion duplex II. These type (B) conformations include the following: • 10(R)-(+)-cis-adduct in the fully complementary 11mer/11-mer duplex I • 10(S)-(-)-cis-adduct in the fully complementary 11mer/11-mer duplex I • 10(S)-(+)-trans-adduct in the 11-mer/10-mer deletion duplex II • 10(R)-(-)-trans-anti-[BP]-N2-dG lesion in the 11-mer/ 10-mer deletion duplex3 II


• 10(R)-(+)-cis-adduct in the 11-mer/10-mer deletion duplex II In the case of the 10(R)-(+)-cis-adduct, a second, minor conformation was also observed that displayed features of a minor groove structure in that the base pairing at the lesion site was intact (13). However, this minor conformer could not be characterized in detail because of its low abundance.5 4.6.2. Similarity of Torsion Angles r′ and β′ in Minor Groove and Base-Displaced Intercalative Conformers. In spite of the strikingly different conformations, the experimentally determined minor groove and base-displaced intercalative adducts appear to be structurally related because the sets of torsion angles R′ and β′ are similar in value (Table 1). Interestingly, a similar relationship between these two sets of torsion angles is evident in the computed structures (A) and (B) for the 10(S)-(+)- and 10(R)-(-)-trans-anti-[BP]-N2-dG adducts, respectively (100; Table 5). It is evident that, in going from the minor groove structure (A) to the basedisplaced intercalated structure (B), the glycosidic torsion angle χ changes from anti (253-255°) to high anti (317323°). However, angles R′ and β′ are remarkably similar for structures (A) and (B), except in the case of the (-)trans-adduct, where the angle β′ changes from 71° to 131°; these two values, however, are still in domain 1 of β′ though at the opposite ends of the allowed range of β′ values (Figure 20). The structural relationships between the minor groove and base-displaced intercalative structures can be further analyzed by comparing the values of adduct torsion angles in the experimentally based (Table 1) and the computationally predicted models (Table 5). The details are provided in the Supporting Information; it is shown there that the similarities in the torsion angles R′ and β′ are a common element that structurally links adducts that have the same 10(R) or the same 10(S) absolute configurations at the C10 PAH-base linkage sites, regardless of their minor groove or base-displaced intercalative conformations. 4.6.3. Equilibrium between Minor Groove and Base-Displaced Intercalative Conformers. Because of the similarities in R′ and β′ values, it appears that the 10(S)-(+)-trans-anti-[BP]-N2-dG minor groove adduct in duplex I can be rearranged to a base-displaced/intercalation structure like that observed for the same lesion in the 11-mer/10-mer deletion duplex II, and the 10(S)(-)-cis-anti-[BP]-N2-dG adduct in duplex I; this rearrangement can be achieved by maintaining similar values of R′ and β′, and by changing the glycosidic torsion angle χ of the modified nucleoside, or making other small changes within the nearby backbone torsion angles within the B-DNA domain. A similar scenario can be envisioned for rearrangement of the 10(R)-(-)-trans-anti[BP]-N2-dG minor groove conformer in duplex I to a basedisplaced/intercalative conformer like that of the predominant 10(R)-(+)-cis-anti-adduct in the full 11-mer/11mer duplex I, or the 10(R)-(-)-trans-anti-[BP]-N2-dG deletion duplex II. These considerations suggest the concept of interconversion between minor groove and base-displaced/intercalative conformers. This concept can be visualized in terms of a rearrangement of base-displaced/intercalation conformers with 10(R)-(+)-cis stereochemistry, to minor groove conformations like that of the 10(R)-(-)-trans-anti[BP]-N2-dG adduct in which the pyrenyl moiety is directed along the 3′-direction of the modified strand.


Similarly, the oppositely positioned base-displaced/ intercalation structure of the 10(S)-(-)-cis-anti adduct could rearrange to a minor groove conformer with the pyrenyl moiety directed toward the 5′-end of the modified strand, like in the 10(S)-(+)-trans-anti-[BP]-N2-dG adduct (12). Thus, an equilibrium between minor groove and base-displaced/intercalation conformers can be proposed for all four anti-[BP]-N2-dG adducts, as depicted below:

minor groove conformer F R (conformer A) base-displaced/intercalative conformer (1) (conformer B) The left side of the equilibrium represents conformers in which the pyrenyl residue points toward the 5′-end of the modified strand in the 10(S)-(+)-trans- and 10(S)(-)-cis-adduct pairs, and toward the 3′-end in the 10(R)(-)-trans- and 10(R)-(+)-cis-anti-[BP]-N2-dG adduct pairs. The right side of the equilibrium represents conformers with the benzylic ring positioned in the major groove for 10(S)-(+)-trans- and (-)-cis-adducts, and in the minor groove for the 10(R)-(-)-trans- and the (+)-cis-adducts. Such an equilibrium may exist in the case of the 10(R)-(+)-cis-anti-[BP]-N2-dG 11-mer/11-mer duplex I, with conformer B constituting the major form and conformer A the minor one. In the case of the 10(S)-(-)-cis-adduct, conformer B also is dominant, and other minor forms are observed experimentally as well, although their conformations were not established (20). Since only minor groove conformers are observed in the base sequence context of duplex I (12, 21), the equilibrium is heavily in favor of conformers A in the 10(S)-(+)-trans- and 10(R)(-)-trans-anti-[BP]-N2-dG lesions in the full 11-mer/11mer duplexes I, but in the 11-mer/10-mer deletion duplexes II conformer B is heavily favored. On the other hand, in the 10(S)-(+)-trans-anti-[BP]-N2-dG 11-mer/11mer duplex IV, with a dT:dA base pair on the 5′-side of the BP-modified dG* residue, the major conformer is still the normal minor groove structure (A), but a minor conformer, suggested to have an intercalated pyrenyl residue, is also observed (23). Fountain and Krugh (23) proposed that a conformational equilibrium exists between the predominant minor groove conformer and the less abundant intercalative conformer; the latter could be a conformer of type (B), although this remains to be demonstrated. The observation of a single conformer type (A) in the 10(S)-(+)-trans-anti-[BP]-N2-dG adduct in the full 11-mer/11-mer duplex I (12), and a conformational equilibrium for the same lesion in duplex IV (23), suggests that the increased flexibility in the vicinity of the lesion in duplex IV can be attributed to the 5′flanking dA:dT base pair (124). Thus, the likelihood of observing multiple conformers in equilibrium with one another in (1) is greater when dA:dT base pairs (sequence IV) are substituted for dG:dC base pairs (sequence I) adjacent to the modified dG* residues. 4.6.4. The Torsion Angles in the 11-mer/10-mer Deletion Duplexes. Removal of only one base from the complementary strand, the dC base opposite the modified G6* residue in the 10(S)-(+)-trans-anti-[BP]-N2-dG in duplex I, results in a change in adduct conformation from the minor groove conformer (A) in the 11-mer/11-mer duplex I to the base-displaced intercalative conformer of type (B) in the 11-mer/10-mer duplex II. However, the torsion angles R′ and β′ are almost the same in these two duplexes (Table 1)! This conformational difference is

Geacintov et al.

accompanied by a change in the glycosidic angle χ from anti (257°) in duplex I to high anti (301°) in duplex II. Moreover, the orientation of the benzylic ring in this case is, in fact, like that in the 10(S)-(-)-cis-anti-[BP]-N2-dG adduct, i.e., directed toward the major groove. These results further support the notion that base-displaced intercalative conformers can arise in the case of the 10(S)-(+)-trans-anti-[BP]-N2-dG lesions. The equilibrium in (1) is shifted to the left in the full 11-mer/11-mer duplex, and to the right in the absence of the partner base dC opposite the modified guanine residue in the 11mer/10-mer deletion duplex. A similar phenomenon is observed in the 10(R)-(-)-trans-anti-[BP]-N2-dG adducts in the 11-mer/11-mer duplexes I and in the 11-mer/10mer deletion duplexes3 II. In the case of the 10(R)-(+)-cis-anti-[BP]-N2-dG adduct in duplexes I and II, the conformation of the pyrenyl residues is base-displaced/intercalative even though substantial differences in χ and β′ are observed, and conformer (B) dominates in the equilibrium (1) in both cases. 4.7. Base Stacking and Hydrogen Bonding in the Classically Intercalated trans-anti-[BPh]-N6-dA Adducts. In the intercalative 1(R)-(+)- and 1(S)-(-)-transanti-[BPh]-N6-dA adduct 11-mer/11-mer duplexes VI, carcinogen-base stacking and Watson-Crick base pairing appear to be optimized. This is achieved by positioning the polycyclic aromatic BPh residues between two adjacent bases on the complementary partner strands, and by maintaining the torsion angle R′ in the vicinity of 180° (so that the single N6 proton at the modified A6* base can maintain at least some extent of hydrogen bonding with T17 in duplex VI). Just as in the case of the anti-[BP]-N2-dG adducts, the range of β′ values allowed is limited by steric clashes between the BPh and the covalently linked A6* residues (primary steric hindrance effects). In the case of the 1(S)(-)-trans-anti-[BPh]-N6-dA mononucleoside adduct, for the experimentally observed values of R′ and χ (Table 3), the sterically allowed ranges of β′ are between 43° and 84° (domain 1), and 223° and 276° (domain 2), as determined with the facility “bump-check” in INSIGHT 2.0. In the case of the isomeric 1(R)-(+)-trans-anti-[BPh]N6-dA adduct, the steric crowding is much more severe and only values near β′ ≈ 104° are possible for the experimentally observed values of R′ and χ . Primary Steric Hindrance Effects and the 5′- and 3′Intercalation Motifs. The preferred intercalative insertions of the BPh residues on the 3′- or 5′-side of the modified A6* residues in the 1(S)- or 1(R)-adducts, respectively, may be rationalized by viewing the possible relative orientations of the dA and BPh residues at various values of β′ within the permitted ranges, and at the values of χ and R′ in the structural models shown in Figures 16 and 17. These simple modeling studies suggest that, at these fixed values of R′ and χ, intercalation can be achieved successfully only as observed experimentally, and with the observed values of β′ (Table 3). We also examined the effects of varying the angle R′ to determine if it is possible to orient the intercalated BPh residues on sides opposite to those observed, relative to the modified A6* residues in duplex VI. We first note that the torsion angle R′ ) 154° for the 1(R)-(+)-trans11-mer/11-mer adducts (Table 3) corresponds to a tilt of the N6-C1 dA-BPh bond into the 3′-direction. Furthermore, this bond is tilted out of the plane that defines perfect hydrogen bonding between the A6*:T17 comple

Invited Review

mentary bases with R′ ) 180° (Figures 16 and 17). In the 1(S)-(-)-trans-11-mer/11-mer adduct, R′ ) 229°, which corresponds to a tilt of this N6-C1 dA-BPh bond into the 5′-direction. These two R′ angles (Table 3) in the 1(R)-(+)-trans- and 1(S)-(-)-trans-anti-[BP]-N6-dA adducts, respectively, help to achieve the experimentally observed opposite intercalative positions of the BPh ring systems relative to the modified deoxyadenosyl residue A6* (Figures 16 and 17). The question arises why these particular 5′ or 3′ orientations are favored for the 1(R)- and 1(S)-adducts, respectively. We carried out simple modeling studies in which the values of R’ of the 1(R)-adduct were switched from the observed 154° to a hypothetical value of 229°, while R′ of the 1(S)-adduct was switched from the observed 229° to a hypothetical value of 154°. These studies (discussed in detail in the Supporting Information) suggest that adducts with BPh insertions on opposite sides to those observed experimentally (Figures 16 and 17) are not favored because of diminished carcinogen-base stacking interactions of the aromatic BPh residues with bases on the complementary strand. Finally, we observed that altering the torsion angle R′ to a value of 180° (perfect hydrogen bonding between A6* and T17 in duplex VI) would lead to orientations of the BPh ring system (at all allowed values of β′) that are also not favorable for efficient carcinogen-base stacking interactions. This effect accounts for the substantial deviations of the values of R′ from 180° which, of course, leads to a weakening of one of the hydrogen bonds in the A6*:T17 base pairs. However, this deviation leads to a more favorable alignment of the planar portion of the BPh residue for stacking between the dG and dT bases of the complementary strand in duplex VI. Twist and Buckle. Other interesting implications of the computed model structures of the two trans-anti[BPh]-N6-dA 11-mer/11-mer adducts in the duplexes VI (Figures 16 and 17) are the orientations adopted by the A6*:T17 base pairs, which optimize the stacking interactions between two of the three aromatic rings of the BPh residue and the adjacent dT and dG moieties on the complementary strand. In order to accommodate and align the BPh residue on the 5′-side for optimal base stacking, the A6*:T17 base pair is buckled toward the 3′-direction (-29.5°, Table 3) and propeller-twisted (by +15.0°) in the 1(R)-(+)-trans-anti-adduct. In the isomeric 1(S)-(-)-trans-anti-adduct, the buckle is in the opposite, 5′-direction (32.1°) in order to accommodate the BPh residue on the 3′-side of the modified A6* residue; the A6*:T17 base pair is propeller-twisted in the opposite direction (-22.7°) in order to align the somewhat twisted plane of the aromatic BPh residue nearly parallel to dG and dT base moieties on the complementary strand for optimal base stacking. Furthermore, in order to maximize base-stacking interactions, the BPh dihedral angles C4B-C6B-C8B-C12, which describe the deviation from planarity of the BPh aromatic region, are distorted from ideal values of 0° in opposite directions in the 1(R)-(+)-trans-anti- and the 1(S)-(-)-trans-anti-adducts (+18.1° and -15.8°, respectively). Finally, our analysis of the permitted values of the torsion angle β′ for the experimentally observed values of R′ (Table 3) have shown that the steric crowding in the 1(R)-(+)-trans-anti-[BPh]-N6-dA at the mononucleoside adduct level is considerably more severe than it is in the 1(S)-(-)-trans-anti-adduct (see above). This may account for the unusual benzylic ring conformation


(the H3 and H4 protons are quasi-diequatorial, Table 2) adopted by the BPh residue in the 1(R)-(+)-trans-antiadduct (14, 17, 125). This combination of changes in the local DNA conformation and the planarity of the aromatic portion of the BPh residue apparently permit the minimization of the overall energy in order to generate the nearly classical intercalative adduct conformations depicted in Figures 16-18. 4.8. The Importance of Secondary Steric Hindrance Effects. The influence of steric hindrance effects between the BP residues and nearby DNA groups and their possible effects on the conformations of [BP]-N6dA and [BP]-N2-dG adducts in duplex DNA were discussed by Schurter et al. (28). The interactions between the three OH groups at the chiral C7, C8, and C9 carbon centers with neighboring DNA groups depend on the two possible benzylic ring conformations (e.g., Table 2) and the positioning of the benzylic ring in either the major or the minor groove. These interactions fall within the secondary steric hindrance category as defined in section 4.5.1. A number of the observed conformations of adducts with (R)- and (S)-adduct stereochemistry at C10 of the BPDE residues were interpreted in terms of steric clashes of the benzylic ring substituents with nearby DNA moieties. The important point was made by Schurter et al. that secondary steric clashes are more acute in the sterically crowded minor groove than in the major groove (28). However, the primary steric hindrance effects appear to govern the allowed conformational domains which define the relative orientations of the PAH and covalently linked purine moieties. The evidence based on the variety of structural motifs discovered until now suggests that unfavorable secondary steric clashes are partially alleviated by the inherent flexibility of the DNA, thus giving rise to the observed base sequence contextdependent diversity of PAH diol epoxide-DNA adduct conformations (Table 4). The secondary steric hindrance effects no doubt contribute to the enthalpic and entropic balance which energetically favors either external (minor or major groove) or intercalated adduct conformations in the equilibrium defined in section 4.6.3.

5. Adduct Conformations: Biological and Biochemical Implications The elucidation of the three-dimensional structures of PAH diol epoxide-DNA adducts and correlation of their structural features with their biochemical characteristics and biological end points have long been important goals in the fields of mutagenesis and chemical carcinogenesis. The availability of site-specifically modified PAH diol epoxide-oligonucleotide adducts within the last few years, and the elucidation of their solution structures by NMR techniques, has brought these goals into the realm of feasibility. However, structure-biological function correlation studies using site-specifically modified PAH diol epoxide-DNA adducts are now only in their beginning stages. Here we provide a brief perspective on this growing field of research. 5.1. Detection and Identification of [BP]-DNA Adducts in Vitro and in Vivo Correlations with Previous Low-Resolution Spectroscopic Studies. When racemic anti-BPDE reacts with DNA either in vitro or in vivo, a variety of chemically and stereochemically distinct covalent adducts are formed with the dG and dA residues (73). Previous spectroscopic studies have clearly shown that a multiplicity of adduct


conformations are observable (85, 91, 92-98). The combined NMR (Table 4) and spectroscopic studies (99) with stereochemically defined site-specific BPDE-oligodeoxyribonucleotide adduct duplexes have shown that anti-[BP]-N2-dG adducts with cis stereochemistry at the linkage sites, and all [BP]-N6-dA lesions, are characterized by site I-type intercalative conformations. The trans-anti-[BP]-N2-dG adducts, on the other hand, are usually characterized mainly by site II external adduct conformations in normal duplexes. When (+)-anti-BPDE is reacted with DNA in vitro, the linear dichroism characteristics indicate that the BPDE-DNA adducts are predominantly of the site II-type (91, 92, 98); this is understandable because the dominant reaction products of (+)-anti-BPDE with native DNA are the (+)-trans-anti[BP]-N2-dG lesions (72, 73) that are positioned predominantly externally, in the minor groove of DNA in a normal duplex (12, 21, 23). When (-)-anti-BPDE reacts with native DNA, significantly greater proportions of cisanti-[BP]-N2-dG and anti-[BP]-N6-dA adducts are formed, all of which lead to site I intercalative adduct conformations (13, 20, 22, 28, 30-32); indeed, the linear dichroism spectra are consistent with greater proportions of intercalative site I-type adducts than in the case of adducts derived from the binding of (+)-anti-BPDE to native DNA (91, 92). Thus, spectroscopic methods can provide quick, qualitative information on the site I/site II adduct distributions in native DNA exposed to BPDE stereoisomers. Identifying Different BPDE-DNA Adduct Conformers in Vivo. While the NMR method provides highly detailed structural information, it is not sufficiently sensitive for detecting or identifying PAH adducts in biological samples. The low-resolution UV absorption (99, 111) and fluorescence (99) methods are quite sensitive and can crudely distinguish between minor groove trans- and intercalated cis-anti-[BP]-N2-dG adducts in double-stranded oligonucleotides (95, 96, 99). The absorption and fluorescence emission spectra of the predominantly base-displaced intercalative cis-adducts are red-shifted by 5-10 nm with respect to those of the minor groove trans-anti-[BP]-N2dG adducts in duplexes I. Thus, the low-resolution, but high-sensitivity spectroscopic methods, especially fluorescence techniques (96), can be used to detect adducts of preferred intercalative or minor groove conformations even in cellular DNA (131, 132). Low-temperature fluorescence techniques are particularly suitable for detecting low levels of adduct concentrations because the efficient fluorescence quenching processes that greatly diminish the yield of [BP]-N2-dG-adducts in native DNA at ambient temperatures are suppressed at temperatures below about 100 K (131, 133). Low-temperature fluorescence line narrowing techniques developed by Jankowiak and Small (134) are extraordinarily sensitive and can distinguish between the quasi-intercalative cis-adducts and the externally bound trans-adducts (96). These methods have been used to follow the fates of these structurally distinct anti-[BP]-N2-dG adducts in mouse skin DNA (132). Adduct Stereochemistry and Detection by Immunological Techniques. High-sensitivity immunologic methods for detecting trace amounts of PAH diol epoxide-DNA adducts have been developed for determining human exposure to PAH compounds in the environment (see, for example, refs 135 and 136), and for quantitatively assessing the formation and repair of adducts in cellular DNA (137, 138). Venkatachalam and Wani (139) found that polyclonal and monoclonal antibodies developed

Geacintov et al.

against racemic anti-BPDE-modified DNA exhibit remarkable stereospecific selectivities in the recognition of site-specific (+)- and (-)-trans-anti-[BP]-N2-dG adducts in single- and double-stranded sequence I. While a polyclonal antibody (Pab BP1) recognized the (+)-transadduct with a 40-fold preference over the (-)-transadduct, the monoclonal antibody (Mab 5D2) displayed an overwhelming preference for the (-)-trans-adduct. These differences are attributed to the accidental selection of one or the other stereoisomeric adduct in the development of these antibodies with antigens derived from the covalent binding of racemic anti-BPDE to DNA (139). Considering these results in relation to duplex adduct conformations in solution, it is evident that antibodies can distinguish the subtle differences in adduct orientations within the minor groove in the (-)-trans- and (+)trans-anti-[BP]-N2-dG adducts in duplexes I; this assumes that the orientations of the pyrenyl residues toward the 3′- and 5′-directions of the modified strands, respectively, are maintained in the presence of the antibody. In general, antisera derived from racemic antiBPDE-modified DNA antigens react significantly less with cis-anti-[BP]-N2-dG 11-mer/11-mer adducts than with their trans isomers (140). These results indicate that the stereochemical adduct configurations are an important variable in immunologic assays. The differences in the conformations of stereoisomeric PAH-DNA adduct conformation must be taken into account in risk assessment biomonitoring studies based on immunologic approaches (139). Some of the stereoisomeric adduct forms that are not recognized by antisera could be as harmful biologically as the ones that are. 5.2. Stereoselective Digestion of [BP]-DNA Adducts by Exonucleases. Exonucleases are enzymes that are of great importance in a variety of cellular processes and are also routinely used in the characterization of PAH diol epoxide-DNA adducts. The resistance of various PAH-DNA adducts to digestion by exonucleases was reported some time ago (141-143). For example, Cheh et al. (142) noted a stereoselective resistance of stereochemically different PAH diol epoxideDNA adducts to digestion by phosphodiesterases I and II. This effect was studied in detail by Mao et al. (144) using (+)- and (-)-trans-anti-[BP]dG lesions in sitespecific single-stranded oligonucleotides. In both stereoisomeric adducts, enzyme digestion-resistant fragments were identified; the exonucleases snake venom phosphodiesterase I and spleen phosphodiesterase II stalled one base before the BPDE-modified guanines on the 3′side or the 5′-side of the lesions, respectively. These results suggest that the pyrenyl residues are tilted toward the 5′-side of the modified dG in the (+)-transanti-[BP]-N2-dG adducts (10(S)-adduct stereochemistry), and toward the 3′-side in the case of the 10(R)-(-)-transanti-[BP]-N2-dG adducts (144). A very interesting implication of these results is that the tilt of the pyrenyl residues relative to the modified guanosine residue and the 5′- or 3′-ends of the modified strands can be similar in single-stranded (144) and in double-stranded oligonucleotides (12, 21; Table 4). A stereoselective resistance to digestion by phosphodiesterase I is also exhibited by the 4(R)-(+)- and 4(S)(-)-anti-[MC]-N2-dG adducts in single-stranded oligonucleotides (145); the digestion patterns are consistent with orientations of the MC aromatic ring system on the 5′-side of the modified dG residue in the case of the (+)-, and on the 3′-side in the case of the (-)-anti-[MC]-N2-

Invited Review

dG adducts. Thus, the orientation of the polycyclic MC residue in (-)-anti-[MC]-N2-dG adducts appears to be similar in the single-stranded (145) and in the doublestranded (18) oligonucleotides, just as in the case of the stereoisomeric (-)-trans-anti-[BP]-N2-dG adducts (144); however, the NMR solution structure of the (+)-anti[MC]-N2-dG adducts has not yet been established, and thus analogous comparisons of adduct orientations cannot be made in this case. Similar correlations between stereochemistry-dependent resistance to digestion by exonucleases of the 10(R)(+)- and the 10(S)-(-)-trans-anti-[BPh]-N6-dA lesions (125), and adduct conformations in duplexes VI determined by NMR techniques (14, 17), have also been reported. The differences in the rates of digestion by phosphodiesterases I and II suggest that the BPh residues are oriented on the 5′-side of the modified dA* residue in the 1(R)-(+)-trans-anti-[BPh]-N6-dA, and on the 3′-side in the 1(S)-(-)-trans-anti-[BPh]-N6-dA adducts in single-stranded oligonucleotides (125). Again, the tilts of the BPh polycyclic aromatic residues relative to the modified dA residues appear to be similar in the singlestranded and the double-stranded oligonucleotides. The influence of stereochemistry on resistance to digestion by phosphodiesterases I and II of six stereoisomeric [BP]-N6-dA lesions in site-specifically modified single-stranded oligonucleotides was investigated in detail by Chary and Lloyd (146). The stereoselective digestion patterns were consistent with 3′-orientations in the case of the 10(R)-adducts (e.g., the (-)-trans- and (+)-cis-anti-[BP]-N6-dA lesions), and 5′-orientations in the case of the 10(S)-adducts (e.g., the (+)-trans-anti-[BP]N6-dA adduct). However, these orientations are opposite to those deduced by NMR methods for BPDE-N6-dA adducts in various duplexes and in other sequence contexts (Table 4). Therefore, these series of adducts do not follow the pattern that indicates that the tilts of the PAH residues relative to the modified bases are similar in single-stranded and in double-stranded oligonucleotides (125, 144, 145). These results suggest either that adduct tilts are not always similar in single- and doublestranded oligonucleotides or that exonuclease digestion patterns may not always be indicative of adduct orientations in single-stranded DNA (146), possibly because of base sequence-dependent effects. Finally, we note that the orientations of the covalently bound PAH moieties relative to the modified bases inferred from stereoselective resistance to enzyme digestion may not necessarily reflect the adduct orientations at single strand-double strand junctions relevant to DNA replication. For example, the conformation of the (+)-trans-anti-[BP]-N2-dG adduct at a primer/template junction is different from that found in duplexes (Table 4) or in single-stranded DNA (144). In summary, pairs of chiral enantiomers of PAH diol epoxides give rise to covalent DNA adducts with either (R) or (S) absolute configurations at the PAH-nucleoside residue linkage sites. These stereochemically distinct lesions are processed at different rates by phosphodiesterases I and II, in some cases stalling on opposite sides of the lesions (144). These results provide one clear example of how a pair of enantiomeric PAH diol epoxides, after binding to DNA, can give rise to significantly different enzymatic reaction characteristics. 5.3. Transcription. The effects of the four stereoisomeric anti-[BP]-N2-dG lesions (Figure 3) on transcription catalyzed by T7 RNA polymerase was investigated


by Choi et al. (147). The BP lesions were site-specifically placed at site +16 following the promoter initiation site, in a 58-mer duplex. The adducts clearly inhibited, but did not entirely block RNA synthesis. While differing amounts of full-length transcripts were formed in all cases, a series of truncated transcripts was also observed. This suggests that the polymerase stalls when it encounters a lesion, either releasing an incomplete transcript or eventually bypassing the adduct, which results in a full-length, normal transcript (148). The order of inhibition of RNA synthesis by the [BP]-N2-dG adducts was (+)-trans > (-)-trans > (+)-cis > (-)-cis. Interestingly, the first three lesions stall elongation beginning at the modified guanine residue; the (-)-cis adduct, on the other hand, inhibited transcription at the site three bases prior to the actual adduct. There is a clear effect of adduct stereochemistry on transcription, which may have implications for adduct stereochemistry-dependent transcription-coupled repair processes. However, because of a limited data base, and because the actual adduct conformations may well be different in transcription bubbles than in duplexes, the specific influence of adduct orientations on transcription is difficult to evaluate at this time. 5.4. DNA Replication and Influence of Adduct Stereochemistry. The differences in the orientations of bulky stereoisomeric PAH lesions positioned in template strands in the immediate vicinity of the 3′-hydroxyl end of the growing primer strand can strongly influence DNA replication. The effects of site-specifically placed and stereochemically defined PAH diol epoxide-DNA lesions on DNA synthesis in vitro (115, 117, 146, 149155) and in vivo (152, 156-158) have been studied. Hruszkyewicz et al. (150) reported that both (+)-transanti- and (+)-cis-anti-[BP]-N2-dG lesions (Figure 3) in a 5′-d(...T-G*-T...) sequence context inhibit primer extension catalyzed by Sequenase 2.0 or human polymerase R, but that the cis-adducts were significantly more inhibitory than the trans-adducts (the star designates the modified base). Shibutani et al. (117) investigated the efficiencies of primer extension by Escherichia coli Pol I (Klenow fragment) of all four stereoisomeric anti-[BP]N2-dG lesions (Figure 3) in 5′-d(...C-G*-C...) and 5′-d(...TG*-C...) sequence contexts. The dominant mutations involve the insertion of dA opposite the modified guanines, and one- and two-base deletions. Li (115) studied the Michaelis-Menten primer extension kinetics of insertion of single nucleotides, one at a time, in the vicinity of the (+)-trans- and (-)-trans-anti[BP]-N2-dG lesions in the 13-mer template sequence III (Figure 4) with complementary primer strands 8, 9, or 10 bases long; the primer extension reactions were catalyzed by the Klenow fragment of Pol I (exonucleasefree). The Michaelis-Menten parameter Km is about 400 times larger for one-base primer extension opposite the lesion G4* than in the case of a 13-mer template strand opposite the unmodified G4. In single-base primer extension reactions using either the 5′- or 3′-flanking C3 or C5 bases as templates, Km is only 4-7 times larger than in the unmodified 13-mer template strand. With G4* ) 10(R)-(-)-trans-anti-[BP]-N2-dG, the MichaelisMenten parameter Vmax for addition of the natural nucleotides to the primer strand opposite the 5′- and 3′flanking bases and opposite G4* was uniformly lower than in the case of the isomeric (+)-trans-adduct (G4* ) 10(S)-(+)-trans-anti-[BP]-N2-dG). The uniformly lower Vmax in the case of the (-)-trans-anti-[BP]-N2-dG lesion


is consistent with the structural features of this primer/ template complex determined by NMR methods in the absence of the enzyme; the (-)-trans-anti-[BP]-N2-dG lesion in sequence III causes a significant disturbance over at least three base pairs flanking the modified G4* residue on the 3′-side (19). This suggests that the pyrenyl residue may be oriented toward the 3′-end of the modified template strand, thus significantly disturbing the template/primer complex structure at all positions in the immediate vicinity of the lesion. In contrast, the same three base pairs flanking the 10(S)-(+)-trans-anti-[BP]N2-dG lesion on the 3′-side of the template strand exhibit almost ideal Watson-Crick base pairing, and a minimal disturbance of the template/primer strand on the 3′-side of the lesion. This difference in the integrity of the template/primer strands may thus account for the uniformly lower kinetic parameters Vmax in the primer extension kinetic experiments using the 13-mer strand (sequence III) with G4* ) (-)-trans-anti-[BP]-N2-dG. The dependence of DNA polymerase-catalyzed primer extension in vitro on adduct stereochemistry has been studied more extensively in the case of [BP]-N6-dA than [BP]-N2-dG lesions. Christner et al. (151) employed several different polymerases, including the Klenow fragment of Pol I (exo-), to study in vitro primer extension in two sequence contexts: 5′-d(...T-A*-G...) and 5′-d(...GA*-T...), with A* ) (+)- or (-)-trans-anti- or (+)- or (-)trans-syn-[BP]-N6-dA. Each adduct was a block to primer extension. While the efficiencies of primer extension depended on the polymerase and sequence context, blockage of primer extension was more a function of the C10 (R) or (S) absolute configurations of the adducts; the configurations of the OH groups at the other three chiral carbon atoms appear to be less important. In general, adducts with (S) stereochemistry blocked nucleotide insertion opposite A* more efficiently than those with (R) stereochemistry. Similar patterns of blockage of polymerases were observed by Lloyd and co-workers (152154), who studied primer extension using six different polymerases and six different stereoisomeric [BP]-N6-dA adducts at position 2 in the N-ras codon 61 5′-d(...C-A*A...). Termination of DNA synthesis 3′ to the lesion and termination opposite the BP-modified adenine residues were the dominant effects observed in all cases (153). A striking effect of the absolute (R) or (S) configuration at the C10 atom of covalently bound BPDE residues was also noted (152, 153). In particular, primer extension assays with the Klenow fragment showed that the three [BP]N6-dA adducts with 10(R) stereochemistry strongly blocked DNA synthesis at the base flanking the lesion on the 3′side; in the case of the three 10(S)-adducts, on the other hand, primer extension was blocked at the base flanking the lesion on the 5′-side (152). Similar patterns of replication blockage were observed with HIV-1 reverse transcriptase (146). Li (115) studied the effects of the 1(R)-(+)- and the 1(S)(-)-trans-anti-[BPh]-N6-dA lesions in the sequence context 5′-d(...C-A*-C...) on the Michaelis-Menten polymerase kinetic parameters for single nucleotide addition to the primer opposite the 3′-flanking base, opposite the 5′-flanking base, or opposite the lesion. Consistent with results obtained with [BP]-N6-dA lesions (146, 150-153), primer extension on the 3′-side of the lesion was more facile for the 1(R)- than for the 1(S)-[BPh]-N6-dA adduct. With the BPh-modified adenine residue as the template, the overall efficiency of primer extension was also significantly lower in the case of the 1(S)- than the 1(R)-

Geacintov et al.

[BPh]-N6-dA adduct. In double-stranded DNA, adducts with R or S stereochemistry at the PAH-N6-dA linkage site are characterized by intercalative insertions of the covalently bound PAH residues on the 5′-side or 3′-side, respectively, of the modified dA residues (Table 4). The effects of PAH-N6-dA adduct stereochemistry on DNA replication can be rationalized if it is assumed that the orientations of the PAH lesions, especially in the case of the (S)-adducts, are similar in double-stranded DNA and at the primer-template junctions. In adducts with (S) stereochemistry, the BP or BPh residues are oriented on the 3′-sides of the modified adenine residues at the template/primer junction, as they are in duplexes (Table 4). In that case, as in the (-)-trans-anti-[BP]-N2-dG adducts in the primer/template sequence III (19), the double-stranded region adjacent to, and on the 3′-side of the PAH-N6-dA lesion may be disordered; such a disturbance in the local DNA structure should lead to a lower DNA synthesis efficiency, as observed experimentally (115, 146, 151-153). The (R) and (S) absolute configurations at adduct linkage sites also play a role in the in vitro replication of adenine N6 adducts of styrene oxide situated in the major grooves within codons 60-62 of the human N-ras gene, catalyzed by various prokaryotic and eukaryotic polymerases (154). In the (R)-R-[SO]-N6-dA adducts, the phenyl rings are situated on the 5′-side of the adducted adenine residue (33), while in the (S)-R-[SO]-N6-dA they are positioned on the 3′-side (34). The (R)-adducts were bypassed by all of the polymerases assayed, while translesional bypass was poor for the (S)-adduct in the case of some, though not all, polymerases (154). Interestingly, the lesions are effectively bypassed by HIV-1 reverse transcriptase, although prominent, polymerase-specific stall sites are observed 3-5 nucleotides on the 5′-side of R-[SO]-N6-dA lesions (155). Overall, with the noted exception of HIV-1 reverse transcriptase, there is a greater tendency of the 3′-oriented (S)- than the 5′oriented (R)-adducts to hinder primer extension; these characteristics of R-[SO]-N6-dA adducts are thus similar to those observed with trans-anti-[BP]-N2-dG (115) and [BP]-N6-dA lesions (146, 151-153). The amount of data available from site-directed mutagenesis experiments in vivo is still limited at the present time (152, 156-158). It is clear, however, that the mutational frequencies and specificities are a function of the type of adduct, its stereochemical configuration, sequence context, and the cell system in which the mutagenic activities of the lesions are expressed. It is evident that a greater data base will be needed to correlate the stereochemical features of individual PAHDNA lesions with their mutagenic potentials in vivo. Furthermore, possible stereochemical effects on the differential rates of removal of these lesions by cellular repair systems will need to be considered. 5.5. Stereochemical Effects on Excision of anti[BP]-N2-dG Lesions by the UvrABC Nuclease System. The mutagenic potential of a particular PAHmodified DNA base is expressed only if this lesion escapes repair before replication. The interactions of repair proteins with site-specific and stereochemically defined lesions are therefore of great interest. Zou et al. (159) ligated the 10(S)-(+)- and 10(R)-(-)-trans- and the 10(R)-(+)- and 10(S)-(-)-cis-anti-[BP]-N2-dG 11-mer/11-mer duplexes I to two other oligonucleotides to generate a 50mer duplex containing a single, site-specifically placed and stereochemically and configurationally defined le-

Invited Review

sion. The interactions of these 50-mer duplexes with the E. coli UvrABC nuclease system were then investigated. This enzyme produces bimodal incisions at the 8th phosphodiester bond on the 5′-side of the modified guanine residue in all four cases, but the patterns of incision on the 3′-side depend on the stereochemistry of the adducts and occur at the 5th, 6th, and 7th phosphodiester bond (P5, P6, and P7, respectively). In the (+)-trans-adduct, incision at P5 predominates, while in the case of the (-)-trans-adduct, the incision probability is greater at P6 than at P5; in both cases, minor extents of incision are observed at P7. This difference in the incision patterns seems to be correlated with orientation of the bulky pyrenyl residues. The pyrenyl residue is tilted toward the 3′-end of the modified strand (21), and the highest probability of incision is shifted by one nucleotide to the 3′-side (P6) in the (-)-trans-adducts. In the (+)-trans-adducts with an opposite tilt of the pyrenyl residue toward the 5′-side (12), incision at P5 is favored by a factor of ∼7 with respect to incision at P6. There are no marked differences in the relative incision rates at these two sites in the case of the two cis-adducts, which is consistent with the rather similar adduct orientations with respect to the 5′f3′ strand orientation (13, 20). The overall incision rates are arranged in the order (+)-cis- > (+)-trans- g (-)-cis- . (-)-trans. Thus, adducts arising from (+)-anti-BPDE are more easily excised than adducts arising from the (-)-enantiomer. The enzyme seems to recognize and excise the base-displaced intercalated cis-adducts more readily than the minor groove trans-adducts. The difference in excision rates observed with the (+)-trans- and the (-)-trans-adducts may be due to the observed prominent bend observed only in the (+)-trans-[BP]-N2-dG adduct case by gel electrophoresis techniques (11, 160). Recent detailed thermodynamic studies have shown that the pyrenyl moiety in (+)-trans-anti-[BP]-N2-dG 11-mer/11-mer duplex I is significantly more exposed to the aqueous solvent than in the (-)-trans-anti-[BP]-N2-dG 11-mer/11-mer duplex I (161), even though the pyrenyl residue is positioned in the minor groove in both cases (12, 21); this effect was also related to the greater extent of bending of the (+)trans-anti-[BP]-N2-dG 11-mer/11-mer duplex I. Zou et al. (159) suggest that the formation of the UvrB preincision complex promotes local bending; thus adducts with (+)-trans-lesions which are already characterized by greater degrees of bending than the (-)-trans-adducts could promote the formation of the UvrB-DNA preincision complex. Finally, UvrC binds to this preincision complex and induces an allosteric change in UvrB, activating a nuclease that incises DNA on the 3′-side of the carcinogen-modified base. Since the pyrenyl residue in the (-)-trans-adducts is situated also on the 3′-side of the damaged base, it is reasonable to assume that it can sterically interfere with this allosteric change or subsequent nuclease activity. The differences in incision rates between the (+)-trans- and (-)-trans-adducts thus can be rationalized in terms of the different conformations in these two stereochemically related adducts. The results presented by Zou et al. suggest that the UvrABC nuclease system recognizes the overall conformational character of the damaged DNA duplexes, rather than recognizing solely the damaged base, as has been discussed by Van Houten (162). Thus, further experiments with site-specifically modified and stereochemically defined PAH diol epoxide-guanine and -adenine lesions of defined conformations in DNA duplexes should greatly


enhance our understanding of the mechanistic details of the interactions of these lesions with DNA repair proteins. 5.6. Slipped Frameshift Intermediates during DNA Replication. Our NMR results on the solution structures of the deletion duplexes (+)-trans- and (+)cis-anti-[BP]-N2-dG 11-mer/10-mer deletion duplexes II (Figure 4), with complementary strands missing a single base opposite the lesion, exhibit base-displaced intercalative adduct structures. Even though these structures have 10(S) and 10(R) absolute configurations at the [BP]-DNA linkage site, they do share the common feature of base-displaced intercalation. The remarkable influence of the intercalated bulky polycyclic aromatic hydrocarbon residues on stabilizing the 11-mer/10-mer duplexes is demonstrated by increases in the duplex melting points from 24 °C (unmodified deletion duplex) to 30 or 47 °C for the (+)-trans- and (+)-cis-adduct deletion duplexes II, respectively (Table 1). Thus, stalling of the polymerases at the bulky adducts, a process that has been amply documented (112-115, 117, 146, 151-155), should allow for the formation of misaligned primer-template intermediates during DNA replication (112-114). The conformational features of the two deletion duplexes (15, 16) provide key insights into the stabilizing properties of such slipped frameshift structures. The 11-mer/10-mer duplexes II represent the structures that might occur in primer/template segments in which the primer has been extended by five bases beyond the lesion and has suffered a -1 deletion mutation in the process. The PAH-base stacking interactions which contribute to the stabilities of the deletion duplexes are, no doubt, dependent on the base sequence of the complementary strand. Thus, the exploration of effects of base sequence on the structures, stabilities, and probabilities of formation of stable frameshift intermediates with PAH lesions of different stereochemical characteristics would be very interesting. The possibility of correlating experimental observations of this type with PAH-DNA lesions of known conformations should provide rich opportunities for establishing structure-activity correlations, and for reaching a better understanding of base sequence effects in mutagenesis.

6. Summary and Future Outlook The NMR structural studies have elucidated a variety of strikingly diverse conformational themes among adducts of polycyclic aromatic compounds with DNA. Three basic types of conformers have been discovered for PAHDNA adducts (Table 4, Figures 11, 12, and 18): (1) minor groove external binding sites, (2) base-displaced intercalative structures, and (3) intercalative insertions of the bulky PAH residues into the helix without base displacement (modified classical intercalation). An additional conformational motif, a major groove adduct structure in the case of R-(N6-adenyl)styrene oxide adducts, has been observed (Table 4). The opposite orientations relative to the modified bases of chemically identical adduct pairs derived from chiral pairs of diol epoxide stereoisomers are noteworthy. Remarkably, this theme of opposite orientations persists regardless of (1) the conformer type (minor groove, base-displaced/intercalation, classical intercalation), or (2) the nature of the polycyclic aromatic parent compound (5-MeC, BPh, BP, R-[SO]) and adduct site (guanine or adenine group, see Table 4). The conformational theme of base-displaced


intercalation, observed in the cases of the cis-anti-[BP]N2-dG adducts, is also found for adducts derived from polycyclic aromatic amines. The possibility that the minor groove and base-displaced intercalated [BP]-N2dG adducts may exist in equilibrium with one another is proposed, and the structural basis and experimental evidence for such equilibria are discussed in detail. In the aromatic amine series, base-displaced/intercalation structures have been observed for N-acetyl-2-aminofluorene (39), 2-aminofluorene (40-42, 44, 45), and 1-aminopyrene (46), and an equilibrium between such states was also observed in the 2-aminofluorene case (40-42). An equilibrium is also indicated in the solution structure of a 4-aminobiphenyl adduct (43). We hypothesize that such equilibria may prevail in other adducts derived from polycyclic aromatic hydrocarbons and amines, as well. The concept of an equilibrium between inserted and external conformers had also been foreshadowed in early computations and was coupled with the suggestion that inserted conformers might be mutagenic (163-165), an idea that has since been advanced also by Eckel and Krugh (41, 42) on the basis of their experimental work on 2-aminofluorene. A number of critical objectives and important issues remain to be addressed. Among these are the effects of base sequence context on adduct conformations. Several existing results point to the importance of the flanking bases on adduct conformations, and this issue needs to be explored in greater detail. The effects of flanking bases on the types of structural deformations caused by PAH lesions at single strand-double strand junctions, relevant to biochemical events during replication, appear to be particularly important for gaining insight into the molecular events underlying hotspot phenomena in mutagenesis. Ultimately, the complex problem of the role of the protein or enzyme environment in PAH-DNA adduct conformation and function will need to be addressed. There is no doubt that this issue is one of the most important goals for achieving a better understanding of structure-biological function relationships.

Acknowledgment. We are grateful to Dr. Bing Mao for preparing the stereoview and color figures, and to Xiaoming Xie for his assistance in preparing some of the other figures. This research was supported by NIH Grant CA-46533 to D.J.P., by NIH CA-20851 and DOE Grant DE-FGO2-88ER60405 to N.E.G., by NIH Grant CA-28038, NIH Grant RR-06458, and DOE Grant DEFGO2-90ER60931 to S.B., by DOE Contract DE-ACO5960R22464 with Lockheed Martin Energy Research and DOE-OHER Field Work Proposal ERKP931 to B.E.H., and by NIH Grant CA 17613 to S.A. Computations were carried out at NERSC (DOE) and SDSC (NSF). Coordinates of all structures solved by the present authors are available from Suse Broyde, email address [email protected].

Supporting Information Available: Supporting information to the indicated section in the main text is available (3 pages). Ordering information can be found on any current masthead.

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