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This paper reports on the solution structure of the (+)-cis-anti-[BP]dG adduct positioned opposite dC in a DNA oligomer duplex which provides the first experimentally based solution structure of an intercalative complex of a polycyclic aromatic hydrocarbon covalently bound to the N2 of deoxyguanosine. The combined NMR-energy minimization computation studies were undertaken on the (+)-cis-anti-[BP]dG adduct embedded in the same d(C5-[BP]G6-C7).d(G16-C17-G18) trinucleotide segment of the complementary 11-mer duplex studied previously with the stereoisomeric trans adducts. The exchangeable and nonexchangeable protons of the benzo[a]pyrenyl moiety and the nucleic acid were assigned following analysis of two-dimensional NMR data sets in H2O and D2O solution. The solution structure of the (+)-cis-anti-[BP]dG-dC 11-mer duplex has been determined by incorporating intramolecular and intermolecular proton-proton distances defined by upper and lower bounds deduced from NOESY data sets as restraints in energy minimization computations. The benzo[a]pyrene ring of [BP]dG6 is intercalated between intact Watson-Crick dC5.dG18 and dC7.dG16 base pairs in a right-handed DNA helix. The benzylic ring is in the minor groove while the pyrenyl ring sacks with flanking dC5 and dC7 bases on the same strand. The deoxyguanosine ring of [BP]dG6 is not Watson-Crick base paired but displaced into the minor groove with its plane parallel to the helix axis and stacks over the sugar ring of dC5. The dC17 base on the partner strand is displaced from the center of the helix toward the major groove by the intercalated benzo[a]pyrene ring. This intercalative structure of the (+)-cis-anti-[BP]dG-dC 11-mer duplex exhibits several unusually shifted proton resonances which can be readily accounted for by the ring current contributions of the deoxyguanosine and pyrenyl rings of the [BP]dG6 adduct. Several phosphorus resonances are shifted to low and high field of the unperturbed phosphorus spectral region and have been assigned to internucleotide phosphates centered about the [BP]dG6 modification site. These studies define the changes in the helix at the central trinucleotide segment needed to generate the intercalation site for the covalently bound (+)-cis-anti-[BP]dG adduct.(ABSTRACT TRUNCATED AT 400 WORDS)

Benzo[a]pyrene (BP) is an environmental genotoxin, which, following metabolic activation to 7,8-diol 9,10-epoxide (BPDE) derivatives, forms covalent adducts with cellular DNA. A major fraction of adducts are derived from the binding of N2 of guanine to the C10 position of BPDE. The mutagenic and carcinogenic potentials of these adducts are strongly dependent on the chirality at the four asymmetric benzylic carbon atoms. We report below on the combined NMR-energy minimization refinement characterization of the solution conformation of (-)-trans-anti-[BP]G positioned opposite C and flanked by G.C base pairs in the d(C1-C2-A3-T4-C5-[BP]G6-C7-T8-A9-C10-C11).d(G12-G13-T14++ +-A15-G16-C17- G18-A19-T20-G21-G22) duplex. Two-dimensional NMR techniques were applied to assign the exchangeable and non-exchangeable protons of the benzo[a]pyrenyl moiety and the nucleic acid in the modified duplex. These results establish Watson-Crick base pair alignment at the [BP]G6.C17 modification site, as well as the flanking C5.G18 and C7.G16 pairs within a regular right-handed helix. The solution structure of the (-)-trans-anti-[BP]G.C 11-mer duplex has been determined by incorporating intramolecular and intermolecular proton-proton distances defined by lower and upper bounds deduced from NOE buildup curves as constraints in energy minimization computations. The BP ring spans both strands of the duplex in the minor groove and is directed toward the 3'-end of the modified strand in the refined structure. One face of the BP ring of [BP]G6 stacks over the C17 residue across from it on the partner strand while the other face is exposed to solvent.(ABSTRACT TRUNCATED AT 250 WORDS)

We have synthesized, separated, and purified approximately 10 mg of a deoxyundecanucleotide duplex containing a single centrally positioned covalent adduct between (+)-anti-benzo[a]pyrene (BP) diol epoxide and the exocyclic amino group of guanosine. Excellent proton NMR spectra are observed for the (+)-trans-anti-BP diol epoxide-N2-dG adduct positioned opposite dC and flanked by G.C pairs in the d[C1-C2-A3-T4-C5-(BP)G6-C7-T8-A9-C10-C11].d[12- G13-T14-A15-G16-C17-G18-A19-T20-G 21-G22] duplex +ADdesignated (BP)G.C 11-mer+BD. We have determined the solution structure centered about the BP covalent adduct site in the (BP)G.C 11-mer duplex by incorporating intramolecular and intermolecular proton-proton distance bounds deduced from the NMR data sets as constraints in energy minimization computations. The BP ring is positioned in the minor groove and directed toward the 5' end of the modified strand. One face of the BP ring of (BP)G6 is stacked over the G18 and A19 sugar-phosphate backbone on the partner strand and the other face is exposed to solvent. A minimally perturbed B-DNA helix is observed for the d[T4-C5-(BP)G6-C7-T8].d[A15-G16-C17-G18-A19] segment centered about the adduct site with Watson-Crick alignment for both the (BP)G6.C17 pair and flanking G.C pairs. A widening of the minor groove at the adduct site is detected that accommodates the BP ring whose long axis makes an angle of approximately 45 degrees with the average direction of the DNA helix axis. Our study holds future promise for the characterization of other steroisomerically pure adducts of BP diol epoxides with DNA to elucidate the molecular basis of structure-activity relationships associated with the stereoisomer-dependent spectrum of mutational and carcinogenic activities.

The conformations of adducts derived from the covalent binding of four different isomeric diol epoxide derivatives of 5- or 6-methylchrysene to native double-stranded calf thymus DNA were studied by linear dichroism techniques. Out of four isomers investigated here, only the R,S,S,R enantiomer of anti-1,2-dihydroxy-3,4-epoxy-1,2,3,4-tetrahydro-5-methylchrysene, (+)-5-MeCDE, is highly tumorigenic and mutagenic toward Salmonella typhimurium TA100; the S,R,R,S enantiomer, (-)-5-MeCDE, and the corresponding R,S,S,R and S,R,R,S enantiomers of anti-1,2,-dihydroxy-3,4-epoxy-1,2,3,4-tetrahydro-6-methylchrysene are non-tumorigenic and much less mutagenic than (+)-5-MeCDE. [Melikian et al., (1988) Cancer Res., 48, 1781-1787] Only the DNA adducts derived from the binding of (+)-5-MeCDE are characterized by a pronounced positive linear dichroism signal at 308 nm due to the phenanthrenyl residue which is tilted at an angle of 45-48 degrees with respect to the average orientations of the axes of unoriented DNA segments. The phenanthrenyl residues derived from the covalent binding to DNA of the other three inactive or less active isomers appear to be unoriented. The defined orientation of the covalently bound phenanthrenyl residues derived from (+)-5-MeCDE corresponds to adduct conformations which are similar to those obtained from the binding of the highly tumorigenic trans-7,8-dihydroxy-anti-9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene stereoisomer and other highly active bay-region diol epoxide derivatives to DNA. These findings provide further evidence that there is a correlation between DNA adduct conformation and biological activities for these series of polycyclic aromatic diol epoxide derivatives with R,S,S,R absolute configuration and which are known to bind predominantly to N2 of guanine.

The conformations of the adducts derived from the covalent binding of the two enantiomeric forms of 9,10-epoxy-9,10,11,12-tetrahydrobenzo(e)pyrene (BePE) with native DNA were investigated by the electric linear dichroism technique. Both enantiomers give rise to two major adducts, one of which appears to be a quasi-intercalative site (I) while the other one is an external binding site (II). While the overall linear dichroism spectra are similar, in the case of the (-) enantiomer there is a greater contribution of site II adducts. These results are markedly different from the ones obtained with the two enantiomers of anti-benzo(a)pyrene-7,8-diol-9,10-epoxide (BaPDE), where the (+) enantiomer gives rise almost exclusively to site II binding, while the (-) enantiomer gives rise to both site I and site II covalent binding. The differences in the heterogeneity of binding between BePE and anti-BaPDE enantiomers may be due to the absence of hydroxyl groups in BePE which, in the case of BaPDE, are an important factor in determining the stereoselective properties of the covalent binding to double-stranded DNA.

Spectroscopic techniques including absorption, fluorescence excitation and emission spectra, fluorescence decay profiles (determined by single photon counting techniques), and electric linear dichroism are applied to a study of the conformation of covalent adducts derived from a reaction of 9,10-dihydroxy-11,12-epoxy-9,10,11,12-tetrahydro[e]pyrene (B[e]PDE) with DNA. The characteristics of non-covalent adducts obtained from the intercalative binding of 9,10,11,12-tetrahydroxytetrahydrobenzo[e]pyrene (B[e]PT) (derived from the hydrolysis of B[e]PDE) with DNA are compared to those of the covalent B[e]PDE--DNA adducts. It is shown that there are two types of binding sites in B[e]PDE--DNA adducts: (1) an exterior binding site similar to the one observed with the isomeric 7,8-dihydroxy-9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (B[a]PDE)--DNA adducts, and (2) a quasi-intercalative type of binding site in which the properties of the pyrene chromophore are similar to those of an intercalated pyrene moiety, but in which the red shift in the absorption maximum, and fluorescence quenching are less pronounced. This latter conformation is not observed in covalent B[a]PDE--DNA adducts. It is shown that the DNA concentration is an important parameter in determining the relative number of pyrene chromophores at these two binding sites. The extent of covalent binding of B[e]PDE is 4-8 times less than the binding of B[a]PDE to DNA under the same experimental conditions. The reduced reactivity of B[e]PDE is tentatively attributed to steric hindrance due to quasi-diaxial conformations of the two hydroxyl groups in one of the two bay-regions of B[e]PDE.

The linear dichroism spectra of complexes of tetrakis(N-methyl-4-pyridinio)prophine (H2TMpyP) and its zinc(II) derivative (ZnTMpyP) with DNA oriented in a flow gradient have been investigated. The dichroism of H2TMpyP determined within the Soret band and the Qy band system is consistent with an intercalative conformation in which the plane of the porphyrin ring system is nearly parallel to the planes of the DNA bases. In the case of ZnTMpyP on the other hand, the porphyrin ring system is inclined at angles of 62-67 degrees with respect to the axis of the DNA helix. The pyridyl groups in both cases are characterized by a low degree of orientation with respect to the axis of the helix. In contrast to H2TMpyP which does not significantly affect the degree of alignment of the DNA in the flow gradient, the binding of ZnTMpyP causes a significant decrease (about 50% for a base pair/ZnTMpyP ratio of 20) in the intrinsic dichroism at 260 nm due to the oriented DNA bases; the binding of ZnTMpyP to DNA either gives rise to regions of higher flexibility or causes bends or kinks at the binding sites. Increasing the ionic strength has little influence on the linear dichroism of the ZnTMpyP-DNA complexes, but the number of molecules bound at intercalation sites diminishes in the case of the H2TMpyP-DNA complexes; the accompanying changes in the linear dichroism characteristics suggest that external H2TMpyP complexes are formed at the expense of intercalation complexes.(ABSTRACT TRUNCATED AT 250 WORDS)

In the presence of native DNA the hydrolysis of benzo[a]pyrene-7,8-diol 9,10-epoxide (BPDE) to tetrols (BPT) is markedly accelerated (by a factor of up to approximately 80 at 25 degrees C, pH 7.0, in 5 mM sodium cacodylate buffer solution). When stopped-flow kinetic techniques are utilized, it is shown that the pseudo-first-order hydrolysis rate constant kH is smaller by a factor of approximately 3 in the presence of equivalent concentrations of denatured DNA, by a factor of 8-25 in the presence of nucleotides, and by a factor of 35-45 in the presence of nucleosides (depending on the nucleotide or nucleoside). In the presence of native DNa, kH increases with increasing DNA concentration and reaches a limiting value of kH = 0.684 +/- 0.04 s-1 at DNA concentrations in excess of approximately 5 x 10(-4) M (expressed in concentration of nucleotides). A kinetic model based on (1) rapid formation of a noncovalent BPDE-DNA complex followed by (2) slower hydrolysis of BPDE to BPT at these binding sites is consistent with the experimental data. It is shown furthermore that the DNA concentration dependence of kH and of noncovalent intercalative binding of BPDE to DNA is similar and that addition of magnesium ions (which is known to reduce intercalative binding of planar aromatic molecules to DNA) also reduces kH. These results suggest, but do not necessarily prove, that the DNA binding sites at which the hydrolysis of BPDE (to BPT) is catalyzed are intercalative in nature.

The conformation of adducts derived from the reactions and covalent binding of the (+) and (-) enantiomers of 7 beta, 8 alpha-dihydroxy-9 alpha, 10 alpha-epoxy-7,8,9,10-tetrahydrobenzo(a)pyrene (anti-BaPDE) with double-stranded calf thymus DNA in vitro were investigated utilizing the electric linear dichroism technique. The linear dichroism and absorption spectra of the covalent DNA complexes are interpreted in terms of a superposition of two types of binding sites. One of these conformations (site I) is a complex in which the plane of the pyrene residue is close to parallel (within 30 degrees) to the planes of the DNA bases (quasi-intercalation), while the other (site II) is an external binding site; this latter type of adduct is attributed to the covalent binding of anti-BaPDE to the exocyclic amino group of deoxyguanine (N2-dG), while site I adducts are attributed to the O6-deoxyguanine and N6-deoxyadenine adducts identified in the product analysis of P. Brookes and M.R. Osborne (Carcinogenesis (1982) 3, 1223-1226). Site II adducts are dominant (approximately 90% in the covalent complexes derived from the (+) enantiomer), but account for only 50 +/- 5% of the adducts in the case of the (-)-enantiomer. The orientation of site II complexes is different by 20 +/- 10 degrees in the adducts derived from the binding of the (+) and the (-) enantiomers to DNA, the long axis of the pyrene chromophore being oriented more parallel to the axis of the DNA helix in the case of the (+) enantiomer. These findings support the proposals by Brookes and Osborne that the difference in spatial orientation of the N2-dG adducts of (-)-anti-BaPDE together with their lower abundance may account for the lower biological activity of the (-) enantiomer. The external site II adducts, rather than site I adducts, appear to be correlated with the biological activity of these compounds.

Linear dichroism and absorption methods are used to study the orientations of transition moments of absorption bands of polycyclic aromatic epoxide derivatives which overlap with those of the DNA band in the 240-300 nm region. Both the short and long axes of the pyrene residues of 1-oxiranylpyrene (1-OP) and the (+) and (-) enantiomers of trans-7,8-dihydroxy-anti-9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (BPDE) noncovalently bound to double-stranded native DNA are oriented approximately perpendicular to the axis of the DNA helix, consistent with intercalative modes of binding. The covalent binding of these three epoxide derivatives to DNA is accompanied by reorientations of both the short and long axes of the pyrene residues. Covalent adducts derived from the highly mutagenic (+)-anti-BPDE are characterized by tilts of the short axis within 35 degrees or less, and of the long axis by more than 60-80 degrees, with respect to the planes of the DNA bases. In the adducts derived from the binding of the less mutagenic (-)-anti-BPDE and 1-OP epoxide derivatives to DNA, the long axes of the pyrenyl rings are predominantly oriented within 25 degrees of the planes of the DNA bases; however, in the case of the (-) enantiomer of BPDE, there is significant heterogeneity of conformations. In the case of the 1-OP covalent DNA adducts, the short axis of the pyrene ring system is tilted away from the planes of the DNA bases, and the pyrene ring system is not intercalated between DNA base-pairs as in the noncovalent complexes. The stereochemical properties of the saturated 7,8,9,10-ring in BPDE, or the lack of the 7 and 8 carbon atoms in 1-OP, do not seem to affect noncovalent intercalative complex formation which, most likely, is influenced mainly by the flat pyrenyl residues. These structural features, however, strongly influence the conformations of the covalent adducts, which in turn may be responsible for the differences in the mutagenic activities of these molecules.

Kinetic flow dichroism studies indicate that the (+) enantiomer of 7 beta, 8 alpha-dihydroxy-9 alpha, 10 alpha-epoxy-7,8,9,10-tetrahydrobenzo(a)pyrene physically bound at intercalative-type sites in double-stranded DNA undergoes covalent binding reactions to form adducts at external binding sites. The conformation of the non-covalent complex derived from the (-) stereoisomer is also intercalative in nature, but the conformations of the covalent adducts are heterogeneous and are characterized by both intercalative-type and external conformations. It is suggested that the distinctly higher biological activity of the (+) enantiomer relative to the activity of the (-) enantiomer may be related to the preponderance of 7,8,9-triol benzo(a)pyrene residues covalently linked to deoxyguanine and located at external binding sites in the DNA adducts.

The physical and chemical reaction pathways of the metabolite model compound benzo(a)pyrene-7,8-diol-9,10-epoxide (BPDE) in aqueous (double-stranded) DNA solutions was investigated as a function of temperature (0-30 degrees C), pH (7.0-9.5), sodium chloride concentration (0-1.5M) and DNA concentration in order to clarify the relationships between the multiple reaction mechanisms of this diol epoxide in the presence of nucleic acids. The reaction pathways are (1) noncovalent intercalative complex formation with DNA, characterized by the equilibrium constant K, and Xb the fraction of molecules physically bound; (2) accelerated hydrolysis of BPDE bound to DNA; (3) covalent binding to DNA; and (4) hydrolysis of free BPDE(kh). The DNA-induced hydrolysis of BPDE to tetraols and the covalent binding to DNA are parallel pseudo-first-order reactions. Following the rapid (millisecond time scale) noncovalent complex formation between BPDE and DNA, a much slower (approximately minutes) H+-dependent (either specific or general acid catalysis) formation of a DNA-bound triol carbonium ion (rate constant k3) occurs. At pH 7.0 the activation energy of k3 is 8.7 +/- 0.9 kcal/mol, which is lower than the activation energy of hydrolysis of free BPDE in buffer solution (14.2 +/- 0.7 kcal/mol), and which thus partially accounts for the acceleration of hydrolysis of BPDE upon complexation with DNA. The formation of the triol carbonium ion is followed by a rapid reaction with either water to form tetraols (rate constant kT), or covalent binding to DNA (kc). The fraction of BPDE molecules which undergo covalent binding is fcov approximately equal to kc/(kc + kT) = 0.10 and is independent of the overall BPDE reaction rate constant k = kh(1 - Xb) + k3Xb if Xb----1.0, or is independent of Xb as long as k3Xb much greater than kh(1 - Xb). Thus, at Xb = 0.9, fcov is independent of pH (7.0-9.5) even though k exhibits a 70-fold variation in this pH range and k----kh above pH 9 (k3 = kh). Similarly, fcov is independent of temperature (0-30 degrees C), while k varies by a factor of approx. 3. In the range of 0-1.5 M NaCl, fcov decreases from 0.10 to 0.04. These variations are attributed to a combination of salt-induced variations in the factors k3, Xb and the ratio kc/kT.

The spectroscopic absorption and fluorescence properties of adducts derived from the covalent binding of (+/-)trans-7,8-dihydroxy-anti-9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyre ne (BPDE) to DNA are re-examined in view of conflicting interpretations regarding the conformations of these adducts which currently exist in the literature. The fluorescence decay profiles were accurately determined utilizing synchrotron-pulsed light source excitation and the time-correlated single photon counting technique. The conformational properties of the adducts were probed by determining their accessibilities to acrylamide, a known fluorescence quencher, and by comparing the accessibilities of the BPDE-DNA adducts with those of known model systems with intercalative, partially intercalative and minor groove binding conformations. In contrast to any of these model systems, the fluorescence of the aromatic pyrenyl residues in the covalent BPDE-DNA adducts exhibit significant sensitivity to acrylamide, suggesting that these residues are located at binding sites with significant solvent exposure. A quantitative analysis of the acrylamide fluorescence quenching according to a dynamic Stern-Volmer quenching model suggests the following characteristics: the major (65%) component (1.4 ns lifetime) is characterized by significant exposure to the solvent environment; the second component (6-7 ns lifetime) can be subdivided into a solvent-accessible and a solvent-inaccessible component, the inaccessible fraction being attributed to minor adducts, possibly with a quasi-intercalative conformation. The amplitude of the third, long-lived (200-ns) component is variable; it arises from the photochemical decomposition of the adducts which gives rise to tetraols (7,8,9,10-tetrahydro-tetrahydroxybenzo[a]pyrene). The variable content of these degradation products accounts for most discrepancies in the fluorescence properties of the covalent BPDE-DNA adducts previously reported.

The conformation of covalent adducts derived from the reactions of racemic 7 beta,8 alpha-dihydroxy-9 alpha, 10 alpha-epoxy-7,8,9,10-tetrahydrobenzo [a] pyrene (BaPDE), 9,10-epoxy-7,8,9,10-tetrahydrobenzo [a] pyrene (BaPE), and 9,10-epoxy-9,10,11,12-tetrahydrobenzo [e] pyrene (BePE) with calf thymus DNA in aqueous buffer solution (25 degrees C, pH 7.0) were investigated and compared by means of absorption, fluorescence and electric linear dichroism techniques. Two types of conformations are recognized. Site I is characterized by a red shift (approximately 10 nm) in the absorption maximum of the pyrene nucleus, a significantly reduced fluorescence yield, and a negative electric linear dichroism signal (delta A); this site is presumed to involve a near-parallel (within 25 degrees) orientation of the planar pyrene residue with the planes of the DNA bases, and a relatively strong interaction between the phi electrons of the nucleic acid bases and the pyrene residue. In site II, there is only a small red-shift in the absorption maximum (approximately 2 nm), a non-zero fluorescence yield, and a positive delta A throughout the absorption region of the pyrene residue; in this conformation the pyrene residue is presumed to lie on the outside of the DNA molecule, possibly in one of the grooves. The BaPDE-DNA complex displays predominantly a site II-type conformation while the BaPE- and BePE-DNA complexes display both site I and site II adducts, with site I conformations predominating. The lack of hydroxyl groups in BaPE and BePE lead to a loss in stereospecificity in covalent adduct formation. The 7 and 8 hydroxyl groups in covalent adduct formation. The 7 and 8 hydroxyl groups in BaPDE appear to reduce the probability of formation of site I-type of covalent adducts, and appear to be, at least in part, responsible for the enantiomeric stereospecificity in the covalent reaction between BaPDE and DNA.