Solution structure of the minor conformer of a DNA duplex containing a dG mismatch opposite a benzo[a]pyrene diol epoxide/dA adduct: glycosidic rotation from syn to anti at the modified deoxyadenosine

Polycyclic aromatic hydrocarbons (PAHs) are widespread environmental contaminants whose metabolism in mammals results in deleterious cell transformation. Covalent modification of DNA by diol epoxides metabolically formed from PAHs such a benzo[a]pyrene (BaP) provides a mechanism for the genotoxicity, mutagenicity, and carcinogenicity of PAHs. We had previously reported NMR evidence for a minor conformer of the duplex d(G1G2T3C4A5*C6G7A8G9).d(C10T11C12G13G14G15A16C17C18) containing a dG14 mismatch opposite a dA5* residue modified at the exocyclic amino group by trans addition to (+)-(7R,8S,9S,10R)-7,8-dihydroxy-9,10-epoxy-7,8,9,10-tetrahydrobenzo[a] pyrene [Yeh, H.J.C., Sayer, J.M., Liu, X., Altieri, A.S., Byrd, R.A., Lashman, M.K., Yagi, H., Schurer, E.J., Gorenstein, D.G., & Jerina, D.M. (1995) Biochemistry 34, 13570-13581]. In the present work, we describe the structure of this minor conformer (ca. 17% of the total conformer population). This represents the first structural determination of a minor conformer of a carcinogen-lesion DNA adduct. Two-dimensional NOESY, ROESY, TOCSY, and exchange-only spectra at 750 MHz allowed nearly complete sequential assignment of both conformers. In the minor conformer, the adducted base assumes an anti-glycosidic torsion angle whereas in the major conformer it assumes an unusual syn-glycosidic torsion angle. The aromatic hydrocarbon in the minor conformer is intercalated between dG13 and dG14, preserving the energetically favorable stacking interactions found in the major conformer. The major structural differences between the two conformers appear to be near the lesion site as evidenced by the large chemical shift differences between major and minor conformer protons near the lesion site; away from this site, the chemical shifts of the major and minor conformer protons are nearly identical. Because any of the conformations of benzo[a]pyrene diol epoxide-modified DNA may contribute to tumorigenic activity, structural determination of all conformations is essential for the elucidation of the mechanism of cell transformation initiated by covalent modification of DNA by PAHs.     

Solution conformation of the N-(deoxyguanosin-8-yl)-1-aminopyrene ([AP]dG) adduct opposite dC in a DNA duplex

Combined NMR-molecular mechanics computational studies were undertaken on the C8-deoxyguanosine adduct formed by the carcinogen 1-nitropyrene embedded in the d(C5-[AP]G6-C7).d(G16-C17-G18) sequence context in a 11-mer duplex, with dC opposite the modified deoxyguanosine. The exchangeable and nonexchangeable protons of the aminopyrene moiety and the nucleic acid were assigned following analysis of two-dimensional NMR data sets in H2O and D2O solution. There was a general broadening of several proton resonances for the three nucleotide d(G16-C17-G18) segment positioned opposite the [AP]dG6 lesion site resulting in weaker NOEs involving these protons in the adduct duplex. The solution conformation of the [AP]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 spectra as restraints in molecular mechanics computations in torsion angle space. The aminopyrene ring of [AP]dG6 is intercalated into the DNA helix between intact Watson-Crick dC5.dG18 and dC7.dG16 base pairs. The modified deoxyguanosine ring of [AP]dG6 is displaced into the major groove and stacks with the major groove edge of dC5 in the adduct duplex. Both carbon and proton chemical shift data for the sugar resonances of the modified deoxyguanosine residue are consistent with a syn glycosidic torsion angle for the [AP]dG6 residue. The dC17 base on the partner strand is displaced from the center of the helix toward the major groove as a consequence of the aminopyrene ring intercalation into the helix. This base-displaced intercalative structure of the [AP]dG.dC 11-mer duplex exhibits several unusually shifted proton resonances which can be accounted for by the ring current contributions of the deoxyguanosinyl and pyrenyl rings of the [AP]dG6 adduct. In summary, intercalation of the aminopyrene moiety is accompanied by displacement of both [AP]dG6 and the partner dC17 into the major groove in the [AP]dG.dC 11-mer duplex.     

Solution structure of the aminofluorene-intercalated conformer of the syn [AF]-C8-dG adduct opposite a--2 deletion site in the NarI hot spot sequence context

This paper addresses structural issues related to the capacity of aminofluorene [AF] for frameshift mutations of the -2 type on C8 covalent adduct formation at the G3 site in the d(C-G1-G2-C-G3-C-C) NarI hot spot sequence. This problem has been approached from a combined NMR and relaxation matrix analysis computational structural study of the [AF]dG adduct in the d(C-G-G-C-[AF]G-C-C).d(G-G-C-C-G) sequence context at the 12/10-mer adduct level (designated [AF]dG.del(-2) 12/10-mer). The proton spectra of this system are of exceptional quality and are consistent with the formation of an AF-intercalated conformer with the modified guanine in a syn alignment displaced along with the 5'-flanking cytosine residue into the major groove. The solution structure has been determined by initially incorporating intramolecular and intermolecular proton-proton distances defined by lower and upper bound deduced from NOESY spectra as restraints in molecular mechanics computations in torsion angle space and subsequently refined through restrainted molecular dynamics calculations based on a NOE distance and intensity refinement protocol. Strikingly, the [AF]dG.del(-2) 12/10-mer duplex adopts only one of two potential AF-intercalation alignments for the [AF]dG adduct opposite the -2 deletion site in the NarI sequence context with the extrusion of the dC-[AF]dG step favored completely over extrusion of the [AF]dG-dC step at the lesion site. This polarity establishes that the structural perturbation extends 5' rather than 3' to the [AF]dG lesion site in the adduct duplex. This structure of the [AF]dG adduct opposite a -2 deletion site shows distinct differences with conclusions reported on the alignment of the related acetylaminofluorene [AAF]dG adduct opposite a -2 deletion site in the identical NarI sequence context [Milhe, C., Fuchs, R. P. P., and Lefevre, J. F. (1996) Eur. J. Biochem. 235, 120-127]. In that study, qualitative NMR data without computational analysis were employed to conclude that the extrusion at the lesion site occurs at the [AAF]dG-dC step for the AAF-intercalated conformer of the adduct duplex. The structure of the [AF]dG adduct opposite a -2 deletion site determined in our group provides molecular insights into the architecture of extended slipped mutagenic intermediates involving aromatic amine intercalation and base-displaced syn modified guanines in AF and, by analogy, AAF-induced mutagenesis in the NarI hot spot sequence context.     

Induced circular dichroism of benzo[a]pyrene-7,8-dihydrodiol 9,10-epoxide stereoisomers covalently bound to deoxyribooligonucleotides used to probe equilibrium distribution between groove binding and intercalative adduct conformations

Binding conformations of single anti-BPDE-N2-dG adducts in oligonucleotides of varying base composition have been studied by induced circular dichroism (ICD). The sign of the ICD around 350 nm of single-stranded oligonucleotide adducts and the sign of an exciton type of CD component at 260 nm in both single strand and duplex forms of adducts correlate with the absolute configuration of the cyclohexyl moiety of the adduct. Changes in magnitude and sign of the ICD around 350 nm were observed upon duplex formation. The results show that adducts displaying external (minor groove) binding characteristics are associated with a significant positive ICD. Conversely, adducts displaying intercalation binding characteristics were found to have a positive or negative ICD. The magnitude of the ICD is dependent on the sequence context and the particular adduct isomer studied. Duplexes with (+)-trans-anti-BPDE-N2-dG in 5'-d(CCTATCGCTATCC) or 5'-d(CCTATAGATATCC) exhibit a relatively strong positive ICD. In contrast, the duplexes with (+)-trans-anti-BPDE-N2-dG in 5'-d(CCTATTGCTATCC) and 5'-d(CCTATTGTTATCC) display a small positive and negative ICD, respectively, in both cases suggesting conformational heterogeneity. Partially complementary duplexes (dA, dT, or dG) localized opposite the (+)-trans-anti-BPDE-N2-dG adduct in 5'-d(CCTATCGCTATCC) or 5'-d(CCTATAGATATCC) also demonstrated negative ICD. These results together with light absorption characteristics suggest a preferred conformation of intercalation for the mismatched duplexes. Evidence of an equilibrium between the external and intercalative adduct conformation is provided by the results from the temperature dependence of the near-UV absorption and ICD characteristics of (+)-trans-anti-BPDE-N2-dG complex in a 5'-d(CCTATAGATATCC) duplex.