Influence of benzo[a]pyrenediol epoxide chirality on solution conformations of DNA covalent adducts: the (-)-trans-anti-[BP]G.cntdot.C adduct structure and comparison with the (+)-trans-anti[BP]G.cntdot.C enantiomer

Abstract

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)

Methylation of Cytosine at C5 in a CpG Sequence Context Causes a Conformational Switch of a Benzo[a]pyrene diol epoxide-N2-guanine Adduct in DNA from a Minor Groove Alignment to Intercalation with Base Displacement

“…Such upfield imino proton shifts (b) are characteristic of intercalation of the pyrenyl ring into the helix in the meC- [BP]G-C (b) sequence context. The lack of upfield-shifted imino protons in the unmethylated C- [BP]G-C sequence context (a) is in agreement with the previous results and minor groove structural assignment of the same 10R (−)-trans-anti- [BP]G adduct in the same duplex reported by de los Santos et al 29 However, the presence of the 5-methyl group at the C flanking the same adduct on the 5′-side is unusual and resembles the imino spectrum of the stereoisomeric 10R (+)-cis-adduct in the same unmethylated 11-mer duplex that has a base-displaced intercalated conformation. 30 Expanded regions of the NOESY contour plot (200 ms mixing time) for the 10R (−)-transanti- [BP]G adduct in the meC- [BP]G-C sequence context at the duplex level in H 2 O buffer at 0 °C are shown in Figure 3.…”

“…The imino proton assignments are shown in (b) and were determined as described for the same sequence context in earlier publications. [28][29][30][31]33 The partially resolved imino protons resonating between 12.5 ppm and 14 ppm are characteristic of Watson-Crick basepairing in these 11-mer duplexes. There are dramatic differences in the imino proton spectra on proceeding from the C- [BP]G-C (a) to the meC- [BP]G-C (b) sequence context.…”

“…This is in sharp contrast to the external, minor groove alignment of the aromatic BP ring system of the same 10R (−)-trans-anti-adduct in the unmethylated 11-mer duplex. 29 …”

“…65,66 One was created from the basedisplaced-intercalative solution structure of the 10R (+)-cis-anti- [BP]G adduct, 30 by remodeling the 10R (+)-cis-anti isomer to the 10R (−)-trans-anti adduct (these two adducts differ from one another only by the absolute configurations at the C7, C8, and C9 carbon atoms, Figure 1), and by adding the methyl group to the 5-position of cytosine residue 5. The second model was created from the NMR solution structure of the minor groove 10R (−)-trans-anti- [BP]G adduct, 29 again by addition of the 5-methyl to cytosine residue 5 ( Figure 1). A model of the 10S (+)-trans-anti- [BP]G adduct in a base displaced-intercalative conformation in the meC-G-C sequence context was obtained with DUPLEX, by remodeling the NMR solution structure of the 10S (−)-cis-anti- [BP]G adduct, 31 with addition of the 5-methyl group to C5 and energy minimization.…”

“…The 10S (+)-and 10R (−)-trans-anti adducts are aligned with the bulky pyrenyl ring system in the minor groove, pointing either towards the 5′-end of the modified strand (the (+)-trans-adduct), or towards the 3′-end (the (−)-trans-adduct). 28,29 Both the 10S (−)-cis-and the 10R (+)-cis-anti adducts are intercalated, but with the modified guanine residue displaced into the major and minor grooves, respectively, and with the partner dC group in the complementary strand displaced towards the major groove in both cases. 30,31 Specifically, we hypothesized earlier that the 10S (+)-trans-and (−)-cis-adduct conformers, on the one hand, and the 10R (−)-trans-and 10R (+)-cis-adduct conformations, on the other, can interconvert because they have the identical absolute configurations about the C10-N 2 -dG bond, and that the equilibrium between these two conformations could depend on the base sequence context.…”