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.

In the supF gene, most (+)-anti-benzo[a]pyrene diol epoxide ((+)-anti-B[a]PDE) mutagenesis hot spots in Escherichia coli are in 5'-GG sequences [Rodriguez and Loechler (1993) Carcinogenesis 14, 373-383]. A major hot spot was detected at G1 in the sequence 5'-GCG1G2-CCAAAG, whereas G2 yielded very few mutants. In order to investigate the details of such sequence context effects of (+)-anti-B[a]PDE mutagenesis, we have constructed 25-mer oligonucleotides and single-stranded M13 genomes containing the above decamer sequence, in which the trans-N2-dG adduct induced by (+)-anti-B[a]PDE [(+)-trans-anti-B[a]P-N2-dG] at G1 or G2 was introduced. In vitro DNA synthesis on the adducted 25-mers was strongly blocked at each site, although the 3'-->5' exonuclease-deficient Klenow fragment could incorporate a nucleotide opposite the adduct in the presence of Mn2+. For both sites purine nucleotides were preferred. The ratio Vmax/K(m) indicated that the efficiency of incorporation of dGTP opposite these sites was very similar, but dATP incorporation opposite the adduct at G1 was five-fold more efficient than that at G2. For each site, further extension beyond the adducted nucleotide was investigated by annealing four different primers, in which only the nucleotide opposite the adducted deoxyguanosine was altered. Significant extension was only observed when deoxyadenosine was located opposite adducted G1. When the M13 genomes containing the (+)-trans-anti-B[a]P-N2-dG were replicated in E. coli, survival of each adducted genome was less than 1% as compared to the unadducted genome. Upon induction of SOS, viability increased 2-6-fold. DNA sequencing showed no base substitutions in the progeny from SOS-uninduced cells, although small deletions in a quasipalindromic sequence occurred with the adduct being located at either site. However, following SOS induction, up to 40% targeted base substitutions were detected when the adduct was located at G1, while approximately 12% of the progeny were mutants with the adduct at G2. Most base substitutions were targeted G-->T transversions. We conclude that (+)-trans-anti-B[a]P-N2-dG is a highly mutagenic and replication blocking lesion. In addition, the biological consequence of this adduct depends on whether it is located at G1 or G2, suggesting that sequence context plays a major role in the mutagenic processing of this adduct.