Sequence Specific Mutagenesis of the Major (+)-anti-Benzo[a]pyrene Diol Epoxide−DNA Adduct at a Mutational Hot Spot in Vitro and in Escherichia coli Cells

Abstract

In the supF gene, most (+)-anti-benzo[a]pyrene diol epoxide ((+)-anti-B[a]PDE) mutagenesis hot spots in Escherichiacoli are in 5‘-GG sequences [Rodriguez and Loechler (1993) Carcinogenesis14, 373−383]. A major hot spot was detected at G₁ in the sequence 5‘-GCG₁G₂CCAAAG, 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 V_max/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 ∼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.