Sequence context modulation of polycyclic aromatic hydrocarbon-induced mutagenesis

DNA structural perturbations that are induced by site specifically and stereospecifically defined benzo[a]pyrene-7,8-dihydrodiol-9,10-epoxide (BPDE) adducts are directly correlated with mutagenesis, leading to cellular transformation. Although previous investigations had established that replication of DNAs containing N⁶-BPDE dA adducts at the second position in the N-ras codon 61(CAA) (61²) resulted exclusively in A to G transitions, NMR analyses not only established the structural basis for this transition mutation but also predicted that if the adduct were positioned at the third position in the same codon, an expanded spectra of mutations was possible. To test this prediction, replication of DNAs containing C₁₀S-BPDE and C₁₀R-BPDE lesions linked through the N⁶ position of adenine in the sequence context N-ras codon 61, position 3 (C₁₀S-BPDE and C₁₀R-BPDE at 61³) was carried out in Escherichia coli, and these data revealed a wide mutation spectrum. In addition to A to G transitions produced by replication of both lesions, replication of the C₁₀S-BPDE and C₁₀R-BPDE adducts also yielded A to C and A to T transversions, respectively. Analyses of single nucleotide incorporation using Sequenase 2.0 and exonuclease-deficient E. coli Klenow fragment and pol II not only revealed high fidelity synthesis but also demonstrated the same hierarchy of preference opposite a particular lesion, independent of the sequence context. Primer extension assays with the two lesions at N-ras 61³ resulted in truncated products, with the C₁₀S-BPDE adducts being more blocking than C₁₀R-BPDE lesions, and termination of synthesis was more pronounced at position 61³ than at 61² for each of the lesions.