Site-specific frame-shift mutagenesis by the 1-nitropyrene-DNA adduct N-(deoxyguanosin-8-y1)-1-aminopyrene located in the (CG)(3) sequence: Effects of SOS, proofreading, and mismatch repair

1-Nitropyrene (1-NP), the predominant nitropolycyclic hydrocarbon found in diesel exhaust, is a mutagen and tumorigen. Nitroreduction is a major pathway by which 1-NP is metabolized. Reductively activated 1-NP forms a major DNA adduct, N-(deoxyguanosin-8-yl)-1-aminopyrene (dG(AP)), both in vitro and in vivo. In Salmonella typhimurium 1-NP induces a CPG deletion in a CGCGCGCG sequence. In Escherichia coli, however, mostly -1 and +1 frame-shifts are observed, which occur predominantly in 5'-CG, 5'-GC, and 5'-GG sequences. In order to determine the mechanism of mutagenesis by dG(AP) in a CpG repetitive sequence, we constructed a single-stranded M13 genome containing the adduct at the underscored deoxyguanosine of an inserted CGCGCG-sequence. In E. coli strains with normal repair capability the adduct induced approximately 2% CpG deletions, which was 20-fold that of the control. With SOS, the frequency of frame-shift mutations increased to 2.6%, even though the frequency of CpG deletion accompanied 50% reduction. The enhancement in mutagenesis was due to a +1 frame-shift that occurred at a high frequency. In strains with a defect in methyl-directed mismatch repair, 50-70% increase in mutation frequency was observed. When these strains were SOS induced, frame-shift mutagenesis increased by approximately 100%. When transfections were carried Out in dnaQ strains that are impaired in 3'-->5' exonuclease activity of DNA polymerase III, frame-shift mutagenesis increased 5-7-fold. dG(AP)-induced frame-shifts in the (CG)(3) sequence, therefore, varied from 2% to 17% depending on the state of repair of the host cells. We conclude that dG(AP) induces both -2 and +1 frame-shifts in a CpG repetitive sequence and that these two mutagenic events are competing pathways. The CpG deletion does not require SOS functions, whereas the +1 frame-shifts are SOS-dependent. On the basis of the data in repair-deficient strains, it appears that both types of frame-shifts occurred as a result of misalignment, which are corrected primarily by the proofreading exonuclease of the DNA polymerase. Misaligned structures that escape the exonuclease are repaired by the methyl-directed mismatch repair, albeit with limited efficiency.