SITE-SPECIFIC FRAME-SHIFT MUTAGENESIS BY THE 1-NITROPYRENE-DNA ADDUCTN-(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 foun d in diesel exhaust, is a mutagen and tumorigen. Nitroreduction is a m ajor 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, mo stly -1 and +1 frame-shifts are observed, which occur predominantly in 5'-CG, 5'-GC, and 5'-GG sequences. In order to determine the mechanis m of mutagenesis by dG(AP) in a CpG repetitive sequence, we constructe d a single-stranded M13 genome containing the adduct at the underscore d deoxyguanosine of an inserted CGCGCG-sequence. In E. coli strains wi th normal repair capability the adduct induced approximately 2% CpG de letions, which was 20-fold that of the control. With SOS, the frequenc y of frame-shift mutations increased to 2.6%, even though the frequenc y of CpG deletion accompanied 50% reduction. The enhancement in mutage nesis was due to a +1 frame-shift that occurred at a high frequency. I n strains with a defect in methyl-directed mismatch repair, 50-70% inc rease 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 mutagen esis increased 5-7-fold. dG(AP)-induced frame-shifts in the (CG)(3) se quence, therefore, varied from 2% to 17% depending on the state of rep air 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 o f frame-shifts occurred as a result of misalignment, which are correct ed primarily by the proofreading exonuclease of the DNA polymerase. Mi saligned structures that escape the exonuclease are repaired by the me thyl-directed mismatch repair, albeit with limited efficiency.