MUTAGENESIS AT A SITE-SPECIFICALLY MODIFIED NARI SEQUENCE BY ACETYLATED AND DEACETYLATED AMINOFLUORENE ADDUCTS

A hotspot for mutagenesis by N-acetyl-2-aminofluorene (AAF) was site-s pecifically modified with 2-aminofluorene (AF) and AAF adducts, and th e mutation frequencies and specificities were determined and compared. Previous work has shown that the presence of an AAF adduct in a NarI sequence (GGCGCC) results a high mutation frequency for a CG double ba se pair deletion. In the present study, an M13 derivative was construc ted that contained a NarI recognition sequence in the beta-galactosida se gene of bacteriophage M13mp9. This derivative was site-specifically modified with either an AF or an AAF adduct, the products were charac terized, and these templates were then transformed into Escherichia co li wild-type strain JM103 or uvrA strain SMH12. The levels and mutatio n spectra were determined either with or without SOS induction. It was found that, with SOS functions induced, the measured mutation frequen cies were substantially higher in all cases. More importantly, the typ es of mutations induced by the AAF and AF adducts were very different: AAF adducts induced almost exclusively CG double base deletion mutati ons, whereas AF adducts gave rise specifically to base-substitution mu tations. The AF-derived mutation spectrum included both G to T and G t o A mutations. The results are discussed in light of the current views on the relationship between the DNA structure and mutagenesis.