MUTATIONS INDUCED BY AROMATIC AMINE DNA-ADDUCTS IN PBR322

A 276 bp region from the tetracycline resistance gene of the plasmid p BR322 was modified with 2-acetylaminofluorene (AAF), 2-aminofluorene ( AF), 4-aminobiphenyl (ABP), N'-acetylbenzidine or 1-aminopyrene (AP) i n order to determine the effect of adduct structure upon mutation indu ction. Each modification reaction gave one major adduct and these addu cts had chromatographic properties, as determined by P-32-postlabeling , identical to those in which substitution had occurred at C8 of deoxy guanosine through the amine or amide nitrogen. The types and distribut ion of mutations were then characterized following introduction of the modified plasmids into SOS-induced Escherichia coli using Hanahan et al.'s procedure (Methods Enzymol., 204, 63-113, 1991). With AAF-modifi ed plasmid, 60% of the mutations were deletions or additions, and thes e were detected primarily at NarI sites or in repetitive G sequences. Modification with AF gave -G deletions, primarily in runs of Gs, and b ase substitution mutations, which were mainly G to T transversions. Su bstitution with ABP or N'-acetylbenzidine resulted in G to T and G to C transversions, the latter being a mutation not detected with AF; in addition, -G deletions were detected at only very low frequency. AP mo dification gave both -G frameshift and base substitution mutations, of which G to T transversions predominated. A comparison of the mutation frequencies per adduct indicated that the mutagenic efficiencies of t he adducts decreased in the order AP > AF > AAF similar to ABP similar to N'-acetylbenzidine. AAF- and ABP-modified pBR322 were also introdu ced with a CaCl2 method. The mutation frequency per adduct increased w ith this transformation procedure, and this appeared to be a reflectio n of a greater percentage of frameshift mutations. These data indicate that a series of structurally related aromatic amines will induce bot h base substitution and frameshift mutations when incorporated into pB R322, but that frameshift mutations occur almost exclusively with the planar derivatives. Furthermore, the ability to induce frameshift muta tions increases the mutagenic efficiency of an adduct.