MUTAGENESIS BY SITE-SPECIFIC ARYLAMINE ADDUCTS IN PLASMID DNA - ENHANCING REPLICATION OF THE ADDUCTED STRAND ALTERS MUTATION FREQUENCY

Site specifically modified plasmids were used to determine the mutagenic effects of single arylamine adducts in bacterial cells. A synthetic heptadecamer bearing a single N-(guanin-8-yl)-2-aminofluorene (AF) or N-(guanin-8-yl)-2-(Acctylamino)fluorene (AAF) adduct was used to introduce the adducts into a specific site in plasmid DNA that contained a 17-base single-stranded region complementary to the modified oligonucleotide. Following transformation of bacterial cells with the adduct-bearing DNA, putative mutants were detected by colony hybridization techniques that allowed unbiased detection of all mutations at or near the site of the adduct. The site-specific AF or AAF adducts were also placed into plasmid DNA that contained uracil residues on the strand opposite that bearing the lesions. The presence of uracil in one strand of the DNA decreases the ability of the bacterial replication system to use the uracil-containing strand, thereby favoring the use of the strand bearing the adducts. In a comparison of the results obtained with site specifically modified DNA, either with or without uracil, the presence of the uracil increased the mutation frequencies of the AF adduct by >7-fold to 2.9% and of the AAF adduct by > 12-fold to 0.75%. The mutation frequency of the AF adduct was greatly reduced in a uvrA−~ strain while no mutations occurred with the AAF adduct in this strain. The sequence changes resulting from these treatments were dependent on adduct structure and the presence or absence of uracil on the strand opposite the adducts. The AF adduct produced primarily single-base deletions in the absence of uracil but only base substitutions in the uracil-containing constructs. The AAF adduct produced mutations only in the uracil-containing DNA, which included both frame shifts and base substitutions. Mutations produced by both adducts were SOS dependent. © 1990, American Chemical Society. All rights reserved.