Various organic agents that alkylate DNA are known to induce mutations in bacterial and animal cells. The precise nature and location of modified DNA sequences in such mutants are often difficult to ascertain. In this report, a 10-base-pair oligomer (BamHI linker) is treated with (±)-trans-benzo [alpyrene-7,8-dihydrodiol-9,10-epoxide and inserted into replicative form DNA of phage M13 by ligation at a specific restriction site. Escherichia coli are transfected with the recombinant DNA containing the alkylated target, progeny viral plaques are selected, and their DNAs are subjected to DNA sequence analysis at the region of oligomer insertion. For the alkylated inserts used in this study, the DNA sequence analysis of progeny viral DNA showed that nucleotide deletions were present in every clone examined. These deletions occurred primarily, but not exclusively, at G-C cluster regions, varied from 1 to 24 base pairs in length, and included both target and nontarget nucleotides. A second type of repair, which restores most of the original nucleotide bases in the alkylated insert, is also implied by the DNA sequence data obtained.It is generally believed that polycyclic aromatic hydrocarbons (PAH) exert their mutagenic effects by metabolic activation to reactive intermediates that interact with cellular nucleic acids. For benzo [a]pyrene (BP), the activated metabolite has been identified as (+)-trans-benzo[a]pyrene-7,8-dihydrodiol-9,10-epoxide (BPDE) (1). The reversion, or back mutation, of bacterial mutants to their wild-type state is a widely used assay for determination of the mutagenic capacity of chemical agents (2, 3). Such assays, however, offer little information about specific alterations in the DNA sequence arrangement, nor do they indicate whether the mutational site is the same as or different from the site of lesion. Base substitution mutations induced by BPDE, 3,4-epoxycyclopenta[cd]pyrene, and activated aflatoxin B1 in the lacI gene of Escherichia coli have been identified (4, 5). By DNA sequence analysis, activated PAH have been found to induce G-C and T-A base pair insertions and deletions (6, 7). In all of these studies, the precise nature and sites of the DNA lesion were not firmly established.In this report, a model system is described for the detection of sequence modifications at a specific location in viral DNA induced by BPDE. A 10-base-pair (bp) oligonucleotide is alkylated with BPDE and inserted into replicative form (RF) DNA of phage M13. After transfection of host cells with the recombinant DNA, viral clones are selected, and
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