The work described in this proposal seeks to answer the question--why is benzo[a]pyrene (BP) a potent mutagen? The proximate mutagen of BP, namely, benzo[a]pyrene 7,8-diol 9,10-oxide (BPDE) will be studied. Much of the work will involve a comparison of BPDE with a structural analogue, benzene 1,2-diol 3,4 oxide (bde). bde is approximately 10,000 times less mutagenic than BPDE, and provides a standard by which to assess the potency of BPDE as a mutagen. Four factors are expected to influence this difference in potency: (i) chemical reactivity; (ii) the ability to form stable, non-covalent interactions with DNA prior to adduction; (iii) the inherent mutagenicity of the DNA adduct(s) responsible for BPDE-induced mutagenesis; and (iv) the ability of E. coli cells to accurately repair this adduct(s). The major BPDE/DNA adduct N2-BPDE-Gua will be built into a defined site in the E. coli bacteriophage, M13mp18, using chemical synthetic and recombinant DNA techniques. This vector will be used to study mutagenesis by N2-BPDE-Gua in isolation from all other BPDE/DNA lesions. Ultimately, by using this vector (and a variety of other techniques) the lesion(s) responsible for BPDE-induced mutations will be determined. Following the determination of the structure of the mutagenic adduct, the factors affecting its formation in DNA will be assessed--in particular the effects of chemical reactivity and non-covalent, DNA interactions. Similar experiments will be performed with bde. Ultimately, a quantitative apportionment of the 10,000 difference in mutagenicity between BPDE and bde among the four factors listed above will be done. This will allow an assessment of the chemical and structural basis of the high mutagenic potency of BPDE.
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