The long-term objective of this proposal is to elucidate the molecular mechanisms by which environmental chemicals cause cancer. The specific concentration in this project is on the mechanisms of mutagenesis by the fungal metabolite aflatoxin B1, a common food contaminant and a suspected human carcinogen. The experimental system primarily involves prokaryotic model genomes (phage PhiX174 and plasmid pBR322) although certain experiments involve cultured primate cell lines. Both single and multiple AFB1 adducts will be introduced into the model genomes using in vitro DNA manipulations and their mutagenic consequences in vivo under selected repair conditions will be studied. All mutational consequences in selected PhiX genes will be isolated by the strategy of plasmid-mediated complementation. Representative mutants will be analyzed at the sequence level to answer the following types of questions: (1) Are AFB1-induced DNA modifications mutagenic? (2) If they are, what principal types of mutations are produced? (3) Is there a positive correlation between site of AFB1 adduct and mutation? (4) What are the mechanisms for the reported frameshift mutagenesis by AFB1? If deletion or insertion, how many bases are involved and are there patterns? Essentially similar questions will also be addressed in a highly specific in vitro mutagenesis system. In addition, we will examine certain aspects of the interaction of AFB1 with DNA in vitro and in vivo. Some of these experiments were suggested by our recent finding that the rate of AFB1 modification of G residues is strongly and predictably influenced by the sequence context. a) Are the in vitro sequence-specific effects mimicked in vivo? b) How does DNA in chromatin react with AFB1? c) Does AFB1 prefer actively expressed genes? d) How does DNA superhelicity affect sequence specificity? and e) What is the mechanistic basis for the specificity?
