The goal of this work is to investigate the mechanisms by which simple alkylating agents and the potent human liver carcinogen aflatoxin B 1 induce mutations. Natural bases in the genomes of viruses or plasmids will be replaced by DNA adducts known to be formed by alkylating agents and by aflatoxin. The genomes containing adducts at specific sites will be constructed by using a combination of chemical synthesis and recombinant DNA techniques. Following their construction, the modified genomes will be introduced into bacterial or mammalian cells, where the adducts will be exposed to and processed by the natural repair and replicative systems of the host. Progeny will be isolated and characterized for the type and frequency of mutation induced at or near the original site of the adduct. The genetic requirements for mutagenesis will be characterized as will the ability of the adduct to affect survival. This study will help to rank the genetic threats posed by the various DNA adducts formed by DNA damaging agents. With aflatoxin, the hypothesis being tested is that some feature of the mutational spectrum of aflatoxin will correlate with the type of mutations induced by one or more of the DNA adducts. With alkylating agents, previous work has defined the types of mutations induced by the most common mutagenic adducts. We have now progressed to a deeper level of biochemical detail in which we are probing the extent to which the context (the neighboring sequence of nucleotides) of a lesion determines the likelihood that the lesion will be repaired or mutate. We are also examining the mutagenic properties of less-well studied adducts, including DNA-DNA crosslinks and N1- and N3 substituted purines and pyrimidines, respectively, which could be important as progenitors to genetic change in humans.
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