Mechanisms for induction and suppression of spontaneous and induced mutations

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Alkylating agents are potent mutagens and carcinogens and sometimes cause cell death. These effects of alkylating agents are mainly attributed to the formation of various alkylated bases in DNA. Among them, O^6-methylguanine appears to be most responsible for induction of mutations as well as of cancers. Many organisms possess an enzyme, O^6-methylguanine-DNA methyltransferase, that repairs O^6-methylguanine in DNA. It transfers a methylgroup from methylated DNA to a cysteine residue of the enzyme molecule itself. In Escherichia coli the methyltransferase enzyme (Ada protein) and other enzymes involved in repair of alkylation lesions in DNA are induced on response of cell to a relatively low dose of alkylating agent. This process, termed the adaptive response, is controlled by the ada gene, whose product is a repair enzyme as well as a transcriptional regulator.It has been demonstrated that about one-fifth of human tumor cell lines show the Mer^- phenotype, and that the Mer^- strains are deficient in the methyltransferase activity. By cDNA transfection, we were able to obtain a cell line possessing an extremely high level of O^6-methylguanine-DNA methyltransferase activity. The cDNA for methyltransferase was recovered from the secondary transformant by the polymerase chain reaction and its nucleotide sequence was determined.To elucidate molecular basis of the chemical mutagenesis by acetylaminofluorene(AAF), effects of this compound on the DNA replication were studied. Dynamics of E. coli DNA polymerase III on template DNA with a single AAF adduct lesion at a particular site was analized by a method newly developed in this work. We found that the DNA polymerase III stopped DNA synthesis at one nucleotide before the adduct site on the template and no obvious bypass replication occured. Further, we obtained evidence that a structural alteration of the template DNA by the AAF adduct attenuates the chain elongation.