Site-Specific Mutagenesis:: A New Approach for Studying the Molecular Mechanisms of Mutation by Carcinogens

Summary

Most chemical carcinogens are also mutagens. Usually, they are also electrophilic reagents that react at high electron density regions in DNA forming covalent adducts. It is generally believed that these adducts occasionally lead to mutations during replication and/or repair. There is no direct experimental evidence bearing on the molecular mechanisms that are responsible for producing mutations from these adducts. To complicate matters, there are at least 18 distinct sites where adducts can form. Not all sites react with a given carcinogen in vivo under a given set of conditions, but many different adducts are almost always formed. In order to gain a better understanding of how carcinogens produce mutations, and, hopefully, to obtain some insight into the relationship between mutagenesis and carcinogenesis, we need to answer the following questions: (1) Which of the covalent adducts that form when a carcinogen reacts with DNA actually produce mutations? (2) What kind of mutation does each different premutational lesion produce? (3) What role do the various DNA repair systems play in producing these mutations?

We have developed a site-specific mutagenesis system that is capable of answering these questions directly and unambiguously. The system involves gene G of bacteriophage 0X174. Through a combination of chemical and enzymatic procedures we are able to introduce the covalent adduct to be studied at a single preselected site in this essential gene. The site-modified DNA produced is studied in vivo by transfection of spheroplasts. Since one of the strands in the modified RF DNA is wild type, all of the normal viral proteins are produced in the spheroplast and infectious mutant viruses are assembled even when the mutation is lethal. Mutants that are produced, regardless of their nature, are identified and propagated using a host cell carrying a functional copy of #x00F8;X gene G on a plasmid. DNA isolated from different mutants is sequenced in the region that carried the original covalent adduct in order to identify the nature of the mutation unambiguously: By studying the same type of adduct (e.g., a methyl group) at different positions in the purine or pyrimidine rings, one at a time, it should be possible to determine which adducts are mutagenic and which are not. Furthermore, the same site-specific DNA adduct can be studied in spheroplasts derived from cells carrying mutations that produce defects in various DNA repair systems. By comparing mutant frequencies and mutant types produced in these different repair backgrounds, important information concerning the role of DNA repair and producing mutations from different kinds of lesions should be obtained.