In this project the mechanisms of mutagenesis are investigated through the detailed study of its specificity. This is pursued by DNA sequencing of mutations in the Escherichia coli lacI gene, occurring either spontaneously in defined genetic backgrounds or after treatment with a mutagen. The specificity of mutation provides a way to dissect the components of mutagenesis since different pathways and mechanisms produce characteristic patterns of mutation with regard to type (base substitution, frameshift, deletion, duplication, insertion element, etc.) and DNA sequence dependence. The lacI gene encodes the repressor of the lac operon and forward mutations to lacI- can be scored based on their constitutive expression of the operon. The mutant genes (several hundreds at a time), are then transferred by in vivo recombination to a single-stranded phage vector and sequenced. In order to understand the pathways that contribute to spontaneous mutations, we have analyzed spectra of mutations in the E. coli wild-type strain and in its mutH, mutL, mutS, mutT, mutD and mutY derivatives. This has allowed us to determine not only the nature of spontaneous mutations, but also the nature of primary DNA replication errors and the specific contributions of exonucleolytic proofreading and DNA mismatch repair to preventing mutations resulting from replication errors. In most recent research, we have initiated a search for E. coli antimutator strains, i.e., strains that have a lower mutation frequency than the wild-type strain. The determination of the genetic defect in such strains is likely to provide pivotal insights into the pathways that contribute to mutation. One set of antimutators that we have isolated is affected in the dnaE gene, which encodes DNA polymerase III, responsible for the replication of the bacterial chromosome. These strains may allow us to define the specific contribution of DNA replication errors to spontaneous mutation. Analogously, we will attempt to isolate antimutators that are affected in other defined pathways that are assumed to contribute to spontaneous mutation, such as the occurrence of DNA damage.
