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 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 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. 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 the nature of spontaneous mutations, as well as the nature of primary DNA replication errors. These studies also defined the specific contributions of exonucleolytic proofreading and DNA mismatch repair during DNA replication. For induced mutagenesis, we have focused on mutagenesis by ultraviolet light (UV). The nature and location of the UV-induced mutations should provide insights into the responsible mechanisms. We, and others, have shown that the large majority of UV-induced mutations occurs at adjacent pyrimidines in the DNA sequence, implicating cyclobutane pyrimidine dimers and (6-4) pyrimidine-pyrimidone lesions (the two predominant UV-induced lesions in DNA) as premutagenic lesions. Such analysis has been performed in excision-repair proficient and deficient strains as well as mfd strains, deficient in the factor that stimulates repair of lesions in the transcribed DNA strand. These studies allow a further understanding of the pathways by which UV-induced lesions are processed through transcription, repair and replication, ultimately leading to a mutation.
