In this project the mechanisms of mutagenesis are investigated through a detailed study of its specificity. In this approach, DNA sequence information is gathered on all the classes of mutations that occur under a given condition, e.g., base substitutions, frameshifts, deletions, duplications, insertion elements, complex rearrangements, etc. These classes have their own dependence on the local DNA sequence and generally result from different mutational pathways. The specificity of mutation can thus be used to separate and analyze the various contributing components of mutation. Our system uses the lacI gene of the bacterium E. coli. This gene codes for the repressor of the lac operon. Forward mutations to lacI-are selected on the basis of the constitutive expression of the operon. The lacI- genes (typically several hundreds at a time) are transferred by in vivo recombination to a single-stranded recombinant-phage vector and sequenced, yielding the mutational spectrum of interest. The power of the system can be increased by the comparison of spectra in strains that are affected in DNA repair or replication pathways, such as mutator or antimutator strains. When the affected enzymatic pathways are known (e.g. mutD or mutH,L,S strains) the observed spectra can be used to directly correlate mutational classes with specific enzymatic pathways. In case of induced mutagenesis the specificity of mutation can, in addition, be used to identify the nature of the premutagenic lesions. Examples of current interest to our lab are mutagenesis by UV light and the chemical carcinogen N- acetoxyacetylaminofluorene (NAAAF). In these cases correlation of DNA damage spectra with mutational spectra, in number of different repair or replication backgrounds, provides insights into the responsible mutagenic lesion(s) as well as the mechanisms by which they are converted into mechanisms.
