The SOS system of Escherichia coli plays a crucial role in many aspects of mutagenesis in the organism. The system is not normally present in the cell but becomes induced upon blockage of DNA replication by DNA damage. Its induction entails the expression of a large number of new gene products, several of which are thought to interact with the process of DNA replication, rendering it error prone and producing mutations on both damaged and undamaged DNA. The evidence for the existence of these components rests largely on genetic experiments. However, the elucidation of the nature of these components and their mechanisms of action requires a more direct biochemical approach. We have designed an in vitro DNA replication system in which the existence of the error-prone replication components may be tested. The system uses the conversion of single-stranded bacteriophage M13 DNA into its double-stranded form (ss RF conversion) by crude extracts derived from either normal or SOS-induced cells. After replication, the product DNA is transfected to produce intact bacteriophage. The accuracy of the in vitro replication step is then assayed by accuracy of DNA replication in crude extracts is extremely high and resembles the in vivo accuracy. Our early efforts have already demonstrated that an understanding of the accuracy of DNA replication in SOS-induced cells requires a full understanding of the factors that are involved in maintaining normal accuracy. This aspect is therefore pursued simultaneously. E. coli mutator and antimutator strains with known (or presumed) defects in the process of DNA replication are essential tools in these studies.
