The SOS system of Escherichia coli plays a central role in mutagenesis in this 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 gene products, several of which are postulated 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. It uses the conversion of single-stranded bacteriophage M13 DNA into its double- stranded (RF) form by cell-free extracts. After replication, the product DNA is transfected to produce bacteriophage, and the accuracy of the in vitro replication step is then determined from the mutant frequency-among phage before and after replication. We have found DNA replication in E. coli extracts to be extremely accurate, with error rates approaching (or identical to) estimated in vivo rates. Extracts derived from two different E. coli mutator strains, mutD and mutT, display greatly enhanced error rates, indicating the validity of the system for probing in vivo error rates. The system is currently being used to determine the accuracy of extracts derived from SOS-induced cells. In a parallel approach, we are attempting to isolate new E. coli derivatives that have reduced capacity for SOS mutagenesis, focusing in particular on the dnaE and dnaQ genes. These genes encode, respectively, the DNA polymerase (alpha subunit) and the exonucleolytic proofreading activity (epsilon subunit) of Pol III holoenzyme, the enzyme responsible for the replication of the bacterial chromosome and involved in the fixation of mutations during SOS mutagenesis. Such mutants are expected to yield, by a combination of in vivo and in vitro approaches, new insights into the mechanisms of SOS mutagenesis.
