The objective of this project is to study the molecular events that lead to mutation in the bacterium E. coli after induction of the SOS-response. The error-prone type of DNA replication that is presumed to be responsible for SOS-mutagenesis will be studied in an in vitro replication system. The accuracy with which crude extracts of E. coli cells copy normal or damaged single-stranded bacteriophage M13 DNA will be used as an indicator for in vitro SOS-expression. Characterization of the components involved is important for the study of SOS-mutagenesis and for the question of the regulation of mutation rates in general. The crude-extract replication system was developed to the point that reliable estimates can be made about the accuracy of phage DNA replication in vitro. The accuracy is extremely high and resembles the values expected for in vivo replication. The system is currently used to further define the parameters that determine this accuracy and for a comparison of these parameters in normal and SOS-induced extracts.
