Analysis of DNA replication during the SOS response in Escherichia coli.

Recovery of DNA synthesis in UV-irradiated E. coli. E. coli undergoing the SOS response to DNA damage (e.g. UV-irradiation) displays transient inhibition of DNA synthesis. The mechanism of the inhibition and of the recovery of DNA synthesis following UV-irradition was analysed in several mutants defective in repair or in the regulation of the RecA-LexA dependent SOS response. The results indicated that inhibition is not an inducible function and is probably due to the direct effect of lesions in the template blocking replisome movement. In contrast, recovery of DNA synthesis after UV required protein synthesis and was clearly shown to be an SOS function under RecA control. In addition, analyses involving a recA200 (temperature sensitive RecA) and recA constitutive mutants demonstrated that RecA protein was directly required for recovery in addition to its regulatory role. These experiments however also suggested that an inducible Irr (inducible replisome reactivation) factor under RecA control was also required for post UV-recovery. In vitro mutagenesis. In this part of my study attempts were also made to establish an vitro system for untargeted mutagenesis based upon a plasmid replication system. This involved establishing optimum conditions for extracting plasmid DNA (replicated vitro) and a highly efficient transformation system, in order to analyse directly mutagenic events.