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. An in vivo mutational assay was developed which demonstrated a strong mutational response when depurinated M13 molecules were transfected in SOS-induced cells. To allow the in vitro analog of this system to be tested, procedures were developed for the efficient replication of M13 ssDNA by crude E. coli extracts to yield RFI DNA, and the subsequent recovery of the product DNA for transfection to yield sufficient numbers of infective centers for an analysis of mutation. Preliminary results indicate that DNA replication as it occurs in the crude extract system is extremely accurate and may approach values expected in vivo.
