In vitro replication and mutagenesis of ColE1 plasmid DNA in extracts from repair deficient Escherichia coli mutants.
We have investigated conditions in vitro for the analysis of replication of ultraviolet-irradiated ColE1 DNA in cell extracts from Escherichia coli. In wild-type extracts substantial replication was obtained; however, this could be greatly reduced when the irradiated plasmid was incubated in extracts prepared from a uvrA recB strain. Modest stimulation of DNA replication was then obtained by addition of extracts from the same strain previously ultraviolet-irradiated. However, this stimulating activity proved to be highly unstable and has so far proved unsuitable as a basis for purification of specific factors involved in replication on irradiated templates. We also investigated the mutagenesis of pBR325 DNA replicated in cell extracts from a strain expressing the SOS response constitutively. Conditions for efficient recovery and transformation by plasmid DNA replicated in vitro were determined and, using this system, a more than 10-fold increase in reversion frequency of a mutation in the tet gene, compared to that with wild-type extracts, was obtained. This mutagenesis appeared to be independent of replication, indicating the presence of an error-prone repair system in the extract. This effect was not enhanced by the presence of the muc gene products in the extracts. This suggests that the observed mutagenesis is also independent of the lexA-controlled umuCD genes.
Casaregola
S
S
Department of Genetics, University of Leicester, U.K.
Khidhir
M
M
Holland
I B
IB
D003086:Bacteriocin Plasmids / Q000528:radiation effects; D004260:DNA Repair; D004261:DNA Replication / Q000528:radiation effects; D004269:DNA, Bacterial / Q000096:biosynthesis / Q000302:isolation & purification; D004926:Escherichia coli / Q000235:genetics* / Q000528:radiation effects; D007700:Kinetics; D009154:Mutation; D010957:Plasmids / Q000528:radiation effects; D013014:SOS Response, Genetics; D014466:Ultraviolet Rays
D004269:DNA, Bacterial