Replication of M13 single-stranded DNA bearing a site-specific ethenocytosine lesion by Escherichia coil cell extracts

Previous investigation on the mutagenic effects of 3, N⁴-Ethenocytosine (εC), a nonpairing DNA lesion, revealed the existence of a novel SOS-independent inducible mutagenic mechanism in E. coli termed UVM for UV modulation of mutagenesis. To investigate whether UVM is mediated by an alteration of DNA replication, we have set up an in vitro replication system in which phage M13 viral single-stranded DNA bearing a single site-specific (εC) residue is replicated by soluble protein extracts from E. coli cells. Replication products were analyzed by agarose gel electrophoresis and the frequency of translesion synthesis was determined by restriction endonuclease analyses. Our data indicate that DNA replication is strongly inhibited by εC, but that translesion DNA synthesis does occur in about 14% of the replicated DNA molecules. These results are very similar to those observed previously in vivo, and suggest that this experimental system may be suitable for evaluating alterations in DNA replication in UVM-induced cells.