The Escherichia coli UVM response is accompanied by an SOS-independent error-prone DNA replication activity demonstrablein vitro.

UVM is an SOS-independent inducible response characterized by elevated mutagenesis at a sitespecific 3, N[sup 4]-ethenocytosine (εC) residue borne on M13 single-stranded DNA transfected into Escherichia coli cells pretreated with DNA-damaging agents. By constructing and using E. coli strain AM124 (polA polB umuDC dinB lexA1[Ind-]), we show here that the UVM response is manifested in cells deficient for SOS induction, as well as for all four of the 'nonreplicative' DNA polymerases, namely DNA polymerase I (polA), II (polB), IV (dinB) and V (umuDC). These results confirm that UVM represents a novel, previously unidentified cellular response to DNAdamaging agents. To address the question as to whether the UVM response is accompanied by an error-prone DNA replication activity, we applied a newly developed in vitro replication assay coupled to an in vitro mutation analysis system. In the assay, circular M13 single-stranded DNA bearing a sitespecific lesion is converted to circular doublestranded replicative-form DNA in the presence of cell extracts and nucleotide precursors under conditions that closely mimic M13 replication in viva. The newly synthesized (minus) DNA strand is selectively amplified by ligation-mediated polymerase chain reaction (LM-PCR), followed by a multiplex sequence analysis to determine the frequency and specificity of mutations. Replication of DNA bearing a site-specific εC lesion by cell extracts from uninduced E. coli AM124 cells results in a mutation frequency of about 13%. Mutation frequency is elevated fivefold (to 58%) in cell extracts from UVM-induced AM124 cells, with C → A mutations predominating over C → T mutations, a specificity similar to that observed in viva. These results, together with previously reported data, suggest that the UVM response is mediated through the induction of a transient error-prone DNA replication activity and that a modification of DNA polymerase III or the expression of a pre... [ABSTRACT FROM AUTHOR]