Mutator activity and specificity of Escherichia coli dnaQ49 allele – effect of umuDC products

Abstract

The high fidelity of DNA replication in Escherichia coli is ensured by the α (DnaE) and ɛ (DnaQ) subunits of DNA polymerase providing insertion fidelity, 3′ → 5′ exonuclease proofreading activity, and by the dam-directed mismatch repair system. dnaQ49 is a recessive allele that confers a temperature-sensitive proofreading phenotype resulting in a high rate of spontaneous mutations and chronic induction of the SOS response. The aim of this study was to analyse the mutational specificity of dnaQ49 in umuDC and Δ umuDC backgrounds at 28 and 37 °C in a system developed by J.H. Miller. We confirmed that the mutator activity of dnaQ49 was negligible at 28 °C and fully expressed at 37 °C. Of the six possible base pair substitutions, only GC → AT transitions and GC → TA and AT → TA transversions were appreciably increased. However, the most numerous mutations were frameshifts, −1G deletions and +1A insertions. All mutations which increased in response to dnaQ49 damage were to a various extent umuDC-dependent, especially −1G deletions. This type of mutations decreased in CC108 dnaQ49Δ umuDC to 10% of the value found in CC108 dnaQ49umuDC + and increased in the presence of plasmids producing UmuD′C or UmuDC proteins. In the recovery of dnaQ49 mutator activity the plasmid harbouring umuD′ C genes was more effective than the one harbouring umuDC. Analysis of mutational specificity of pol III with defective ɛ subunit indicates that continuation of DNA replication is allowed past G:T, C:T, T:T (or C:A, G:A, A:A) mismatches but does not allow for acceptance of T:C, C:C, A:C (or A:G, G:G, T:G) (the underlined base is in the template strand).