Figure 2. Spectra of spontaneously arising rpoB mutations in recA730 lexA(Def) ΔumuDC ΔdinB strains expressing umuC_Y11A and proficient- or deficient- in MMR and RNase HII- mediated RER.

(A), Types of base-pair substitutions generated in the rpoB gene of mismatch repair defective recA730 lexA(Def) ΔdinB ΔumuDC mutL rnhB+/− strains. The arrows indicate mutagenic hot spots within rpoB. The spectra for the rnhB ⁺ strain (RW710) are taken from [14] and are shown for direct comparison with the spectra for the isogenic ΔrnhB strain (RW942). The forward mutation rates in the MMR⁻ strains was assayed by measuring resistance to rifampicin and were calculated as 1.22±0.2×10⁻⁶ for RW710 and 1.82±0.2×10⁻⁶ for RW942. As expected, because of defects in MMR, the spectra are dominated by transition mutations. However, we have previously reported that low-fidelity pol V makes a significant number of transversion mutations compared to other E.coli DNA polymerases [25] and we note a 4-fold increase in the number of transversion mutations in the ΔrnhB mutL strain expressing umuC_Y11A compared to the mutL rnhB ⁺ strain (see Table S1) consistent with pol V-dependent errors. The rnhB ⁺ mutL strain lacks these transversion mutations, as it undergoes active pol I-dependent RER and as a consequence, the spectrum generated in this strain actually reflects uncorrected pol I-dependent errors, rather than pol V-dependent errors. (B), Types of base-pair substitutions generated in the rpoB gene of mismatch repair proficient recA730 lexA(Def) ΔdinB ΔumuDC rnhB+/− strains. The forward mutation rates in the MMR⁺ strains assayed by measuring resistance to rifampicin and were calculated as 4.8±0.8×10⁻⁸ for RW698 and 2.5±0.4×10⁻⁷ for RW838. As expected, because the strains are proficient in MMR and transitions are repaired efficiently, the spectra are dominated by the poorly repaired transversion mutations. Approximately 300 Rif^R mutants were analyzed for each strain (Table S1). However, the spectrum of the rnhB ⁺ strain has been normalized to reflect the overall ∼6-fold lower mutation rate compared to the ΔrnhB strain. One can clearly observe that the spectra are very different in the two strains, with changes in both the types of mutations and locations of mutagenic hot-spots. Again, we believe the data support the model that in the MMR⁺ strains, pol I is responsible for low-levels of mutagenic events generated during RNase HII-pol I dependent RER, whilst in the absence of RER, umuC_Y11A mutations characterized by frequent transversion events persist and lead to a 6-fold increase in mutation rate.