Mechanisms Employed by <em>Escherichia coli</em> to Prevent Ribonucleotide Incorporation into Genomic DNA by Pol V

<div><p><em>Escherichia coli</em> pol V (UmuD′₂C), the main translesion DNA polymerase, ensures continued nascent strand extension when the cellular replicase is blocked by unrepaired DNA lesions. Pol V is characterized by low sugar selectivity, which can be further reduced by a Y11A “steric-gate” substitution in UmuC that enables pol V to preferentially incorporate rNTPs over dNTPs <em>in vitro.</em> Despite efficient error-prone translesion synthesis catalyzed by UmuC_Y11A <em>in vitro</em>, strains expressing <em>umuC</em>_Y11A exhibit low UV mutability and UV resistance. Here, we show that these phenotypes result from the concomitant dual actions of Ribonuclease HII (RNase HII) initiating removal of rNMPs from the nascent DNA strand and nucleotide excision repair (NER) removing UV lesions from the parental strand. In the absence of either repair pathway, UV resistance and mutagenesis conferred by <em>umuC</em>_Y11A is significantly enhanced, suggesting that the combined actions of RNase HII and NER lead to double-strand breaks that result in reduced cell viability. We present evidence that the Y11A-specific UV phenotype is tempered by pol IV <em>in vivo</em>. At physiological ratios of the two polymerases, pol IV inhibits pol V–catalyzed translesion synthesis (TLS) past UV lesions and significantly reduces the number of Y11A-incorporated rNTPs by limiting the length of the pol V–dependent TLS tract generated during lesion bypass <em>in vitro</em>. In a <em>recA730 lexA</em>(Def) Δ<em>umuDC</em> Δ<em>dinB</em> strain, plasmid-encoded wild-type pol V promotes high levels of spontaneous mutagenesis. However, <em>umuC</em>_Y11A-dependent spontaneous mutagenesis is only ∼7% of that observed with wild-type pol V, but increases to ∼39% of wild-type levels in an isogenic Δ<em>rnhB</em> strain and ∼72% of wild-type levels in a Δ<em>rnhA</em> Δ<em>rnhB</em> double mutant. Our observations suggest that errant ribonucleotides incorporated by pol V can be tolerated in the <em>E. coli</em> genome, but at the cost of higher levels of cellular mutagenesis.</p>