Abstract
Escherichia coli 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 in vitro. Despite efficient error-prone translesion synthesis catalyzed by UmuC_Y11A in vitro, strains expressing umuC_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 umuC_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 in vivo. 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 in vitro. In a recA730 lexA(Def) ΔumuDC ΔdinB strain, plasmid-encoded wild-type pol V promotes high levels of spontaneous mutagenesis. However, umuC_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 ΔrnhB strain and ∼72% of wild-type levels in a ΔrnhA ΔrnhB double mutant. Our observations suggest that errant ribonucleotides incorporated by pol V can be tolerated in the E. coli genome, but at the cost of higher levels of cellular mutagenesis.
Highlights
Translesion synthesis (TLS) allows living organisms to tolerate DNA damage to their genome
By taking a genetic approach, supported by in vitro biochemical data, we show that SOS mutations triggered by pol V–catalyzed errant ribonucleotide incorporation are kept in check by the action of nucleotide excision repair operating in conjunction with RNase HII and, unexpectedly, by another error-prone Y-family polymerase, pol IV
Our studies provide new insight into a growing field investigating the processing of ribonucleotides that are misincorporated by DNA polymerases and how these basic mechanisms contribute to cell survival and mutagenesis
Summary
Translesion synthesis (TLS) allows living organisms to tolerate DNA damage to their genome. The vast majority of TLS in Escherichia coli is catalyzed by the LexA-regulated damageinducible polymerases II, IV and V, which alone, or in various combinations, are recruited to the sites of DNA damage [1]. Pol II-dependent replication of both undamaged and damaged DNA is quite accurate with the exception of an N2acetylaminofluorene adducts, where it promotes 22 frameshifts [3]. Pol IV is remarkably accurate when replicating past certain DNA lesions, such as N2-dG adducts [9]. While pol II and pol IV each appear to facilitate TLS of a narrow range of damaged substrates, pol V is able to accommodate a diverse spectrum of DNA lesions in its active site and bears the greatest burden of TLS in E. coli [1,6,10]. Pol V-dependent TLS is highly error-prone causing the majority of cellular mutagenesis after DNA damage [6,11]
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