Stringent steric exclusion mechanisms limit the misincorporation of ribonucleotides by high-fidelity DNA polymerases into genomic DNA. In contrast, low-fidelity Escherichia coli DNA polymerase V (pol V) has relatively poor sugar discrimination and frequently misincorporates ribonucleotides. Substitution of a steric gate tyrosine residue with alanine (umuC_Y11A) reduces sugar selectivity further and allows pol V to readily misincorporate ribonucleotides as easily as deoxynucleotides, whilst leaving its poor base-substitution fidelity essentially unchanged. However, the mutability of cells expressing the steric gate pol V mutant is very low due to efficient repair mechanisms that are triggered by the misincorporated rNMPs. Comparison of the mutation frequency between strains expressing wild-type and mutant pol V therefore allows us to identify pathways specifically directed at ribonucleotide excision repair (RER). We previously demonstrated that rNMPs incorporated by umuC_Y11A are efficiently removed from DNA in a repair pathway initiated by RNase HII. Using the same approach, we show here that mismatch repair and base excision repair play minimal back-up roles in RER in vivo. In contrast, in the absence of functional RNase HII, umuC_Y11A-dependent mutagenesis increases significantly in ΔuvrA, uvrB5 and ΔuvrC strains, suggesting that rNMPs misincorporated into DNA are actively repaired by nucleotide excision repair (NER) in vivo. Participation of NER in RER was confirmed by reconstituting ribonucleotide-dependent NER in vitro. We show that UvrABC nuclease-catalyzed incisions are readily made on DNA templates containing one, two, or five rNMPs and that the reactions are stimulated by the presence of mispaired bases. Similar to NER of DNA lesions, excision of rNMPs proceeds through dual incisions made at the 8th phosphodiester bond 5′ and 4th–5th phosphodiester bonds 3′ of the ribonucleotide. Ribonucleotides misinserted into DNA can therefore be added to the broad list of helix-distorting modifications that are substrates for NER.

The active form of Escherichia coli DNA polymerase V responsible for damage-induced mutagenesis is a multiprotein complex (UmuD′ 2 C-RecA-ATP), called pol V Mut. Optimal activity of pol V Mut in vitro is observed on an SSB-coated single-stranded circular DNA template in the presence of the β/γ complex and a trans activated RecA nucleoprotein filament, RecA*. Remarkably, under these conditions, wild-type pol V Mut efficiently incorporates ribonucleotides into DNA. A Y11A substitution in the ‘steric gate’ of UmuC further reduces pol V sugar selectivity and converts pol V Mut into a primer-dependent RNA polymerase that is capable of synthesizing long RNAs with a processivity comparable to that of DNA synthesis. Despite such properties, Y11A only promotes low levels of spontaneous mutagenesis in vivo . While the Y11F substitution has a minimal effect on sugar selectivity, it results in an increase in spontaneous mutagenesis. In comparison, an F10L substitution increases sugar selectivity and the overall fidelity of pol V Mut. Molecular modeling analysis reveals that the branched side-chain of L10 impinges on the benzene ring of Y11 so as to constrict its movement and as a consequence, firmly closes the steric gate, which in wild-type enzyme fails to guard against ribonucleoside triphosphates incorporation with sufficient stringency.

We have investigated whether DNA polymerase IV (Pol IV; the dinB gene product) contributes to the error rate of chromosomal DNA replication in Escherichia coli. We compared mutation frequencies in mismatch repair-defective strains that were either dinB positive or dinB deficient, using a series of mutational markers, including lac targets in both orientations on the chromosome. Virtually no contribution of Pol IV to the chromosomal mutation rate was observed. On the other hand, a significant effect of dinB was observed for reversion of a lac allele when the lac gene resided on an F(pro-lac) episome.Several mechanisms control the fidelity of the DNA replication process. These include correct base selection by the DNA polymerase, removal of base insertion errors by 3Ј-exonucleolytic proofreading, and correction by DNA mismatch repair (29). In Escherichia coli, base selection and proofreading are performed by the DNA polymerase III (Pol III) holoenzyme, the enzyme that replicates the bacterial chromosome. It is generally considered a highly accurate enzyme (29). Mismatch repair is performed by the mutHLS mismatch repair system (17). In combination, these three processes yield an error rate of 10 Ϫ9 to 10 Ϫ11 error per base pair replicated per cell division (6,29).In addition to Pol III, E. coli possesses four other DNA polymerases, Pol I, Pol II, Pol IV, and Pol V, whose precise functions are still being defined. Pol IV and Pol V belong to the recently described Y family of DNA polymerases

We investigated the mutator effect resulting from overproduction of Escherichia coli DNA polymerase IV. Using lac mutational targets in the two possible orientations on the chromosome, we observed preferential mutagenesis during lagging strand synthesis. The mutator activity likely results from extension of mismatches produced by polymerase III holoenzyme.