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.

Low fidelity Escherichia coli DNA polymerase V (pol V/UmuD′2C) is best characterized for its ability to perform translesion synthesis (TLS). However, in recA730 lexA(Def) strains, the enzyme is expressed under optimal conditions allowing it to compete with the cell’s replicase for access to undamaged chromosomal DNA and leads to a substantial increase in spontaneous mutagenesis. We have recently shown that a Y11A substitution in the “steric gate” residue of UmuC reduces both base and sugar selectivity of pol V, but instead of generating an increased number of spontaneous mutations, strains expressing umuC_Y11A are poorly mutable in vivo. This phenotype is attributed to efficient RNase HII-initiated repair of the misincorporated ribonucleotides that concomitantly removes adjacent misincorporated deoxyribonucleotides. We have utilized the ability of the pol V steric gate mutant to promote incorporation of large numbers of errant ribonucleotides into the E. coli genome to investigate the fundamental mechanisms underlying ribonucleotide excision repair (RER). Here, we demonstrate that RER is normally facilitated by DNA polymerase I (pol I) via classical “nick translation”. In vitro, pol I displaces 1–3 nucleotides of the RNA/DNA hybrid and through its 5′→3′ (exo/endo) nuclease activity releases ribo- and deoxyribonucleotides from DNA. In vivo, umuC_Y11A-dependent mutagenesis changes significantly in polymerase-deficient, or proofreading-deficient polA strains, indicating a pivotal role for pol I in ribonucleotide excision repair (RER). However, there is also considerable redundancy in the RER pathway in E. coli. Pol I’s strand displacement and FLAP- exo/endonuclease activities can be facilitated by alternate enzymes, while the DNA polymerization step can be assumed by high-fidelity pol III. We conclude that RNase HII and pol I normally act to minimize the genomic instability that is generated through errant ribonucleotide incorporation, but that the “nick-translation” activities encoded by the single pol I polypeptide can be undertaken by a variety of back-up enzymes.

We have developed a series of plasmid vectors for the soluble expression and subsequent purification of recombinant proteins that have historically proven to be extremely difficult to purify from Escherichia coli. Instead of dramatically overproducing the target protein, it is expressed at a low basal level that facilitates the correct folding of the recombinant protein and increases its solubility. Highly active recombinant proteins that are traditionally difficult to purify are readily purified using standard affinity tags and conventional chromatography. To demonstrate the utility of these vectors, we have expressed and purified full-length human DNA polymerases η, ι, and ν from E. coli and show that the purified DNA polymerases are catalytically active in vitro.

Gearhart and collaborators address the long-standing question of the roles of error-prone DNA polymerases in somatic hypermutation of antibody genes.

The integrating conjugative element ICE391 (formerly known as IncJ R391) harbors an error-prone DNA polymerase V ortholog, polVICE391, encoded by the ICE391 rumAB operon. polV and its orthologs have previously been shown to be major contributors to spontaneous and DNA damage-induced mutagenesis in vivo. As a result, multiple levels of regulation are imposed on the polymerases so as to avoid aberrant mutagenesis. We report here, that the mutagenesis-promoting activity of polVICE391 is additionally regulated by a transcriptional repressor encoded by SetRICE391, since Escherichia coli expressing SetRICE391 demonstrated reduced levels of polVICE391–mediated spontaneous mutagenesis relative to cells lacking SetRICE391. SetRICE391 regulation was shown to be specific for the rumAB operon and in vitro studies with highly purified SetRICE391 revealed that under alkaline conditions, as well as in the presence of activated RecA, SetRICE391 undergoes a self-mediated cleavage reaction that inactivates repressor functions. Conversely, a non-cleavable SetRICE391 mutant capable of maintaining repressor activity, even in the presence of activated RecA, exhibited low levels of polVICE391-dependent mutagenesis. Electrophoretic mobility shift assays revealed that SetRICE391 acts as a transcriptional repressor by binding to a site overlapping the −35 region of the rumAB operon promoter. Our study therefore provides evidence indicating that SetRICE391 acts as a transcriptional repressor of the ICE391-encoded mutagenic response.