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Homologs of the mutagenic Escherichia coli DNA polymerase V (pol V) are encoded by numerous pathogens and mobile elements. We have used Rum pol (RumA′2B), from the integrative conjugative element (ICE), R391, as a model mobile element-encoded polymerase (MEPol). The highly mutagenic Rum pol is transferred horizontally into a variety of recipient cells, including many pathogens. Moving between species, it is unclear if Rum pol can function on its own or requires activation by host factors. Here, we show that Rum pol biochemical activity requires the formation of a physical mutasomal complex, Rum Mut, containing RumA′2B-RecA-ATP, with RecA being donated by each recipient bacteria. For R391, Rum Mut specific activities in vitro and mutagenesis rates in vivo depend on the phylogenetic distance of host-cell RecA from E. coli RecA. Rum pol is a highly conserved and effective mobile catalyst of rapid evolution, with the potential to generate a broad mutational landscape that could serve to ensure bacterial adaptation in antibiotic-rich environments leading to the establishment of antibiotic resistance.

The Escherichia coli dnaE gene encodes the α‐catalytic subunit (pol IIIα) of DNA polymerase III, the cell’s main replicase. Like all high‐fidelity DNA polymerases, pol III possesses stringent base and sugar discrimination. The latter is mediated by a so‐called “steric gate” residue in the active site of the polymerase that physically clashes with the 2′‐OH of an incoming ribonucleotide. Our structural modeling data suggest that H760 is the steric gate residue in E.coli pol IIIα. To understand how H760 and the adjacent S759 residue help maintain genome stability, we generated DNA fragments in which the codons for H760 or S759 were systematically changed to the other nineteen naturally occurring amino acids and attempted to clone them into a plasmid expressing pol III core (α‐θ‐ε subunits). Of the possible 38 mutants, only nine were successfully sub‐cloned: three with substitutions at H760 and 6 with substitutions at S759. Three of the plasmid‐encoded alleles, S759C, S759N, and S759T, exhibited mild to moderate mutator activity and were moved onto the chromosome for further characterization. These studies revealed altered phenotypes regarding deoxyribonucleotide base selectivity and ribonucleotide discrimination. We believe that these are the first dnaE mutants with such phenotypes to be reported in the literature., The accurate replication of every living organisms’ genome is achieved by high fidelity DNA polymerases. The enzymes not only need to choose the nucleotide with the correctly paired base (G, A, T or C), but also the right sugar (deoxyribonucleotide, not ribonucleotide). We report here, the construction and characterization of three novel active site mutants of the α‐catalytic subunit of Escherichia coli DNA polymerase III that have altered phenotypes with regard to base and sugar discrimination.

When subcloned into low-copy-number expression vectors, rumAB, encoding polVR391 (RumA'2 B), is best characterized as a potent mutator giving rise to high levels of spontaneous mutagenesis in vivo. This is in dramatic contrast to the poorly mutable phenotype when polVR391 is expressed from the native 88.5 kb R391, suggesting that R391 expresses cis-acting factors that suppress the expression and/or the activity of polVR391 . Indeed, we recently discovered that SetRR391 , an ortholog of λ cI repressor, is a transcriptional repressor of rumAB. Here, we report that CroSR391 , an ortholog of λ Cro, also serves as a potent transcriptional repressor of rumAB. Levels of RumA are dependent upon an interplay between SetRR391 and CroSR391 , with the greatest reduction of RumA protein levels observed in the absence of SetRR391 and the presence of CroSR391 . Under these conditions, CroSR391 completely abolishes the high levels of mutagenesis promoted by polVR391 expressed from low-copy-number plasmids. Furthermore, deletion of croSR391 on the native R391 results in a dramatic increase in mutagenesis, indicating that CroSR391 plays a major role in suppressing polVR391 mutagenesis in vivo. Inactivating mutations in CroSR391 therefore have the distinct possibility of increasing cellular mutagenesis that could lead to the evolution of antibiotic resistance of pathogenic bacteria harboring R391.

High transmissivity aquifers typically have low hydraulic gradients (i.e., a flat water table). Measuring low gradients using water levels can be problematic because measurement error may be greater than the true difference in water levels (i.e., a low signal-to-noise ratio). In this study, the feasibility of measuring a hydraulic gradient in the range of 10-6 to 10-5 m/m was demonstrated. The study was performed at a site where the depth to water from land surface ranged from 40.1 to 94.2 m and the aquifer transmissivity was estimated at 41,300 m2 /d (hydraulic conductivity of 18,800 m/d). The goals of the study were to reduce measurement error as much as practicable and assess the importance of factors affecting water level measurement accuracy. Well verticality was the largest source of error (0.000 to 0.168 m; median of 0.014 m), and geodetic survey of casing elevations was the next most important source of error (0.002 to 0.013 m; median of 0.005 m). Variability due to barometric pressure fluctuations was not an important factor at the site. Hydraulic heads were measured to an accuracy of ±0.0065 m, and the average hydraulic gradient was estimated to be 8.0 × 10-6 (±0.9 × 10-6 ) m/m. The improvement in accuracy allowed for two reversals in the groundwater flow direction to be identified, after which the gradient averaged 2.5 × 10-5 (±0.4 × 10-5 ) m/m. This study showed it is possible to sufficiently control sources of error to measure hydraulic gradients in the 10-6 to 10-5 m/m range.

When subcloned into low‐copy‐number expression vectors, rumAB, encoding polVR391 (RumA′2B), is best characterized as a potent mutator giving rise to high levels of spontaneous mutagenesis in vivo. This is in dramatic contrast to the poorly mutable phenotype when polVR391 is expressed from the native 88.5 kb R391, suggesting that R391 expresses cis‐acting factors that suppress the expression and/or the activity of polVR391. Indeed, we recently discovered that SetRR391, an ortholog of λ cI repressor, is a transcriptional repressor of rumAB. Here, we report that CroSR391, an ortholog of λ Cro, also serves as a potent transcriptional repressor of rumAB. Levels of RumA are dependent upon an interplay between SetRR391 and CroSR391, with the greatest reduction of RumA protein levels observed in the absence of SetRR391 and the presence of CroSR391. Under these conditions, CroSR391 completely abolishes the high levels of mutagenesis promoted by polVR391 expressed from low‐copy‐number plasmids. Furthermore, deletion of croS R391 on the native R391 results in a dramatic increase in mutagenesis, indicating that CroSR391 plays a major role in suppressing polVR391 mutagenesis in vivo. Inactivating mutations in CroSR391 therefore have the distinct possibility of increasing cellular mutagenesis that could lead to the evolution of antibiotic resistance of pathogenic bacteria harboring R391., When subcloned from R391 into low‐copy‐number expression vectors, polVR391 (RumA′2B) encoded by rumAB, is a potent mutator giving rise to high levels of spontaneous mutagenesis in vivo. This is in contrast to the poorly mutable phenotype when polVR391 is expressed from the native R391. Here, we report that CroSR391, an ortholog of λ Cro, serves as a potent transcriptional repressor of rumAB that plays a major role in suppressing polVR391 mutagenesis in vivo.

Several functions have been proposed for the Escherichia coli DNA polymerase IV (pol IV). Although much research has focused on a potential role for pol IV in assisting pol III replisomes in the bypass of lesions, pol IV is rarely found at the replication fork in vivo. Pol IV is expressed at increased levels in E. coli cells exposed to exogenous DNA damaging agents, including many commonly used antibiotics. Here we present live-cell single-molecule microscopy measurements indicating that double-strand breaks induced by antibiotics strongly stimulate pol IV activity. Exposure to the antibiotics ciprofloxacin and trimethoprim leads to the formation of double strand breaks in E. coli cells. RecA and pol IV foci increase after treatment and exhibit strong colocalization. The induction of the SOS response, the appearance of RecA foci, the appearance of pol IV foci and RecA-pol IV colocalization are all dependent on RecB function. The positioning of pol IV foci likely reflects a physical interaction with the RecA* nucleoprotein filaments that has been detected previously in vitro. Our observations provide an in vivo substantiation of a direct role for pol IV in double strand break repair in cells treated with double strand break-inducing antibiotics.

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.

pol V(ICE391) (RumA′(2)B) is a low-fidelity polymerase that promotes considerably higher levels of spontaneous “SOS-induced” mutagenesis than the related E. coli pol V (UmuD′2C). The molecular basis for the enhanced mutagenesis was previously unknown. Using single molecule fluorescence microscopy to visualize pol V enzymes, we discovered that the elevated levels of mutagenesis are likely due, in part, to prolonged binding of RumB to genomic DNA leading to its increased levels of DNA synthesis compared to UmuC. We have generated a steric gate pol V(ICE391) variant (pol V(ICE391)_Y13A) that readily misincorporates ribonucleotides into the E. coli genome and have used the enzyme to investigate the molecular mechanisms of Ribonucleotide Excision Repair (RER) under conditions of increased ribonucleotide-induced stress. To do so, we compared the extent of spontaneous mutagenesis promoted by pol V and pol V(ICE391) to that of their respective steric gate variants. Levels of mutagenesis promoted by the steric gate variants that are lower than that of the wild-type enzyme are indicative of active RER that removes misincorporated ribonucleotides, but also misincorporated deoxyribonucleotides from the genome. Using such an approach, we confirmed that RNase HII plays a pivotal role in RER. In the absence of RNase HII, Nucleotide Excision Repair (NER) proteins help remove misincorporated ribonucleotides. However, significant RER occurs in the absence of RNase HII and NER. Most of the RNase HII and NER-independent RER occurs on the lagging strand during genome duplication. We suggest that this is most likely due to efficient RNase HI-dependent RER which recognizes the polyribonucleotide tracts generated by pol V(ICE391)_Y13A. These activities are critical for the maintenance of genomic integrity when RNase HII is overwhelmed, or inactivated, as ΔrnhB or ΔrnhB ΔuvrA strains expressing pol V(ICE391)_Y13A exhibit genome and plasmid instability in the absence of RNase HI.

It is now close to 40 years since the isolation of non-mutable umu/uvm strains of Escherichia coli and the realization that damage induced mutagenesis in E.coli is not a passive process. Early models of mutagenesis envisioned the Umu proteins as accessory factors to the cell's replicase that not only reduced its normally high fidelity, but also allowed the enzyme to traverse otherwise replication-blocking lesions in the genome. However, these models underwent a radical revision approximately 15 years ago, with the discovery that the Umu proteins actually encode for a DNA polymerase, E.coli pol V. The polymerase lacks 3'→5' exonucleolytic proofreading activity and is inherently error-prone when replicating both undamaged and damage DNA. So as to limit any "gratuitous" mutagenesis, the activity of pol V is strictly regulated in the cell at multiple levels. This review will summarize our current understanding of the myriad levels of regulation imposed on pol V including transcriptional control, posttranslational modification, targeted proteolysis, activation of the catalytic activity of pol V through protein-protein interactions and the very recently described intracellular spatial regulation of pol V. Remarkably, despite the multiple levels at which pol V is regulated, the enzyme is nevertheless able to contribute to the genetic diversity and evolutionary fitness of E.coli.

Under many conditions the killing of bacterial cells by antibiotics is potentiated by damage induced by reactive oxygen species (ROS). In most bacteria, ROS primarily target biomolecules such as proteins and DNA. Damage to DNA, particularly in the form of double-strand breaks (DSBs), is a major contributor to cell death. DNA polymerase IV (pol IV), an error-prone DNA polymerase produced at elevated levels in cells experiencing DNA damage, has been implicated both in ROS-dependent killing and in DSB repair (DSBR). Here, we show using single-molecule fluorescence microscopy that ROS-induced DSBs promote pol IV activity in two ways. First, exposure to the DNA-damaging antibiotics ciprofloxacin and trimethoprim triggers an SOS-mediated increase in intracellular pol IV concentration that is strongly dependent on both ROS and DSBR. Second, in cells that constitutively express pol IV, co-treatment with a ROS mitigator dramatically reduces the number of DSBs as well as pol IV foci formed, indicating a role of pol IV in the repair of ROS-induced DSBs. Significance Many antibiotics induce an accumulation of reactive oxygen species (ROS) in bacterial cells. ROS-induced damage to DNA, in particular formation of double-strand breaks (DSBs), potentiates killing by several bactericidal antibiotics. Here we used single-molecule fluorescence microscopy to reveal new links between ROS-induced DSBs and the activity of error-prone DNA polymerase IV (pol IV). We found that antibiotic-induced up-regulation of pol IV production requires active formation of DSB intermediates and can be supressed by ROS mitigators. The formation of pol IV foci, which reflect DNA-binding events, also requires DSB repair. Our findings support a major role for pol IV in DSB intermediates and reveal new details of how antibiotic treatment can potentially drive the development of antibiotic resistance in bacteria.

The integrating conjugative element ICE391 (formerly known as IncJ R391) harbors an error-prone DNA polymerase V ortholog, polV(ICE391), 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 polV(ICE391) is additionally regulated by a transcriptional repressor encoded by SetR(ICE391), since Escherichia coli expressing SetR(ICE391) demonstrated reduced levels of polV(ICE391)–mediated spontaneous mutagenesis relative to cells lacking SetR(ICE391). SetR(ICE391) regulation was shown to be specific for the rumAB operon and in vitro studies with highly purified SetR(ICE391) revealed that under alkaline conditions, as well as in the presence of activated RecA, SetR(ICE391) undergoes a self-mediated cleavage reaction that inactivates repressor functions. Conversely, a non-cleavable SetR(ICE391) mutant capable of maintaining repressor activity, even in the presence of activated RecA, exhibited low levels of polV(ICE391)-dependent mutagenesis. Electrophoretic mobility shift assays revealed that SetR(ICE391) 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 SetR(ICE391) acts as a transcriptional repressor of the ICE391-encoded mutagenic response.

In Escherichia coli, damage to the chromosomal DNA induces the SOS response, setting in motion a series of different DNA repair and damage tolerance pathways. DNA polymerase IV (pol IV) is one of three specialised DNA polymerases called into action during the SOS response to help cells tolerate certain types of DNA damage. The canonical view in the field is that pol IV primarily acts at replisomes that have stalled on the damaged DNA template. However, the results of several studies indicate that pol IV also acts on other substrates, including single-stranded DNA gaps left behind replisomes that re-initiate replication downstream of a lesion, stalled transcription complexes and recombination intermediates. In this study, we use single-molecule time-lapse microscopy to directly visualize fluorescently labelled pol IV in live cells. We treat cells with the DNA-damaging antibiotic ciprofloxacin, Methylmethane sulfonate (MMS) or ultraviolet light and measure changes in pol IV concentrations and cellular locations through time. We observe that only 5–10% of foci induced by DNA damage form close to replisomes, suggesting that pol IV predominantly carries out non-replisomal functions. The minority of foci that do form close to replisomes exhibit a broad distribution of colocalisation distances, consistent with a significant proportion of pol IV molecules carrying out postreplicative TLS in gaps behind the replisome. Interestingly, the proportion of pol IV foci that form close to replisomes drops dramatically in the period 90–180 min after treatment, despite pol IV concentrations remaining relatively constant. In an SOS-constitutive mutant that expresses high levels of pol IV, few foci are observed in the absence of damage, indicating that within cells access of pol IV to DNA is dependent on the presence of damage, as opposed to concentration-driven competition for binding sites., Author summary Translesion DNA polymerases play a critical role in DNA damage tolerance in all cells. In Escherichia coli, the translesion polymerases include DNA polymerases II, IV, and V. At stalled replication forks, DNA polymerase IV is thought to compete with, and perhaps displace the polymerizing subunits of DNA polymerase III to facilitate translesion replication. The results of the current fluorescence microscopy study challenge that view. The results indicate that DNA polymerase IV acts predominantly at sites away from the replisome. These sites may include recombination intermediates, stalled transcription complexes, and single-stranded gaps left in the wake of DNA polymerase III replisomes that re-initiate replication downstream of a lesion.

The cDNA encoding human DNA polymerase ι (POLI) was cloned in 1999. At that time, it was believed that the POLI gene encoded a protein of 715 amino acids. Advances in DNA sequencing technologies led to the realization that there is an upstream, in-frame initiation codon that would encode a DNA polymerase ι (polι) protein of 740 amino acids. The extra 25 amino acid region is rich in acidic residues (11/25) and is reasonably conserved in eukaryotes ranging from fish to humans. As a consequence, the curated Reference Sequence (RefSeq) database identified polι as a 740 amino acid protein. However, the existence of the 740 amino acid polι has never been shown experimentally. Using highly specific antibodies to the 25 N-terminal amino acids of polι, we were unable to detect the longer 740 amino acid (ι-long) isoform in western blots. However, trace amounts of the ι-long isoform were detected after enrichment by immunoprecipitation. One might argue that the longer isoform may have a distinct biological function, if it exhibits significant differences in its enzymatic properties from the shorter, well-characterized 715 amino acid polι. We therefore purified and characterized recombinant full-length (740 amino acid) polι-long and compared it to full-length (715 amino acid) polι-short in vitro. The metal ion requirements for optimal catalytic activity differ slightly between ι-long and ι-short, but under optimal conditions, both isoforms exhibit indistinguishable enzymatic properties in vitro. We also report that like ι-short, the ι-long isoform can be monoubiquitinated and polyubiuquitinated in vivo, as well as form damage induced foci in vivo. We conclude that the predominant isoform of DNA polι in human cells is the shorter 715 amino acid protein and that if, or when, expressed, the longer 740 amino acid isoform has identical properties to the considerably more abundant shorter isoform.

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.

In 2003, we reported that 129-derived strains of mice carry a naturally occurring nonsense mutation at codon 27 of the Poli gene that would produce a polι peptide of just 26 amino acids, rather then the full-length 717 amino acid wild-type polymerase. In support of the genomic analysis, no polι protein was detected in testes extracts from 129X1/SvJmice, where wild-type polι is normally highly expressed. The early truncation in polι occurs before any structural domains of the polymerase are synthesized and as a consequence, we reasoned that 129-derived strains of mice should be considered as functionally defective in polι activity. However, it has recently been reported that during the maturation of the Poli mRNA in 129-derived strains, exon- 2 is sometimes skipped and that an exon-2-less polι protein of 675 amino acids is synthesized that retains catalytic activity in vitro and in vivo . From a structural perspective, we found this idea untenable, given that the amino acids encoded by exon-2 include residues critical for the coordination of the metal ions required for catalysis, as well as the structural integrity of the DNA polymerase. To determine if the exon-2-less polι isoform possesses catalytic activity in vitro , we have purified a glutathione-tagged full-length exon-2-less (675 amino acid) polι protein from baculovirus infected insect cells and compared the activity of the isoform to full-length (717 amino acid) GST-tagged wild-type mouse polι in vitro . Reaction conditions were performed under a range of magnesium or manganese concentrations, as well as different template sequence contexts. Wild-type mouse polι exhibited robust characteristic properties previously associated with human polι’s biochemical properties. However, we did not detect any polymerase activity associated with the exon-2-less polι enzyme under the same reaction conditions and conclude that exon-2-less polι is indeed rendered catalytically inactive in vitro .

Human DNA polymerases η and ι are best characterized for their ability to facilitate translesion DNA synthesis (TLS). Both polymerases (pols) co-localize in ‘replication factories’ in vivo after cells are exposed to ultraviolet light and this co-localization is mediated through a physical interaction between the two TLS pols. We have mapped the polη-ι interacting region to their respective ubiquitin-binding domains (UBZ in polη and UBM1 and UBM2 in polι), and demonstrate that ubiquitination of either TLS polymerase is a prerequisite for their physical and functional interaction. Importantly, while monoubiquitination of polη precludes its ability to interact with proliferating cell nuclear antigen (PCNA), it enhances its interaction with polι. Furthermore, a polι-ubiquitin chimera interacts avidly with both polη and PCNA. Thus, the ubiquitination status of polη, or polι plays a key regulatory function in controlling the protein partners with which each polymerase interacts, and in doing so, determines the efficiency of targeting the respective polymerase to stalled replication forks where they facilitate TLS.

Escherichia coli polymerase V (pol V/UmuD 2 ′C) is a low-fidelity DNA polymerase that has recently been shown to avidly incorporate ribonucleotides (rNTPs) into undamaged DNA. The fidelity and sugar selectivity of pol V can be modified by missense mutations around the “steric gate” of UmuC. Here, we analyze the ability of three steric gate mutants of UmuC to facilitate translesion DNA synthesis (TLS) of a cyclobutane pyrimidine dimer (CPD) in vitro , and to promote UV-induced mutagenesis and cell survival in vivo . The pol V (UmuC_F10L) mutant discriminates against rNTP and incorrect dNTP incorporation much better than wild-type pol V and although exhibiting a reduced ability to bypass a CPD in vitro , does so with high-fidelity and consequently produces minimal UV-induced mutagenesis in vivo . In contrast, pol V (UmuC_Y11A) readily misincorporates both rNTPs and dNTPs during efficient TLS of the CPD in vitro . However, cells expressing umuD ′ C (Y11A) were considerably more UV-sensitive and exhibited lower levels of UV-induced mutagenesis than cells expressing wild-type umuD ′ C or umuD ′ C (Y11F). We propose that the increased UV-sensitivity and reduced UV-mutability of umuD ′ C (Y11A) is due to excessive incorporation of rNTPs during TLS that are subsequently targeted for repair, rather than an inability to traverse UV-induced lesions.

This study examines how entrepreneurial marketing dimensions (proactiveness, opportunity focused, leveraging, innovativeness, risk taking, value creation, and customer intensity) are related to qualitative and quantitative outcome measures for the SME and the entrepreneur (including company success, customer success, financial success, satisfaction with return goals, satisfaction with growth goals, excellence, and the entrepreneurʼs standard of living). Using factor analysis, three success outcome variables (financial, customer, and strong company success) emerged together. A separate factor analysis identified satisfactory growth and return goals. Stepwise regression revealed entrepreneurial marketing impacts outcome variables, particularly value creation. Implications for entrepreneurs and areas for research are included.

Purge and pump samples from screened wells reflect concentration averaging and contaminant redistribution by wellbore flow. These issues were assessed in a screened well at the Hanford Site by investigating the vertical profile of a technetium-99 plume in a conventional well under static and pumped conditions. Specific conductance and technetium-99 concentrations were well correlated, and this enabled measurement of specific conductance to be used as a surrogate for technetium-99 concentration. Time-series measurements were collected during purging from three specific conductance probes installed in the well at 1.2, 3.1, and 4.9 m below the static water level in a 7.7-m-deep screened well. The vertical contaminant profile adjacent to the well in the aquifer was calculated using the concentration profile in the well during pumping, the pumping flow rate, and a wellbore flow and mixing model. The plume was found to be stratified in the aquifer—the highest concentrations occurred adjacent to the upper part of the screened interval. The purge and pump sample concentrations were 41% to 58% of the calculated peak concentration in the aquifer. Plume stratification in the aquifer adjacent to the well screen became more pronounced as pumping continued. Extended pumping may have partially reversed the effect of contaminant redistribution in the aquifer by wellbore flow and allowed the stratification of the plume to be more observable. It was also found that the vertical profile of contamination in the well under static (i.e., nonpumping conditions) was not representative of the profile in the aquifer. Thus, passive or micropurge sampling techniques, which sample the wellbore water at different depths, would not yield results representative of the aquifer in this well.

Escherichia coli possesses five known DNA polymerases (pols). Pol III holoenzyme is the cell's main replicase, while pol I is responsible for the maturation of Okazaki fragments and filling gaps generated during nucleotide excision repair. Pols II, IV and V are significantly upregulated as part of the cell's global SOS response to DNA damage and under these conditions, may alter the fidelity of DNA replication by potentially interfering with the ability of pols I and III to complete their cellular functions. To test this hypothesis, we determined the spectrum of rpoB mutations arising in an isogenic set of mutL strains differentially expressing the chromosomally encoded pols. Interestingly, mutagenic hot spots in rpoB were identified that are susceptible to the actions of pols I-V. For example, in a recA730 lexA(Def) mutL background most transversions were dependent upon pols IV and V. In contrast, transitions were largely dependent upon pol I and to a lesser extent, pol III. Furthermore, the extent of pol I-dependent mutagenesis at one particular site was modulated by pols II and IV. Our observations suggest that there is considerable interplay among all five E. coli polymerases that either reduces or enhances the mutagenic load on the E. coli chromosome.

N3-methyl-adenine (3MeA) is the major cytotoxic lesion formed in DNA by S(N)2 methylating agents. The lesion presumably blocks progression of cellular replicases because the N3-methyl group hinders interactions between the polymerase and the minor groove of DNA. However, this hypothesis has yet to be rigorously proven, as 3MeA is intrinsically unstable and is converted to an abasic site, which itself is a blocking lesion. To circumvent these problems, we have chemically synthesized a 3-deaza analog of 3MeA (3dMeA) as a stable phosphoramidite and have incorporated the analog into synthetic oligonucleotides that have been used in vitro as templates for DNA replication. As expected, the 3dMeA lesion blocked both human DNA polymerases alpha and delta. In contrast, human polymerases eta, iota and kappa, as well as Saccharomyces cerevisiae poleta were able to bypass the lesion, albeit with varying efficiencies and accuracy. To confirm the physiological relevance of our findings, we show that in S. cerevisiae lacking Mag1-dependent 3MeA repair, poleta (Rad30) contributes to the survival of cells exposed to methyl methanesulfonate (MMS) and in the absence of Mag1, Rad30 and Rev3, human polymerases eta, iota and kappa are capable of restoring MMS-resistance to the normally MMS-sensitive strain.

Spatial regulation is often encountered as a component of multi-tiered regulatory systems in eukaryotes, where processes are readily segregated by organelle boundaries. Well-characterized examples of spatial regulation are less common in bacteria. Low-fidelity DNA polymerase V (UmuD′2C) is produced in Escherichia coli as part of the bacterial SOS response to DNA damage. Due to the mutagenic potential of this enzyme, pol V activity is controlled by means of an elaborate regulatory system at transcriptional and posttranslational levels. Using single-molecule fluorescence microscopy to visualize UmuC inside living cells in space and time, we now show that pol V is also subject to a novel form of spatial regulation. After an initial delay (~ 45 min) post UV irradiation, UmuC is synthesized, but is not immediately activated. Instead, it is sequestered at the inner cell membrane. The release of UmuC into the cytosol requires the RecA* nucleoprotein filament-mediated cleavage of UmuD→UmuD′. Classic SOS damage response mutants either block [umuD(K97A)] or constitutively stimulate [recA(E38K)] UmuC release from the membrane. Foci of mutagenically active pol V Mut (UmuD′2C-RecA-ATP) formed in the cytosol after UV irradiation do not co-localize with pol III replisomes, suggesting a capacity to promote translesion DNA synthesis at lesions skipped over by DNA polymerase III. In effect, at least three molecular mechanisms limit the amount of time that pol V has to access DNA: (1) transcriptional and posttranslational regulation that initially keep the intracellular levels of pol V to a minimum; (2) spatial regulation via transient sequestration of UmuC at the membrane, which further delays pol V activation; and (3) the hydrolytic activity of a recently discovered pol V Mut ATPase function that limits active polymerase time on the chromosomal template., Author Summary Escherichia coli, and many other bacteria, respond to high levels of DNA damage with an inducible system called the SOS response. In this response, bacteria first try to restart replication using non-mutagenic DNA repair strategies. If that fails, replication can be restored using DNA polymerases that simply replicate over DNA lesions, a desperation strategy that results in mutations. DNA polymerase V (pol V) is responsible for most mutagenesis that accompanies the SOS response. Because of the risk inherent to elevated mutation levels, pol V activation is tightly constrained. This report introduces a new layer of regulation on pol V activation, with a novel spatial component. After synthesis, the UmuC subunit of pol V is sequestered transiently at the membrane. Release into the cytosol and final activation depends on the activity of RecA protein and the autocatalytic cleavage of UmuD to generate the UmuD' subunit of pol V. The resulting delay in activation represents an additional molecular mechanism that limits the amount of time that this sometimes necessary but potentially detrimental enzyme spends on the DNA.

DNA polymerase η (pol η) is best characterized for its ability to perform accurate and efficient translesion DNA synthesis (TLS) through cyclobutane pyrimidine dimers (CPDs). To ensure accurate bypass the polymerase is not only required to select the correct base, but also discriminate between NTPs and dNTPs. Most DNA polymerases have a conserved “steric gate” residue which functions to prevent incorporation of NMPs during DNA synthesis. Here, we demonstrate that the Phe35 residue of Saccharomyces cerevisiae pol η functions as a steric gate to limit the use of ribonucleotides during polymerization both in vitro and in vivo. Unlike the related pol ι enzyme, wild-type pol η does not readily incorporate NMPs in vitro. In contrast, a pol η F35A mutant incorporates NMPs on both damaged and undamaged DNA in vitro with a high degree of base selectivity. An S.cerevisiae strain expressing pol η F35A (rad30-F35A) that is also deficient for nucleotide excision repair (rad1Δ) and the TLS polymerase, pol ζ (rev3Δ), is extremely sensitive to UV-light. The sensitivity is due, in part, to RNase H2 activity, as an isogenic rnh201Δ strain is roughly 50-fold more UV-resistant than its RNH201+ counterpart. Interestingly the rad1Δ rev3Δ rad30-F35A rnh201Δ strain exhibits a significant increase in the extent of spontaneous mutagenesis with a spectrum dominated by 1bp deletions at runs of template Ts. We hypothesize that the increased mutagenesis is due to rA incorporation at these sites and that the short poly rA tract is subsequently repaired in an error-prone manner by a novel repair pathway that is specifically targeted to polyribonucleotide tracks. These data indicate that under certain conditions, pol η can compete with the cell’s replicases and gain access to undamaged genomic DNA. Such observations are consistent with a role for pol η in replicating common fragile sites (CFS) in human cells.

For many years, Taq polymerase has served as the stalwart enzyme in the PCR amplification of DNA. However, a major limitation of Taq is its inability to amplify damaged DNA, thereby restricting its usefulness in forensic applications. In contrast, Y-family DNA polymerases, such as Dpo4 from Sulfolobus solfataricus, can traverse a wide variety of DNA lesions. Here, we report the identification and characterization of five novel thermostable Dpo4-like enzymes from Acidianus infernus, Sulfolobus shibatae, Sulfolobus tengchongensis, Stygiolobus azoricus and Sulfurisphaera ohwakuensis, as well as two recombinant chimeras that have enhanced enzymatic properties compared with the naturally occurring polymerases. The Dpo4-like polymerases are moderately processive, can substitute for Taq in PCR and can bypass DNA lesions that normally block Taq. Such properties make the Dpo4-like enzymes ideally suited for the PCR amplification of damaged DNA samples. Indeed, by using a blend of Taq and Dpo4-like enzymes, we obtained a PCR amplicon from ultraviolet-irradiated DNA that was largely unamplifyable with Taq alone. The inclusion of thermostable Dpo4-like polymerases in PCRs, therefore, augments the recovery and analysis of lesion-containing DNA samples, such as those commonly found in forensic or ancient DNA molecular applications.

The birth of the slogan dates back to the start of history itself. Today, the slogan remains visible in virtually every segment of society, attesting to its unique staying power. As much as the slogan has been able to influence people throughout the ages, however, very little research has been conducted on the topic. Of the substantive material that does exist, most focuses on slogans used by advertisers, with a few minor exceptions found in the political slogan area (e.g., Sherif, 1937), The purpose of this paper is to trace the empirical work which has been conducted in the area of advertising slogan effectiveness.

Based upon phylogenetic relationships, the broad Y-family of DNA polymerases can be divided into various subfamilies consisting of UmuC (polV)-like; DinB (polIV/polkappa)-like; Rev1-like, Rad30A (poleta)-like and Rad30B (poliota)-like polymerases. The polIV/polkappa-like polymerases are most ubiquitous, having been identified in bacteria, archaea and eukaryotes. In contrast, the polV-like polymerases appear restricted to bacteria (both Gram positive and Gram negative). Rev1 and poleta-like polymerases are found exclusively in eukaryotes, and to date, poliota-like polymerases have only been identified in higher eukaryotes. In general, the in vitro properties of polymerases characterized within each sub-family are quite similar. An exception to this rule occurs with the poliota-like polymerases, where the enzymatic properties of Drosophila melanogaster poliota are more similar to that of Saccharomyces cerevisiae and human poleta than to the related human poliota. For example, like poleta, Drosophila poliota can bypass a cis-syn thymine-thymine dimer both accurately and efficiently, while human poliota bypasses the same lesion inefficiently and with low-fidelity. Even in cases where human poliota can efficiently insert a base opposite a lesion (such as a synthetic abasic site, the 3'T of a 6-4-thymine-thymine pyrimidine-pyrimidone photoproduct or opposite benzo[a]pyrene diol epoxide deoxyadenosine adducts), further extension is often limited. Thus, although poliota most likely arose from a genetic duplication of poleta millions of years ago as eukaryotes evolved, it would appear that poliota from humans (and possibly all mammals) has been further subjected to evolutionary pressures that have "tailored" its enzymatic properties away from lesion bypass and towards other function(s) specific for higher eukaryotes. The identification of such functions and the role that mammalian poliota plays in lesion bypass in vivo, should hopefully be forthcoming with the construction of human cell lines deleted for poliota and the identification of mice deficient in poliota.

A hallmark of human DNA polymerase iota (poliota) is the asymmetric fidelity of replication at template A and T when the enzyme extends primers annealed to a single-stranded template. Here, we report on the efficiency and accuracy of poliota-dependent replication at a nick, a gap, the very end of a template and from a mispaired primer. Poliota cannot initiate synthesis on a nicked DNA substrate, but fills short gaps efficiently. Surprisingly, poliota's ability to blunt-end a 1 bp recessed terminus is dependent upon the template nucleotide encountered and is highly erroneous. At template G, both C and T are inserted with roughly equal efficiency, whilst at template C, C and A are misinserted 8- and 3-fold more often than the correct base, G. Using substrates containing mispaired primer termini, we show that poliota can extend all 12 mispairs, but with differing efficiencies. Poliota can also extend a tandem mispair, especially when it is located within a short gap. The enzymatic properties of poliota appear consistent with that of a somatic hypermutase and suggest that poliota may be one of the low-fidelity DNA polymerases hypothesized to participate in the hypermutation of immunoglobulin variable genes in vivo.

The human RAD30B gene has recently been shown to encode a novel DNA polymerase, DNA polymerase i (poli). The role of poli within the cell is presently unknown, and the only clues to its cellular function come from its biochemical characterization in vitro. The aim of this short review is, therefore, to summarize the known enzymic activities of poli and to speculate as to how these biochemical properties might relate to its in vivo function.

Predictions of four risk assessment models — RESRAD, PRESTO, MMSOILS, and MEPAS — for a test scenario involving the migration of a single, rapidly transforming radionuclide, 90Sr, and a persistent, long radionuclide chain, 234U and its progeny, in groundwater are compared. All four models make comparable predictions for the plume centerline concentrations of the primary contaminants in the aquifer for a distance of up to about 300 m from the source. MEPAS, MMSOILS, and RESRAD make similar predictions for the transverse concentration profiles in the aquifer. The four models make considerably different predictions for the temporal concentration profiles of the progeny in the aquifer. The profiles differ in shape, magnitude of the peak, and in width. The differences are a result of the simplifying assumptions underlying each of the models.

TheSaccharomyces cerevisiae RAD30gene encodes DNA polymerase η. Humans possess two Rad30 homologs. One (RAD30A/POLH) has previously been characterized and shown to be defective in humans with the Xeroderma pigmentosum variant phenotype. Here, we report experiments demonstrating that the second human homolog (RAD30B), also encodes a novel DNA polymerase that we designate polι. polι, is a distributive enzyme that is highly error-prone when replicating undamaged DNA. At template G or C, the average error frequency was ∼1 × 10−2. Our studies revealed, however, a striking asymmetry in misincorporation frequency at template A and T. For example, template A was replicated with the greatest accuracy, with misincorporation of G, A, or C occurring with a frequency of ∼1 × 10−4to 2 × 10−4. In dramatic contrast, most errors occurred at template T, where the misincorporation of G was, in fact, favored ∼3:1 over the correct nucleotide, A, and misincorporation of T occurred at a frequency of ∼6.7 × 10−1. These findings demonstrate that polι is one of the most error-prone eukaryotic polymerases reported to date and exhibits an unusual misincorporation spectrum in vitro.

Multimedia modelers from the US Environmental Protection Agency (EPA) and US Department of Energy (DOE) are collaborating to conduct a comprehensive and quantitative benchmarking analysis of four intermedia models: MEPAS, MMSOILS, PRESTO, and RESRAD. These models represent typical analytically based tools that are used in human-risk and endangerment assessments at installations containing radioactive and hazardous contaminants. The objective is to demonstrate an approach for developing an adequate source term by simplifying an existing, real-world, 90 Sr plume at DOE's Hanford installation in Richland, WA, for use in a multimedia benchmarking exercise between MEPAS, MMSOILS, PRESTO, and RESRAD. Source characteristics and a release mechanism are developed and described; also described is a typical process and procedure that an analyst would follow in developing a source term for using this class of analytical tool in a preliminary assessment.

The UmuD-like proteins are best characterized for their role in damage-induced SOS mutagenesis. An essential step in this process is the enzymatic self-processing of the UmuD-like proteins. This reaction is thought to occur either via an intramolecular or intermolecular self-cleavage mechanism. Here, we demonstrate that it can also occur via an heterologous intermolecular cleavage reaction. The Escherichia coli UmuD enzyme demonstrated the broadest substrate specificity, cleaving both E. coli and Salmonella typhimurium UmuD substrates in vivo. In comparison, the wild-type S. typhimurium UmuD (UmuDSt) and MucA enzymes catalyzed intermolecular self-cleavage, but did not facilitate heterologous cleavage. Heterologous cleavage by the UmuDSt enzyme was, however, observed with chimeric UmuD substrates that possess residues 30-55 of UmuDSt. We have further localized the residue predominantly responsible for UmuDSt-catalyzed heterologous cleavage to Ser50 in the substrate molecule. We hypothesize that changes at this residue affect the positioning of the cleavage site of a substrate molecule within the catalytic cleft of the UmuDSt enzyme by affecting the formation of a so-called UmuD "filament-dimer". This hypothesis is further supported by the observation that mutations known to disrupt an E. coli UmuD' filament dimer also block intermolecular UmuDEc cleavage.

A comprehensive benchmarking is being performed between three multimedia risk assessment models: RESRAD, MMSOILS, and MEPAS. Each multimedia model is comprised of a suite of modules (e.g., groundwater, air, surface water, exposure, and risk/hazard), all of which can impact the estimation of the human health risk. As a component of the comprehensive benchmarking exercise, the saturated-zone modules of each model were applied to an environmental release scenario, where uranium-234 was released from the waste site to a saturated zone. Uranium-234 time-varying emission rates exiting from the source and concentrations at three downgradient locations (0, 150, and 1500 m) are compared for each multimedia model. Time-varying concentrations for uranium-234 decay products (e.g., thorium-230, radium-226, and lead-210) at the 1500-m location are also presented. Different results reported for RESRAD, MMSOILS, and MEPAS are solely due to the assumptions and mathematical constructs inherently built into each mo...

An essential step in SOS mutagenesis is the RecA-mediated posttranslational processing of UmuD-like proteins to the shorter, but mutagenically active, UmuD′-like proteins. Interestingly, the UmuD-like proteins undergo posttranslational processing at different rates. For example, although the Escherichia coli UmuD (UmuD Ec) and the Salmonella typhimurium UmuD (UmuD St) proteins are 73% identical, UmuD Stis processed in vivo at a significantly faster rate than the UmuD Ec protein. Here, we report experiments aimed at investigating the molecular basis of these phenotypic differences. The faster rate of UmuDSt cleavage probably does not result solely from a better interaction with RecA, since we observed that, in vitro, UmuDStundergoes RecA-independent autocatalytic processing about four-times faster than UmuDEc. By constructing chimeric UmuD proteins, we determined that the amino-terminal tail of the UmuD proteins proximal to the Cys24-Gly25 cleavage site is mainly responsible for the difference in UmuDStand UmuDEc cleavage rates. Site-directed mutagenesis of the UmuDEc protein suggests that most of the enhanced cleavage observed with the UmuDSt protein can be attributed to the presence of a Pro23 residue, juxtaposed to the cleavage site in UmuDSt. Furthermore, this proline residue appears to result in a UmuD protein that is a much better substrate for intermolecular cleavage. These findings clearly implicate the N-terminal tail of the UmuD-like proteins as playing an important and unexpected regulatory function in the maturation of the mutagenically active UmuD′-like mutagenesis proteins.

Although it has been 10 years since the discovery that the Escherichia coli UmuD protein undergoes a RecA-mediated cleavage reaction to generate mutagenically active UmuD′, the function of UmuD′ has yet to be determined. In an attempt to elucidate the role of UmuD′ in SOS mutagenesis, we have utilized a colorimetric papillation assay to screen for mutants of a hydroxylamine-treated, low-copy-number umuD ′ plasmid that are unable to promote SOS-dependent spontaneous mutagenesis. Using such an approach, we have identified 14 independent umuD ′ mutants. Analysis of these mutants revealed that two resulted from promoter changes which reduced the expression of wild-type UmuD′, three were nonsense mutations that resulted in a truncated UmuD′ protein, and the remaining nine were missense alterations. In addition to the hydroxylamine-generated mutants, we have subcloned the mutations found in three chromosomal umuD1 , umuD44 , and umuD77 alleles into umuD ′. All 17 umuD ′ mutants resulted in lower levels of SOS-dependent spontaneous mutagenesis but varied in the extent to which they promoted methyl methanesulfonate-induced mutagenesis. We have attempted to correlate these phenotypes with the potential effect of each mutation on the recently described structure of UmuD′.

(1998). Commentary on Fate and Exposure Models: Description of the Multimedia Environmental Pollutant Assessment System (MEPAS®) Version 3.2, with Application to a Hypothetical Soil Contamination Scenario. Journal of Soil Contamination: Vol. 7, No. 3, pp. 283-300.

The Escherichia coli Umu proteins are best characterized by their role in damage inducible mutagenesis. Recently, we discovered that the intracellular levels of the UmuD and UmuC proteins are kept to a minimum by the Lon serine protease. Studies with the Salmonella typhimurium UmuD protein (which is 73% homologous with its E. coli counterpart) revealed that it too is degraded by Lon, suggesting that both UmuD proteins share conserved structural motifs. In contrast, E. coli UmuD' is removed from the cell by the ClpXP serine protease, but only when it is in a heterodimer complex with UmuD. We have generated deletion mutants of UmuD' and have coexpressed the mutant proteins with UmuD1 (a non-cleavable UmuD protein). By assaying the sensitivity of the mutant UmuD'-UmuD1 complex to ClpXP, we have been able to map regions of UmuD' that appear essential for efficient UmuD'-UmuD heterodimer formation. Previous experiments have suggested that the in vivo posttranslational processing of UmuD to UmuD' is inefficient. We have, however, discovered that limited cleavage occurs in an undamaged cell, but that these small amounts of UmuD' are rapidly degraded by ClpXP, thus giving rise to the appearance of inefficient cleavage. The ClpXP protease therefore plays dual roles in regulating SOS mutagenesis: it keeps the basal levels of UmuD' to a minimum in undamaged cells but it also acts in damaged cells to reduce the elevated levels of mutagenically active UmuD' protein, thereby returning the cell to a resting non-mutable state.

The activity of a number of proteins is regulated by self-processing reactions. Elegant examples are the cleavage of the prokaryotic LexA and λCI transcriptional repressors and the UmuD-like mutagenesis proteins. Various studies support the hypothesis that LexA and λCI cleavage reactions are predominantly intramolecular in nature. The recently described crystal structure of the Escherichia coli UmuD′ protein (the posttranslational cleavage product of the UmuD protein) suggests, however, that the region of the protein corresponding to the cleavage site is at least 50 Å away from the catalytic active site. We considered the possibility, therefore, that the UmuD-like proteins might undergo self-processing that, in contrast to LexA and λCI, occurs via an intermolecular rather than intramolecular reaction. To test this hypothesis, we introduced into E. coli compatible plasmids with mutations at either the cleavage or the catalytic site of three UmuD-like proteins. Cleavage of these proteins only occurs in the presence of both plasmids, indicating that the reaction is indeed intermolecular in nature. Furthermore, this intermolecular reaction is completely dependent upon the multifunctional RecA protein and leads to the restoration of cellular mutagenesis in nonmutable E. coli strains. Intermolecular cleavage of a biotinylated UmuD active site mutant was also observed in vitro in the presence of the wild-type UmuD′ protein, indicating that in addition to the intact UmuD protein, the normal cleavage product (UmuD′) can also act as a classical enzyme.

Bubbles entrained in the plunge pools of hydraulic structures are subjected to the hydrostatic pressure of the water column. The hydrostatic pressure increases the saturation concentration of gases within the bubbles and correspondingly alters the rates of gas transfer. An “effective saturation concentration” is introduced to account for the elevated pressure within the bubbles. The effective saturation concentration must be incorporated into gas-transfer calculations to accurately predict downstream concentrations of dissolved gases. A method of measuring the effective saturation concentration from simultaneous measurements of dissolved oxygen and methane is presented. The method is verified through similar measurements of dissolved nitrogen and methane. Applications demonstrate the use of the effective saturation concentration in the prediction of dissolved-gas levels downstream of hydraulic structures and the use of dissolved-gas measurements in the determination of the effective saturation concentration.

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., Author Summary Most DNA polymerases differentiate between ribo- and deoxyribonucleotides quite effectively, thereby deterring insertion of nucleotides with the “wrong” sugar into chromosomes. Nevertheless, a significant number of ribonucleotides still get stably incorporated into genomic DNA. E.coli pol V is among the most inaccurate DNA polymerases in terms of both sugar selectivity and base substitution fidelity. The umuC_Y11A steric gate variant of pol V is even less discriminating when selecting sugar of the incoming nucleotide while keeping a similar capacity to form non-Watson-Crick base pairs. In the present study, we describe mechanisms employed by E. coli to excise rNMPs from DNA and to concomitantly reduce the extent of spontaneous mutagenesis induced by umuC_Y11A. The first line of defense comes from Ribonuclease HII, which initiates the ribonucleotide excision repair pathway. In the absence of RNase HII, alternate repair pathways help remove the misincorporated ribonucleotides. Here, we present the first direct evidence that nucleotide excision repair (NER) has the capacity to recognize both correctly and incorrectly paired rNMPs embedded in DNA. The combined actions of RNase HII and NER thereby reduce the mutagenic potential of ribonucleotides errantly incorporated into prokaryotic genomes.

FOR life to be sustained, mistakes in DNA repair must be tolerated when damage obscures the genetic information. In bacteria such as Escherichia coli, DNA damage elicits the well regulated 'SOS response' (reviewed in ref. 1). For the extreme case of damage that cannot be repaired by conventional enzymes, there are proteins that allow the replication of DNA through such lesions, but with a reduction in the fidelity of replication2. Essential proteins in this mutagenic process are RecA, DNA polymerase III, UmuD, UmuD′ and UmuC (umu: UV mutagen-esis)1–3. Regulation of this response involves a RecA-mediated self-cleavage of UmuD to produce UmuD′ To understand this system in more detail, we have determined the crystal structure of the E. coli UmuD′ mutagenesis protein at 2.5 A resolution. Globular heads folded in an unusual β-structure associate to form molecular dimers, and extended ammo-terminal tails associate to produce crystallized filaments. The structure provides insight into the mechanism of the self-cleavage reaction that UmuD-like proteins undergo as part of the global SOS response4–8.

All living organisms are continually exposed to agents that damage their DNA, which threatens the integrity of their genome. As a consequence, cells are equipped with a plethora of DNA repair enzymes to remove the damaged DNA. Unfortunately, situations nevertheless arise where lesions persist, and these lesions block the progression of the cell's replicase. In these situations, cells are forced to choose between recombination-mediated "damage avoidance" pathways or a specialized DNA polymerase (pol) to traverse the blocking lesion. The latter process is referred to as Translesion DNA Synthesis (TLS). As inferred by its name, TLS not only results in bases being (mis)incorporated opposite DNA lesions but also bases being (mis)incorporated downstream of the replicase-blocking lesion, so as to ensure continued genome duplication and cell survival. Escherichia coli and Salmonella typhimurium possess five DNA polymerases, and while all have been shown to facilitate TLS under certain experimental conditions, it is clear that the LexA-regulated and damage-inducible pols II, IV, and V perform the vast majority of TLS under physiological conditions. Pol V can traverse a wide range of DNA lesions and performs the bulk of mutagenic TLS, whereas pol II and pol IV appear to be more specialized TLS polymerases.