Host cell RecA activates a mobile element-encoded mutagenic DNA polymerase.

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.

CroSR391 , an ortholog of the λ Cro repressor, plays a major role in suppressing polVR391 -dependent mutagenesis.

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.

Novel Escherichia coli active site dnaE alleles with altered base and sugar selectivity.

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.

Single-molecule live-cell imaging reveals RecB-dependent function of DNA polymerase IV in double strand break repair.

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.

DNA polymerase η contributes to genome-wide lagging strand synthesis.

DNA polymerase η (pol η) is best known for its ability to bypass UV-induced thymine-thymine (T-T) dimers and other bulky DNA lesions, but pol η also has other cellular roles. Here, we present evidence that pol η competes with DNA polymerases α and δ for the synthesis of the lagging strand genome-wide, where it also shows a preference for T-T in the DNA template. Moreover, we found that the C-terminus of pol η, which contains a PCNA-Interacting Protein motif is required for pol η to function in lagging strand synthesis. Finally, we provide evidence that a pol η dependent signature is also found to be lagging strand specific in patients with skin cancer. Taken together, these findings provide insight into the physiological role of DNA synthesis by pol η and have implications for our understanding of how our genome is replicated to avoid mutagenesis, genome instability and cancer.

DNA polymerase IV primarily operates outside of DNA replication forks in Escherichia coli.

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.

Regulation of Mutagenic DNA Polymerase V Activation in Space and Time.

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.

Removal of misincorporated ribonucleotides from prokaryotic genomes: an unexpected role for nucleotide excision repair.

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 8(th) phosphodiester bond 5' and 4(th)-5(th) 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.

Ubiquitin mediates the physical and functional interaction between human DNA polymerases η and ι.

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.

Mechanisms employed by Escherichia coli to prevent ribonucleotide incorporation into genomic DNA by Pol V.

Escherichia coli pol V (UmuD'(2)C), 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.

Investigating the mechanisms of ribonucleotide excision repair in Escherichia coli.

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.

Critical amino acids in Escherichia coli UmuC responsible for sugar discrimination and base-substitution fidelity.

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 ß/γ complex and a transactivated 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.

Pressure Transducer Measurement Variability in Deep Wells Screened Across the Water Table.

Automated water level measurements collected using vented pressure transducers in deep wells screened across the water table may exhibit a greater response to barometric pressure changes than the true water level. The cause was hypothesized to be disequilibrium in barometric pressure between the wellbores and land surface due to air exchange with the deep vadose zone. In this study, vented and nonvented pressure transducers were installed and operated simultaneously in two deep wells screened across the water table. A vent tube open to the atmosphere at land surface allowed for barometric compensation of the vented transducers. Two nonvented transducers were installed in each well, one submerged in the water and one above the water surface. The difference in readings allowed for barometric compensation. Manual measurements were also collected. It was confirmed that measurements from the vented transducers exhibited greater variability in response to barometric pressure changes than the nonvented transducers and manual measurements. Comparison of the downhole barometric pressure measurements to values from a nearby meteorology station showed the response in the wells to changes in barometric pressure was time-lagged and attenuated. Thus, the reference pressure from land surface supplied to the vented transducers was not representative of the air pressure within the wells. This caused fluctuations of the transducer readings in response to barometric pressure changes to be greater than the true water level change. This issue can be resolved by the use of nonvented pressure transducers.

Integration of molecular modelling and in vitro studies to inhibit LexA proteolysis.

Introduction: As antibiotic resistance has become more prevalent, the social and economic impacts are increasingly pressing. Indeed, bacteria have developed the SOS response which facilitates the evolution of resistance under genotoxic stress. The transcriptional repressor, LexA, plays a key role in this response. Mutation of LexA to a non-cleavable form that prevents the induction of the SOS response sensitizes bacteria to antibiotics. Achieving the same inhibition of proteolysis with small molecules also increases antibiotic susceptibility and reduces drug resistance acquisition. The availability of multiple LexA crystal structures, and the unique Ser-119 and Lys-156 catalytic dyad in the protein enables the rational design of inhibitors. Methods: We pursued a binary approach to inhibit proteolysis; we first investigated ß-turn mimetics, and in the second approach we tested covalent warheads targeting the Ser-119 residue. We found that the cleavage site region (CSR) of the LexA protein is a classical Type II ß-turn, and that published 1,2,3-triazole compounds mimic the ß-turn. Generic covalent molecule libraries and a ß-turn mimetic library were docked to the LexA C-terminal domain using molecular modelling methods in FlexX and CovDock respectively. The 133 highest-scoring molecules were screened for their ability to inhibit LexA cleavage under alkaline conditions. The top molecules were then tested using a RecA-mediated cleavage assay. Results: The ß-turn library screen did not produce any hit compounds that inhibited RecA-mediated cleavage. The covalent screen discovered an electrophilic serine warhead that can inhibit LexA proteolysis, reacting with Ser-119 via a nitrile moiety. Discussion: This research presents a starting point for hit-to-lead optimisation, which could lead to inhibition of the SOS response and prevent the acquisition of antibiotic resistance.

A strategy for the expression of recombinant proteins traditionally hard to purify.

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.

A real-time fluorescence method for enzymatic characterization of specialized human DNA polymerases.

Specialized DNA polymerases are involved in DNA synthesis during base-excision repair and translesion synthesis across a wide range of chemically modified DNA templates. Notable features of these enzymes include low catalytic efficiency, low processivity and low fidelity. Traditionally, in vitro studies of these enzymes have utilized radiolabeled substrates and gel electrophoretic separation of products. We have developed a simple homogeneous fluorescence-based method to study the enzymology of specialized DNA polymerases in real time. The method is based on fluorescent reporter strand displacement from a tripartite substrate containing a quencher-labeled template strand, an unlabeled primer and a fluorophore-labeled reporter. With this method, we could follow the activity of human DNA polymerases beta, eta, iota and kappa under different reaction conditions, and we investigated incorporation of the aberrant nucleotide, 8-oxodGTP, as well as bypass of an abasic site or 8-oxoG DNA template lesion in different configurations. Lastly, we demonstrate that the method can be used for small molecule inhibitor discovery and characterization in highly miniaturized settings, and we report the first nanomolar inhibitors of Y-family DNA polymerases iota and eta. The fluorogenic method presented here should facilitate mechanistic and inhibitor investigations of these polymerases and is also applicable to the study of highly processive replicative polymerases.

Measuring a Low Horizontal Hydraulic Gradient in a High Transmissivity Aquifer.

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.

Corrigendum: Identification and Characterization of Thermostable Y-Family DNA Polymerases η, ι, κ and Rev1 From a Lower Eukaryote, Thermomyces lanuginosus.

[This corrects the article DOI: 10.3389/fmolb.2021.778400.].

Identification and Characterization of Thermostable Y-Family DNA Polymerases η, ι, κ and Rev1 From a Lower Eukaryote, Thermomyces lanuginosus.

Y-family DNA polymerases (pols) consist of six phylogenetically separate subfamilies; two UmuC (polV) branches, DinB (pol IV, Dpo4, polκ), Rad30A/POLH (polη), and Rad30B/POLI (polι) and Rev1. Of these subfamilies, DinB orthologs are found in all three domains of life; eubacteria, archaea, and eukarya. UmuC orthologs are identified only in bacteria, whilst Rev1 and Rad30A/B orthologs are only detected in eukaryotes. Within eukaryotes, a wide array of evolutionary diversity exists. Humans possess all four Y-family pols (pols η, ι, κ, and Rev1), Schizosaccharomyces pombe has three Y-family pols (pols η, κ, and Rev1), and Saccharomyces cerevisiae only has polη and Rev1. Here, we report the cloning, expression, and biochemical characterization of the four Y-family pols from the lower eukaryotic thermophilic fungi, Thermomyces lanuginosus. Apart from the expected increased thermostability of the T. lanuginosus Y-family pols, their major biochemical properties are very similar to properties of their human counterparts. In particular, both Rad30B homologs (T. lanuginosus and human polÉ©) exhibit remarkably low fidelity during DNA synthesis that is template sequence dependent. It was previously hypothesized that higher organisms had acquired this property during eukaryotic evolution, but these observations imply that polι originated earlier than previously known, suggesting a critical cellular function in both lower and higher eukaryotes.

129-derived strains of mice are deficient in DNA polymerase iota and have normal immunoglobulin hypermutation.

Recent studies suggest that DNA polymerase eta (poleta) and DNA polymerase iota (poliota) are involved in somatic hypermutation of immunoglobulin variable genes. To test the role of poliota in generating mutations in an animal model, we first characterized the biochemical properties of murine poliota. Like its human counterpart, murine poliota is extremely error-prone when catalyzing synthesis on a variety of DNA templates in vitro. Interestingly, when filling in a 1 base-pair gap, DNA synthesis and subsequent strand displacement was greatest in the presence of both pols iota and eta. Genomic sequence analysis of Poli led to the serendipitous discovery that 129-derived strains of mice have a nonsense codon mutation in exon 2 that abrogates production of poliota. Analysis of hypermutation in variable genes from 129/SvJ (Poli-/-) and C57BL/6J (Poli+/+) mice revealed that the overall frequency and spectrum of mutation were normal in poliota-deficient mice. Thus, either poliota does not participate in hypermutation, or its role is nonessential and can be readily assumed by another low-fidelity polymerase.