Impairment of the non-catalytic subunit Dpb2 of DNA Pol ɛ results in increased involvement of Pol δ on the leading strand.

The generally accepted model assumes that leading strand synthesis is performed by Pol ε, while lagging-strand synthesis is catalyzed by Pol δ. Pol ε has been shown to target the leading strand by interacting with the CMG helicase [Cdc45 Mcm2-7 GINS(Psf1-3, Sld5)]. Proper functioning of the CMG-Pol ɛ, the helicase-polymerase complex is essential for its progression and the fidelity of DNA replication. Dpb2p, the essential non-catalytic subunit of Pol ε plays a key role in maintaining the correct architecture of the replisome by acting as a link between Pol ε and the CMG complex. Using a temperature-sensitive dpb2-100 mutant previously isolated in our laboratory, and a genetic system which takes advantage of a distinct mutational signature of the Pol δ-L612M variant which allows detection of the involvement of Pol δ in the replication of particular DNA strands we show that in yeast cells with an impaired Dpb2 subunit, the contribution of Pol δ to the replication of the leading strand is significantly increased.

Strand specificity of ribonucleotide excision repair in Escherichia coli.

In Escherichia coli, replication of both strands of genomic DNA is carried out by a single replicase-DNA polymerase III holoenzyme (pol III HE). However, in certain genetic backgrounds, the low-fidelity TLS polymerase, DNA polymerase V (pol V) gains access to undamaged genomic DNA where it promotes elevated levels of spontaneous mutagenesis preferentially on the lagging strand. We employed active site mutants of pol III (pol IIIα_S759N) and pol V (pol V_Y11A) to analyze ribonucleotide incorporation and removal from the E. coli chromosome on a genome-wide scale under conditions of normal replication, as well as SOS induction. Using a variety of methods tuned to the specific properties of these polymerases (analysis of lacI mutational spectra, lacZ reversion assay, HydEn-seq, alkaline gel electrophoresis), we present evidence that repair of ribonucleotides from both DNA strands in E. coli is unequal. While RNase HII plays a primary role in leading-strand Ribonucleotide Excision Repair (RER), the lagging strand is subject to other repair systems (RNase HI and under conditions of SOS activation also Nucleotide Excision Repair). Importantly, we suggest that RNase HI activity can also influence the repair of single ribonucleotides incorporated by the replicase pol III HE into the lagging strand.

Mutation spectrum data for Saccharomyces cerevisiae psf1-1 pol2-M644G mutants.

DNA replication in Saccharomyces cerevisiae and other eukaryotes is performed mainly by polymerase epsilon (Pol ε) on the leading strand and polymerase delta (Pol δ) on the lagging strand. Using a mutant form of a DNA polymerase enables tracking its signature in the replicated DNA. Here, we used the pol2-M644G allele encoding the catalytic subunit of Pol ε to analyse its contribution to DNA replication in yeast with the psf1-1 allele of an essential gene encoding a subunit of the GINS complex. GINS is involved in the recruitment of Pol ε, the major leading strand replicase. Thus, its defective functioning can affect the involvement of Pol ε in DNA replication. Together with Cdc45 and Mcm2-7, GINS forms the CMG helicase complex. Our DNA sequencing data enable the observation of changes in the mutational spectra in the URA3 reporter gene cloned in two orientations regarding the nearest ARS. The data presented in this article support the study "Increased contribution of DNA polymerase delta to the leading strand replication in yeast with an impaired CMG helicase complex" [1].

Increased contribution of DNA polymerase delta to the leading strand replication in yeast with an impaired CMG helicase complex.

DNA replication is performed by replisome proteins, which are highly conserved from yeast to humans. The CMG [Cdc45-Mcm2-7-GINS(Psf1-3, Sld5)] helicase unwinds the double helix to separate the leading and lagging DNA strands, which are replicated by the specialized DNA polymerases epsilon (Pol ε) and delta (Pol δ), respectively. This division of labor was confirmed by both genetic analyses and in vitro studies. Exceptions from this rule were described mainly in cells with impaired catalytic polymerase ε subunit. The central role in the recruitment and establishment of Pol ε on the leading strand is played by the CMG complex assembled on DNA during replication initiation. In this work we analyzed the consequences of impaired functioning of the CMG complex for the division labor between DNA polymerases on the two replicating strands. We showed in vitro that the GINSPsf1-1 complex poorly bound the Psf3 subunit. In vivo, we observed increased rates of L612M Pol δ-specific mutations during replication of the leading DNA strand in psf1-1 cells. These findings indicated that defective functioning of GINS impaired leading strand replication by Pol ε and necessitated involvement of Pol δ in the synthesis on this strand with a possible impact on the distribution of mutations and genomic stability. These are the first results to imply that the division of labor between the two main replicases can be severely influenced by a defective nonpolymerase subunit of the replisome.

Recombination and Pol ζ Rescue Defective DNA Replication upon Impaired CMG Helicase-Pol ε Interaction.

The CMG complex (Cdc45, Mcm2-7, GINS (Psf1, 2, 3, and Sld5)) is crucial for both DNA replication initiation and fork progression. The CMG helicase interaction with the leading strand DNA polymerase epsilon (Pol ε) is essential for the preferential loading of Pol ε onto the leading strand, the stimulation of the polymerase, and the modulation of helicase activity. Here, we analyze the consequences of impaired interaction between Pol ε and GINS in Saccharomyces cerevisiae cells with the psf1-100 mutation. This significantly affects DNA replication activity measured in vitro, while in vivo, the psf1-100 mutation reduces replication fidelity by increasing slippage of Pol ε, which manifests as an elevated number of frameshifts. It also increases the occurrence of single-stranded DNA (ssDNA) gaps and the demand for homologous recombination. The psf1-100 mutant shows elevated recombination rates and synthetic lethality with rad52Δ. Additionally, we observe increased participation of DNA polymerase zeta (Pol ζ) in DNA synthesis. We conclude that the impaired interaction between GINS and Pol ε requires enhanced involvement of error-prone Pol ζ, and increased participation of recombination as a rescue mechanism for recovery of impaired replication forks.

Defects in the GINS complex increase the instability of repetitive sequences via a recombination-dependent mechanism.

Faithful replication and repair of DNA lesions ensure genome maintenance. During replication in eukaryotic cells, DNA is unwound by the CMG helicase complex, which is composed of three major components: the Cdc45 protein, Mcm2-7, and the GINS complex. The CMG in complex with DNA polymerase epsilon (CMG-E) participates in the establishment and progression of the replisome. Impaired functioning of the CMG-E was shown to induce genomic instability and promote the development of various diseases. Therefore, CMG-E components play important roles as caretakers of the genome. In Saccharomyces cerevisiae, the GINS complex is composed of the Psf1, Psf2, Psf3, and Sld5 essential subunits. The Psf1-1 mutant form fails to interact with Psf3, resulting in impaired replisome assembly and chromosome replication. Here, we show increased instability of repeat tracts (mononucleotide, dinucleotide, trinucleotide and longer) in yeast psf1-1 mutants. To identify the mechanisms underlying this effect, we analyzed repeated sequence instability using derivatives of psf1-1 strains lacking genes involved in translesion synthesis, recombination, or mismatch repair. Among these derivatives, deletion of RAD52, RAD51, MMS2, POL32, or PIF1 significantly decreased DNA repeat instability. These results, together with the observed increased amounts of single-stranded DNA regions and Rfa1 foci suggest that recombinational mechanisms make important contributions to repeat tract instability in psf1-1 cells. We propose that defective functioning of the CMG-E complex in psf1-1 cells impairs the progression of DNA replication what increases the contribution of repair mechanisms such as template switch and break-induced replication. These processes require sequence homology search which in case of a repeated DNA tract may result in misalignment leading to its expansion or contraction.

Role of RNase H enzymes in maintaining genome stability in Escherichia coli expressing a steric-gate mutant of pol VICE391.

pol VICE391 (RumA'2B) 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 increased levels of DNA synthesis compared to UmuC. We have generated a steric gate pol VICE391 variant (pol VICE391_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 VICE391 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 VICE391_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 VICE391_Y13A exhibit genome and plasmid instability in the absence of RNase HI.

Replication fidelity in E. coli: Differential leading and lagging strand effects for dnaE antimutator alleles.

DNA Pol III holoenzyme (HE) is the major DNA replicase of Escherichia coli. It is a highly accurate enzyme responsible for simultaneously replicating the leading- and lagging DNA strands. Interestingly, the fidelity of replication for the two DNA strands is unequal, with a higher accuracy for lagging-strand replication. We have previously proposed this higher lagging-strand fidelity results from the more dissociative character of the lagging-strand polymerase. In support of this hypothesis, an E. coli mutant carrying a catalytic DNA polymerase subunit (DnaE915) characterized by decreased processivity yielded an antimutator phenotype (higher fidelity). The present work was undertaken to gain deeper insight into the factors that influence the fidelity of chromosomal DNA replication in E. coli. We used three different dnaE alleles (dnaE915, dnaE911, and dnaE941) that had previously been isolated as antimutators. We confirmed that each of the three dnaE alleles produced significant antimutator effects, but in addition showed that these antimutator effects proved largest for the normally less accurate leading strand. Additionally, in the presence of error-prone DNA polymerases, each of the three dnaE antimutator strains turned into mutators. The combined observations are fully supportive of our model in which the dissociative character of the DNA polymerase is an important determinant of in vivo replication fidelity. In this model, increased dissociation from terminal mismatches (i.e., potential mutations) leads to removal of the mismatches (antimutator effect), but in the presence of error-prone (or translesion) DNA polymerases the abandoned terminal mismatches become targets for error-prone extension (mutator effect). We also propose that these dnaE alleles are promising tools for studying polymerase exchanges at the replication fork.

The SOS system: A complex and tightly regulated response to DNA damage.

Genomes of all living organisms are constantly threatened by endogenous and exogenous agents that challenge the chemical integrity of DNA. Most bacteria have evolved a coordinated response to DNA damage. In Escherichia coli, this inducible system is termed the SOS response. The SOS global regulatory network consists of multiple factors promoting the integrity of DNA as well as error-prone factors allowing for survival and continuous replication upon extensive DNA damage at the cost of elevated mutagenesis. Due to its mutagenic potential, the SOS response is subject to elaborate regulatory control involving not only transcriptional derepression, but also post-translational activation, and inhibition. This review summarizes current knowledge about the molecular mechanism of the SOS response induction and progression and its consequences for genome stability. Environ. Mol. Mutagen. 60:368-384, 2019. © 2018 The Authors. Environmental and Molecular Mutagenesis published by Wiley Periodicals, Inc. on behalf of Environmental Mutagen Society.

High-accuracy lagging-strand DNA replication mediated by DNA polymerase dissociation.

The fidelity of DNA replication is a critical factor in the rate at which cells incur mutations. Due to the antiparallel orientation of the two chromosomal DNA strands, one strand (leading strand) is replicated in a mostly processive manner, while the other (lagging strand) is synthesized in short sections called Okazaki fragments. A fundamental question that remains to be answered is whether the two strands are copied with the same intrinsic fidelity. In most experimental systems, this question is difficult to answer, as the replication complex contains a different DNA polymerase for each strand, such as, for example, DNA polymerases δ and ε in eukaryotes. Here we have investigated this question in the bacterium Escherichia coli, in which the replicase (DNA polymerase III holoenzyme) contains two copies of the same polymerase (Pol III, the dnaE gene product), and hence the two strands are copied by the same polymerase. Our in vivo mutagenesis data indicate that the two DNA strands are not copied with the same accuracy, and that, remarkably, the lagging strand has the highest fidelity. We postulate that this effect results from the greater dissociative character of the lagging-strand polymerase, which provides additional options for error removal. Our conclusion is strongly supported by results with dnaE antimutator polymerases characterized by increased dissociation rates.

The importance of an interaction network for proper DNA polymerase ζ heterotetramer activity.

Precisely controlled mechanisms have been evolved to rescue impeded DNA replication resulting from encountered obstacles and involve a set of low-fidelity translesion synthesis (TLS) DNA polymerases. Studies in recent years have brought new insights into those TLS polymerases, especially concerning the structure and subunit composition of DNA polymerase zeta (Pol ζ). Pol ζ is predominantly involved in induced mutagenesis as well as the bypass of noncanonical DNA structures, and it is proficient in extending from terminal mismatched nucleotides incorporated by major replicative DNA polymerases. Two active forms of Pol ζ, heterodimeric (Pol ζ2) and heterotetrameric (Pol ζ4) ones, have been identified and studied. Here, in the light of recent publications regarding induced and spontaneous mutagenesis and diverse interactions within Pol ζ holoenzyme, combined with Pol ζ binding to the TLS polymerase Rev1p, we discuss the subunit composition of Pol ζ in various cellular physiological conditions. Available data show that it is the heterotetrameric form of Pol ζ that is involved both during spontaneous and induced mutagenesis, and underline the importance of interactions within Pol ζ when an increased Pol ζ recruitment occurs. Understanding Pol ζ function in the bypass of DNA obstacles would give a significant insight into cellular tolerance of DNA damage, genetic instability and the onset of cancer progression.

The CysB motif of Rev3p involved in the formation of the four-subunit DNA polymerase ζ is required for defective-replisome-induced mutagenesis.

Eukaryotic DNA replication is performed by high-fidelity multi-subunit replicative B-family DNA polymerases (Pols) α, δ and ɛ. Those complexes are composed of catalytic and accessory subunits and organized in multicomplex machinery: the replisome. The fourth B-family member, DNA polymerase zeta (Pol ζ), is responsible for a large portion of mutagenesis in eukaryotic cells. Two forms of Pol ζ have been identified, a hetero-dimeric (Pol ζ2 ) and a hetero-tetrameric (Pol ζ4 ) ones and recent data have demonstrated that Pol ζ4 is responsible for damage-induced mutagenesis. Here, using yeast Pol ζ mutant defective in the assembly of the Pol ζ four-subunit form, we show in vivo that [4Fe-4S] cluster in Pol ζ catalytic subunit (Rev3p) is also required for spontaneous (wild-type cells) and defective-replisome-induced mutagenesis - DRIM (pol3-Y708A, pol2-1 or psf1-100 cells), when cells are not treated with any external damaging agents.

Diverse roles of Dpb2, the non-catalytic subunit of DNA polymerase ε.

Timely progression of living cells through the cell cycle is precisely regulated. This involves a series of phosphorylation events which are regulated by various cyclins, activated in coordination with the cell cycle progression. Phosphorylated proteins govern cell growth, division as well as duplication of the genetic material and transcriptional activation of genes involved in these processes. A subset of these tightly regulated genes, which depend on the MBF transcription factor and are mainly involved in DNA replication and cell division, is transiently activated at the transition from G1 to S phase. A Saccharomyces cerevisiae mutant in the Dpb2 non-catalytic subunit of DNA polymerase ε (Polε) demonstrates abnormalities in transcription of MBF-dependent genes even in normal growth conditions. It is, therefore, tempting to speculate that Dpb2 which, as described previously, participates in the early stages of DNA replication initiation, has an impact on the regulation of replication-related genes expression with possible implications for genomic stability.

Mutations in the Non-Catalytic Subunit Dpb2 of DNA Polymerase Epsilon Affect the Nrm1 Branch of the DNA Replication Checkpoint.

To preserve genome integrity, the S-phase checkpoint senses damaged DNA or nucleotide depletion and when necessary, arrests replication progression and delays cell division. Previous studies, based on two pol2 mutants have suggested the involvement of DNA polymerase epsilon (Pol ε) in sensing DNA replication accuracy in Saccharomyces cerevisiae. Here we have studied the involvement of Pol ε in sensing proper progression of DNA replication, using a mutant in DPB2, the gene coding for a non-catalytic subunit of Pol ε. Under genotoxic conditions, the dpb2-103 cells progress through S phase faster than wild-type cells. Moreover, the Nrm1-dependent branch of the checkpoint, which regulates the expression of many replication checkpoint genes, is impaired in dpb2-103 cells. Finally, deletion of DDC1 in the dpb2-103 mutant is lethal supporting a model of strand-specific activation of the replication checkpoint. This lethality is suppressed by NRM1 deletion. We postulate that improper activation of the Nrm1-branch may explain inefficient replication checkpoint activation in Pol ε mutants.

Suppression of the E. coli SOS response by dNTP pool changes.

The Escherichia coli SOS system is a well-established model for the cellular response to DNA damage. Control of SOS depends largely on the RecA protein. When RecA is activated by single-stranded DNA in the presence of a nucleotide triphosphate cofactor, it mediates cleavage of the LexA repressor, leading to expression of the 30(+)-member SOS regulon. RecA activation generally requires the introduction of DNA damage. However, certain recA mutants, like recA730, bypass this requirement and display constitutive SOS expression as well as a spontaneous (SOS) mutator effect. Presently, we investigated the possible interaction between SOS and the cellular deoxynucleoside triphosphate (dNTP) pools. We found that dNTP pool changes caused by deficiencies in the ndk or dcd genes, encoding nucleoside diphosphate kinase and dCTP deaminase, respectively, had a strongly suppressive effect on constitutive SOS expression in recA730 strains. The suppression of the recA730 mutator effect was alleviated in a lexA-deficient background. Overall, the findings suggest a model in which the dNTP alterations in the ndk and dcd strains interfere with the activation of RecA, thereby preventing LexA cleavage and SOS induction.

Fidelity consequences of the impaired interaction between DNA polymerase epsilon and the GINS complex.

DNA polymerase epsilon interacts with the CMG (Cdc45-MCM-GINS) complex by Dpb2p, the non-catalytic subunit of DNA polymerase epsilon. It is postulated that CMG is responsible for targeting of Pol ɛ to the leading strand. We isolated a mutator dpb2-100 allele which encodes the mutant form of Dpb2p. We showed previously that Dpb2-100p has impaired interactions with Pol2p, the catalytic subunit of Pol ɛ. Here, we present that Dpb2-100p has strongly impaired interaction with the Psf1 and Psf3 subunits of the GINS complex. Our in vitro results suggest that while dpb2-100 does not alter Pol ɛ's biochemical properties including catalytic efficiency, processivity or proofreading activity - it moderately decreases the fidelity of DNA synthesis. As the in vitro results did not explain the strong in vivo mutator effect of the dpb2-100 allele we analyzed the mutation spectrum in vivo. The analysis of the mutation rates in the dpb2-100 mutant indicated an increased participation of the error-prone DNA polymerase zeta in replication. However, even in the absence of Pol ζ activity the presence of the dpb2-100 allele was mutagenic, indicating that a significant part of mutagenesis is Pol ζ-independent. A strong synergistic mutator effect observed for transversions in the triple mutant dpb2-100 pol2-4 rev3Δ as compared to pol2-4 rev3Δ and dpb2-100 rev3Δ suggests that in the presence of the dpb2-100 allele the number of replication errors is enhanced. We hypothesize that in the dpb2-100 strain, where the interaction between Pol ɛ and GINS is weakened, the access of Pol δ to the leading strand may be increased. The increased participation of Pol δ on the leading strand in the dpb2-100 mutant may explain the synergistic mutator effect observed in the dpb2-100 pol3-5DV double mutant.

Proper functioning of the GINS complex is important for the fidelity of DNA replication in yeast.

The role of replicative DNA polymerases in ensuring genome stability is intensively studied, but the role of other components of the replisome is still not fully understood. One of such component is the GINS complex (comprising the Psf1, Psf2, Psf3 and Sld5 subunits), which participates in both initiation and elongation of DNA replication. Until now, the understanding of the physiological role of GINS mostly originated from biochemical studies. In this article, we present genetic evidence for an essential role of GINS in the maintenance of replication fidelity in Saccharomyces cerevisiae. In our studies we employed the psf1-1 allele (Takayama et al., 2003) and a novel psf1-100 allele isolated in our laboratory. Analysis of the levels and specificity of mutations in the psf1 strains indicates that the destabilization of the GINS complex or its impaired interaction with DNA polymerase epsilon increases the level of spontaneous mutagenesis and the participation of the error-prone DNA polymerase zeta. Additionally, a synergistic mutator effect was found for the defects in Psf1p and in the proofreading activity of Pol epsilon, suggesting that proper functioning of GINS is crucial for facilitating error-free processing of terminal mismatches created by Pol epsilon.

Effect of dNTP pool alterations on fidelity of leading and lagging strand DNA replication in E. coli.

The fidelity with which organisms replicate their chromosomal DNA is of considerable interest. Detailed studies in the bacterium Escherichia coli have indicated that the fidelity of leading- and lagging-strand DNA replication is not the same, based on experiments in which the orientation of certain mutational targets on the chromosome was inverted relative to the movement of the replication fork: different mutation rates for several base-pair substitutions were observed depending on this orientation. While these experiments are indicative of differential replication fidelity in the two strands, a conclusion whether leading or lagging strand is the more accurate depends on knowledge of the primary mispairing error responsible for the base substitutions in question. A broad analysis of in vitro base-pairing preferences of DNA polymerases led us to propose that lagging-strand is the more accurate strand. In the present work, we present more direct in vivo evidence in support of this proposal. We determine the orientation dependence of mutant frequencies in ndk and dcd strains, which carry defined dNTP pool alterations. As these pool alterations lead to predictable effects on the array of possible mispairing errors, they mark the strands in which the observed errors occur. The combined results support the proposed higher accuracy of lagging-strand replication in E. coli.

Defect of Dpb2p, a noncatalytic subunit of DNA polymerase ɛ, promotes error prone replication of undamaged chromosomal DNA in Saccharomyces cerevisiae.

The Saccharomyces cerevisiae DNA polymerase epsilon holoenzyme (Pol ɛ HE) is composed of four subunits: Pol2p, Dpb2p, Dpb3p and Dpb4p. The biological functions of Pol2p, the catalytic subunit of Pol ɛ, are subject of active investigation, while the role of the other three, noncatalytic subunits, is not well defined. We showed previously that mutations in Dpb2p, a noncatalytic but essential subunit of Pol ɛ HE, influence the fidelity of DNA replication in yeast cells. The strength of the mutator phenotype due to the different dpb2 alleles was inversely proportional to the strength of protein-protein interactions between Pol2p and the mutated forms of Dpb2p. To understand better the mechanisms of the contribution of Dpb2p to the controlling of the level of spontaneous mutagenesis we undertook here a further genetic analysis of the mutator phenotype observed in dpb2 mutants. We demonstrate that the presence of mutated forms of Dpb2p in the cell not only influences the intrinsic fidelity of Pol ɛ but also facilitates more frequent participation of error-prone DNA polymerase zeta (Pol ζ) in DNA replication. The obtained results suggest that the structural integrity of Pol ɛ HE is a crucial contributor to accurate chromosomal DNA replication and, when compromised, favors participation of error prone DNA Pol ζ in this process.

DNA replication fidelity in Escherichia coli: a multi-DNA polymerase affair.

High accuracy (fidelity) of DNA replication is important for cells to preserve the genetic identity and to prevent the accumulation of deleterious mutations. The error rate during DNA replication is as low as 10(-9) to 10(-11) errors per base pair. How this low level is achieved is an issue of major interest. This review is concerned with the mechanisms underlying the fidelity of the chromosomal replication in the model system Escherichia coli by DNA polymerase III holoenzyme, with further emphasis on participation of the other, accessory DNA polymerases, of which E. coli contains four (Pols I, II, IV, and V). Detailed genetic analysis of mutation rates revealed that (1) Pol II has an important role as a back-up proofreader for Pol III, (2) Pols IV and V do not normally contribute significantly to replication fidelity, but can readily do so under conditions of elevated expression, (3) participation of Pols IV and V, in contrast to that of Pol II, is specific to the lagging strand, and (4) Pol I also makes a lagging-strand-specific fidelity contribution, limited, however, to the faithful filling of the Okazaki fragment gaps. The fidelity role of the Pol III τ subunit is also reviewed.