Escherichia coli DNA polymerase IV (Pol IV), but not Pol II, dynamically switches with a stalled Pol III* replicase

The dnaN159 allele encodes a temperature-sensitive mutant form of the β sliding clamp (β159). SOS-induced levels of DNA polymerase IV (Pol IV) confer UV sensitivity upon the dnaN159 strain, while levels of Pol IV ～4-fold higher than those induced by the SOS response severely impede its growth. Here, we used mutations in Pol IV that disrupted specific interactions with the β clamp to test our hypothesis that these phenotypes were the result of Pol IV gaining inappropriate access to the replication fork via a Pol III*-Pol IV switch relying on both the rim and cleft of the clamp. Our results clearly demonstrate that Pol IV relied on both the clamp rim and cleft interactions for these phenotypes. In contrast to the case for Pol IV, elevated levels of the other Pols, including Pol II, which was expressed at levels ～8-fold higher than the normal SOS-induced levels, failed to impede growth of the dnaN159 strain. These findings suggest that the mechanism used by Pol IV to switch with Pol III* is distinct from those used by the other Pols. Results of experiments utilizing purified components to reconstitute the Pol III*-Pol II switch in vitro indicated that Pol II switched equally well with both a stalled and an actively replicating Pol III* in a manner that was independent of the rim contact required by Pol IV. These results provide compelling support for the Pol III*-Pol IV two-step switch model and demonstrate important mechanistic differences in how Pol IV and Pol II switch with Pol III*.     

Polyphosphate kinase regulates error-prone replication by DNA polymerase IV in Escherichia coli

The ppk gene encodes polyphosphate kinase (Ppk), an enzyme that catalyses the polymerization of inorganic phosphate into long chains of polyphosphate (polyP). An insertion mutation in ppk causes a decrease in adaptive mutation in Escherichia coli strain FC40. Adaptive mutation in FC40 mostly results from error-prone DNA polymerase IV (Pol IV), encoded by dinB; most of the antimutagenic phenotype of the ppk mutant disappears in a dinB mutant strain. In addition, the ppk mutant causes a decrease in growth-dependent mutations produced by overexpressing Pol IV. However, the amount of Pol IV protein is unchanged in the ppk mutant strain, indicating that the activity or fidelity of Pol IV is altered. Adaptive mutation is inhibited both by the absence of Ppk, which results in low amounts of polyP, and by overproduction of Ppk, which results in high amounts of polyP, suggesting that an optimal level of polyP is necessary. Taken together, these results suggest a novel mechanism involving polyP that directly or indirectly regulates DNA polymerase activity or fidelity.     

Escherichia coli DNA polymerase IV contributes to spontaneous mutagenesis at coding sequences but not microsatellite alleles

Slipped strand mispairing during DNA synthesis is one proposed mechanism for microsatellite or short tandem repeat (STR) mutation. However, the DNA polymerase(s) responsible for STR mutagenesis have not been determined. In this study, we investigated the effect of the Escherichia colidinB gene product (Pol IV) on mononucleotide and dinucleotide repeat stability, using an HSV-tk gene episomal reporter system for microsatellite mutations. For the control vector (HSV-tk gene only) we observed a statistically significant 3.5-fold lower median mutation frequency in dinB(-) than dinB(+) cells (p<0.001, Wilcoxon Mann Whitney Test). For vectors containing an in-frame mononucleotide allele ([G/C](10)) or either of two dinucleotide alleles ([GT/CA](10) and [TC/AG](11)) we observed no statistically significant difference in the overall HSV-tk mutation frequency observed between dinB(+) and dinB(-) strains. To determine if a mutational bias exists for mutations made by Pol IV, mutational spectra were generated for each STR vector and strain. No statistically significant differences between strains were observed for either the proportion of mutational events at the STR or STR specificity among the three vectors. However, the specificity of mutational events at the STR alleles in each strain varied in a statistically significant manner as a consequence of microsatellite sequence. Our results indicate that while Pol IV contributes to spontaneous mutations within the HSV-tk coding sequence, Pol IV does not play a significant role in spontaneous mutagenesis at [G/C](10), [GT/CA](10), or [TC/AG](11) microsatellite alleles. Our data demonstrate that in a wild type genetic background, the major factor influencing microsatellite mutagenesis is the allelic sequence composition.     

MutS deficiency and activity of the error-prone DNA polymerase IV are crucial for determining mucA as the main target for mucoid conversion in Pseudomonas aeruginosa

Pseudomonas aeruginosa colonizes the respiratory tract of cystic fibrosis (CF) patients, where mutators along with mucoid variants emerge leading to chronic infection. Mucoid conversion generally involves mutations inactivating the mucA gene. This study correlates the frequency and nature of mucA mutations with the activity of factors determining the mutation rate, such as MutS and polymerase IV (Pol IV). Results show that: (i) the emergence frequency of mucoid variants was higher in isolates arising from mutS populations compared with the wild-type strain; (ii) in both strains mucoid conversion occurred mainly by mucA mutations; (iii) however, the mutator strain harboured mostly mucA22 (a common allele in CF isolates), while the wild type showed a wider spectrum of mucA mutations with low incidence of mucA22; (iv) disruption of dinB in the wild-type and mutS strains decreased drastically the emergence frequency of mucoid variants; (v) furthermore, the incidence of mucA mutations diminished in the mutS dinB double mutant strain which consisted only in mucA22; (vi) finally, the mucoid isolates obtained from the dinB strain showed an unexpected absence of mucA mutations. Taken together results demonstrate the implication of both MutS and Pol IV in determining mucA as the main target for conversion to mucoidy.     

Escherichia coli Rep DNA helicase and error-prone DNA polymerase IV interact physically and functionally

Escherichia coli DNA polymerase IV, encoded by the dinB gene, is a member of the Y family of specialized DNA polymerases. Pol IV is capable of synthesizing past DNA lesions and may help to restart stalled replication forks. However, Pol IV is error-prone, contributing to both DNA damage-induced and stress-induced (adaptive) mutations. Here we demonstrate that Pol IV interacts in vitro with Rep DNA helicase and that this interaction enhances Rep's helicase activity. In addition, Pol IV polymerase activity is stimulated by interacting with Rep, and Pol IV β clamp-binding motif appears to be required for this stimulation. However, neither Rep's helicase activity nor its ability to bind DNA is required for it to stimulate Pol IV's polymerase activity. The interaction between Rep and Pol IV is biologically significant in vivo as Rep enhances Pol IV's mutagenic activity in stationary-phase cells. These data indicate a new role for Rep in contributing to Pol IV-dependent adaptive mutation. This functional interaction also provides new insight into how the cell might control or target Pol IV's mutagenic activity.     

Polymerases leave fingerprints: analysis of the mutational spectrum in Escherichia coli rpoB to assess the role of polymerase IV in spontaneous mutation

We compared the distribution of mutations in rpoB that lead to rifampin resistance in strains with differing levels of polymerase IV (Pol IV), including strains with deletions of the Pol IV-encoding dinB gene, strains with a chromosomal copy of dinB, strains with the F'128 plasmid, and strains with plasmid amplification of either the dinB operon (dinB-yafNOP) or the dinB gene alone. This analysis identifies several hot spots specific to Pol IV which are virtually absent from the normal spontaneous spectrum, indicating that Pol IV does not contribute significantly to mutations occurring during exponential growth in liquid culture.     

Error-prone DNA polymerase IV is regulated by the heat shock chaperone GroE in Escherichia coli

An insertion in the promoter of the operon that encodes the molecular chaperone GroE was isolated as an antimutator for stationary-phase or adaptive mutation. The groE operon consists of two genes, groES and groEL; point mutations in either gene conferred the same phenotype, reducing Lac+ adaptive mutation 10- to 20-fold. groE mutant strains had 1/10 the amount of error-prone DNA polymerase IV (Pol IV). In recG+ strains, the reduction in Pol IV was sufficient to account for their low rate of adaptive mutation, but in recG mutant strains, a deficiency of GroE had some additional effect on adaptive mutation. Pol IV is induced as part of the SOS response, but the effect of GroE on Pol IV was independent of LexA. We were unable to show that GroE interacts directly with Pol IV, suggesting that GroE may act indirectly. Together with previous results, these findings indicate that Pol IV is a component of several cellular stress responses.     

The Escherichia coli histone-like protein HU has a role in stationary phase adaptive mutation

Stationary phase adaptive mutation in Escherichia coli is thought to be a mechanism by which mutation rates are increased during stressful conditions, increasing the possibility that fitness-enhancing mutations arise. Here we present data showing that the histone-like protein, HU, has a role in the molecular pathway by which adaptive Lac(+) mutants arise in E. coli strain FC40. Adaptive Lac(+) mutations are largely but not entirely due to error-prone DNA polymerase IV (Pol IV). Mutations in either of the HU subunits, HUalpha or HUbeta, decrease adaptive mutation to Lac(+) by both Pol IV-dependent and Pol IV-independent pathways. Additionally, HU mutations inhibit growth-dependent mutations without a reduction in the level of Pol IV. These effects of HU mutations on adaptive mutation and on growth-dependent mutations reveal novel functions for HU in mutagenesis.     

Escherichia coli DNA polymerase IV mutator activity: genetic requirements and mutational specificity

The dinB gene of Escherichia coli is known to be involved in the untargeted mutagenesis of lambda phage. Recently, we have demonstrated that this damage-inducible and SOS-controlled gene encodes a novel DNA polymerase, DNA Pol IV, which is able to dramatically increase the untargeted mutagenesis of F' plasmid. At the amino acid level, DNA Pol IV shares sequence homologies with E. coli UmuC (DNA Pol V), Rev1p, and Rad30p (DNA polymerase eta) of Saccharomyces cerevisiae and human Rad30A (XPV) proteins, all of which are involved in translesion DNA synthesis. To better characterize the Pol IV-dependent untargeted mutagenesis, i.e., the DNA Pol IV mutator activity, we analyzed the genetic requirements of this activity and determined the forward mutation spectrum generated by this protein within the cII gene of lambda phage. The results indicated that the DNA Pol IV mutator activity is independent of polA, polB, recA, umuDC, uvrA, and mutS functions. The analysis of more than 300 independent mutations obtained in the wild-type or mutS background revealed that the mutator activity clearly promotes single-nucleotide substitutions as well as one-base deletions in the ratio of about 1:2. The base changes were strikingly biased for substitutions toward G:C base pairs, and about 70% of them occurred in 5'-GX-3' sequences, where X represents the base (T, A, or C) that is mutated to G. These results are discussed with respect to the recently described biochemical characteristics of DNA Pol IV.     

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.     

The beta clamp targets DNA polymerase IV to DNA and strongly increases its processivity

The recent discovery of a new family of ubiquitous DNA polymerases involved in translesion synthesis has shed new light onto the biochemical basis of mutagenesis. Among these polymerases, the dinB gene product (Pol IV) is involved in mutagenesis in Escherichia coli. We show here that the activity of native Pol IV is drastically modified upon interaction with the β subunit, the processivity factor of DNA Pol III. In the absence of the β subunit Pol IV is strictly distributive and no stable complex between Pol IV and DNA could be detected. In contrast, the β clamp allows Pol IV to form a stable initiation complex (t_1/2 ≈ 2.3 min), which leads to a dramatic increase in the processivity of Pol IV reaching an average of 300–400 nucleotides. In vivo, the β processivity subunit may target DNA Pol IV to its substrate, generating synthesis tracks much longer than previously thought.     

Plant nuclear RNA polymerase IV mediates siRNA and DNA methylation-dependent heterochromatin formation

All eukaryotes have three nuclear DNA-dependent RNA polymerases, namely, Pol I, II, and III. Interestingly, plants have catalytic subunits for a fourth nuclear polymerase, Pol IV. Genetic and biochemical evidence indicates that Pol IV does not functionally overlap with Pol I, II, or III and is nonessential for viability. However, disruption of the Pol IV catalytic subunit genes NRPD1 or NRPD2 inhibits heterochromatin association into chromocenters, coincident with losses in cytosine methylation at pericentromeric 5S gene clusters and AtSN1 retroelements. Loss of CG, CNG, and CNN methylation in Pol IV mutants implicates a partnership between Pol IV and the methyltransferase responsible for RNA-directed de novo methylation. Consistent with this hypothesis, 5S gene and AtSN1 siRNAs are essentially eliminated in Pol IV mutants. The data suggest that Pol IV helps produce siRNAs that target de novo cytosine methylation events required for facultative heterochromatin formation and higher-order heterochromatin associations.     

Roles of RNA polymerase IV in gene silencing

Eukaryotes typically have three multi-subunit enzymes that decode the nuclear genome into RNA: DNA-dependent RNA polymerases I, II and III (Pol I, II and III). Remarkably, higher plants have five multi-subunit nuclear RNA polymerases: the ubiquitous Pol I, II and III, which are essential for viability; plus two non-essential polymerases, Pol IVa and Pol IVb, which specialize in small RNA-mediated gene silencing pathways. There are numerous examples of phenomena that require Pol IVa and/or Pol IVb, including RNA-directed DNA methylation of endogenous repetitive elements, silencing of transgenes, regulation of flowering-time genes, inducible regulation of adjacent gene pairs, and spreading of mobile silencing signals. Although biochemical details concerning Pol IV enzymatic activities are lacking, genetic evidence suggests several alternative models for how Pol IV might function.     

Role of damage-specific DNA polymerases in M13 phage mutagenesis induced by a major lipid peroxidation product trans-4-hydroxy-2-nonenal

One of the major lipid peroxidation products trans-4-hydroxy-2-nonenal (HNE), forms cyclic propano- or ethenoadducts bearing six- or seven-carbon atom side chains to G > C ? A > T. To specify the role of SOS DNA polymerases in HNE-induced mutations, we tested survival and mutation spectra in the lacZα gene of M13mp18 phage, whose DNA was treated in vitro with HNE, and which was grown in uvrA^? Escherichia coli strains, carrying one, two or all three SOS DNA polymerases. When Pol IV was the only DNA SOS polymerase in the bacterial host, survival of HNE-treated M13 DNA was similar to, but mutation frequency was lower than in the strain containing all SOS DNA polymerases. When only Pol II or Pol V were present in host bacteria, phage survival decreased dramatically. Simultaneously, mutation frequency was substantially increased, but exclusively in the strain carrying only Pol V, suggesting that induction of mutations by HNE is mainly dependent on Pol V. To determine the role of Pol II and Pol IV in HNE induced mutagenesis, Pol II or Pol IV were expressed together with Pol V. This resulted in decrease of mutation frequency, suggesting that both enzymes can compete with Pol V, and bypass HNE-DNA adducts in an error-free manner. However, HNE-DNA adducts were easily bypassed by Pol IV and only infrequently by Pol II.Mutation spectrum established for strains expressing only Pol V, showed that in uvrA^? bacteria the frequency of base substitutions and recombination increased in relation to NER proficient strains, particularly mutations at adenine sites. Among base substitutions A:T → C:G, A:T → G:C, G:C → A:T and G:C → T:A prevailed.The results suggest that Pol V can infrequently bypass HNE-DNA adducts inducing mutations at G, C and A sites, while bypass by Pol IV and Pol II is error-free, but for Pol II infrequent.     

DNA polymerase beta is critical for genomic stability of sperm cells

Maintaining genome integrity in germ cells is important, given that the germ cells produce the next generation of offspring. Base excision repair is a DNA repair pathway that is responsible for the repair of most endogenous DNA damage. A key enzyme that functions in this repair pathway is DNA polymerase beta (Pol β). We previously used conditional gene targeting to engineer mice with sperm deleted of the Pol B gene, which encodes Pol β. We characterized mutagenesis in the sperm of these mice and compared it to wild-type and mice heterozygous for the Pol B gene. We found that sperm obtained that were heterozygously or homozygously deleted of the Pol B gene exhibited increased mutation frequencies compared to wild-type sperm. We identified an increase in transition mutations in both heterozygously and homozygously deleted sperm, and the types of mutations induced suggest that a polymerase other than Pol β functions in its absence. Interestingly, most of the transversions we observed were induced only in heterozygous, compared with wild-type sperm. Our results suggest that haploinsufficiency of Pol β leads to increased frequencies and varieties of mutations. Our study also shows that Pol β is critical for genome stability in the germline.     

Interaction between Escherichia coli DNA polymerase IV and single-stranded DNA-binding protein is required for DNA synthesis on SSB-coated DNA

DNA polymerase IV (Pol IV) is one of three translesion polymerases in Escherichia coli. A mass spectrometry study revealed that single-stranded DNA-binding protein (SSB) in lysates prepared from exponentially-growing cells has a strong affinity for column-immobilized Pol IV. We found that purified SSB binds directly to Pol IV in a pull-down assay, whereas SSBΔC8, a mutant protein lacking the C-terminal tail, failed to interact with Pol IV. These results show that the interaction between Pol IV and SSB is mediated by the C-terminal tail of SSB. When polymerase activity was tested on an SSBΔC8-coated template, we observed a strong inhibition of Pol IV activity. Competition experiments using a synthetic peptide containing the amino acid sequence of SSB tail revealed that the chain-elongating capacity of Pol IV was greatly impaired when the interaction between Pol IV and SSB tail was inhibited. These results demonstrate that Pol IV requires the interaction with the C-terminal tail of SSB to replicate DNA efficiently when the template ssDNA is covered with SSB. We speculate that at the primer/template junction, Pol IV interacts with the tail of the nearest SSB tetramer on the template, and that this interaction allows the polymerase to travel along the template while disassembling SSB.