Translesion-synthesis DNA polymerases participate in replication of the telomeres in Streptomyces

Linear chromosomes and linear plasmids of Streptomyces are capped by terminal proteins that are covalently bound to the 5'-ends of DNA. Replication is initiated from an internal origin, which leaves single-stranded gaps at the 3'-ends. These gaps are patched by terminal protein-primed DNA synthesis. Streptomyces contain five DNA polymerases: one DNA polymerase I (Pol I), two DNA polymerases III (Pol III) and two DNA polymerases IV (Pol IV). Of these, one Pol III, DnaE1, is essential for replication, and Pol I is not required for end patching. In this study, we found the two Pol IVs (DinB1 and DinB2) to be involved in end patching. dinB1 and dinB2 could not be co-deleted from wild-type strains containing a linear chromosome, but could be co-deleted from mutant strains containing a circular chromosome. The resulting ΔdinB1 ΔdinB2 mutants supported replication of circular but not linear plasmids, and exhibited increased ultraviolet sensitivity and ultraviolet-induced mutagenesis. In contrast, the second Pol III, DnaE2, was not required for replication, end patching, or ultraviolet resistance and mutagenesis. All five polymerase genes are relatively syntenous in the Streptomyces chromosomes, including a 4-bp overlap between dnaE2 and dinB2. Phylogenetic analysis showed that the dinB1-dinB2 duplication occurred in a common actinobacterial ancestor.     

Roles of chromosomal and episomal dinB genes encoding DNA pol IV in targeted and untargeted mutagenesis in Escherichia coli

DNA polymerase IV (pol IV) in Escherichia coli is a member of a novel family of DNA polymerases (the DinB/UmuC/Rad30/Rev1 super-family or the DNA polymerase Y family). Although expression of the dinB gene encoding DNA pol IV is known to result in an enhancement of untargeted mutagenesis, it remains uncertain whether DNA pol IV is involved in a variety of lesion-induced mutagenesis (targeted mutagenesis), and the relationship between expression levels of dinB and the mutagenesis that DNA pol IV promotes has not been investigated thoroughly. Here, we report that DNA pol IV is involved in -1 frameshift mutagenesis induced by 4-nitroquinoline N-oxide (4-NQO) and that the expression level of the chromosomal pol IV gene is 6-12 times higher than those for other SOS-inducible DNA polymerases in E. coli, i.e., DNA pol II (PolB) or DNA pol V (UmuDC), respectively. Interestingly, the dinB gene is present not only on the chromosome but also on the F' plasmid in the E. coli CC108 strain. In this strain, 750 molecules of DNA pol IV are expressed from the F' dinB gene in the uninduced state and 250 molecules are expressed from the chromosomal gene. These cellular expression levels strongly affect -1 frameshifts induced by 4-NQO in runs of six guanine bases: mutagenicity was highest in the strain CC108, followed by strains YG2242 (chromosome deltadinB/F' dinB+), YG2247 (chromosome dinB+/F' deltadinB) and FC1243 (chromosome deltadinB/F' deltadinB). The incidence of untargeted -1 frameshifts was reduced by two-thirds on deletion of dinB from the F' episome. The chromosomal dinB gene appeared to have little or no effect on the untargeted mutagenesis. These results suggest that DNA pol IV efficiently mediates targeted mutagenesis by 4-NQO, and that the cellular levels of expression substantially affect targeted and untargeted mutagenesis.     

Overproduction of Escherichia coli DNA polymerase DinB (Pol IV) inhibits replication fork progression and is lethal

Escherichia coli dinB encodes the specialized DNA polymerase DinB (Pol IV), which is induced as part of the SOS stress-response system and functions in translesion synthesis (TLS) to relieve the replicative Pol III that is stalled at DNA lesions. As the number of DinB molecules, even in unstressed cells, is greater than that required to accomplish TLS, it is thought that dinB plays some additional physiological role. Here, we overexpressed dinB under the tightly regulable arabinose promoter and looked for a distinct phenotype. Upon induction of dinB expression, progression of the replication fork was immediately inhibited at random genomic positions, and the colony-forming ability of the cells was reduced. Overexpression of mutated dinB alleles revealed that the structural requirements for these two inhibitory effects and for TLS were distinct. The extent of in vivo inhibition displayed by a mutant DinB matched the extent of its in vitro impedance, at near-physiological concentration, of a moving Pol III. We suggest that DinB targets Pol III, thereby acting as a brake on replication fork progression. Because the brake operates when cells have excess DinB, as they do under stress conditions, it may serve as a checkpoint that modulates replication to safeguard genome stability.     

Epistatic roles for Pseudomonas aeruginosa MutS and DinB (DNA Pol IV) in coping with reactive oxygen species-induced DNA damage

Pseudomonas aeruginosa is especially adept at colonizing the airways of individuals afflicted with the autosomal recessive disease cystic fibrosis (CF). CF patients suffer from chronic airway inflammation, which contributes to lung deterioration. Once established in the airways, P. aeruginosa continuously adapts to the changing environment, in part through acquisition of beneficial mutations via a process termed pathoadaptation. MutS and DinB are proposed to play opposing roles in P. aeruginosa pathoadaptation: MutS acts in replication-coupled mismatch repair, which acts to limit spontaneous mutations; in contrast, DinB (DNA polymerase IV) catalyzes error-prone bypass of DNA lesions, contributing to mutations. As part of an ongoing effort to understand mechanisms underlying P. aeruginosa pathoadaptation, we characterized hydrogen peroxide (H(2)O(2))-induced phenotypes of isogenic P. aeruginosa strains bearing different combinations of mutS and dinB alleles. Our results demonstrate an unexpected epistatic relationship between mutS and dinB with respect to H(2)O(2)-induced cell killing involving error-prone repair and/or tolerance of oxidized DNA lesions. In striking contrast to these error-prone roles, both MutS and DinB played largely accurate roles in coping with DNA lesions induced by ultraviolet light, mitomycin C, or 4-nitroquinilone 1-oxide. Models discussing roles for MutS and DinB functionality in DNA damage-induced mutagenesis, particularly during CF airway colonization and subsequent P. aeruginosa pathoadaptation are discussed.     

Roles of YqjH and YqjW,homologs of the Escherichia coli UmuC/DinB or Y superfamily of DNA polymerases,in stationary-phase mutagenesis and UV-induced mutagenesis of Bacillus subtilis

YqjH and YqjW are Bacillus subtilis homologs of the UmuC/DinB or Y superfamily of DNA polymerases that are involved in SOS-induced mutagenesis in Escherichia coli. While the functions of YqjH and YqjW in B. subtilis are still unclear, the comparisons of protein structures demonstrate that YqjH has 36% identity to E. coli DNA polymerase IV (DinB protein), and YqjW has 26% identity to E. coli DNA polymerase V (UmuC protein). In this report, we demonstrate that both YqjH and the products of the yqjW operon are involved in UV-induced mutagenesis in this bacterium. Furthermore, resistance to UV-induced damage is significantly reduced in cells lacking a functional YqjH protein. Analysis of stationary-phase mutagenesis indicates that absences of YqjH, but not that of YqjW, decreases the ability of B. subtilis to generate revertants at the hisC952 allele via this system. These data suggest a role for YqjH in the generation of at least some types of stationary-phase-induced mutagenesis.     

Escherichia coli DinB inhibits replication fork progression without significantly inducing the SOS response

The SOS response is readily triggered by replication fork stalling caused by DNA damage or a dysfunctional replicative apparatus in Escherichia coli cells. E. coli dinB encodes DinB DNA polymerase and its expression is upregulated during the SOS response. DinB catalyzes translesion DNA synthesis in place of a replicative DNA polymerase III that is stalled at a DNA lesion. We showed previously that DNA replication was suppressed without exogenous DNA damage in cells overproducing DinB. In this report, we confirm that this was due to a dose-dependent inhibition of ongoing replication forks by DinB. Interestingly, the DinB-overproducing cells did not significantly induce the SOS response even though DNA replication was perturbed. RecA protein is activated by forming a nucleoprotein filament with single-stranded DNA, which leads to the onset of the SOS response. In the DinB-overproducing cells, RecA was not activated to induce the SOS response. However, the SOS response was observed after heat-inducible activation in strain recA441 (encoding a temperature-sensitive RecA) and after replication blockage in strain dnaE486 (encoding a temperature-sensitive catalytic subunit of the replicative DNA polymerase III) at a non-permissive temperature when DinB was overproduced in these cells. Furthermore, since catalytically inactive DinB could avoid the SOS response to a DinB-promoted fork block, it is unlikely that overproduced DinB takes control of primer extension and thus limits single-stranded DNA. These observations suggest that DinB possesses a feature that suppresses DNA replication but does not abolish the cell's capacity to induce the SOS response. We conclude that DinB impedes replication fork progression in a way that does not activate RecA, in contrast to obstructive DNA lesions and dysfunctional replication machinery.     

Requirement of DNA polymerase activity of yeast Rad30 protein for its biological function.

The RAD30 gene of Saccharomyces cerevisiae encodes a DNA polymerase, Poleta. The Rad30 protein shares homology with the yeast Rev1 and the Escherichia coli DinB and UmuC proteins. Although these proteins contain several highly conserved motifs, only Rad30 has been shown to possess a DNA polymerase activity. To determine whether the DNA polymerase activity of Rad30 was essential for its biological function, we made a mutation in the highly conserved SIDE sequence in Rad30, in which the aspartate and glutamate residues have each been changed to alanine. The mutant Rad30 protein lacks the DNA polymerase activity, and the mutant gene does not complement the rad30Delta mutation. These findings indicate that DNA polymerase activity is indispensable for the biological function of RAD30.     

Y-family DNA polymerases respond to DNA damage-independent inhibition of replication fork progression

In Escherichia coli, the Y-family DNA polymerases Pol IV (DinB) and Pol V (UmuD2'C) enhance cell survival upon DNA damage by bypassing replication-blocking DNA lesions. We report a unique function for these polymerases when DNA replication fork progression is arrested not by exogenous DNA damage, but with hydroxyurea (HU), thereby inhibiting ribonucleotide reductase, and bringing about damage-independent DNA replication stalling. Remarkably, the umuC122::Tn5 allele of umuC, dinB, and certain forms of umuD gene products endow E. coli with the ability to withstand HU treatment (HUR). The catalytic activities of the UmuC122 and DinB proteins are both required for HUR. Moreover, the lethality brought about by such stalled replication forks in the wild-type derivatives appears to proceed through the toxin/antitoxin pairs mazEF and relBE. This novel function reveals a role for Y-family polymerases in enhancing cell survival under conditions of nucleotide starvation, in addition to their established functions in response to DNA damage.     

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.     

What limits the efficiency of double-strand break-dependent stress-induced mutation in Escherichia coli?

Stress-induced mutation is a collection of molecular mechanisms in bacterial, yeast and human cells that promote mutagenesis specifically when cells are maladapted to their environment, i.e. when they are stressed. Here, we review one molecular mechanism: double-strand break (DSB)-dependent stress-induced mutagenesis described in starving Escherichia coli. In it, the otherwise high-fidelity process of DSB repair by homologous recombination is switched to an error-prone mode under the control of the RpoS general stress response, which licenses the use of error-prone DNA polymerase, DinB, in DSB repair. This mechanism requires DSB repair proteins, RpoS, the SOS response and DinB. This pathway underlies half of spontaneous chromosomal frameshift and base substitution mutations in starving E. coli [Proc Natl Acad Sci USA 2011;108:13659-13664], yet appeared less efficient in chromosomal than F' plasmid-borne genes. Here, we demonstrate and quantify DSB-dependent stress-induced reversion of a chromosomal lac allele with DSBs supplied by I-SceI double-strand endonuclease. I-SceI-induced reversion of this allele was previously studied in an F'. We compare the efficiencies of mutagenesis in the two locations. When we account for contributions of an F'-borne extra dinB gene, strain background differences, and bypass considerations of rates of spontaneous DNA breakage by providing I-SceI cuts, the chromosome is still ～100 times less active than F. We suggest that availability of a homologous partner molecule for recombinational break repair may be limiting. That partner could be a duplicated chromosomal segment or sister chromosome.     

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.     

Spontaneous mutagenesis is elevated in protease-defective cells

As a first step towards describing the role of proteolysis in maintaining genomic integrity, we have determined the effect of the loss of ClpXP, a major energy-dependent cytoplasmic protease that degrades truncated proteins as well as a number of regulatory proteins, on spontaneous mutagenesis. In a rifampicin-sensitive to rifampicin-resistance assay that detects base substitution mutations in the essential rpoB gene, there is a modest, but appreciable increase in mutagenesis in Δ( clpP-clpX ) cells relative to wild-type cells. A colony papillation analysis using a set of lacZ strains revealed that genetic −1 frameshift mutations are strongly elevated in Clp-defective cells. A quantitative analysis using a valine-sensitive to valine-resistance assay that detects frameshift mutations showed that mutagenesis is elevated 50-fold in Clp-defective cells. Elevated frameshift mutagenesis observed in Clp-deficient cells is essentially abolished in lexA1 [Ind^-] (SOS-uninducible) cells, and in cells deleted for the SOS gene dinB , which codes for DNA polymerase IV. In contrast, mutagenesis is unaffected or stimulated in cells deleted for umuC or umuD , which code for critical components of DNA polymerase V. Loss of rpoS , which codes for a stress-response sigma factor known to upregulate dinB expression in stationary phase, does not affect mutagenesis. We propose that elevated DinB expression, as well as stabilization of UmuD/UmuD' heterodimers in Δ( clpP-clpX ) cells, contributes to elevated mutagenesis. These findings suggest that in normal cells, Clp-mediated proteolysis plays an important role in preventing gratuitous mutagenesis.     

The Escherichia coli dnaN159 mutant displays altered DNA polymerase usage and chronic SOS induction

The Escherichia coli beta sliding clamp, which is encoded by the dnaN gene, is reported to interact with a variety of proteins involved in different aspects of DNA metabolism. Recent findings indicate that many of these partner proteins interact with a common surface on the beta clamp, suggesting that competition between these partners for binding to the clamp might help to coordinate both the nature and order of the events that take place at a replication fork. The purpose of the experiments discussed in this report was to test a prediction of this model, namely, that a mutant beta clamp protein impaired for interactions with the replicative DNA polymerase (polymerase III [Pol III]) would likewise have impaired interactions with other partner proteins and hence would display pleiotropic phenotypes. Results discussed herein indicate that the dnaN159-encoded mutant beta clamp protein (beta159) is impaired for interactions with the alpha catalytic subunit of Pol III. Moreover, the dnaN159 mutant strain displayed multiple replication and repair phenotypes, including sensitivity to UV light, an absolute dependence on the polymerase activity of Pol I for viability, enhanced Pol V-dependent mutagenesis, and altered induction of the global SOS response. Furthermore, epistasis analyses indicated that the UV sensitivity of the dnaN159 mutant was suppressed by (not epistatic with) inactivation of Pol IV (dinB gene product). Taken together, these findings suggest that in the dnaN159 mutant, DNA polymerase usage, and hence DNA replication, repair, and translesion synthesis, are altered. These findings are discussed in terms of a model to describe how the beta clamp might help to coordinate protein traffic at the replication fork.     

UmuD and RecA directly modulate the mutagenic potential of the Y family DNA polymerase DinB

DinB is the only translesion Y family DNA polymerase conserved among bacteria, archaea, and eukaryotes. DinB and its orthologs possess a specialized lesion bypass function but also display potentially deleterious -1 frameshift mutagenic phenotypes when overproduced. We show that the DNA damage-inducible proteins UmuD(2) and RecA act in concert to modulate this mutagenic activity. Structural modeling suggests that the relatively open active site of DinB is enclosed by interaction with these proteins, thereby preventing the template bulging responsible for -1 frameshift mutagenesis. Intriguingly, residues that define the UmuD(2)-interacting surface on DinB statistically covary throughout evolution, suggesting a driving force for the maintenance of a regulatory protein-protein interaction at this site. Together, these observations indicate that proteins like RecA and UmuD(2) may be responsible for managing the mutagenic potential of DinB orthologs throughout evolution.     

An active site aromatic triad in Escherichia coli DNA Pol IV coordinates cell survival and mutagenesis in different DNA damaging agents

DinB (DNA Pol IV) is a translesion (TLS) DNA polymerase, which inserts a nucleotide opposite an otherwise replication-stalling N(2)-dG lesion in vitro, and confers resistance to nitrofurazone (NFZ), a compound that forms these lesions in vivo. DinB is also known to be part of the cellular response to alkylation DNA damage. Yet it is not known if DinB active site residues, in addition to aminoacids involved in DNA synthesis, are critical in alkylation lesion bypass. It is also unclear which active site aminoacids, if any, might modulate DinB's bypass fidelity of distinct lesions. Here we report that along with the classical catalytic residues, an active site "aromatic triad", namely residues F12, F13, and Y79, is critical for cell survival in the presence of the alkylating agent methyl methanesulfonate (MMS). Strains expressing dinB alleles with single point mutations in the aromatic triad survive poorly in MMS. Remarkably, these strains show fewer MMS- than NFZ-induced mutants, suggesting that the aromatic triad, in addition to its role in TLS, modulates DinB's accuracy in bypassing distinct lesions. The high bypass fidelity of prevalent alkylation lesions is evident even when the DinB active site performs error-prone NFZ-induced lesion bypass. The analyses carried out with the active site aromatic triad suggest that the DinB active site residues are poised to proficiently bypass distinctive DNA lesions, yet they are also malleable so that the accuracy of the bypass is lesion-dependent.     

Structure-function studies of the herpes simplex virus type 1 DNA polymerase.

The analysis of the deduced amino acid sequence of the herpes simplex virus type 1 (HSV-1) DNA polymerase reported here suggests that the polymerase structure consists of domains carrying separate biological functions. The HSV-1 enzyme is known to possess 5'-3'-exonuclease (RNase H), 3'-5'-exonuclease, and DNA polymerase catalytic activities. Sequence analysis suggests an arrangement of these activities into distinct domains resembling the organization of Escherichia coli polymerase I. In order to more precisely define the structure and C-terminal limits of a putative catalytic domain responsible for the DNA polymerization activity of the HSV-1 enzyme, we have undertaken in vitro mutagenesis and computer modeling studies of the HSV-1 DNA polymerase gene. Sequence analysis predicts that the major DNA polymerization domain of the HSV-1 enzyme will be contained between residues 690 and 1100, and we present a three-dimensional model of this region, on the basis of the X-ray crystallographic structure of the E. coli polymerase I. Consistent with these structural and modeling studies, deletion analysis by in vitro mutagenesis of the HSV-1 DNA polymerase gene expressed in Saccharomyces cerevisiae has confirmed that certain amino acids from the C terminus (residues 1073 to 1144 and 1177 to 1235) can be deleted without destroying HSV-1 DNA polymerase catalytic activity and that the extreme N-terminal 227 residues are also not required for this activity.     

UV- and MMS-induced mutagenesis of lambdaO(am)8 phage under nonpermissive conditions for phage DNA replication

Mutagenesis in Escherichia coli, a subject of many years of study is considered to be related to DNA replication. DNA lesions nonrepaired by the error-free nucleotide excision repair (NER), base excision repair (BER) and recombination repair (RR), stop replication at the fork. Reinitiation needs translesion synthesis (TLS) by DNA polymerase V (UmuC), which in the presence of accessory proteins, UmuD', RecA and ssDNA-binding protein (SSB), has an ability to bypass the lesion with high mutagenicity. This enables reinitiation and extension of DNA replication by DNA polymerase III (Pol III). We studied UV- and MMS-induced mutagenesis of lambdaO(am)8 phage in E. coli 594 sup+ host, unable to replicate the phage DNA, as a possible model for mutagenesis induced in nondividing cells (e.g. somatic cells). We show that in E. coli 594 sup+ cells UV- and MMS-induced mutagenesis of lambdaO(am)8 phage may occur. This mutagenic process requires both the UmuD' and C proteins, albeit a high level of UmuD' and low level of UmuC seem to be necessary and sufficient. We compared UV-induced mutagenesis of lambdaO(am)8 in nonpermissive (594 sup+) and permissive (C600 supE) conditions for phage DNA replication. It appeared that while the mutagenesis of lambdaO(am)8 in 594 sup+ requires the UmuD' and C proteins, which can not be replaced by other SOS-inducible protein(s), in C600 supE their functions may be replaced by other inducible protein(s), possibly DNA polymerase IV (DinB). Mutations induced under nonpermissive conditions for phage DNA replication are resistant to mismatch repair (MMR), while among those induced under permissive conditions, only about 40% are resistant.