DNA polymerase III requirement for repair of DNA damage caused by methyl methanesulfonate and hydrogen peroxide.

The pcbA1 mutation allows DNA replication dependent on DNA polymerase I at the restrictive temperature in polC(Ts) strains. Cells which carry pcbA1, a functional DNA polymerase I, and a temperature-sensitive DNA polymerase III gene were used to study the role of DNA polymerase III in DNA repair. At the restrictive temperature for DNA polymerase III, these strains were more sensitive to the alkylating agent methyl methanesulfonate (MMS) and hydrogen peroxide than normal cells. The same strains showed no increase in sensitivity to bleomycin, UV light, or psoralen at the restrictive temperature. The sensitivity of these strains to MMS and hydrogen peroxide was not due to the pcbAl allele, and normal sensitivity was restored by the introduction of a chromosomal or cloned DNA polymerase III gene, verifying that the sensitivity was due to loss of DNA polymerase III alpha-subunit activity. A functional DNA polymerase III is required for the reformation of high-molecular-weight DNA after treatment of cells with MMS or hydrogen peroxide, as demonstrated by alkaline sucrose sedimentation results. Thus, it appears that a functional DNA polymerase III is required for the optimal repair of DNA damage by MMS or hydrogen peroxide.     

Replisome pausing in mutagenesis

E. coli cells containing a temperature-sensitivednaE mutation, in the α-subunit of holoenzyme DNA polymerase III, do not survive at the restrictive temperature. Such cells may survive in the presence of thepcbA1 mutation, an allele of thegyrB gene. Such survival is dependent on an active DNA polymerase I. Evidence indicates that DNA polymerase I interacts directly in the replisome (REP·A). Despite normal survival for cells using thepcbA replication pathway after some type of DNA damage, we have noted a failure of damage-induced mutagenesis. Here we present evidence supporting a model of replisome pausing in cells dependent upon thepcbA replication pathway. The model argues that the (REP·A) complex pauses longer at the site of the lesion, allowing excision repair to occur completely. In the normal replication pathway (REP·E) bypass of the lesion occurs, fixing the mutation.     

Alternate pathways of DNA replication in Escherichia coli

We have described the pcbA1 mutation which enables E. coli cells to replicate DNA in the absence of a functional dnaE gene product if DNA polymerase I (the polA gene product) is present. The pcbA1 mutation phenotypically suppresses multiple dnaEts and dnaEam alleles. The pcbA1/PolI replication pathway differs from normal in sensitivity to certain DNA-damaging agents such as methylmethane sulfonate (MMS) and a lack of damage-directed mutagenesis. We report here cloning of the pcbA1 gene in a multicopy plasmid. The pcbA1 mutation is detected only in cis; therefore, cloning necessitated gene eviction. The pcbA1 gene lies closely- linked to gyrB. We have demonstrated the physical presence of DNA polymerase I in the replicating holoenzyme complex by immunoblotting using dnaEam strains. We conclude that E. coli has two alternate replisome structures: REP-A, in which DNA polymerase I is the functional synthetic subunit; and REP-E, in which the alpha-subunit, product of the dnaE gene, is functional. To investigate further the role of individual DNA polymerases in replication, we have isolated the polB gene on multicopy plasmids.     

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.     

Isolation and characterization of mutants with deletions in dnaQ, the gene for the editing subunit of DNA polymerase III in Salmonella typhimurium.

dnaQ (mutD) encodes the editing exonuclease subunit (epsilon) of DNA polymerase III. Previously described mutations in dnaQ include dominant and recessive mutator alleles as well as leaky temperature-sensitive alleles. We describe the properties of strains bearing null mutations (deletion-substitution alleles) of this gene. Null mutants exhibited a growth defect as well as elevated spontaneous mutation. As a consequence of the poor growth of dnaQ mutants and their high mutation rate, these strains were replaced within single colonies by derivatives carrying an extragenic suppressor mutation that compensated the growth defect but apparently not the mutator effect. Sixteen independently derived suppressors mapped in the vicinity of dnaE, the gene for the polymerization subunit (alpha) of DNA polymerase III, and one suppressor that was sequenced encoded an altered alpha polypeptide. Partially purified DNA polymerase III containing this altered alpha subunit was active in polymerization assays. In addition to their dependence on a suppressor mutation affecting alpha, dnaQ mutants strictly required DNA polymerase I for viability. We argue from these data that in the absence of epsilon, DNA replication falters unless secondary mechanisms, including genetically coded alteration in the intrinsic replication capacity of alpha and increased use of DNA polymerase I, come into play. Thus, epsilon plays a role in DNA replication distinct from its known role in controlling spontaneous mutation frequency.     

Suppression of dnaE nonsense mutations by pcbA1.

DNA polymerase III has been recognized as the required replication enzyme in Escherichia coli. The synthesis subunit of DNA polymerase III holoenzyme (alpha subunit) is encoded by the dnaE gene. We have reported that E. coli cells can survive and grow in the absence of a functional dnaE gene product if DNA polymerase I and the pcbA1 mutation are present. Existing mutations in the dnaE gene have been conditionally defective thermolabile mutations. We report here construction of nonsense mutations in the dnaE gene by use of a temperature-sensitive suppressor mutation to permit survival at the permissive temperature (32 degrees C). Introduction of the pcbA1 mutation eliminated the temperature-sensitive phenotype. We confirmed by immunoblotting the lack of detectable alpha subunit at 43 degrees C.     

The role of DNA polymerase I, II and III in the replication of the bacteriocinogenic factor Clo DF13

Summary The replication of the bacteriocinogenic factor Clo DF13 was studied in Escherichia coli mutants which lack either DNA polymerase I (polA1 and resA1 mutants), DNA polymerase II (polB1 mutant) or DNA polymerase III (dnaE mutant). DNA polymerase I is required for Clo DF13 replication. The Clo DF13 factor, however, can be maintained in a strain carrying the polA107 mutation and thus lacking the 53 exonucleolytic activity of DNA polymerase I. DNA polymerase II is not required for transfer replication and maintenance of the Clo DF13 plasmid. In the temperature sensitive dnaE mutant, Clo DF13 can replicate at the nonpermissive temperature during the first two hours after the temperature shift from 30°C to 43°C. During this period DNA polymerase III seems not to be essential for Clo DF13 replication.     

Bacillus subtilis DNA polymerase III is required for the replication of DNA of bacteriophages SPP-1 and phi 105.

The replication of the Bacillus subtilis bacteriophages SPP-1 and phi 105 is sensitive to 6-(p-hydroxyphenylazo)-uracil (HPUra), a selective inhibitor of replicative DNA synthesis of B. subtilis which acts specifically at the levels of a replication-specific polymerase, DNA polymerase III (pol III). The origin of the HPUra-sensitive polymerase required for phage replication was examined by comparison of the drug sensitivity of phage development in a normosensitive host with that in a host carrying azp-12, a polC mutation that specifies production of an HPUra-resistant pol III. azp-12 specified HPUra-resistant phage host pol III. The host polIII requirement for SPP-1 replication also was confirmed by the demonstration that phage development was temperature sensitive in a host mutant carrying the polC mutation mut-1 (ts). Examination of the pol III activity of crude and purified cell-free preparations derived from phage-infected cells did not indicate any detectable changes in the specific activity, purification behavior, or drug sensitivity of the enzyme.     

A point mutation within conserved region VI of herpes simplex virus type 1 DNA polymerase confers altered drug sensitivity and enhances replication fidelity

Herpes simplex virus type 1 (HSV-1) DNA polymerase contains several conserved regions within the polymerase domain. The conserved regions I, II, III, V, and VII have been shown to have functional roles in the interaction with deoxynucleoside triphosphates (dNTPs) and DNA. However, the role of conserved region VI in DNA replication has remained unclear due, in part, to the lack of a well-characterized region VI mutant. In this report, recombinant viruses containing a point mutation (L774F) within the conserved region VI were constructed. These recombinant viruses were more susceptible to aphidicolin and resistant to both foscarnet and acyclovir, compared to the wild-type KOS strain. Marker transfer experiments demonstrated that the L774F mutation conferred the altered drug sensitivities. Furthermore, mutagenesis assays demonstrated that L774F recombinant viruses containing the supF marker gene, which was integrated within the thymidine kinase locus (tk), exhibited increased fidelity of DNA replication. These data indicate that conserved region VI, together with other conserved regions, forms the polymerase active site, has a role in the interaction with deoxyribonucleotides, and regulates DNA replication fidelity. The possible effect of the L774F mutation in altering the polymerase structure and activity is discussed.     

Isolation of temperature-sensitive DNA polymerase III from Saccharomyces cerevisiae cdc2-2.

DNA polymerase III of the yeast Saccharomyces cerevisiae has been reported to be encoded at the CDC2 locus based on two observations. First, the CDC2 gene has homology to known DNA polymerase genes [Boulet et al. (1989) EMBO J. 8, 1849-1854], and second, the mutants cdc2-1 and cdc2-2 yield little or no DNA polymerase III activity in vitro [Boulet et al. (1989); Sitney et al. (1989) Cell 56, 599-605]. We describe here the isolation of temperature-sensitive DNA polymerase III from cdc2-2 strains. Our results provide direct experimental confirmation of the previously inferred gene/enzyme relationship and verify the conclusion that DNA polymerase III is required to replicate the genome. We isolated DNA polymerase III from two cdc2-2 strains, one containing the wild-type allele for DNA polymerase I (CDC17) and the other a mutant DNA polymerase I allele (cdc17-1). Yields from cdc2-2 cells of both DNA polymerase III activity and an associated 3'-5'-exonuclease activity [exonuclease III; Bauer et al. (1988) J. Biol. Chem. 263, 917-924] were decreased relative to yields from CDC2 cells. DNA polymerase III activity from cdc2-2 strains is thermolabile, displaying at least a 4-fold reduction in half-life at 44 degrees C. The activity is also labile at 37 degrees C, a temperature which is restrictive for growth of cdc2-2 but not CDC2 strains. At 23 degrees C, a temperature which is permissive for growth of both cdc2-2 and CDC2 strains, the mutant and wild-type DNA polymerase III activities display equal stability. These observations provide a demonstrable biochemical basis for the thermosensitive phenotype of cdc2-2 cells.     

DNA polymerase III, a second essential DNA polymerase, is encoded by the S. cerevisiae CDC2 gene

Three nuclear DNA polymerases have been described in yeast: DNA polymerases I, II, and III. DNA polymerase I is encoded by the POL1 gene and is essential for DNA replication. Since the S. cerevisiae CDC2 gene has recently been shown to have DNA sequence similarity to the active site regions of other known DNA polymerases, but to nevertheless be different from DNA polymerase I, we examined cdc2 mutants for the presence of DNA polymerases II and III. DNA polymerase II was not affected by the cdc2 mutation. DNA polymerase III activity was significantly reduced in the cdc2-1 cell extracts. We conclude that the CDC2 gene encodes yeast DNA polymerase III and that DNA polymerase III, therefore, represents a second essential DNA polymerase in yeast.     

DNA replication defect in Salmonella typhimurium mutants lacking the editing (epsilon) subunit of DNA polymerase III.

In Salmonella typhimurium, dnaQ null mutants (encoding the epsilon editing subunit of DNA polymerase III [Pol III]) exhibit a severe growth defect when the genetic background is otherwise wild type. Suppression of the growth defect requires both a mutation affecting the alpha (polymerase) subunit of DNA polymerase III and adequate levels of DNA polymerase I. In the present paper, we report on studies that clarify the nature of the physiological defect imposed by the loss of epsilon and the mechanism of its suppression. Unsuppressed dnaQ mutants exhibited chronic SOS induction, indicating exposure of single-stranded DNA in vivo, most likely as gaps in double-stranded DNA. Suppression of the growth defect was associated with suppression of SOS induction. Thus, Pol I and the mutant Pol III combined to reduce the formation of single-stranded DNA or accelerate its maturation to double-stranded DNA. Studies with mutants in major DNA repair pathways supported the view that the defect in DNA metabolism in dnaQ mutants was at the level of DNA replication rather than of repair. The requirement for Pol I was satisfied by alleles of the gene for Pol I encoding polymerase activity or by rat DNA polymerase beta (which exhibits polymerase activity only). Consequently, normal growth is restored to dnaQ mutants when sufficient polymerase activity is provided and this compensatory polymerase activity can function independently of Pol III. The high level of Pol I polymerase activity may be required to satisfy the increased demand for residual DNA synthesis at regions of single-stranded DNA generated by epsilon-minus pol III. The emphasis on adequate polymerase activity in dnaQ mutants is also observed in the purified alpha subunit containing the suppressor mutation, which exhibits a modestly elevated intrinsic polymerase activity relative to that of wild-type alpha.     

DNA polymerase epsilon: the latest member in the family of mammalian DNA polymerases

DNA polymerase epsilon is a mammalian polymerase that has a tightly associated 3'----5' exonuclease activity. Because of this readily detectable exonuclease activity, the enzyme has been regarded as a form of DNA polymerase delta, an enzyme which, together with DNA polymerase alpha, is in all probability required for the replication of chromosomal DNA. Recently, it was discovered that DNA polymerase epsilon is both catalytically and structurally distinct from DNA polymerase delta. The most striking difference between the two DNA polymerases is that processive DNA synthesis by DNA polymerase delta is dependent on proliferating cell nuclear antigen (PCNA), a replication factor, while DNA polymerase epsilon is inherently processive. DNA polymerase epsilon is required at least for the repair synthesis of UV-damaged DNA. DNA polymerases are highly conserved in eukaryotic cells. Mammalian DNA polymerases alpha, delta and epsilon are counterparts of yeast DNA polymerases I, III and II, respectively. Like DNA polymerases I and III, DNA polymerase II is also essential for the viability of cells, which suggests that DNA polymerase II (and epsilon) may play a role in DNA replication.     

Plasmid rolling circle replication: identification of the RNA polymerase-directed primer RNA and requirement for DNA polymerase I for lagging strand synthesis.

Plasmid rolling circle replication involves generation of single-stranded DNA (ssDNA) intermediates. ssDNA released after leading strand synthesis is converted to a double-stranded form using solely host proteins. Most plasmids that replicate by the rolling circle mode contain palindromic sequences that act as the single strand origin, sso. We have investigated the host requirements for the functionality of one such sequence, ssoA, from the streptococcal plasmid pLS1. We used a new cell-free replication system from Streptococcus pneumoniae to investigate whether host DNA polymerase I was required for lagging strand synthesis. Extracts from DNA polymerase I-deficient cells failed to replicate, but this was corrected by adding purified DNA polymerase I. Efficient DNA synthesis from the pLS1-ssoA required the entire DNA polymerase I (polymerase and 5'-3' exonuclease activities). ssDNA containing the pLS1-ssoA was a substrate for specific RNA polymerase binding and a template for RNA polymerase-directed synthesis of a 20 nucleotide RNA primer. We constructed mutations in two highly conserved regions within the ssoA: a six nucleotide conserved sequence and the recombination site B. Our results show that the former seemed to function as a terminator for primer RNA synthesis, while the latter may be a binding site for RNA polymerase.     

Loss and restoration of viability of E. coli due to combinations of mutations affecting DNA polymerase I and repair activities

Summary We have found that the cells possessing the polA6 mutation affecting DNA polymerase I are unable to accept another mutation (uvr502) leading to UV-sensitivity. The introduction of the polA12 mutation determining the synthesis of a temperature sensitive DNA polymerase I into the uvr502 mutant results in the temperature sensitivity of colony forming ability of the double mutant. These data show that the uvr502 derivatives lacking DNA polymerase I are inviable. Reversions to temperature resistance in the population of the double mutant uvr502 polA12 may occur because of reverse mutations at one of the mutated sites or because of mutations suppressing DNA polymerase I deficiency but not UV- or MMS-sensitivity of revertants. DNA and protein synthesis in uvr502 polA12 cells continues after a shift to 45°C with rates almost indistinguishable from those in single mutants or wild type cells. No differences in DNA degradation were observed during incubation of single and double mutants at 45°C. The single strand molecular weight distribution of parent DNA from the double mutant as well as that from wild type cells is not affected by the shift to 45°C and 3 hours incubation at this temperature. We suggest that DNA polymerase I and/or the product altered by the uvr502 mutation are required for some step(s) of discontinuous DNA replication nonessential for the formation of acid insoluble DNA. The DNA polymerase I and the uvr gene product seem to be able to substitute for each other in accomplishing this process.     

A 'gram-negative-type' DNA polymerase III is essential for replication of the linear chromosome of Streptomyces coelicolor A3(2)

The Streptomyces coelicolor dnaE gene, encoding the catalytic alpha-subunit of DNA polymerase III (pol III) was isolated by genetic complementation of a temperature-sensitive DNA replication mutant, S. coelicolor ts-38. The deduced protein sequence (1179 residues) is highly similar to the Escherichia coli-type pol III alpha-subunit, rather than to the PolC-type alpha-subunit that is known to be essential for replication in the 'low G + C' Gram-positive bacteria such as Bacillus subtilis. The dnaE gene is able to restore replication to a 'slow stop' mutant (ts-38) and a 'fast stop' mutant (ts-114); the dnaE gene of ts-38 carries a single amino acid substitution (Glu-802 to Lys), and the mutation in ts-114 has been mapped between codons 697 and 1062 of dnaE. Mutant ts-38 is considered to be defective in assembly of the multisubunit pol III holoenzyme and, hence, in initiation of replication, whereas ts-114 is defective in chain elongation. This study provides the first evidence that a DnaE-type pol III is essential for replication in a Gram-positive bacterium. In addition, the complementation studies suggest that the C-terminal 117 residues are not essential for DnaE function in S. coelicolor. When integrated at a distant site on the chromosome, a fragment containing the 3' half of dnaE(codons 697-1179) is capable of rescuing ts-38 (but not ts-114) at the restrictive temperature; it was demonstrated that homogenotization was responsible for this phenomenon.     

The Escherichia coli UVM response is accompanied by an SOS-independent error-prone DNA replication activity demonstrable in vitro

UVM is an SOS-independent inducible response characterized by elevated mutagenesis at a site-specific 3, N4-ethenocytosine (epsilonC) residue borne on M13 single-stranded DNA transfected into Escherichia coli cells pretreated with DNA-damaging agents. By constructing and using E. coli strain AM124 (polA polB umuDC dinB lexA1[Ind-]), we show here that the UVM response is manifested in cells deficient for SOS induction, as well as for all four of the 'non-replicative' DNA polymerases, namely DNA polymerase I (polA), II (polB), IV (dinB) and V (umuDC). These results confirm that UVM represents a novel, previously unidentified cellular response to DNA-damaging agents. To address the question as to whether the UVM response is accompanied by an error-prone DNA replication activity, we applied a newly developed in vitro replication assay coupled to an in vitro mutation analysis system. In the assay, circular M13 single-stranded DNA bearing a site-specific lesion is converted to circular double-stranded replicative-form DNA in the presence of cell extracts and nucleotide precursors under conditions that closely mimic M13 replication in vivo. The newly synthesized (minus) DNA strand is selectively amplified by ligation-mediated polymerase chain reaction (LM-PCR), followed by a multiplex sequence analysis to determine the frequency and specificity of mutations. Replication of DNA bearing a site-specific epsilonC lesion by cell extracts from uninduced E. coli AM124 cells results in a mutation frequency of about 13%. Mutation frequency is elevated fivefold (to 58%) in cell extracts from UVM-induced AM124 cells, with C --> A mutations predominating over C --> T mutations, a specificity similar to that observed in vivo. These results, together with previously reported data, suggest that the UVM response is mediated through the induction of a transient error-prone DNA replication activity and that a modification of DNA polymerase III or the expression of a previously unidentified DNA polymerase may account for the UVM phenotype.     

Replication fork reversal in DNA polymerase III mutants of Escherichia coli: a role for the beta clamp

Certain replication mutations lead in Escherichia coli to a specific reaction named replication fork reversal: at blocked forks, annealing of the nascent strands and pairing of the template strands form a four-way junction. RuvABC-catalysed resolution of this Holliday junction causes chromosome double-strand breaks (DSBs) in a recBC context and therefore creates a requirement for the recombination proteins RecBC for viability. In the present work, two mutants were tested for replication fork reversal: a dnaEts mutant and a dnaNts mutant, affected in the alpha (polymerase) and beta (processivity clamp) subunits of DNA polymerase III holoenzyme respectively. In the dnaEts recB strain, RuvABC-dependent DSBs caused by the dnaEts mutation occurred at 37 degrees C or 42 degrees C, indicating the occurrence of replication fork reversal upon partial or complete inactivation of the DNA polymerase alpha subunit. DSB formation was independent of RecA, RecQ and the helicase function of PriA. In the dnaNts recB mutant, RuvABC-dependent DSB caused by the dnaNts mutation occurred only at semi-permissive temperature, 37 degrees C, indicating the occurrence of replication fork reversal in conditions in which the remaining activity of the beta clamp is sufficient for viability. In contrast, the dnaNts mutation did not cause chromosome breakage at 42 degrees C, a temperature at which DnaN is totally inactive and the dnaNts mutant is inviable. We propose that a residual activity of the DNA polymerase III beta clamp is required for replication fork reversal in the dnaNts mutant.     

Analysis of unassisted translesion replication by the DNA polymerase III holoenzyme

DNA damage-induced mutations are formed when damaged nucleotides present in single-stranded DNA are replicated. We have developed a new method for the preparation of gapped plasmids containing site-specific damaged nucleotides, as model DNA substrates for translesion replication. Using these substrates, we show that the DNA polymerase III holoenzyme from Escherichia coli can bypass a synthetic abasic site analogue with high efficiency (30% bypass in 16 min), unassisted by other proteins. The theta and tau subunits of the polymerase were not essential for bypass. No bypass was observed when the enzyme was assayed on a synthetic 60-mer oligonucleotide carrying the same lesion, and bypass on a linear gapped plasmid was 3-4-fold slower than on a circular gapped plasmid. There was no difference in the bypass when standing-start and running-start replication were compared. A comparison of translesion replication by DNA polymerase I, DNA polymerase II, the DNA polymerase III core, and the DNA polymerase III holoenzyme clearly showed that the DNA polymerase III holoenzyme was by far the most effective in performing translesion replication. This was not only due to the high processivity of the pol III holoenzyme, because increasing the processivity of pol II by adding the gamma complex and beta subunit, did not increase bypass. These results support the model that SOS regulation was imposed on a fundamentally constitutive translesion replication reaction to achieve tight control of mutagenesis.