Size classes of products synthesized processively by DNA polymerase III and DNA polymerase III holoenzyme of Escherichia coli.
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DNA Polymerase III↗
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Phage like it HOT: solution structure of the bacteriophage P1-encoded HOT protein, a homolog of the theta subunit of E. coli DNA polymerase III.
DNA polymerase III, the main replicative polymerase of E. coli, contains a small subunit, theta, that binds to the epsilon proofreading subunit and appears to enhance the enzyme's proofreading function--especially under extreme conditions. It was recently discovered that E. coli bacteriophage P1 encodes a theta homolog, named HOT. The (1)H-(15)N HSQC spectrum of HOT exhibits more uniform intensities and less evidence of conformational exchange than that of theta; this uniformity facilitates a determination of the HOT solution structure by NMR. The structure contains three alpha helices, as reported previously for theta; however, the folding topology of the two proteins is very different. Residual dipolar coupling measurements on labeled theta support the conclusion that it is structurally homologous with HOT. As judged by CD measurements, the melting temperature of HOT was 62 degrees C, compared to 56 degrees C for theta, consistent with other data suggesting greater thermal stability of the HOT protein.
Amino Acid Sequence↗
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.
Cell Cycle↗
Purification and characterization of DNA polymerase III'. Identification of tau as a subunit of the DNA polymerase III holoenzyme.
DNA polymerase III', a new form of DNA polymerase III, has been purified 15,000-fold to 90% homogeneity from an Escherichia coli K12 strain. DNA polymerase III's is a subassembly of four subunits of the DNA polymerase III holoenzyme; it has functional and physical properties intermediate between the core DNA polymerase III and holoenzyme. Polyacrylamide gel electrophoresis performed under denaturing conditions indicates DNA polymerase III' to be a complex of the alpha, epsilon, and theta subunits of DNA polymerase III and a newly assigned subunit of the DNA polymerase III holoenzyme, tau (Mr = 83,000). Both gel filtration and phosphocellulose chromatography separate DNA polymerase III from DNA polymerase III'. All enzyme forms can utilize a duplex template containing short gaps. DNA polymerase III', like the DNA polymerase III holoenzyme, can synthesize DNA on a long single-stranded template in the presence of 5 mM spermidine; DNA polymerase III cannot. Alone, DNA polymerase III' is inert in the G4 natural replicative system in which the DNA polymerase III holoenzyme is active. Molecular weight and subunit stoichiometry determinations suggest that DNA polymerase III' contains two units of core DNA polymerase III and two tau subunits.
Chromatography, Gel↗
A separate editing exonuclease for DNA replication: the epsilon subunit of Escherichia coli DNA polymerase III holoenzyme.
DNA polymerase III (polIII) holoenzyme of Escherichia coli has 3'----5' exonuclease ("editing") activity in addition to its polymerase activity, a property shared by other prokaryotic DNA polymerases. The polymerization activity is carried by the large alpha subunit, the product of the dnaE gene. Mutations affecting the fidelity of DNA replication in vivo and the activity of 3'----5' exonuclease assayed in vitro are found in the dnaQ gene, which specifies the epsilon subunit. To determine whether epsilon carries the 3'----5' exonuclease activity, we have used an overproduction protocol to purify epsilon separately from the other subunits of polIII holoenzyme. We find that epsilon has 3'----5' exonuclease activity indistinguishable from that of polIII core, the subassembly of polIII holoenzyme consisting of the alpha, epsilon, and theta subunits. We conclude that the editing and polymerization activities of polIII holoenzyme reside on distinct subunits, in contrast to DNA polymerase I of E. coli and DNA polymerase of phage T4. This functional separation may provide for regulation of exonucleolytic editing independently of polymerization, allowing cellular control of replication fidelity.
DNA Polymerase III↗
Mechanism of DNA elongation catalyzed by Escherichia coli DNA polymerase III, dnaZ protein, and DNA elongation factors I and III.
Elongation of a primed single-stranded DNA template catalyzed by E. coli DNA polymerase III (DNA nucleotidyltransferase, deoxynucleosidetriphosphate:DNA deoxynucleotidyltransferase, EC 2.7.7.7) requires dnaZ protein and two other protein factors, DNA elongation factors I and III. The reaction occurs by the following mechanism: (i) dnaZ protein and DNA elongation factor III together catalyze the transfer of DNA elongation factor I to a primed DNA template. This transfer reaction requires ATP or dATP in addition to dnaZ protein, DNA elongation factors I and III, and primed template; it does not require DNA polymerase III. (ii) DNA polymerase III binds to the complex of DNA elongation factor I with primed template; it does not bind to primed template which is not complexed with DNA elongation factor I. This binding reaction proceeds in the absence of ATP or dATP as cofactor, dnaZ protein, and DNA elongation factor III and without additional DNA elongation factor I. (iii) The complex of DNA polymerase III, DNA elongation factor I, and primed template catalyzes DNA synthesis upon the addition of dNTPs.
Adenosine Triphosphate↗
Complete replication of templates by Escherichia coli DNA polymerase III holoenzyme.
DNA polymerase III holoenzyme (holoenzyme) processively and rapidly replicates a primed single-stranded DNA circle to produce a duplex with an interruption in the synthetic strand. The precise nature of this discontinuity in the replicative form (RF II) and the influence of the 5' termini of the DNA and RNA primers were analyzed in this study. Virtually all (90%) of the RF II products primed by DNA were nicked structures sealable by Escherichia coli DNA ligase; in 10% of the products, replication proceeded one nucleotide beyond the 5' DNA terminus displacing (but not removing) the 5' terminal nucleotide. With RNA primers, replication generally went beyond the available single-stranded template. The 5' RNA terminus was displaced by 1-5 nucleotides in 85% of the products; a minority of products was nicked (9%) or had short gaps (6%). Termination of synthesis on a linear DNA template was usually (85%) one base shy of completion. Thus, replication by holoenzyme utilizes all, or nearly all, of the available template and shows no significant 5'----3' exonuclease action as observed in primer removal by the "nick-translation" activity of DNA polymerase I.
Bacteriophage phi X 174↗
Mechanism of the sliding beta-clamp of DNA polymerase III holoenzyme.
DNA polymerase III holoenzyme (holoenzyme), the multiprotein replicase of Escherichia coli, is essentially unlimited in processive DNA synthesis. Processive activity can be reconstituted from two components. One component, the beta preinitiation complex, is a beta dimer clamped onto primed DNA. The beta preinitiation complex is formed by the five-protein gamma complex, which hydrolyzes ATP to chaperone beta onto primed DNA. The other component is the alpha epsilon polymerase. The alpha epsilon polymerase itself is not processive, but is endowed with extremely high processive activity upon assembly with the beta preinitiation complex. Here we examine the mechanism by which the beta preinitiation complex confers processivity onto the alpha epsilon polymerase. We find the beta preinitiation complex to be mobile on DNA. Diffusion of beta on DNA is specific to duplex DNA, is bidirectional, does not require ATP, and appears to diffuse linearly along the duplex. Furthermore, beta directly binds the alpha epsilon polymerase through contact with alpha, the DNA polymerase subunit. Hence, the high processivity of the holoenzyme is rooted in a "sliding clamp" of beta on DNA that tethers the polymerase to the primed template. Implications for transcription and translation are discussed.
DNA Polymerase III↗
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.
Base Composition↗
Pre-steady state analysis of the assembly of wild type and mutant circular clamps of Escherichia coli DNA polymerase III onto DNA.
The beta protein, a dimeric ring-shaped clamp essential for processive DNA replication by Escherichia coli DNA polymerase III holoenzyme, is assembled onto DNA by the gamma complex. This study examines the clamp loading pathway in real time, using pre-steady state fluorescent depolarization measurements to investigate the loading reaction and ATP requirements for the assembly of beta onto DNA. Two beta dimer interface mutants, L273A and L108A, and a nonhydrolyzable ATP analog, adenosine 5'-O-(3-thiotriphosphate) (ATPgammaS), have been used to show that ATP binding is required for gamma complex and beta to associate with DNA, but that a gamma complex-catalyzed ATP hydrolysis is required for gamma complex to release the beta.DNA complex and complete the reaction. In the presence of ATP and gamma complex, the beta mutants associate with DNA as efficiently as wild type beta. However, completion of the reaction is much slower with the beta mutants because of decreased ATP hydrolysis by the gamma complex, resulting in a much slower release of the mutants onto DNA. The effects of mutations in the dimer interface were similar to the effects of replacing ATP with ATPgammaS in reactions using wild type beta. Thus, the assembly of beta around DNA is coupled tightly to the ATPase activity of the gamma complex, and completion of the assembly process requires ATP hydrolysis for turnover of the catalytic clamp loader.
Adenosine Triphosphate↗
Size classes of products synthesized processively by two subassemblies of Escherichia coli DNA polymerase III holoenzyme.
Two forms of Escherichia coli DNA polymerase III, DNA polymerase III', and DNA polymerase III have been shown to synthesize DNA products via a processive mechanism with product sizes distinctive for each enzyme form. These forms of DNA polymerase III are intermediate in complexity between the core DNA polymerase III and the DNA polymerase III holoenzyme. In a previous publication (Fay, P. J., Johanson K. O., McHenry, C. S., and Bambara, R. A. (1981) J. Biol. Chem. 256, 976-983), we demonstrated that on a randomly primed fd DNA template or on an oligo(dT)10 . poly(dA) template, the DNA polymerase III holoenzyme adds more than 100 nucleotides before dissociation, whereas the core enzyme adds 10 to 15 nucleotides. Now we show that DNA polymerase III' adds 30 to 40 nucleotides before dissociation. This number can be increased to approximately 60 if spermidine is present, but it is insensitive to the presence of E. coli single-stranded DNA-binding protein. DNA polymerase III adds about 50 nucleotides before dissociation, but this value can be increased to 200 nucleotides in the presence of the binding protein. Using measurement of product sizes made on an oligo(dT)10 . poly(dA) template, reconstitution of holoenzyme activity from DNA polymerase III and the beta subunit was monitored. Finally, it is shown that the products obtained from a purified initiation complex of holoenzyme and oligo(dT)10 . poly(dA) derive solely from the holoenzyme.
DNA Polymerase III↗
Dynamics of loading the beta sliding clamp of DNA polymerase III onto DNA.
A "minimal" DNA primer-template system, consisting of an 80-mer template and 30-mer primer, supports processive DNA synthesis by DNA polymerase III core in the presence of the beta sliding clamp, gamma complex clamp loader, and single-stranded binding protein from Escherichia coli. This primer-template system was used to measure the loading of the beta sliding clamp by the gamma complex in an ATP-dependent reaction. Bound protein-DNA complexes were detected by monitoring fluorescence depolarization of DNA. Steady state and time-resolved anisotropies were measured, and stopped-flow pre-steady state fluorescence measurements allowed visualization of the loading reactions in real time. The rate of loading beta onto DNA was 12 s-1, demonstrating that clamp assembly is rapid on the time scale required for lagging strand Okazaki fragment synthesis. The association rate appears to be limited by an intramolecular step occurring prior to the clamp-loading reaction, possibly the opening of the toroidal beta dimer.
Adenosine Triphosphate↗
Lethality of bypass polymerases in Escherichia coli cells with a defective clamp loader complex of DNA polymerase III.
Escherichia coli DNA polymerase III (Pol III) is one of the best studied replicative DNA polymerases. Here we report the properties of an E. coli mutant that lacks one of the subunits of the Pol III clamp loader complex, Psi (psi), as a result of the complete inactivation of the holD gene. We show that, in this mutant, chronic induction of the SOS response in a RecFOR-dependent way leads to lethality at high temperature. The SOS-induced proteins that are lethal in the holD mutant are the specialized DNA polymerases Pol II and Pol IV, combined with the division inhibitor SfiA. Prevention of SOS induction or inactivation of Pol II, Pol IV and SfiA encoding genes allows growth of the holD mutant, although at a reduced rate compared to a wild-type cell. In contrast, the SOS-induced Pol V DNA polymerase does not participate to the lethality of the holD mutant. We conclude that: (i) Psi is essential for efficient replication of the E. coli chromosome; (ii) SOS-induction of specialized DNA polymerases can be lethal in cells in which the replicative polymerase is defective, and (iii) specialized DNA polymerases differ in respect to their access to inactivated replication forks.
Bacterial Proteins↗
Holoenzyme DNA polymerase III fixes mutations.
DNA polymerase III is required for mutagenesis after damage to the chromosome. This effect is not modulated by the presence or absence of DNA polymerase II activity in the cell. In cells containing a temperature-sensitive dnaE mutation, the alpha-subunit of DNA polymerase III is inactivated at the restrictive temperature, resulting in lethality. Cells containing the pcbA1 mutation can continue replication if DNA polymerase I activity is present. When such cells are shifted from the permissive to the restrictive temperature, mutagenesis decreases rapidly after 10 min. These results are compatible with conversion of the replicative apparatus from one containing a functional DNA polymerase III synthetic subunit to one containing DNA polymerase I. We also find that DNA polymerase I dependent replication is markedly sensitive to coumermycin A1. We conclude that DNA polymerase III holoenzyme with the alpha-subunit is required for fixing mutations in the genome.
Aminocoumarins↗
Dysfunctional proofreading in the Escherichia coli DNA polymerase III core.
The epsilon-subunit contains the catalytic site for the 3'-->5' proofreading exonuclease that functions in the DNA pol III (DNA polymerase III) core to edit nucleotides misinserted by the alpha-subunit DNA pol. A novel mutagenesis strategy was used to identify 23 dnaQ alleles that exhibit a mutator phenotype in vivo. Fourteen of the epsilon mutants were purified, and these proteins exhibited 3'-->5' exonuclease activities that ranged from 32% to 155% of the activity exhibited by the wild-type epsilon protein, in contrast with the 2% activity exhibited by purified MutD5 protein. DNA pol III core enzymes constituted with 11 of the 14 epsilon mutants exhibited an increased error rate during in vitro DNA synthesis using a forward mutation assay. Interactions of the purified epsilon mutants with the alpha- and theta;-subunits were examined by gel filtration chromatography and exonuclease stimulation assays, and by measuring polymerase/exonuclease ratios to identify the catalytically active epsilon511 (I170T/V215A) mutant with dysfunctional proofreading in the DNA pol III core. The epsilon511 mutant associated tightly with the alpha-subunit, but the exonuclease activity of epsilon511 was not stimulated in the alpha-epsilon511 complex. Addition of the theta;-subunit to generate the alpha-epsilon511-theta; DNA pol III core partially restored stimulation of the epsilon511 exonuclease, indicating a role for the theta;-subunit in co-ordinating the alpha-epsilon polymerase-exonuclease interaction. The alpha-epsilon511-theta; DNA pol III core exhibited a 3.5-fold higher polymerase/exonuclease ratio relative to the wild-type DNA pol III core, further indicating dysfunctional proofreading in the alpha-epsilon511-theta; complex. Thus the epsilon511 mutant has wild-type 3'-->5' exonuclease activity and associates physically with the alpha- and theta;-subunits to generate a proofreading-defective DNA pol III enzyme.
Alleles↗
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.
Alleles↗
The beta subunit dissociates readily from the Escherichia coli DNA polymerase III holoenzyme.
Purified DNA polymerase III holoenzyme (holoenzyme) was separated by glycerol gradient sedimentation into the beta subunit and the subassembly that lacks it (pol III). In the presence of ATP, beta subunit dimer dissociated from holoenzyme with a KD of 1 nM; in the absence of ATP, the KD was greater than 5 nM. The beta subunit was known to remain tightly associated in the holoenzyme upon formation of an initiation complex with a primed template and during the course of replication. With separation from the template, holoenzyme dissociated into beta and pol III. Cycling to a new template depended on the reformation of holoenzyme. Holoenzyme was in equilibrium with pol III and the beta subunit in crude enzyme fractions as well as in pure preparations.
Adenosine Triphosphate↗
Crystal structure of the catalytic alpha subunit of E. coli replicative DNA polymerase III.
Bacterial replicative DNA polymerases such as Polymerase III (Pol III) share no sequence similarity with other polymerases. The crystal structure, determined at 2.3 A resolution, of a large fragment of Pol III (residues 1-917), reveals a unique chain fold with localized similarity in the catalytic domain to DNA polymerase beta and related nucleotidyltransferases. The structure of Pol III is strikingly different from those of members of the canonical DNA polymerase families, which include eukaryotic replicative polymerases, suggesting that the DNA replication machinery in bacteria arose independently. A structural element near the active site in Pol III that is not present in nucleotidyltransferases but which resembles an element at the active sites of some canonical DNA polymerases suggests that, at a more distant level, all DNA polymerases may share a common ancestor. The structure also suggests a model for interaction of Pol III with the sliding clamp and DNA.
Bacterial Proteins↗