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.     

Nuclear magnetic resonance solution structure of the Escherichia coli DNA polymerase III theta subunit

The catalytic core of Escherichia coli DNA polymerase III holoenzyme contains three subunits: alpha, epsilon, and theta. The alpha subunit contains the polymerase, and the epsilon subunit contains the exonucleolytic proofreading function. The small (8-kDa) theta subunit binds only to epsilon. Its function is not well understood, although it was shown to exert a small stabilizing effect on the epsilon proofreading function. In order to help elucidate its function, we undertook a determination of its solution structure. In aqueous solution, theta yielded poor-quality nuclear magnetic resonance spectra, presumably due to conformational exchange and/or protein aggregation. Based on our recently determined structure of the theta homolog from bacteriophage P1, named HOT, we constructed a homology model of theta. This model suggested that the unfavorable behavior of theta might arise from exposed hydrophobic residues, particularly toward the end of alpha-helix 3. In gel filtration studies, theta elutes later than expected, indicating that aggregation is potentially responsible for these problems. To address this issue, we recorded 1H-15N heteronuclear single quantum correlation (HSQC) spectra in water-alcohol mixed solvents and observed substantially improved dispersion and uniformity of peak intensities, facilitating a structural determination under these conditions. The structure of theta in 60/40 (vol/vol) water-methanol is similar to that of HOT but differs significantly from a previously reported theta structure. The new theta structure is expected to provide additional insight into its physiological role and its effect on the epsilon proofreading subunit.     

The polymerase subunit of DNA polymerase III of Escherichia coli. II. Purification of the alpha subunit, devoid of nuclease activities

The alpha subunit (140 kDa) of DNA polymerase III (pol III) holoenzyme has been purified to near-homogeneity from a plasmid-carrying Escherichia coli strain which overproduced the alpha subunit about 20-fold. Pol III core (containing only the alpha, epsilon, and theta subunits), produced at twice the normal level, was also purified in good yield. The isolated alpha subunit has DNA polymerase activity, which is completely inhibited by 10 mM N-ethylmaleimide or 150 mM KCl as observed in the pol III core or holoenzyme. The alpha subunit has an apparent turnover number of 7.7 nucleotides polymerized per s, compared to 20 for pol III core, and is more thermolabile. The alpha subunit lacks the 3'----5' exonuclease (proofreading) activity of pol III core; neither alpha subunit nor core (nor holoenzyme) possesses any of the previously reported 5'----3' exonuclease activity. Thus, the alpha polypeptide is the polymerase subunit and epsilon (27 kDa) is the proofreading subunit (Scheuermann, R. H., and Echols, H. (1984) Proc. Natl. Acad. Sci. U. S. A. 81, 7747-7751). Together with the theta polypeptide (10 kDa), of unknown function, they form a pol III core with greater stability and catalytic efficiency.     

The C-terminal domain of dnaQ contains the polymerase binding site

The Escherichia coli dnaQ gene encodes the 3'-->5' exonucleolytic proofreading (epsilon) subunit of DNA polymerase III (Pol III). Genetic analysis of dnaQ mutants has suggested that epsilon might consist of two domains, an N-terminal domain containing the exonuclease and a C-terminal domain essential for binding the polymerase (alpha) subunit. We have created truncated forms of dnaQ resulting in epsilon subunits that contain either the N-terminal or the C-terminal domain. Using the yeast two-hybrid system, we analyzed the interactions of the single-domain epsilon subunits with the alpha and theta subunits of the Pol III core. The DnaQ991 protein, consisting of the N-terminal 186 amino acids, was defective in binding to the alpha subunit while retaining normal binding to the theta subunit. In contrast, the NDelta186 protein, consisting of the C-terminal 57 amino acids, exhibited normal binding to the alpha subunit but was defective in binding to the theta subunit. A strain carrying the dnaQ991 allele exhibited a strong, recessive mutator phenotype, as expected from a defective alpha binding mutant. The data are consistent with the existence of two functional domains in epsilon, with the C-terminal domain responsible for polymerase binding.     

A coproofreading Zn(2+)-dependent exonuclease within a bacterial replicase

The proofreading exonucleases of all DNA replicases contain acidic residues that chelate two Mg(2+) ions that participate in catalysis. DNA polymerase III holoenzymes contain their proofreading activity in a separate subunit, epsilon, which binds the polymerase subunit, alpha, through alpha's N-terminal php domain. Here we demonstrate that the alpha php domain contains a novel Zn(2+)-dependent 3' --> 5' exonuclease that preferentially removes mispaired nucleotides, providing the first example of a coediting nuclease.     

Elucidation of the epsilon-theta subunit interface of Escherichia coli DNA polymerase III by NMR spectroscopy

The DNA polymerase III holoenzyme (HE) is the primary replicative polymerase of Escherichia coli. The epsilon (epsilon) subunit of HE provides the 3'-->5' exonucleolytic proofreading activity for this complex. Epsilon consists of two domains: an N-terminal domain containing the proofreading exonuclease activity (residues 1-186) and a C-terminal domain required for binding to the polymerase (alpha) subunit (residues 187-243). In addition to alpha, epsilon also binds the small (8 kDa) theta (theta) subunit. The function of theta is unknown, although it has been hypothesized to enhance the 3'-->5' exonucleolytic proofreading activity of epsilon. Using NMR analysis and molecular modeling, we have previously reported a structural model of epsilon186, the N-terminal catalytic domain of epsilon [DeRose et al. (2002) Biochemistry 41, 94]. Here, we have performed 3D triple resonance NMR experiments to assign the backbone and C(beta) resonances of [U-(2)H,(13)C,(15)N] methyl protonated epsilon186 in complex with unlabeled theta. A structural comparison of the epsilon186-theta complex with free epsilon186 revealed no major changes in secondary structure, implying that the overall structure is not significantly perturbed in the complex. Amide chemical shift comparisons between bound and unbound epsilon186 revealed a potential binding surface on epsilon for interaction with theta involving structural elements near the epsilon catalytic site. The most significant shifts observed for the epsilon186 amide resonances are localized to helix alpha1 and beta-strands 2 and 3 and to the region near the beginning of alpha-helix 7. Additionally, a small stretch of residues (K158-L161), which previously had not been assigned in uncomplexed epsilon186, is predicted to adopt beta-strand secondary structure in the epsilon186-theta complex and may be significant for interaction with theta. The amide shift pattern was confirmed by the shifts of aliphatic methyl protons, for which the larger shifts generally were concentrated in the same regions of the protein. These chemical shift mapping results also suggest an explanation for how the unstable dnaQ49 mutator phenotype of epsilon may be stabilized by binding theta.     

The theta subunit of Escherichia coli DNA polymerase III: a role in stabilizing the epsilon proofreading subunit

The function of the theta subunit of Escherichia coli DNA polymerase III holoenzyme is not well established. theta is a tightly bound component of the DNA polymerase III core, which contains the alpha subunit (polymerase), the epsilon subunit (3'-->5' exonuclease), and the theta subunit, in the linear order alpha-epsilon-theta. Previous studies have shown that the theta subunit is not essential, as strains carrying a deletion of the holE gene (which encodes theta) proved fully viable. No significant phenotypic effects of the holE deletion could be detected, as the strain displayed normal cell health, morphology, and mutation rates. On the other hand, in vitro experiments have indicated the efficiency of the 3'-exonuclease activity of epsilon to be modestly enhanced by the presence of theta. Here, we report a series of genetic experiments that suggest that theta has a stabilizing role for the epsilon proofreading subunit. The observations include (i) defined DeltaholE mutator effects in mismatch-repair-defective mutL backgrounds, (ii) strong DeltaholE mutator effects in certain proofreading-impaired dnaQ strains, and (iii) yeast two- and three-hybrid experiments demonstrating enhancement of alpha-epsilon interactions by the presence of theta. theta appears conserved among gram-negative organisms which have an exonuclease subunit that exists as a separate protein (i.e., not part of the polymerase polypeptide), and the presence of theta might be uniquely beneficial in those instances where the proofreading 3'-exonuclease is not part of the polymerase polypeptide.     

Structure of the theta subunit of Escherichia coli DNA polymerase III in complex with the epsilon subunit

The catalytic core of Escherichia coli DNA polymerase III contains three tightly associated subunits, the alpha, epsilon, and theta subunits. The theta subunit is the smallest and least understood subunit. The three-dimensional structure of theta in a complex with the unlabeled N-terminal domain of the epsilon subunit, epsilon186, was determined by multidimensional nuclear magnetic resonance spectroscopy. The structure was refined using pseudocontact shifts that resulted from inserting a lanthanide ion (Dy3+, Er3+, or Ho3+) at the active site of epsilon186. The structure determination revealed a three-helix bundle fold that is similar to the solution structures of theta in a methanol-water buffer and of the bacteriophage P1 homolog, HOT, in aqueous buffer. Conserved nuclear Overhauser enhancement (NOE) patterns obtained for free and complexed theta show that most of the structure changes little upon complex formation. Discrepancies with respect to a previously published structure of free theta (Keniry et al., Protein Sci. 9:721-733, 2000) were attributed to errors in the latter structure. The present structure satisfies the pseudocontact shifts better than either the structure of theta in methanol-water buffer or the structure of HOT. satisfies these shifts. The epitope of epsilon186 on theta was mapped by NOE difference spectroscopy and was found to involve helix 1 and the C-terminal part of helix 3. The pseudocontact shifts indicated that the helices of theta are located about 15 A or farther from the lanthanide ion in the active site of epsilon186, in agreement with the extensive biochemical data for the theta-epsilon system.     

Model for the catalytic domain of the proofreading epsilon subunit of Escherichia coli DNA polymerase III based on NMR structural data.

The DNA polymerase III holoenzyme (HE) is the primary replicative polymerase of Escherichia coli. The epsilon subunit of the HE complex provides the 3'-exonucleolytic proofreading activity for this enzyme complex. epsilon consists of two domains: an N-terminal domain containing the proofreading exonuclease activity (residues 1-186) and a C-terminal domain required for binding to the polymerase (alpha) subunit (residues 187-243). Multidimensional NMR studies of (2)H-, (13)C-, and (15)N-labeled N-terminal domains (epsilon186) were performed to assign the backbone resonances and measure H(N)-H(N) nuclear Overhauser effects (NOEs). NMR studies were also performed on triple-lableled [U-(2)H,(13)C,(15)N]epsilon186 containing Val, Leu, and Ile residues with protonated methyl groups, which allowed for the assignment of H(N)-CH(3) and CH(3)-CH(3) NOEs. Analysis of the (13)C(alpha), (13)C(beta), and (13)CO shifts, using chemical shift indexing and the TALOS program, allowed for the identification of regions of the secondary structure. H(N)-H(N) NOEs provided information on the assembly of the extended strands into a beta-sheet structure and confirmed the assignment of the alpha helices. Measurement of H(N)-CH(3) and CH(3)-CH(3) NOEs confirmed the beta-sheet structure and assisted in the positioning of the alpha helices. The resulting preliminary characterization of the three-dimensional structure of the protein indicated that significant structural homology exists with the active site of the Klenow proofreading exonuclease domain, despite the extremely limited sequence homology. On the basis of this analogy, molecular modeling studies of epsilon186 were performed using as templates the crystal structures of the exonuclease domains of the Klenow fragment and the T4 DNA polymerase and the recently determined structure of the E. coli Exonuclease I. A multiple sequence alignment was constructed, with the initial alignment taken from the previously published hidden Markov model and NMR constraints. Because several of the published structures included complexed ssDNA, we were also able to incorporate an A-C-G trinucleotide into the epsilon186 structure. Nearly all of the residues which have been identified as mutators are located in the portion of the molecule which binds the DNA, with most of these playing either a catalytic or structural role.     

A strong mutator effect caused by an amino acid change in the alpha subunit of DNA polymerase III of Escherichia coli

Most potent mutators heretofore detected in Escherichia coli are associated with defects in epsilon subunit of DNA polymerase III, encoded by the dnaQ gene. To elucidate the role of the alpha subunit, the catalytic subunit of the polymerase, in maintaining the high fidelity of DNA replication, we isolated a mutator mutant, the mutation (dnaE173) of which resides on the dnaE gene, encoding the alpha subunit. The dnaE173 mutant was unable to grow in salt-free L broth at temperatures exceeding 44.5 degrees C and exhibited an increased frequency of spontaneous mutations, 1,000 to 10,000-fold the wild type level, at permissive temperatures. The mutator effect of dnaE173 mutation is dominant over the wild type allele. These phenotypes are caused by a single base substitution, resulting in one amino acid change, Glu612 (GAA)----Lys(AAA), in the alpha subunit molecule. DNA polymerase III purified from the dnaE173 mutant contained both alpha and epsilon subunits, in a normal molar ratio. We found no differences between wild type and mutant polymerases in the Vmax, thermolabilities, and salt sensitivities. However, the apparent Km for the substrate nucleotide of the mutant polymerase was 1/6 of that determined with the wild type polymerase. Although the mutant polymerase retained a normal level of 3'----5' exonuclease activity, the proofreading capacity determined by "turnover assay" was significantly lower in the mutant polymerase, as compared with findings in the normal enzyme. It seems likely that the enhanced mutability in the dnaE173 strain results from, at least in part, a defect in the editing function of DNA polymerase III, and further suggests that a portion of the alpha subunit in which the amino acid change resides may be important for the proper setting of the two subunits at the replication fork so as to facilitate efficient editing during the DNA replication.     

A preliminary CD and NMR study of the Escherichia coli DNA polymerase III theta subunit.

The theta subunit of DNA polymerase III, the main replicative polymerase of Escherichia coli, has been examined by circular dichroism and by NMR spectroscopy. The polymerase core consists of three subunits: alpha, epsilon, and theta, with alpha possessing the polymerase activity, epsilon functioning as a proofreading exonuclease, and theta, a small subunit of 8.9 kD, of undetermined function. The theta subunit has been expressed in E. coli, and a CD analysis of theta indicates the presence of a significant amount of secondary structure: approximately 52% alpha helix, 9% beta sheet, 21% turns, and 18% random coil. However, at higher concentrations, theta yields a poorly-resolved 1D proton NMR spectrum in which both the amide protons and the methyl protons show poor chemical shift dispersion. Subsequent 1H-15N HSQC analysis of uniformly-15N-labeled theta supports the conclusion that approximately half of the protein is reasonably well-structured. Another quarter of the protein, probably including some of the N-terminal region, is highly mobile, exhibiting a chemical shift pattern indicative of random coil structure. The remaining amide resonances exhibit significant broadening, indicative of intermolecular and/or intramolecular exchange processes. Improved chemical shift dispersion and greater uniformity of resonance intensities in the 1H-15N HSQC spectra resulted when [U-15N]-theta was examined in the presence of epsilon186--the N-terminal domain of the epsilon-subunit. Further work is currently in progress to define the solution structure of theta and the theta-epsilon186 complex.     

An Escherichia coli dnaE mutation with suppressor activity toward mutator mutD5.

The Escherichia coli mutator mutD5 is a conditional mutator whose strength is moderate when the strain is growing in minimal medium but very strong when it is growing in rich medium. The primary defect of this strain resides in the dnaQ gene, which encodes the epsilon (exonucleolytic proofreading) subunit of the DNA polymerase III holoenzyme. In one of our mutD5 strains we discovered a mutation that suppressed the mutability of mutD5. Interestingly, the level of suppression was strong in minimal medium but weak in rich medium. The mutation was localized to the dnaE gene, which encodes the alpha (polymerase) subunit of the DNA polymerase III holoenzyme. This mutation, termed dnaE910, also conferred improved growth of the mutD5 strain and caused increased temperature sensitivity in both wild-type and dnaQ49 backgrounds. The reduction in mutator strength by dnaE910 was also observed when this allele was placed in a mutL, a mutT, or a dnaQ49 background. The results suggest that dnaE910 encodes an antimutator DNA polymerase whose effect might be mediated by improved insertion fidelity or by increased proofreading via its effect on the exonuclease activity.     

holE, the gene coding for the theta subunit of DNA polymerase III of Escherichia coli: characterization of a holE mutant and comparison with a dnaQ (epsilon-subunit) mutant.

DNA polymerase III holoenzyme is a multiprotein complex responsible for the bulk of chromosomal replication in Escherichia coli and Salmonella typhimurium. The catalytic core of the holoenzyme is an alpha epsilon theta heterotrimer that incorporates both a polymerase subunit (alpha; dnaE) and a proofreading subunit (epsilon; dnaQ). The role of theta is unknown. Here, we describe a null mutation of holE, the gene for theta. A strain carrying this mutation was fully viable and displayed no mutant phenotype. In contrast, a dnaQ null mutant exhibited poor growth, chronic SOS induction, and an elevated spontaneous mutation rate, like dnaQ null mutants of S. typhimurium described previously. The poor growth was suppressible by a mutation affecting alpha which was identical to a suppressor mutation identified in S. typhimurium. A double mutant null for both holE and dnaQ was indistinguishable from the dnaQ single mutant. These results show that the theta subunit is dispensable in both dnaQ+ and mutant dnaQ backgrounds, and that the phenotype of epsilon mutants cannot be explained on the basis of interference with theta function.     

Mutagenic DNA repair in Escherichia coli. XIII. Proofreading exonuclease of DNA polymerase III holoenzyme is not operational during UV mutagenesis

We have introduced a mutD5 mutation (which results in defective 3'-5'-exonuclease activity of the epsilon proofreading subunit of DNA polymerase III holoenzyme) into excision-defective Escherichia coli strains with varying SOS responses to UV light. MutD5 increased the spontaneous mutation frequency in all strains tested, including recA430, umuC122::Tn5, and umuC36 derivatives. It had no effect on UV mutability or immutability in any strain or on misincorporation revealed by delayed photoreversal in UV-irradiated umuC36, umuC122::Tn5, or recA430 bacteria. It is concluded that the epsilon proofreading subunit of DNA polymerase III holoenzyme is excluded, inhibited, or inoperative during misincorporation and mutagenesis after UV.     

Mutator and antimutator effects of the bacteriophage P1 hot gene product

The Hot (homolog of theta) protein of bacteriophage P1 can substitute for the Escherichia coli DNA polymerase III theta subunit, as evidenced by its stabilizing effect on certain dnaQ mutants that carry an unstable polymerase III epsilon proofreading subunit (antimutator effect). Here, we show that Hot can also cause an increase in the mutability of various E. coli strains (mutator effect). The hot mutator effect differs from the one caused by the lack of theta. Experiments using chimeric theta/Hot proteins containing various domains of Hot and theta along with a series of point mutants show that both N- and C-terminal parts of each protein are important for stabilizing the epsilon subunit. In contrast, the N-terminal part of Hot appears uniquely responsible for its mutator activity.     

Analysis of a multicomponent thermostable DNA polymerase III replicase from an extreme thermophile

This report takes a proteomic/genomic approach to characterize the DNA polymerase III replication apparatus of the extreme thermophile, Aquifex aeolicus. Genes (dnaX, holA, and holB) encoding the subunits required for clamp loading activity (tau, delta, and delta') were identified. The dnaX gene produces only the full-length product, tau, and therefore differs from Escherichia coli dnaX that produces two proteins (gamma and tau). Nonetheless, the A. aeolicus proteins form a taudeltadelta' complex. The dnaN gene encoding the beta clamp was identified, and the taudeltadelta' complex is active in loading beta onto DNA. A. aeolicus contains one dnaE homologue, encoding the alpha subunit of DNA polymerase III. Like E. coli, A. aeolicus alpha and tau interact, although the interaction is not as tight as the alpha-tau contact in E. coli. In addition, the A. aeolicus homologue to dnaQ, encoding the epsilon proofreading 3'-5'-exonuclease, interacts with alpha but does not form a stable alpha.epsilon complex, suggesting a need for a brace or bridging protein to tightly couple the polymerase and exonuclease in this system. Despite these differences to the E. coli system, the A. aeolicus proteins function to yield a robust replicase that retains significant activity at 90 degrees C. Similarities and differences between the A. aeolicus and E. coli pol III systems are discussed, as is application of thermostable pol III to biotechnology.     

Constitution of the twin polymerase of DNA polymerase III holoenzyme

It is speculated that DNA polymerases which duplicate chromosomes are dimeric to provide concurrent replication of both leading and lagging strands. DNA polymerase III holoenzyme (holoenzyme), is the 10-subunit replicase of the Escherichia coli chromosome. A complex of the alpha (DNA polymerase) and epsilon (3'-5' exonuclease) subunits of the holoenzyme contains only one of each protein. Presumably, one of the eight other subunit(s) functions to dimerize the alpha epsilon polymerase within the holoenzyme. Based on dimeric subassemblies of the holoenzyme, two subunits have been elected as possible agents of polymerase dimerization, one of which is the tau subunit (McHenry, C. S. (1982) J. Biol. Chem. 257, 2657-2663). Here, we have used pure alpha, epsilon, and tau subunits in binding studies to determine whether tau can dimerize the polymerase. We find tau binds directly to alpha. Whereas alpha is monomeric, tau is a dimer in its native state and thereby serves as an efficient scaffold to dimerize the polymerase. The epsilon subunit does not associate directly with tau but becomes dimerized in the alpha epsilon tau complex by virtue of its interaction with alpha. We have analyzed the dimeric alpha epsilon tau complex by different physical methods to increase the confidence that this complex truly contains a dimeric polymerase. The tau subunit is comprised of the NH2-terminal two-thirds of tau but does not bind to alpha epsilon, identifying the COOH-terminal region of tau as essential to its polymerase dimerization function. The significance of these results with respect to the organization of subunits within the holoenzyme is discussed.     

umuDC-dnaQ Interaction and its implications for cell cycle regulation and SOS mutagenesis in Escherichia coli

The Escherichia coli SOS-regulated umuDC gene products participate in a DNA damage checkpoint control and in translesion DNA synthesis. Specific interactions involving the UmuD and UmuD' proteins, both encoded by the umuD gene, and components of the replicative DNA polymerase, Pol III, appear to be important for regulating these two biological activities of the umuDC gene products. Here we show that overproduction of the epsilon proofreading subunit of Pol III suppresses the cold sensitivity normally associated with overexpression of the umuDC gene products. Our results suggest that this suppression is attributable to specific interactions between UmuD or UmuD' and the C-terminal domain of epsilon.     

The dnaE173 mutator mutation confers on the alpha subunit of Escherichia coli DNA polymerase III a capacity for highly processive DNA synthesis and stable binding to primer/template DNA

The strong mutator mutation dnaE173 which causes an amino-acid substitution in the alpha subunit of DNA polymerase III is unique in its ability to induce sequence-substitution mutations. We showed previously that multiple biochemical properties of DNA polymerase III holoenzyme of Escherichia coli are simultaneously affected by the dnaE173 mutation. These effects include a severely reduced proofreading capacity, an increased resistance to replication-pausing on the template DNA, a capability to readily promote strand-displacement DNA synthesis, a reduced rate of DNA chain elongation, and an ability to catalyze highly processive DNA synthesis in the absence of the beta-clamp subunit. Here we show that, in contrast to distributive DNA synthesis exhibited by wild-type alpha subunit, the dnaE173 mutant form of alpha subunit catalyzes highly processive DNA chain elongation without the aid of the beta-clamp. More surprisingly, the dnaE173 alpha subunit appeared to form a stable complex with primer/template DNA, while no such affinity was detected with wild-type alpha subunit. We consider that the highly increased affinity of alpha subunit for primer/template DNA is the basis for the pleiotropic effects of the dnaE173 mutation on DNA polymerase III, and provides a clue to the molecular mechanisms underlying sequence substitution mutagenesis.