ATP interactions of the tau and gamma subunits of DNA polymerase III holoenzyme of Escherichia coli

The tau and gamma subunits of the DNA polymerase III holoenzyme of Escherichia coli were each isolated in large quantities as oligomers from overproducing cells in which their genes (dnaZ and X) were under the control of a T7 phage promoter. The 52-kDa gamma subunit (encoded by the dnaZ sequence) contains three-forths of the N-terminal residues of the 71-kDa tau subunit (encoded by the dnaX sequence). Both gamma and tau share a binding site for ATP (or dATP). A DNA-dependent ATPase activity (Lee, S.H., and Walker, J.R. (1987) Proc. Natl. Acad. Sci. U. S. A. 84, 2713-2717) exhibited only by the tau subunit, presumably requires a DNA-binding site in the C-terminal domain lacking in the gamma subunit. Among ATPases dependent on single-stranded DNA, the tau activity is remarkable in the failure of homopolymers (e.g. poly(dA) or poly(dT)) to replace natural DNAs. The presumed need for certain secondary structures may reflect a feature of template binding in the crucial contribution that tau makes to the high processivity of polymerase III holoenzyme. Limited tryptic digestion of tau generates a fragment that resembles gamma in: (i) size, (ii) binding of ATP without ATPase activity, and (iii) a level of complementing holoenzyme activity in extracts of dnaZ-mutant cells that is higher than that of tau.     

The Escherichia coli DNA polymerase III holoenzyme contains both products of the dnaX gene, tau and gamma, but only tau is essential.

The replicative polymerase of Escherichia coli, DNA polymerase III, consists of a three-subunit core polymerase plus seven accessory subunits. Of these seven, tau and gamma are products of one replication gene, dnaX. The shorter gamma is created from within the tau reading frame by a programmed ribosomal -1 frameshift over codons 428 and 429 followed by a stop codon in the new frame. Two temperature-sensitive mutations are available in dnaX. The 2016(Ts) mutation altered both tau and gamma by changing codon 118 from glycine to aspartate; the 36(Ts) mutation affected the activity only of tau because it altered codon 601 (from glutamate to lysine). Evidence which indicates that, of these two proteins, only the longer tau is essential includes the following. (i) The 36(Ts) mutation is a temperature-sensitive lethal allele, and overproduction of wild-type gamma cannot restore its growth. (ii) An allele which produced tau only could be substituted for the wild-type chromosomal gene, but a gamma-only allele could not substitute for the wild-type dnaX in the haploid state. Thus, the shorter subunit gamma is not essential, suggesting that tau can be substitute for the usual function(s) of gamma. Consistent with these results, we found that a functional polymerase was assembled from nine pure subunits in the absence of the gamma subunit. However, the possibility that, in cells growing without gamma, proteolysis of tau to form a gamma-like product in amounts below the Western blot (immunoblot) sensitivity level cannot be excluded.     

DNA polymerase III holoenzyme of Escherichia coli. I. Purification and distinctive functions of subunits tau and gamma, the dnaZX gene products

Escherichia coli dnaZX, the gene which when mutant blocks DNA chain elongation, was cloned into a lambda PL promoter-mediated expression vector. In cells carrying this plasmid, the activity that complements a mutant dnaZ extract in replicating a primed single-stranded DNA circle was increased about 20-fold. Two polypeptides of 71 and 52 kDa were overproduced. Upon fractionation, two complementing activities were purified to homogeneity and proved to be the 71- and 52-kDa polypeptides. Immunoassays revealed their respective identities with the tau and gamma subunits of DNA polymerase III holoenzyme. The N-terminal amino acid sequences of the first 12 residues were identical in both subunits, as were their molar specific activities in dnaZ complementation. Thus, the tau subunit complements the defect in the mutant holoenzyme from the dnaZts strain as efficiently as does the gamma subunit. Inasmuch as the 71-kDa subunit (tau) can also overcome the enzymatic defect in a dnaX mutant strain, this polypeptide has dual replication functions, only one of which can be performed by the gamma subunit. Availability of pure tau and gamma subunits for study has provided the basis for proposing an asymmetry in the structure and function of a dimeric DNA polymerase III holoenzyme.     

DNA polymerase III holoenzyme of Escherichia coli. Purification and resolution into subunits.

DNA polymerase III holoenzyme has been purified from Escherichia coli HMS-83, using, as an assay, the conversion of coliphage G4 single-stranded DNA to the duplex replicative form. The holoenzyme consists of at least four different subunits: alpha, beta, gamma, and delta of 140,000, 40,000, 52,000, and 32,000 daltons, respectively. The alpha subunit is DNA polymerase III, the dnaE gene product. The holoenzyme has been resolved by phosphocellulose chromatography into an alpha - gamma - delta complex and a subunit beta (copolymerase III*); neither possesses detectable activity in the G4 system but together reconstitute holoenzyme-like activity. The alpha - gamma - delta complex has been further resolved to yield a gamma - delta complex which reconstitutes alpha - gamma - delta activity when added to DNA polymerase III. The gamma - delta complex contains a product of the dnaZ gene and has been purified from a strain which contains a ColE1-dnaZ hybrid plasmid.     

Escherichia coli DNA polymerase II is stimulated by DNA polymerase III holoenzyme auxiliary subunits

DNA polymerase III of Escherichia coli requires multiple auxiliary factors to enable it to serve as a replicative complex. We demonstrate that auxiliary components of the DNA polymerase III holoenzyme, the gamma delta complex and beta subunit, markedly stimulate DNA polymerase II on long single-stranded templates. DNA polymerase II activity is enhanced by single-stranded DNA binding protein, but the stimulation by gamma delta and beta can be observed either in the absence or presence of single-stranded DNA binding protein. In contrast with DNA polymerase III, the requirement of DNA polymerase II for gamma delta cannot be bypassed by large excesses of the beta subunit at low ionic strength in the absence of the single-stranded DNA binding protein. The product of the DNA polymerase II-gamma delta-beta reaction on a uniquely primed single-stranded circle is of full template length; the reconstituted enzyme apparently is incapable of strand displacement synthesis. The possible biological implications of these observations are discussed.     

dnaX36 Mutator of Escherichia coli: effects of the {tau} subunit of the DNA polymerase III holoenzyme on chromosomal DNA replication fidelity

The Escherichia coli dnaX36 mutant displays a mutator effect, reflecting a fidelity function of the dnaX-encoded τ subunit of the DNA polymerase III (Pol III) holoenzyme. We have shown that this fidelity function (i) applies to both leading- and lagging-strand synthesis, (ii) is independent of Pol IV, and (iii) is limited by Pol II.     

DNA polymerase III holoenzyme of Escherichia coli. III. Distinctive processive polymerases reconstituted from purified subunits

The 10 distinctive polypeptides of DNA polymerase III holoenzyme, purified as individual subunits or complexes, could be reconstituted to generate a polymerase with the high catalytic rate of the isolated intact holoenzyme. Functions and interactions of the subunits can be inferred from partial assemblies of the pol III core (alpha, epsilon, and theta subunits) with auxiliary subunits. The core possesses the polymerase and proofreading activities; the auxiliary subunits provide the core with processivity, the capacity to replicate long stretches of DNA without dissociating from the template. In a sequence of reconstruction steps, the beta subunit binds the primed template in an ATP-dependent manner through the catalytic action of a complex made up of the gamma, delta, delta', chi, and psi polypeptides. With the beta subunit in place, a processive polymerase is produced upon addition of the core. When the tau subunit is lacking, binding of polymerase to the primed template is less efficient and stable. The tau-less reconstituted polymerase is more prone to dissociation upon encountering secondary structures in the template in its path, such as a hairpin region in the single strand or a duplex region formed by a strand annealed to the template. With the tau subunit present, the interaction of the core.beta complex (the basic unit of a processive polymerase) with the primed template is strengthened. The tau-containing reconstituted polymerase can replicate DNA continuously through secondary structures in the template. The two distinctive kinds of processivity demonstrated by the tau-less and tau-containing reconstituted polymerases fit nicely into a scheme in which, organized as an asymmetric dimeric holoenzyme, the tau half is responsible for continuous synthesis of one strand, and the less stable half for discontinuous synthesis of the other.     

The DnaX-binding subunits delta' and psi are bound to gamma and not tau in the DNA polymerase III holoenzyme

The DnaX complex subassembly of the DNA polymerase III holoenzyme is comprised of the DnaX proteins tau and gamma and the auxiliary subunits delta, delta', chi, and psi, which together load the beta processivity factor onto primed DNA in an ATP-dependent reaction. delta' and psi bind directly to DnaX whereas delta and chi bind to delta' and psi, respectively (Onrust, R., Finkelstein, J., Naktinis, V., Turner, J., Fang, L., and O'Donnell, M. (1995) J. Biol. Chem. 270, 13348-13357). Until now, it has been unclear which DnaX protein, tau or gamma, in holoenzyme binds the auxiliary subunits delta, delta', chi,and psi. Treatment of purified holoenzyme with the homobifunctional cross-linker bis(sulfosuccinimidyl)suberate produces covalently cross-linked gamma-delta' and gamma-psi complexes identified by Western blot analysis. Immunodetection of cross-linked species with anti-delta' and anti-psi antibodies revealed that no tau-delta' or tau-psi cross-links had formed, suggesting that the delta' and psi subunits reside only on gamma within holoenzyme.     

DNA and RNA-DNA annealing activity associated with the tau subunit of the Escherichia coli DNA polymerase III holoenzyme.

The DNA polymerase III (pol III)holoenzyme is the 10 subunit replicase of Escherichia coli. The 71 kDa tau subunit, encoded by dnaX, dimerizes the core polymerase (alpha epsilon theta) to form pol III'[(alpha epsilon theta)2 tau 2]. tau is also a single-stranded DNA-dependent ATPase and can substitute for the gamma subunit during initiation complex formation. We show here that tau also possesses a DNA-DNA and RNA-DNA annealing activity that is stimulated by Mg2+, but neither requires ATP nor is inhibited by non-hydrolyzable ATP analogs. This suggests the tau may act to stabilize the primer-template interaction during DNA replication.     

DNA structure requirements for the Escherichia coli gamma complex clamp loader and DNA polymerase III holoenzyme

The Escherichia coli chromosomal replicase, DNA polymerase III holoenzyme, is highly processive during DNA synthesis. Underlying high processivity is a ring-shaped protein, the beta clamp, that encircles DNA and slides along it, thereby tethering the enzyme to the template. The beta clamp is assembled onto DNA by the multiprotein gamma complex clamp loader that opens and closes the beta ring around DNA in an ATP-dependent manner. This study examines the DNA structure required for clamp loading action. We found that the gamma complex assembles beta onto supercoiled DNA (replicative form I), but only at very low ionic strength, where regions of unwound DNA may exist in the duplex. Consistent with this, the gamma complex does not assemble beta onto relaxed closed circular DNA even at low ionic strength. Hence, a 3'-end is not required for clamp loading, but a single-stranded DNA (ssDNA)/double-stranded DNA (dsDNA) junction can be utilized as a substrate, a result confirmed using synthetic oligonucleotides that form forked ssDNA/dsDNA junctions on M13 ssDNA. On a flush primed template, the gamma complex exhibits polarity; it acts specifically at the 3'-ssDNA/dsDNA junction to assemble beta onto the DNA. The gamma complex can assemble beta onto a primed site as short as 10 nucleotides, corresponding to the width of the beta ring. However, a protein block placed closer than 14 base pairs (bp) upstream from the primer 3' terminus prevents the clamp loading reaction, indicating that the gamma complex and its associated beta clamp interact with approximately 14-16 bp at a ssDNA/dsDNA junction during the clamp loading operation. A protein block positioned closer than 20-22 bp from the 3' terminus prevents use of the clamp by the polymerase in chain elongation, indicating that the polymerase has an even greater spatial requirement than the gamma complex on the duplex portion of the primed site for function with beta. Interestingly, DNA secondary structure elements placed near the 3' terminus impose similar steric limits on the gamma complex and polymerase action with beta. The possible biological significance of these structural constraints is discussed.     

Site-directed mutagenesis with Escherichia coli DNA polymerase III holoenzyme

Escherichia coli DNA polymerase III holoenzyme was used to synthesize double-stranded DNA from M13 single-stranded DNA hybridized to a phosphorylated synthetic oligodeoxynucleotide containing a nucleotide substitution. The resulting DNA was transfected into E. coli JM101 without further treatment. Sequence analysis of randomly chosen phage clones revealed that the efficiency of mutagenesis was nearly 50%, which is the theoretical maximum. Treatment with DNA ligase after DNA synthesis was not necessary to obtain high efficiency of mutagenesis. Thus, use of DNA polymerase III holoenzyme provides a simple and efficient procedure for site-directed mutagenesis.     

The dnaZ protein, the gamma subunit of DNA polymerase III holoenzyme of Escherichia coli

The dnaZ protein has been purified to near-homogeneity using an in vitro complementation assay that measures the restoration of activity in a crude enzyme fraction from the dnaZ mutant deficient in the replication of phi X174 DNA. Over 70-fold overproduction of the protein was obtained with a bacteriophage lambda lysogen carrying the dnaZ gene. The purified protein, under reducing and denaturing conditions, has a molecular weight of 52,000 and appears to be a dimer in its native form. The dnaZ protein is judged to be th 52,000-dalton gamma subunit of DNA polymerase III holoenzyme (McHenry, C., and Kornberg, A. (1977) J. Biol. Chem. 252, 6478-6484) for the following reasons: (i) highly purified DNA polymerase III holoenzyme contains a 52,000-dalton polypeptide and has dnaZ-complementing activity; (ii) the 52,000-dalton polypeptide is associated tightly with the DNA polymerase III holoenzyme and can be separated from the DNA polymerase III core only with severe measures; (iii) no other purified replication protein, among 14 tested, contains dnaZ protein activity; and (iv) the abundance of dnaZ protein, estimated at about 10 dimer molecules per Escherichia coli cell, is similar to that of the DNA polymerase III core. Among several circular templates tested in vitro (i.e. single stranded phi X174, G4 and M13 DNAs, and duplex phi X174 DNA), all rely on dnaZ protein for elongation by DNA polymerase III holoenzyme. The protein acts catalytically at a stoichiometry of one dimer per template.     

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.     

Prereplicative complexes of components of DNA polymerase III holoenzyme of Escherichia coli.

Stepwise reconstitution of the subunits of DNA polymerase III holoenzyme of Escherichia coli offers insights into the organization and function of this multisubunit assembly. A highly processive, holoenzyme-like activity can be generated when the gamma complex, in the presence of ATP and a primed template, activates the beta subunit to form a preinitiation complex, and this is then followed by addition of the core polymerase. Further analysis of early replicative complexes has now revealed: 1) that the gamma complex can stably bind a single-stranded DNA binding protein (SSB)-coated template, 2) that neither SSB coating of the template nor a proper primer terminus is required to form the preinitiation complex, and 3) that the gamma complex stabilizes the preinitiation complex in the presence of ATP and destabilizes it in the presence of adenosine 5'-O-(thiotriphosphate). Based on these findings, a sequence of stages can be formulated for an activation of the beta subunit that enables it to bind the template-primer and thereby interact with the core to create a processive polymerase.     

Interplay of clamp loader subunits in opening the beta sliding clamp of Escherichia coli DNA polymerase III holoenzyme

The Escherichia coli beta dimer is a ring-shaped protein that encircles DNA and acts as a sliding clamp to tether the replicase, DNA polymerase III holoenzyme, to DNA. The gamma complex (gammadeltadelta'chipsi) clamp loader couples ATP to the opening and closing of beta in assembly of the ring onto DNA. These proteins are functionally and structurally conserved in all cells. The eukaryotic equivalents are the replication factor C (RFC) clamp loader and the proliferating cell nuclear antigen (PCNA) clamp. The delta subunit of the E. coli gamma complex clamp loader is known to bind beta and open it by parting one of the dimer interfaces. This study demonstrates that other subunits of gamma complex also bind beta, although weaker than delta. The gamma subunit like delta, affects the opening of beta, but with a lower efficiency than delta. The delta' subunit regulates both gamma and delta ring opening activities in a fashion that is modulated by ATP interaction with gamma. The implications of these actions for the workings of the E. coli clamp loading machinery and for eukaryotic RFC and PCNA are discussed.     

DNA polymerase III of Escherichia coli. Purification and identification of subunits.

DNA polymerase III, the core of the DNA polymerase III holoenzyme, has been purified 28,000-fold to 97% homogeneity from Escherichia coli HMS-83. The enzyme contains subunits: alpha, epsilon, and theta of 140,000, 25,000, and 10,000 daltons, respectively. The alpha subunit has been previously shown to be a component of both DNA polymerase III and the more complex DNA polymerase III holoenzyme (Livingston, D.M., Hinkle, D., and Richardson, C. (1975) J. Biol. Chem. 250, 461-469; McHenry, C., and Kornberg, A. (1977) J. Biol. Chem. 252, 6478-6484). It is demonstrated here that the epsilon and theta subunits are also subunits of the DNA polymerase III holoenzyme. Thus, the DNA polymerase III holoenzyme contains at least six different subunits. Our preparation has both the 3' leads to 5' and 5' leads to 3' exonuclease activities previously assigned to DNA polymerase III (Livingston, D., and Richardson, C. (1975) J. Biol. Chem. 250, 470-478).     

DNA polymerase III holoenzyme of Escherichia coli. II. A novel complex including the gamma subunit essential for processive synthesis

Processive DNA synthesis, a property of DNA polymerase III holoenzyme of Escherichia coli, was not achieved by combining the pol III core (alpha, epsilon, and theta subunits) and the beta and gamma subunits. An activity that restored processivity to these subunits was found in crude extracts and was overproduced 4-fold in cells with plasmids amplifying the tau and gamma subunits. Purified to homogeneity, the activity, assayed by reconstitution of processivity, was represented by five polypeptides which were copurified. Judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, these correspond to the known subunits gamma (52 kDa) and delta (35 kDa) and to three new polypeptides: delta' (33 kDa), chi (15 kDa), and psi (12 kDa). The five polypeptides form a tight complex with a native molecular weight of about 200 kDa and a subunit stoichiometry of two gamma subunits to one each of the others. Processive DNA synthesis, now achieved with only three components (pol III core, beta, and the auxiliary complex), provides the opportunity to assess the functions of each and the contribution that the remaining auxiliary tau subunit makes to reconstitute a holoenzyme.     

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.     

Programmed ribosomal frameshifting generates the Escherichia coli DNA polymerase III gamma subunit from within the tau subunit reading frame.

The Escherichia coli dnaX gene encodes both the tau and gamma subunits of DNA polymerase III holoenzyme in one reading frame. The 71.1 kDa tau and the shorter gamma share N-terminal sequences. Mutagenesis of a potential ribosomal frameshift signal located at codons 428-430 without changing the amino acid sequence of the tau product, eliminated detectable synthesis of the gamma subunit, suggesting that the reading frame is shifted at that sequence and gamma is terminated by a nonsense codon located in the -1 frame 3 nucleotides downstream of the signal. This seems to be the first known case of a frameshift which is used, along with the termination codon in the -1 frame, to terminate a peptide within a reading frame. [Mutagenesis of a dibasic peptide (lys-lys) at codons 498-499, the site at which a tau'-'LacZ fusion protein was cleaved in vitro (1) had no effect on gamma formation in vivo, suggesting that cleavage observed in vitro is not the mechanism of gamma formation in vivo.