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.     

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.     

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.     

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.     

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.     

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.     

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.     

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.     

Mechanism of beta clamp opening by the delta subunit of Escherichia coli DNA polymerase III holoenzyme

The beta sliding clamp encircles the primer-template and tethers DNA polymerase III holoenzyme to DNA for processive replication of the Escherichia coli genome. The clamp is formed via hydrophobic and ionic interactions between two semicircular beta monomers. This report demonstrates that the beta dimer is a stable closed ring and is not monomerized when the gamma complex clamp loader (gamma(3)delta(1)delta(1)chi(1)psi(1)) assembles the beta ring around DNA. delta is the subunit of the gamma complex that binds beta and opens the ring; it also does not appear to monomerize beta. Point mutations were introduced at the beta dimer interface to test its structural integrity and gain insight into its interaction with delta. Mutation of two residues at the dimer interface of beta, I272A/L273A, yields a stable beta monomer. We find that delta binds the beta monomer mutant at least 50-fold tighter than the beta dimer. These findings suggest that when delta interacts with the beta clamp, it binds one beta subunit with high affinity and utilizes some of that binding energy to perform work on the dimeric clamp, probably cracking one dimer interface open.     

Isolation and characterization of inhibitory factors of DNA polymerase III holoenzyme from Escherichia coli

Abstract Escherichia coli penicillin-binding protein 5 (PBP5) is anchored to the periplasmic face of the inner membrane via a C-terminal amphiphilic α-helix. The results of washing experiments have suggested an electrostatic contribution to the anchoring mechanism which may involve the cationic region of the C-terminal α-helix. Similarities between this anchor domain and some surface active agents, such as melittin, suggest that the cationic region of the PBP5 anchor may require the presence of anionic phospholipids for membrane interaction. Washing experiments performed on membranes of HDL11, an E. coli mutant in which the expression of the major anionic phospholipids is under lac control, found no such requirement. The results are discussed in relation to the hypothesis that the cationic region may interact with other sources of negative charge, possibly arising from a PBP complex.     

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.     

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 ϵ 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 of 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 ϵ proofreading subunit of DNA polymerase III holoenzyme is excluded, inhibited, or inoperative during misincorporation and mutagenesis after UV.     

Relation of the Escherichia coli dnaX gene to its two products--the tau and gamma subunits of DNA polymerase III holoenzyme.

The Escherichia coli DNA polymerase III holoenzyme 71.1 kDa tau subunit is a 643 amino acid protein encoded by the dnaX gene. This gene also encodes the holoenzyme 56.5 kDa gamma subunit. The tau factor (as a tau'-LacZ' fusion protein) has been isolated and shown to be cleaved in vitro to form gamma and a 135 kda C-terminal cleavage product. The tau'-LacZ' fusion protein, gamma, and the C-terminal cleavage product have been isolated. N-terminal sequencing has demonstrated that tau and gamma share the same N-terminal sequences and that tau is proteolytically cleaved in vitro between residues 498 and 499 to form gamma. In addition, residues 420-440 were shown to be present in both tau and gamma by use of antibody specific for a synthetic peptide corresponding to that sequence. Some mechanism functions in vivo to ensure that tau and gamma are synthesized in a ratio of about one-to-one, as shown by radioimmune precipitation of tau and gamma from cellular extracts.     

RecA protein inhibits in vitro replication of single-stranded DNA with DNA polymerase III holoenzyme of Escherichia coli

Purified RecA protein from Escherichia coli inhibited 5-10-fold the rate of in vitro replication of both unirradiated and UV-irradiated single-stranded DNA (ssDNA) with DNA polymerase III holoenzyme. Maximal inhibition occurred at a ratio of 1 molecule of RecA per 2-4 nucleotides of DNA, and it affected primarily the initiation of elongation on primed ssDNA. Adding single-strand DNA-binding protein (SSB) caused a relief of inhibition. Under conditions when there was enough SSB to cover the ssDNA completely, RecA protein had no effect on initiation, elongation or dissociation steps of replication. These observations together with data from in vivo studies suggest a role for RecA protein in the arrest of DNA replication observed in cells exposed to UV-radiation and a variety of chemical carcinogens.     

ATP activation of DNA polymerase III holoenzyme from Escherichia coli. II. Initiation complex: stoichiometry and reactivity

DNA polymerase III holoenzyme (holenzyme) has an ATPase activity elicited only by a primed DNA template. Reaction of preformed ATP.holoenzyme complex with a primed template results in hydrolysis of the ATP bound to the holoenzyme, release of ADP and Pi, and formation of an initiation complex between holoenzyme and the primed template. Approximately two ATP molecules are hydrolyzed for each initiation complex formed, a value in keeping with the number bound in the ATP.holoenzyme complex. The possibility that the latter and the initiation complex contain two holoenzyme molecules is supported by the presence of two beta monomers in the initiation complex. Holoenzyme action in the absence of ATP resembles that of pol III (the holoenzyme core) or DNA polymerase III (holoenzyme lacking the beta subunit), with or without ATP, in sensitivity to salt and in processivity of elongation. The initiation complex formed by ATP-activated holoenzyme resists a level of KCl (150 mM) that completely inhibits nonactivated holoenzyme and the incomplete forms of the holoenzyme, and displays a processivity at least 20 times greater. Upon completing replication of available template, holoenzyme can dissociate and form an initiation complex with another primed template, provided ATP is available to reactivate the holoenzyme. By inference, no essential subunits are lost in the cycle of initiation, elongation and dissociation.     

Dynamics of DNA polymerase III holoenzyme of Escherichia coli in replication of a multiprimed template

Movements of DNA polymerase III holoenzyme (holoenzyme) in replicating a template multiprimed with synthetic pentadecadeoxynucleotides (15-mers) annealed at known positions on a single-stranded circular or linear DNA have been analyzed. After extension of one 15-mer on a multiprimed template, holoenzyme moves downstream in the direction of chain elongation to the next primer. Holoenzyme readily traverses a duplex, even 400 base pairs long, to exploit its 3'-hydroxyl end as the next available primer. This downstream polarity likely results from an inability to diffuse upstream along single-stranded DNA. These holoenzyme movements, unlike formation of the initial complex with a primer, do not require ATP. Time elapsed between completion of a chain and initiation on the next downstream primer is rapid (1 s or less); dissociation of holoenzyme to form a complex with another primed template is slow (1-2 min). Thus, holoenzyme diffuses rapidly only on duplex DNA, probably in both directions, and forms an initiation complex with the first primer encountered. Based on these findings, schemes can be considered for holoenzyme action at the replication fork of a duplex chromosome.     

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.     

Assembly of DNA polymerase III holoenzyme: co-assembly of gamma and tau is inhibited by DnaX complex accessory proteins but stimulated by DNA polymerase III core.

Although the two alternative Escherichia coli dnaX gene products, tau and gamma, are found co-assembled in purified DNA polymerase III holoenzyme, the pathway of assembly is not well understood. When the 10 subunits of holoenzyme are simultaneously mixed, they rapidly form a nine-subunit assembly containing tau but not gamma. We developed a new assay based on the binding of complexes containing biotin-tagged tau to streptavidin-coated agarose beads to investigate the effects of various DNA polymerase III holoenzyme subunits on the kinetics of co-assembly of gamma and tau into the same complex. Auxiliary proteins in combination with delta' almost completely blocked co-assembly, whereas chipsi or delta' alone slowed the association only moderately compared with the interaction of tau with gamma alone. In contrast, DNA polymerase III core, in the absence of deltadelta' and chipsi, accelerated the co-assembly of tau and gamma, suggesting a role for DNA polymerase III' [tau(2)(pol III core)(2)] in the assembly pathway of holoenzyme.     

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).     

The dimer of the beta subunit of Escherichia coli DNA polymerase III holoenzyme is dissociated into monomers upon binding magnesium(II)

The beta subunit of Escherichia coli DNA polymerase III holoenzyme binds Mg2+. Reacting beta with fluoresceinmaleimide (FM) resulted in one label per beta monomer with full retention of activity. Titration of FM-beta with Mg2+ resulted in a saturable 11% fluorescence enhancement. Analysis indicated that there was one noncooperative magnesium binding site per beta monomer with a dissociation constant of 1.7 mM. Saturable fluorescence enhancement was also observed when titration was with Ca2+ or spermidine(3+) but not with the monovalent cations Na+ and K+. The Mg2+-induced fluorescence enhancement was specific for FM-beta and was not observed with FM-glutathione, dimethoxystilbenemaleimide-beta, or pyrenylmaleimide-beta. Gel filtration studies indicated that the beta dimer-monomer dissociation occurred at physiologically significant beta concentrations and that the presence of 10 mM Mg2+ shifted the dimer-monomer equilibrium to favor monomers. Both the gel-filtered dimers and the gel-filtered monomers were active in the replication assay. These and other results suggested that the fluorescence increase which accompanies beta dissociation is due to a relief from homoquenching of FM when the beta dimer dissociates into monomers.