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J. Biol. Chem., Vol. 277, Issue 16, 13401-13408, April 19, 2002

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DNA Polymerase III Holoenzyme from Thermus thermophilus Identification, Expression, Purification of Components, and Use to Reconstitute a Processive Replicase^*

James M. Bullard, Jennifer C. Williams, Wendy K. Acker, Carsten Jacobi^§, Nebojsa Janjic, and Charles S. McHenry^¶

From  Replidyne, Inc., Denver, Colorado 80206, the ^§ Institute of Microbiology and Genetics, University of Göttingen, Göttingen, Germany, and the ^¶ Department of Biochemistry and Molecular Genetics, University of Colorado Health Sciences Center, Denver, Colorado 80262

Received for publication, November 12, 2001, and in revised form, January 28, 2002

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

DNA replication in bacteria is performed by a specialized multicomponent replicase, the DNA polymerase III holoenzyme, that consist of three essential components: a polymerase, the  sliding clamp processivity factor, and the DnaX complex clamp-loader. We report here the assembly of the minimal functional holoenzyme from Thermus thermophilus (Tth), an extreme thermophile. The minimal holoenzyme consists of  (pol III catalytic subunit),  (sliding clamp processivity factor), and the essential DnaX (/),  and ' components of the DnaX complex. We show with purified recombinant proteins that these five components are required for rapid and processive DNA synthesis on long single-stranded DNA templates. Subunit interactions known to occur in DNA polymerase III holoenzyme from mesophilic bacteria including -' interaction, '-/ complex formation, and - interaction, also occur within the Tth enzyme. As in mesophilic holoenzymes, in the presence of a primed DNA template, these subunits assemble into a stable initiation complex in an ATP-dependent manner. However, in contrast to replicative polymerases from mesophilic bacteria, Tth holoenzyme is efficient only at temperatures above 50 °C, both with regard to initiation complex formation and processive DNA synthesis. The minimal Tth DNA polymerase III holoenzyme displays an elongation rate of 350 bp/s at 72 °C and a processivity of greater than 8.6 kilobases, the length of the template that is fully replicated after a single association event.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

DNA replication in all biological systems is performed by specialized multiprotein replicases (1, 2). Cellular replicases consist of three major subassemblies: a sliding clamp processivity factor, a clamp loader, and a specialized polymerase. Replicases, especially bacterial replicases, are rapid and processive consistent with the requirement for them to synthesize a several megabase genome from a single origin in less than one hour.

In the prototypic Escherichia coli replication system, a key determinant of processive DNA synthesis is the interaction between the  processivity factor and pol III¹ (3, 4). The dimeric  subunit is a bracelet-shaped molecule that clamps around DNA permitting it to rapidly slide along duplex DNA without dissociating (5).  binds to the pol III  subunit through protein-protein contacts preventing the polymerase from dissociating from the template, ensuring high processivity. Efficient loading of the  subunit onto DNA requires ATP-dependent opening and closing of the clamp by the DnaX complex. The DnaX complex contains the essential DnaX,  and ' subunits plus two ancillary proteins,  and  (6-9). The dnaX gene encodes two proteins,  and , by programmed ribosomal frameshifting (10-15). Both  and the shorter  product share ATP-binding domain I, domain II, and domain III that is responsible for DnaX oligomerization, - binding, and binding of -' (16-19).  contains two unique domains.  domain IV forms a link with the DnaB helicase and domain V binds pol III (17, 20, 21). Pol III consists of , the catalytic polymerase subunit associated with the  3'  5' exonuclease, and  (22). Pol III gains its special replicative properties by its ability to associate with  and  through interactions enabled by sequences located in the carboxyl-terminal third of the  subunit (23-26).

The E. coli holoenzyme is held together by multiple protein-protein interactions among subunits with the stoichiometry ()₂₂'(₂)₂² (27). Pol III () forms a stable, isolable complex held together by - and - interactions (22, 28-30). The  and  subunits enhance the activity of one another (30-32). Within the DnaX complex, three DnaX subunits form a pentameric core with structurally related proteins,  and ' (19, 27, 33).  binds - and appears to reside adjacent to ' within the pentameric ring that makes up the core of the DnaX complex (16, 19, 33, 34). ' and  associate with one another free in solution and within DnaX complex through interaction of their COOH-terminal domain IIIs (19, 33, 35, 36).

DNA polymerase I-like polymerases have been well characterized from thermophilic eubacteria (37, 38). Although these enzymes provide the basis for PCR and related methods (39), they are limited in their processivity and repeatedly dissociate and reassociate with intermediate products during the amplification process. The core of a replicase-like polymerase from a thermophile was isolated by pursuing a minor activity that could be resolved chromatographically from Tth DNA polymerase I (40). The purified novel polymerase cross-reacted with a subset of monoclonal antibodies directed against E. coli Pol III and was found associated with two proteins that exhibited strong sequence similarity with the  and  subunits of E. coli holoenzyme, suggesting the existence of a thermophilic counterpart of the characterized mesophilic pol III holoenzymes.

That Tth pol III was found associated with both - and -sized dnaX gene products also suggested conservation of the principle of expression of at least two proteins from one dnaX mRNA (40). A dnaX-like mRNA was independently isolated by a PCR approach and expressed in E. coli, resulting in synthesis of - and -like proteins (41). It was recently discovered that Tth  and  are translated from distinct mRNAs that result from transcriptional slippage at a stretch of adenosine residues located at the same site as the E. coli translational frameshift site (42).

Encouraged by our detection of the core of the Tth replicase we pursued identification of the structural genes that encode the essential subunits of the Tth holoenzyme. In this report, we describe the expression and purification of the , DnaX (/), , ', and  subunits of Tth holoenzyme and their use to reconstitute a processive thermophilic replicase.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Materials-- Antibiotics were purchased from Fisher Scientific. d-Biotin was purchased from Sigma. Tryptone and yeast extract were purchased from Difco. Agarose and reagents for SDS-polyacrylamide gel electrophoresis were purchased from Bio-Rad. Ni-NTA-agarose was purchased from Qiagen. Dialysis membrane (MWCO: 50,000 and 10,000, 22-mm diameter) was purchased from Spectrum Laboratories.

Strains-- E. coli strains MGC1030 (mcrA, mcrB, (), inversion (rrnD-rrnE), lexA3.ompT) and AP1.L1 were transformed with plasmids for protein expression. The parent to the AP1.L1 bacterial strain was Novagen BLR bacterial strain [F-, ompT hsdSB(rB- mB-) gal dcm (srl-recA)306::Tn10]. AP1.L1 is a phage-resistant version of the BLR strain developed by Dr. Arthur Pritchard (University of Colorado Health Sciences Center, Denver, CO). DH5 (F 80lacZM15 (lacZYA-argF)U169 deoR recA1 endA1 hsdR17(r_K, mK⁺) phoA supE44 thi-1 gyrA96 relA1) was transformed with plasmids for amplification of vectors.

Buffers-- Buffer E is 50 mM Tris-HCl (pH 7.5), 40 mM KCl, 7 mM MgCl₂, 10% glycerol, and 7 mM -mercaptoethanol. Buffer W is 50 mM Tris-HCl (pH 7.5), 1 M KCl, 7 mM MgCl₂, 10% glycerol, 7 mM -mercaptoethanol, and 10 mM imidazole. TDB buffer is 50 mM HEPES (pH 7.5), 20% glycerol, 0.02% Nonidet P-40, and 0.2 mg/ml bovine serum albumin. HG.04 buffer is 20 mM HEPES (pH 7.5), 40 mM KCl, 1 mM MgCl₂, 0.1 mM EDTA, 7.0 mM -mercaptoethanol, and 10% glycerol. ICX buffer is 25 mM HEPES (pH 7.5), 100 mM NaCl, 8 mM MgOAc, and 5% glycerol. TBST buffer contained 200 mM Tris-HCl (pH 7.5), 1.5 M NaCl, 0.2% Tween 20 (v/v).

General Molecular Cloning Procedures-- All intermediate plasmids were transformed into DH5. Amp^R colonies were selected and the plasmids were screened for gain or loss of the appropriate restriction sites. The sequences of inserted DNA were confirmed by DNA sequencing. Ligations were performed in the presence of T4 DNA ligase and ATP. Restriction enzymes were used according to the manufacturer's instructions.

Construction of Starting Vectors-- The expression vectors utilized in this study were constructed from two starting vectors: pA1-NB-AvrII and pA1-NB-AgeI. To construct pA1-NB-AvrII, pDRK-N(M), a plasmid designed for expression of proteins with an amino-terminal tag was used as the starting plasmid (24). The amino-terminal tag is composed of a 30-amino acid peptide that is biotinylated in vivo and contains a hexahistidine sequence. The construct also contains a pBR322 origin of replication, a gene expressing the laqI^Q repressor protein, and a semisynthetic E. coli promoter (pA1) that is repressed by the lacI^Q repressor. Oligonucleotides 5'-CTAGGAAAAAAAAAGGTACCAAAAAAAAAGGCCGGCCACTAGTG-3' and 5'-TCGACACTAGTGGCCGGCCTTTTTTTTTGGTACCTTTTTTTTTC-3' were annealed to form a duplex with sticky ends (AvrII and SalI), and inserted into the AvrII/SalI-digested pDRK-N(M). The insertion of these annealed DNA fragments converted the polylinker following the fusion peptide from PstI-AvrII-DraIII-SalI to PstI-AvrII-spacer-KpnI-spacer-FseI-SpeISalI.

Construction of pA1-NB-AgeI was accomplished by modification of pA1-NB-AvrII whereby the polylinker in pA1-NB-AvrII that contained the restriction sites PstI-AvrII-KpnI-FseI-SpeI was replaced with a polylinker containing the restriction sites PstI-spacer-AgeI-BamHISacII-spacer-NcoI-SpeI. First, a BamHI site upstream of the polylinker was destroyed. This was accomplished by digesting pA1-NB-AvrII with BamHI and removing the sticky ends created by filling in with Klenow fragment. The blunted ends of the DNA were resealed. This plasmid was named pA1-NB-AvrII(BamHI) and was digested with PstI/SpeI restriction enzymes. This removed the polylinker containing the restriction sites PstI-AvrII-KpnI-FseI-SpeI. Oligonucleotides 5'-GAAAAAAAAAACCGGTGGATCCGCGGAAAAAAAACCATGGA-3' and 5'-CTAGTCCATGGTTTTTTTTCCGCGGATCCACCGGTTTTTTTTTTCTGCA-3' were annealed and formed a DNA duplex with the sticky ends corresponding to PstI and SpeI restriction sites. This annealed DNA duplex was inserted into the PstI/SpeI-digested pA1-NB-AvrII thereby forming pA1-NB-AgeI.

Construction of Expression Vectors-- Expression of Tth dnaE ( subunit) was accomplished by the insertion of the dnaE gene into pA1-NB-AvrII. The dnaE gene was amplified by PCR of the coding region from a BamHI fragment (4.9 kb) of Tth genomic DNA containing the dnaE gene cloned into a pBluescript IIKS+ phagemid vector. The forward primer 5'-GAATTCCTAGGCCGCAAACTCCGCTTC-3' adds an AvrII site to the 5' end of the dnaE gene so that the actual PCR product excludes the ATG start codon and begins at codon 2. The AvrII restriction site is adjacent to codon 2 and positions the 5' portion of the dnaE gene in the same reading frame with the NH₂-terminal fusion peptide coding sequences. The underlined sequence indicates the region of the primer complementary to the gene of interest here and below. The reverse primer (5'-GTGCTCGCGCAGGATCTCCCGGTCAATC-3') was designed so that it was downstream of a KpnI restriction site within Tth dnaE (the KpnI site is ~320 bases downstream of the start codon). The resulting PCR product was digested with PstI and KpnI and ligated into pAI-NB-AvrII that had been digested with the same two restriction enzymes forming pA1-NB-TE(5'). A 3454-bp KpnI/FseI fragment representing the 3'-end of the Tth dnaE gene was isolated from the 4.9-kb BamHI Tth genomic DNA fragment cloned into the pBluescript IIKS+ plasmid and inserted into the corresponding sites of pA1-NB-TE(5'). This plasmid (pA1-NB-TE) contained the entire Tth dnaE gene fused to a sequence encoding the amino-terminal tag.

Tth dnaX was inserted into an expression vector by the same procedure as used for dnaE. A PCR reaction was designed to amplify a fragment containing the 5' end of dnaX from a plasmid containing a 7.1-kb cloned fragment of Tth genomic DNA which contained the Tth dnaX gene. The forward primer (5'-AACTGCAGAGCGCCCTCTACCG-3') added a PstI site to the 5' end of the dnaX gene so that the actual PCR product excludes the ATG start codon and begins at codon 2. The PstI restriction site positions the 5' portion of the dnaX gene in the same reading frame with the N-terminal fusion peptide coding sequences. The reverse primer (5'-CGGTGGTGGCGAAGACGAAGAG-3') was designed so that it is downstream of the BamHI restriction site within Tth dnaX (the BamHI site is ~318 bases downstream of the start codon). This PCR product was digested with PstI and BamHI and ligated into pAI-NB-AgeI which had been digested with the same two restriction enzymes forming pA1-NB-TX(5'). The 3' region of the dnaX gene was removed from the 7.1-kb fragment by digestion with BamHI and SpeI and inserted into the precursor plasmid pA1-NB TX5' that had been digested with the same two restriction enzymes. This formed the plasmid pA1-NB-TX, which contained the full-length Tth dnaX gene fused to the sequence encoding the amino-terminal tag.

The Tth holA gene that encodes the  subunit was inserted into pA1-NB-AvrII to be expressed fused to the amino-terminal tag. The holA gene was amplified by PCR using Tth genomic DNA as a template. The forward/sense primer (5'-GAATTCTGCAGGTCATCGCCTTCACCG-3') added a PstI site to the 5' end of the gene so that the actual PCR product excludes the ATG start codon and begins at codon 2. The PstI site adjacent to codon 2 places the holA gene in the same reading frame with the NH₂-terminal fusion peptide. The reverse primer (5'-AGATCTGGTACCTCATCAACGGGCGAGGCGGAG-3') added an additional TGA (stop codon) to the end of the gene giving two stop codons in tandem and a KpnI restriction site was added in the non-complementary region of the primer for insertion into the vector. The PCR product was digested with PstI and KpnI restriction enzymes and inserted into pA1-NB-AvrII digested with the same enzymes. This formed the plasmid pA1-NB-TD, which contained the full-length Tth holA gene fused to the DNA sequence encoding the amino-terminal tag.

The Tth holB gene which encodes ' subunit was cloned into pA1-NB-AgeI to be expressed fused to the amino-terminal tag. The holB gene was amplified by PCR using Tth genomic DNA as a template. The forward/sense primer (5'-GAATTCTGCAGGCTCTACACCCGGCTCACCC-3') added a PstI site to the 5' end of the gene so that the actual PCR product excludes the ATG start codon and began at codon 2. The PstI site adjacent to codon 2 places the holA gene in the same reading frame with the NH₂-terminal fusion peptide. The reverse primer (5'-GGACACTAGTTCATCATGTCTCTAAGTCTAA-3') was complementary to the 3' end of the holB gene and added an additional TGA (stop codon). In addition, a SpeI restriction site was added in the non-complementary region of the primer for insertion into the vector. The PCR product containing the entire Tth holB gene was digested with PstI/SpeI restriction enzymes and inserted into pA1-NB-AgeI digested with the same enzymes. This formed the plasmid pA1-NB-TD', which contained the full-length Tth holB gene fused to the DNA sequence encoding the amino-terminal tag.

To express Tth  subunit, a PCR fragment containing the dnaN gene was amplified from genomic DNA. The forward primer (5'-AACTGCAGAACATAACGGTTCCCAAGAAACTCC-3') added a PstI restriction site to the 5' end adjacent to codon 2, so that when this fragment was inserted into pA1-NB-Age1, the dnaN gene was in the same reading frame with the sequence encoding the amino-terminal tag. The reverse primer 5'-GAGCAGCTAGCCTACTAGACCCTGAGGGGCACCAC-3') was designed so that an additional stop codon was added in the non-complementary region producing two stop codons in tandem. The non-complementary region of the reverse primer also contains an NheI restriction site. The PCR reaction resulted in a product that contained the entire Tth dnaN gene. The PCR product was digested with PstI and NheI and inserted into PstI/NheI-digested pA1-NB-AgeI forming pA1-NB-TN. The correct DNA sequence of all Tth genes cloned into expression vectors was confirmed by DNA sequencing.

Cell Growth and Preparation of Fraction I-- Cells were grown, induced, and harvested using a 250-liter fermentor as described (43), with the exception that ampicillin (100 µg/ml) and d-biotin (10 µM) was added at induction. Lysis of 400 g of cells and preparation of Fraction I for each of the subunits was performed as described by Cull and McHenry (44). The Fraction I supernatants contained the following levels of protein:  (25 g),  (22 g), DnaX (25 g), ' (20 g), and  (39 g).

Purification of Proteins-- As an initial purification step, many endogenous E. coli proteins can be removed by adding ammonium sulfate to concentrations that cause the protein of interest (and some endogenous proteins) to precipitate, while other proteins remain in solution. Therefore, the concentration of ammonium sulfate (expressed as percent saturation at 4 °C) in which >80% of Tth target proteins and minimal amounts of E. coli contaminating proteins precipitate was determined for each subunit by SDS-polyacrylamide electrophoresis. The Tth subunits were precipitated using ammonium sulfate concentration of 45% saturation for , 40% for /, , and , and 35% for ' and were collected by centrifugation (23,000 × g, 45 min, 0 °C).

Ammonium sulfate pellets were dissolved in 125 ml of buffer E using a Dounce homogenizer. Samples were clarified by centrifugation (16,000 × g) resulting in Fraction II. Fraction II contained the following levels of protein:  (3000 mg),  (2600 mg), DnaX (1100 mg), ' (620 mg), and  (4400 mg). Fraction II was added to 60 ml of a 50% slurry of Ni-NTA resin in buffer E and agitated for 1.5 h at 4 °C and then poured into a column (2.5 × 5 cm). The column was washed with 300 ml of buffer W at a flow rate of 0.5 ml/min. Tagged Tth proteins were eluted in 300 ml (10 column volumes) of buffer E containing a 10-200 mM imidazole-HCl (pH 7.5) gradient. The eluate was collected in 150 × 2-ml fractions and the protein concentration for each fraction was determined. Fractions making up the upper one-half of the protein concentration peak were pooled. The proteins were >90% pure based on densitometric analysis of Coomassie Blue-stained SDS-polyacrylamide gels. Following purification, the proteins were dialyzed into HG.04 buffer (Spectra/Por dialysis tubing, 11.5-mm diameter, 3000 MWCO), rapidly frozen in liquid nitrogen, and stored at 80 °C. The resulting Fraction IIIs contained the following levels of activity and protein:  (50 mg, 9.0 × 10⁷ units, 1.8 × 10⁶ units/mg),  (325 mg, 5.5 × 10⁸ units, 1.7 × 10⁶ units/mg), DnaX (85 mg, 2.4 × 10⁸ units, 2.8 × 10⁶ units/mg), ' (35 mg, 6.0 × 10⁸ units, 17.0 × 10⁶ units/mg), and  (325 mg, 45.0 × 10⁸ units, 14.0 × 10⁶ units/mg). One unit of activity is 1 pmol of total deoxyribonucleotide incorporated per min at 60 °C in the M13Gori reconstitution assay.

Protein Assays, SDS-PAGE, Biotin, and Western Blots-- Protein concentrations were determined by the method of Bradford (45) using bovine serum albumin as a standard. For SDS-PAGE, proteins were resolved by electrophoresis on 10% SDS-polyacrylamide gels (14 × 16 × 0.75 cm) for 2.5 h at 250 V, and visualized by staining with Coomassie Brilliant Blue. Proteins containing d-biotin were detected by biotin blots using phosphatase-conjugated streptavidin. Proteins resolved on an SDS-polyacrylamide gel were electroblotted onto a nitrocellulose membrane (Immobilon-P, Millipore) using a Hoefer^TM SemiPhor transfer apparatus at constant voltage (30 V) in 12 mM Tris base (pH 8.5), 96 mM glycine, 0.01% SDS (w/v), and 20% methanol (v/v) for 60 min at room temperature. Membranes were blocked in TBST buffer containing 5% non-fat dry milk (w/v) for 1 h at room temperature. Membranes were then rinsed with TBST and incubated in 2 µg/ml alkaline phosphatase-conjugated streptavidin (Pierce Chemical Co.) in TBST for 1 h at room temperature. Following extensive washing with TBST, membranes were developed using the 5-bromo-4-chloro-3-indolyl phosphate/nitro blue tetrazolium One Component System (Pierce Chemical Co.).

Western blot analysis of DnaX proteins was performed using the same method as biotin blots except that monoclonal antibodies elicited to NH₂-terminal-tagged DnaX (Tissue Culture/Monoclonal Antibody Facility, University of Colorado Health Sciences Center) were used instead of streptavidin. The antibodies selected for Western blotting experiments were specific for DnaX protein sequence and did not recognize hexahistidine and biotinylation sequences on other NH₂-terminal-tagged Tth subunits. Membranes were developed using goat anti-mouse antibodies conjugated to alkaline phosphatase (Bio-Rad).

Reconstitution Assays-- We employed a modified version of the M13Gori assay in which DNA synthesis on a long single-stranded circular template (M13Gori) primed by an RNA primer is used to measure the activity of DNA holoenzyme (46). G4 origin-specific primers were formed by the E. coli DnaG primase on E. coli single-stranded DNA-binding protein-coated M13Gori DNA. Each 19-µl assay contained 0.06 pmol of M13Gori DNA (about 500 pmol total nucleotide), 50 ng of DnaG primase, 1.6 µg of single-stranded DNA-binding protein, 10 mM magnesium acetate, 200 µM ATP, GTP, CTP, and UTP, 48 µM dATP, dGTP, and dCTP, and 18 µM [³H]dTTP (100 cpm/pmol). In some cases, M13Gori single-stranded DNA was primed by a 30-mer DNA oligonucleotide (5'-AGATTACTCTTGATGAAGGTCAGCCAGCCT-3') in reactions conducted in the absence of single-stranded DNA-binding protein or DnaG. The DNA-primed M13Gori was prepared by incubating 0.78 µM 30-mer DNA with 0.52 µM M13Gori in 10 mM HEPES buffer (pH 7.5) containing 100 mM NaCl, heating to boiling, and cooling slowly to room temperature. The RNA or DNA primed template mixtures were used in all M13Gori assays and are referred to as the primed template mixture. The Tth subunits , /, , ', and  were diluted to the desired concentration in TDB buffer in a total volume of 6 µl and combined with 19 µl of the primed template mixture to yield a 25-µl reaction. Following mixing, the reaction contents were incubated for 5 min at 60 °C. The reaction was terminated by placing the reaction tube on ice and adding 2 drops of 0.2 M sodium pyrophosphate and 0.5 ml of 10% trichloroacetic acid. The solution was filtered under vacuum through Whatman GF/C glass microfiber filters. The filters were then washed with 3 assay tube volumes (3 × 5 ml) of 1 M HCl, 0.2 M sodium pyrophosphate, and 1 assay tube volume (5 ml) of 95% EtOH and dried using a heat lamp. The amount of radiolabeled nucleotides incorporated was quantified by scintillation counting. One unit of activity is 1 pmol of total deoxyribonucleotide incorporated per min at 60 °C.

Protein-Protein Interactions Determined by Gel Filtration-- Gel filtration analysis of the interaction of DNA pol III holoenzyme subunits was preformed using a Sephacryl^TM S-200 (Amersham Bioscience) column (0.7 × 30 cm) equilibrated with HG.04 buffer. All proteins and protein mixtures were incubated for 5 min at 60 °C in 300 µl of HG.04 buffer prior to loading onto the column. To ensure that the runs were uniform, the sample was gently pipetted onto the resin, pumped into the resin, then 1 ml of buffer was gently added onto the top of the resin and pumped into the resin. The running buffer was then added to the top of the resin and elution was continued. The first three fractions (1 ml each) contained the void volume and all subsequent fractions contained 300 µl. Fractions were analyzed in 10% SDS-polyacrylamide gels stained with Coomassie Brilliant Blue and scanned using a Molecular Dynamics laser densitometer. The stoichiometry of a candidate complex formed on mixing , /, and ' subunits was determined from Coomassie Blue stain intensities corrected for the differences in molecular masses of the subunits (but not differences in subunit-specific dye binding) using 141 kDa for , 61.9 for , 53.6 kDa for , 36.2 kDa for , and 33.0 kDa for '.

Gel filtration analysis of  and ' alone was performed with 200 and 100 µg of protein, respectively (in 300 µl of HG.04 buffer). For  and ' interaction analysis, a mixture containing 150 µg of each protein was used. Gel Filtration of / alone was performed using 170 µg of protein. Analysis of interactions of / with  and ' was performed using 85 µg of , 70 µg of ', and 170 µg of /.  (75 µg) was gel filtered alone. The interaction between  and / was analyzed using 55 µg of  and 170 µg of /. The (/)' complex formation was analyzed using 40 µg of , 100 µg of /, and 55 µg of both  and '. DNA synthesis activity of eluted fractions was determined using holoenzyme reconstitution assays where all of the subunits were present in the reconstitution assay mixture except those that were loaded onto the gel filtration column. 4 µl of a mixture of all subunits except the protein(s) being analyzed were added to 19 µl of the primer/template mixture (see "Reconstitution Assays"). To this mixture, 2 µl taken from the gel filtration fractions were added and the sample was assayed as described above.

Gel filtration of protein standards (Amersham Bioscience) was carried out to determine elution positions of various molecular weight proteins. Each protein standard contained 500 µg of each protein in 300 µl of HG.04 buffer. Standards were chymotrypsinogen A (25 kDa), ovalbumin (43 kDa), bovine serum albumin (67 kDa), aldolase (158 kDa), catalase (232 kDa), and ferritin (440 kDa). Positions of the protein standards in the elution profiles were determined by protein assays.

Initiation Complex Formation-- Initiation complexes were formed by mixing 150 pmol of , 54 pmol of DnaX₄ (tetramer is the presumed molecular species (41)), 264 pmol of , 264 pmol of ', 167 pmol of ₂ (presumed dimer (47)), and 4 pmol of DNA-primed M13Gori single-stranded DNA in 240 µl of ICX buffer containing 10 mM dithiothreitol and 1 mM ATP (or dATP or ATPS). The mixture was incubated for 5 min at 30 or 60 °C and 200 µl was applied to an HR 10/30 Superose 6 column (Amersham Bioscience) equilibrated with ICX buffer. Fractions (0.5 ml) were collected, maintained on ice, and analyzed by SDS-PAGE. Activity assays for the eluted initiation complex were carried out by adding 3 µl of a mixture containing dATP, dGTP, dCTP, and [³H]dTTP (100 cpm/pmol) to 22 µl from each fraction (final concentration of all four deoxynucleotide triphosphates was 48 µM), incubating at 60 °C for 5 min and determining the amount of acid precipitable radioactivity.

Elongation Rate and Processivity Determinations-- The elongation rate of the holoenzyme was measured at various temperatures by determining the time required to fully extend the primer over the entire length of the M13Gori template (8623 nucleotides) starting with pre-formed initiation complexes. M13Gori pre-primed with DnaG primase in the absence of deoxyribonucleotides was used as a template. Initiation complexes were formed by mixing 16 µl of the RNA-primed M13Gori (500 pmol, total nucleotide) with 5 µl of Tth , DnaX₄, , ', and ₂ subunits (2, 1, 2, 2, and 2 pmol, respectively) in TDB buffer and incubating the mixture at 60 °C for 1 min. DNA synthesis was initiated by adding initiation complex (21 µl) to 4 µl of 0.3 mM dGTP, dCTP, dTTP, and [³²P]dATP (12 mCi/mmol) in TDB. To minimize the time required for thermal equilibration, both solutions were incubated at the desired reaction temperature for 1 min prior to mixing. The reaction was quenched by adding 5 µl of ice-cold 250 mM EDTA. Total DNA synthesis was determined by assaying 1 µl of the reaction mixture for acid precipitable radioactivity. DNA products were analyzed by 0.8% alkaline-agarose gel electrophoresis as described (48).

To determine the processivity of Tth holoenzyme, a mixture (22 µl) containing M13Gori (500 pmol, total nucleotide) and Tth , DnaX₄, , ', and ₂ subunits (2, 1, 2, 2, and 2 pmol, respectively) in TDB buffer were incubated at 60 °C for 2 min to allow formation of initiation complex. A mixture (5 µl) containing 1.2 mM dGTP, dCTP, dTTP, and [³²P]dATP (12 mCi/mmol) was combined with 1 µl of 5 mg/ml solution of activated calf thymus DNA (31) in TDB buffer and incubated at 60 °C for 1 min. The reaction was initiated by combining the two solutions followed by incubation at 60 °C for an additional 45 s. The reaction was quenched by adding 5 µl of ice-cold 250 mM EDTA. DNA products were analyzed by 0.8% alkaline-agarose gel electrophoresis as described (48). The positive control contained no activated calf thymus DNA and the negative control contained no M13Gori DNA. In a control to show that activated calf thymus DNA acts as an efficient competitor in initiation complex formation, the activated calf thymus DNA and M13Gori were incubated at the same time with Tth holoenzyme subunits.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Identification, Expression, and Purification of Tth , /, , ', and  Subunits-- Genes encoding the , /, , ', and  subunits of the Tth holoenzyme³ were either identified or confirmed by searching the Tth genome data base⁴ for homology with the corresponding genes from E. coli (dnaE, dnaX, holA, holB, and dnaN). The identification of the gene encoding  from Tth was aided by partial NH₂-terminal and internal peptide fragment sequencing of the pol III  subunit isolated previously from Tth extracts (40). The gene encoding / (dnaX) was previously identified by a similar reverse genetics approach (40, 41). Sequence comparison of genes for dnaE, dnaX, holA, holB, and dnaN between Thermus thermophilus and E. coli revealed 40, 31, 27, 27, and 26% identity, respectively. Each of the genes was amplified by PCR from Tth DNA, cloned into vectors containing an amino-terminal hexahistidine and biotinylation sequence, sequenced to confirm identity, overexpressed in E. coli, and purified to >90% purity as described under "Experimental Procedures." SDS-polyacrylamide gel analysis indicated that the purified subunits migrated with apparent molecular masses of ~135 kDa for , 66 and 58 kDa for  and , respectively, 42 kDa for , and 36 kDa for  and ' (Fig. 1). The minor band between  and  is probably a degradation product of , because it is stained along with the major bands with a monoclonal antibody specific for Tth DnaX proteins (data not shown). These molecular mass values are in agreement with the calculated values for the NH₂-terminal-tagged subunits of 141 kDa for , 61.9 for , 53.6 kDa for , 44.3 kDa for , 36.2 kDa for , and 33.0 kDa for '.

View larger version (87K): [in this window] [in a new window]   Fig. 1.   Holoenzyme subunits required for reconstitution of a minimal Tth replicative polymerase. Coomassie Blue-stained 10% SDS-polyacrylamide gel of Tth  (1.8 µg), / (3.2 µg),  (3.7 µg),  (3.7 µg), and ' (3.4 µg). Molecular mass standards are shown in the lane marked std.

Requirement of , /, , ', and  Subunits for Processive DNA Synthesis-- Our primary goal has been to assemble the minimal thermophilic replicase capable of rapid and processive synthesis of long stretches of DNA. Based on the E. coli precedent, we expected that Tth , DnaX (/), , ', and  would be required. With the availability of these proteins in NH₂-terminal-tagged forms, we used them in initial attempts at reconstitution. We employed a modified form of the standard assay used for the E. coli holoenzyme, DNA synthesis on a long single-stranded circular template primed by an RNA primer (49). To achieve efficient DNA synthesis, comparable with that observed with the E. coli holoenzyme, Tth holoenzyme , DnaX, , ', and  subunits were required (Table I). In addition, reactions needed to be conducted at 50 °C or higher in contrast to 30 °C used for E. coli holoenzyme. The amount of each subunit necessary to achieve maximal DNA synthesis under the assay conditions was determined by titrating each subunit into a reaction mixture containing an excess of the other four subunits (Fig. 2). Using these titration profiles as a guide, in subsequent experiments we have used  concentrations of 0.024 µM (0.6 pmol in 25 µl), DnaX₄ concentrations of 0.02 µM (0.5 pmol in 25 µl, using the anticipated tetrameric form of DnaX (41), and average molecular mass of 57.8 kDa for DnaX monomer),  and ' concentrations of 0.08 µM (2 pmol in 25 µl) and ₂ concentrations of 0.1 µM (2.5 pmol in 25 µl, using the anticipated dimeric form of  (47)).

View larger version (19K): [in this window] [in a new window]   Fig. 2.   Titration of Tth holoenzyme subunits into the M13Gori reconstitution assay. Except when varied as designated in the X axis, , DnaX4, , ', and 2 subunits were held at saturating concentrations containing 0.64, 0.5, 4.0, 2.4, and 2.6 pmol, respectively. DNA synthesis in the absence of the varied subunit (reported in Table I) was subtracted from all plotted values. A, titration of  subunit in the presence of DnaX4, , ', and 2. B, titration of DnaX4 in the presence of , , ' and 2. C, titration of  in the presence of , DnaX4, ' and 2. D, titration of ' in the presence of , DnaX4, , and 2. E, titration of 2 in the presence of , DnaX4, , and '.

Interaction of Tth Holoenzyme Subunits-- We examined protein-protein interactions among holoenzyme subunits by gel filtration. In view of homology between holoenzyme subunits of E. coli and Tth, we investigated whether some of the known interactions between E. coli subunits also occur in Tth.

The  and ' subunits of the Tth DnaX complex interact with each other as they do in E. coli (50). ' eluted with a peak at fraction 22 (Fig. 3A).  eluted with a peak at fraction 21⁵ (Fig. 3B). When  and ' were incubated together at approximately equal molar concentrations, the protein peak was shifted to a -' complex with a peak in fraction 19 (Fig. 3C).  and ' are similar in size and we were unable to resolve them on our polyacrylamide gels, including gradient gels (data not shown). Therefore, we also tested the DNA synthesis activities of eluting fractions in reconstitution assays where all of the subunits were present in the M13Gori reaction mixture except -' (data not shown). These activity data corroborate the protein elution profiles and further support an interaction between the two subunits.

View larger version (20K): [in this window] [in a new window]   Fig. 3.   Protein interactions between subunits of the Tth DnaX complex. Subunits were mixed to a final volume of 300 µl in HG.04 buffer, incubated at 60 °C for 5 min, and then analyzed by gel filtration as described under "Experimental Procedures." Proteins were visualized by Coomassie Blue staining of 10% SDS-polyacrylamide gels. A, ' (3.0 nmol). B,  (5.5 nmol). C, ' +  (4.5 nmol and 4.1 nmol, respectively). D, / (3.0 nmol, using the average molecular mass of 57.8 kDa). E, ' +  + / (2.1, 2.3, and 3.0 nmol, respectively). Fraction numbers are indicated at the top of the gels, the subunits are identified on the right side of the gel, and the elution positions of molecular mass markers are shown at the bottom. In panels C and E, ' and  co-migrate on SDS-polyacrylamide gels.

We next asked whether the Tth dnaX gene products,  and , interact with -'. Alone,  and  eluted together with a peak in fraction 17 (Fig. 3D). When / were incubated with  and ', the elution profile was shifted to a complex with a peak in fraction 16 (Fig. 3E), indicating a modest increase in the size of the DnaX proteins and a significant shift of -' over the size of the complex alone. This indicates -' binds to a DnaX oligomer, just like in E. coli (50). In similar experiments not shown here, ' alone interacted with / whereas an interaction of / with  was not detectable.

Just as in E. coli (23, 51), Tth  and / interact. Tth / and  when filtered alone, both eluted with a protein peak in fraction 17 (Figs. 3D and 4A). The -/ complex eluted with a peak at fraction 14 (Fig. 4B), indicating a significant size increase upon binding of  to . This interaction was observed previously, providing the basis for the original isolation of the DnaX proteins from Tth (40). When , /, , and ' were incubated together, all subunits co-eluted in a broad peak encompassing fractions 14-20 (Fig. 4C). Since the -/ complex (Fig. 4B) and the /-' complex (Fig. 3E) separately eluted with a similar profile, it is not possible from the elution profile alone to conclude that a stable complex involving , /, and ' is formed. However, quantitative analysis of the Coomassie Blue-stained gel showed that the composition of the complex in the lead fraction 14 (Fig. 4C), expressed as the Coomassie Blue stain intensity, normalized to that of  and corrected for molecular masses, but not subunit-specific variation in dye binding, is _{1.01.1 1.8}(')_2.1(total).  and ' subunits could not be quantified separately because of their identical electrophoretic mobilities and were considered as a single species. We also note an enrichment of  over  within bound DnaX protein in fraction 14, suggesting it is  and not  that binds , just like in E. coli. This observation is consistent with the formation of a complex consisting of ·DnaX₃·', although further work will be needed to definitively characterize the composition of this and other complexes within the holoenzyme.

View larger version (30K): [in this window] [in a new window]   Fig. 4.   Protein interactions between  and DnaX proteins and between  and the DnaX complex. Subunits were mixed to a final volume of 300 µl in HG.04 buffer, incubated at 60 °C for 5 min and then analyzed by gel filtration as described under "Experimental Procedures." Proteins were visualized by Coomassie Blue staining of 10% SDS-polyacrylamide gels. A,  (0.5 nmol). B,  + / (0.4 and 3.0 nmol, respectively). C,  + / + ' +  (0.3 nmol, 2.0, 1.5, and 1.5 nmol, respectively). Fraction numbers are indicated at the top of the gel, the subunits are identified on the right side of the gel, and the elution positions of molecular mass markers are shown at the bottom.

Initiation Complex Formation-- In E. coli, an initiation complex stable to gel filtration is formed upon mixing the holoenzyme with a primed template in an ATP-requiring reaction (52-54). To test whether a similar initiation complex can be formed with Tth DNA polymerase III subunits, we incubated , DnaX, , , and ' subunits with M13Gori annealed to a 30-mer DNA primer in the presence of ATP. The calculated T_m of the 30-mer DNA primer at the salt and oligonucleotide concentrations of our assay is ~65 °C (55, 56). Based on activity measurements of each of the eluting fractions, the initiation complex eluted in fractions 14-15 (Fig. 5). In contrast, incubation of subunits with primed template at 30 °C resulted in no initiation complex formation (Fig. 5). The presence of Tth , DnaX, , ', and  subunits in fractions 14-16 following incubation at 60 °C was confirmed by SDS-polyacrylamide gel electrophoresis of these fractions (data not shown). Following gel filtration, Tth replication-competent initiation complex placed on ice loses only about 10% of its DNA synthesis activity over a period of 1 h, providing ample time to complete activity assays. As in E. coli (52, 53, 57), initiation complex formation in Tth requires ATP, dATP, or ATPS (Table II).

View larger version (10K): [in this window] [in a new window]   Fig. 5.   Formation of isolable Tth holoenzyme initiation complex. Components of initiation complexes (Tth holoenzyme subunits and DNA-primed M13Gori) were mixed and incubated at 30 or 60 °C for 5 min and subjected to gel filtration as described under "Experimental Procedures." A, fractions from the HR 10/30 Superose 6 column were assayed for their ability to synthesize DNA following the addition of all four deoxyribonucleoside triphosphates (22 µl of each fraction was assayed). Activity indicates the picomole of nucleotides incorporated during extension of the primer.

Elongation Rate-- To determine the rate at which the minimal Tth holoenzyme synthesizes DNA at different temperatures, we initiated DNA synthesis on pre-formed initiation complex by addition of all four deoxyribonucleoside triphosphates. The reactions were quenched at various times by addition of EDTA. Analysis of the reaction products on alkaline-agarose gels permitted determination of the time required to fully extend the primer over the entire length of the M13Gori template (Fig. 6). At 45 °C, no full-length product (RFII) is formed in 1 min, although 3-5-kb fragments were visible at 45-60 s. At 60 and 72 °C, RFII is formed within 30 and 25 s, respectively. Thus, the minimal Tth DNA holoenzyme exhibits elongation rates of <140 nucleotides/s at 45 °C, 290 nucleotides/s at 60 °C, and 350 nucleotides/s at 72 °C.

View larger version (34K): [in this window] [in a new window]   Fig. 6.   Elongation rate of the minimal Tth holoenzyme. The rate of polymerization was measured by determining the time required for polymerization of the full-length M13Gori template (8623 nucleotides). Reactions were initiated by adding deoxyribonucleoside triphosphates to pre-formed initiation complexes on RNA-primed M13Gori DNA and incubating for the indicated times and then stopping the reaction by adding EDTA. DNA products were analyzed by alkaline-agarose gel electrophoresis. Reactions were carried out at A, 45 °C; B, 60 °C; and C, 72 °C. DNA standards are shown on the left side of the gels, and the full-length product is indicated on the right side of the gels.

Processivity-- To test the processivity of the minimal Tth holoenzyme, we examined the ability of initiation complexes to synthesize DNA in the presence of primed template competitor added as a trap to prevent dissociated holoenzyme from re-associating with the M13Gori template. As a competitor template, we used activated calf thymus DNA in which single-stranded DNA regions were generated with limited nuclease treatment of sheared double-stranded DNA (3, 58). Analysis of the reaction products on alkaline-agarose gels shows that the minimal Tth holoenzyme indeed recognizes both activated calf thymus DNA and primed M13Gori as templates (Fig. 7, lanes 1 and 2). Given the difficulty of accurately estimating the molar concentration of functional primed template sites on heterogeneous species such as activated calf thymus DNA, we empirically determined that 5 µg was sufficient to completely inhibit DNA synthesis on the M13Gori template when added together during the initiation complex formation reaction (Fig. 7, lane 3). Thus, if the Tth holoenzyme dissociated from the M13Gori template, re-formation of initiation complexes on M13Gori would be prevented. When competitor template was added to the reaction mixture after the initiation complex had formed on the M13Gori template, synthesis of full-length M13Gori RFII products occurred, indicating a processivity of at least 8.6 kb (Fig. 7, lane 4). As expected, the excess subunits not associated with M13Gori were available for the synthesis of smaller (<0.5 kb) fragments resulting from their association with the competitor template (Fig. 7, lane 4).

View larger version (30K): [in this window] [in a new window]   Fig. 7.   Processivity of the minimal reconstituted Tth holoenzyme. To determine processivity, initiation complexes were pre-formed on RNA-primed M13Gori DNA using Tth holoenzyme subunits, followed by the addition of competitor DNA (activated calf thymus DNA) and deoxynucleoside triphosphates to initiate DNA synthesis reaction as described under "Experimental Procedures." The reaction was allowed to proceed for 45 s and then stopped by addition of EDTA. DNA products were analyzed by alkaline-agarose gel electrophoresis. Lane 1, the reaction contains only activated calf thymus DNA as a template. Lane 2, the reaction contains only M13Gori DNA as a template. Lane 3, reaction contains both M13Gori DNA and calf thymus DNA in the initiation complex formation reaction prior to the addition of dNTPs. Lane 4, reaction contains M13Gori as a template during the initiation complex formation step and the competitor DNA was added with the deoxyribonucleoside triphosphates.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

In all organisms studied to date, processive and rapid synthesis of DNA by replicative polymerases requires three distinct components: polymerase core, sliding clamp processivity factor, and a clamp-loading complex. Recent identification of the  pol III catalytic subunit from a thermophilic bacterium, Tth, along with the associated DnaX proteins,  and , suggested that the basic design of the replicative apparatus from mesophilic and thermophilic bacteria may indeed be similar. Here, we report the identification of three additional subunits of the DNA holoenzyme:  and ', components of the clamp-loading complex, and the  processivity factor. To obtain sufficient protein for functional analyses, we cloned, overexpressed in E. coli and purified to homogeneity , DnaX, , ', and  subunits from Tth as fusion proteins containing NH₂-terminal hexahistidine and biotinylation sequences. Testing of these proteins for their ability to support efficient DNA synthesis with long circular templates revealed that , DnaX, , ', and  subunits represent the minimal essential components of the Tth holoenzyme. Thus, the minimal holoenzyme from this thermophilic organism contains the same essential subunits as the holoenzyme from mesophilic bacteria such as Gram-negative E. coli or Gram-positive Streptococcus pyogenes (59-61).

Similarity between thermophilic and mesophilic replicases extends to binding interactions between the essential holoenzyme subunits. Like in E. coli and S. pyogenes, Tth DnaX complex proteins, , ', and /, form an assembly stable to gel filtration. The DnaX complex is held together primarily by -' and '-/ binding interactions. As shown previously (40) and confirmed here, Tth  and / subunits bind to each other as they do in E. coli and S. pyogenes. Co-migration of , /, , and ' subunits and analysis of Coomassie Blue stain intensities of the lead fraction are consistent with the formation of a complex with apparent stoichiometry of ^<