In Vivo Assembly of Overproduced DNA Polymerase III OVERPRODUCTION, PURIFICATION, and CHARACTERIZATION of the ␣, ␣-⑀, and ␣-⑀-␪ SUBUNITS*

Home , DnaE, DnaQ, HolE

THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 271, No. 34, Issue of August 23, pp. 20681–20689, 1996 © 1996 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U.S.A. In Vivo Assembly of Overproduced DNA Polymerase III OVERPRODUCTION, PURIFICATION, AND CHARACTERIZATION OF THE ␣, ␣-⑀, AND ␣-⑀-␪ SUBUNITS*

(Received for publication, January 22, 1996)

Deok Ryong Kim and Charles S. McHenry From the Department of Biochemistry, Biophysics and Genetics, University of Colorado Health Sciences Center, Denver, Colorado 80262

The genes for the polymerase core (␣⑀␪) of the DNA 1984). The function of the ␪ subunit in DNA replication is polymerase III holoenzyme map to widely separated loci unclear. on the Escherichia coli chromosome. To enable efficient Pol III is dimerized via the interaction between the ␶ and ␣ overproduction and in vivo assembly of DNA polymerase subunits, resulting in the formation of a dimeric polymerase III core, artificial operons containing the three struc- that enables the coordinated synthesis of the leading and lag- tural genes, dnaE, dnaQ, and holE, were placed in an ging strands (McHenry, 1982; Studwell-Vaughan and expression plasmid. The proteins ␣, ␣⑀ and ␣⑀␪ were O’Donnell, 1991). The C-terminal region of ␣ binds to a ␶ dimer overexpressed and assembled in E. coli and purified to with a high affinity (KD ϭ 70 pM) (Kim and McHenry, 1996). Pol homogeneity. The three purified polymerases had a sim- III itself is distributive but becomes a processive and rapid ilar specific activity of about 6.0 106 units/mg in a -؋ polymerase on a primed template with other accessory sub gap-filling assay. Kinetics studies showed that neither ⑀ units (Fay et al., 1981). The DnaX complex (␶ ␦␦Ј␹␺ or ␥ ␦␦Ј␹␺) nor ␪ influenced the K of ␣ for deoxynucleotide triphos- 4 4 m loads the ␤ sliding clamp onto the primed template by coupling phate and only slightly decreased the K of ␣ for DNA, m ATP hydrolysis (Dallmann and McHenry, 1995; Onrust et al., although ⑀ was absolutely required for maximal DNA synthesis. The rate of DNA synthesis by ␣-reconstituted 1995). The ␤ sliding clamp provides pol III with high proces- holoenzyme using ␶ complex was about 5-fold less than sivity by tethering it to the template (LaDuca et al., 1986; that of ␣⑀ or ␣⑀␪-reconstituted holoenzyme as deter- Stukenberg et al., 1991). mined by a gel analysis. The processivity of ␣-reconsti- Each subunit of pol III works cooperatively and stimulates tuted holoenzyme was very similar to that of ␣⑀␪-recon- the activity of other subunits. For instance, the ␣ subunit can stituted holoenzyme when ␶ complex was used as a stimulate the exonuclease activity of the ⑀ subunit 10–80-fold clamp loader. by increasing the affinity of ⑀ for the 3Ј-hydroxyl terminus (Maki and Kornberg, 1987). The ⑀ exonuclease activity is also slightly stimulated by the ␪ subunit (Studwell-Vaughan and The DNA polymerase III core (pol III)1 of the DNA polymer- O’Donnell, 1993). Additionally, ⑀ induces a 3-fold increase in ase III holoenzyme is a heterotrimer, composed of ␣, ⑀, and ␪ the polymerase activity of the ␣ subunit (Maki and Kornberg, subunits of 129,900, 27,500, and 8,700 daltons, respectively 1987). Thus, three pol III subunits are functionally cooperative. (McHenry and Crow, 1979). The subunits of pol III are ex- In fact, most DNA polymerases contain separate domains for pressed from genes located at separate sites on the Escherichia the polymerase and exonuclease activities in a single polypep- coli chromosome; ␣ is encoded by dnaE (Gefter et al., 1971; tide, suggesting that the two activities are interactive (Blanco Welch and McHenry, 1982), ⑀ by dnaQ (Horiuchi et al., 1981; et al., 1991). Sheuermann et al., 1983), and ␪ by holE (Studwell-Vaughan In this present study, we constructed artificial operons that and O’Donnell, 1993; Carter et al., 1993). The three subunits overexpress either ␣, ␣⑀,or␣⑀␪ complexes assembled in vivo form a very stable complex at a ratio of 1:1:1. ␣ binds ⑀ which and purified them to homogeneity without the denaturation- binds ␪, but a direct ␣-␪ contact has not been observed renaturation step required for the purification of ⑀ due to its (Studwell-Vaughan and O’Donnell, 1993). insolubility. The three purified polymerases were character- Individual subunits of pol III have been overexpressed, pu- ized, and their function and kinetics in DNA replication were rified, and characterized. The ␣ subunit contains catalytic po- compared. lymerase activity and synthesizes DNA at a rate of approximately 10 nucleotides/s (Maki and Kornberg, 1987; Maki and EXPERIMENTAL PROCEDURES Kornberg, 1985). The ⑀ subunit (the dnaQ product) contains 3Ј Strains—E. coli strains HB101 (FϪ, recA13, ara 14, proA2, lacY1, 3 5Ј exonuclease activity for the proofreading function of DNA galK2, rpsl20, xyl5), JM109 (recA1, endA1, gyrA96, thi, hsdR17, replication (Scheuermann and Echols, 1984); thus, dnaQ supE44, relA1, lambda(Ϫ), lac-proAB(DEL)), and MC1061 (araD139, ara LEU769, galU, galK, lac 174(DEL), hsdR2, mcrB1, rpsL) were used (mutD) has a strong mutator phenotype (DiFrancesco et al., for plasmid propagation and protein expression. Cell Growth and Induction—E. coli strains containing overexpressing plasmids were grown in 200 liters of F medium (1.5% yeast extract, * This work was supported by Research Grant NP940 from the Amer- 1% peptone, 1.2% K PO , 0.02% KPO and 1% glucose) plus 50 ␮g/ml ican Cancer Society and facilities support from the Lucille P. Markey 2 4 4 ampicillin at 37 °C. Cells were induced by isopropyl-␤-D-thiogalactoside Charitable Trust. The costs of publication of this article were defrayed (1 mM final concentration) at A 1.0. After 4.5 h (2.5 h for ␣ in part by the payment of page charges. This article must therefore be 600 ϭ hereby marked “advertisement” in accordance with 18 U.S.C. Section expression), cells were harvested by Sharples AS-16 continuous flow 1734 solely to indicate this fact. centrifugation, resuspended (1:1, w/v) in 50 mM Tris-HCl (pH 7.5) and 1 The abbreviations used are: pol III, DNA polymerase III core; ho- 10% sucrose, and immediately frozen in liquid N2. loenzyme, DNA polymerase III holoenzyme; SSB, E. coli single- Chromatographic Supports—Bio-Rex 70 resins were purchased from stranded DNA binding protein; BSA, bovine serum albumin; DTT, Bio-Rad. Sephacryl-300 HR resins were from Pharmacia. Toyopearl dithiothreitol; dNTP, deoxynucleotide triphosphate; bp, base pair(s); Fr, phenyl-650 M resins were obtained from Tosohaas. fraction; kb, kilobase pair(s). Proteins—The ␶ complex (␶4␦␦Ј␹␺) and ␥ complex (␥4␦␦Ј␹␺) were 20681 20682 In Vivo Assembly of Overproduced DNA Pol III

FIG.2. Overexpression of ␣, ⑀, and ␪ from pHN4. Total cell proteins before and after induction of E. coli strain HB101 (pHN4) were prepared as described (Kim and McHenry, 1996), and 20 ␮l of each sample was loaded on a 10–20% gradient SDS-polyacrylamide gel. Proteins were separated at a constant 65 V overnight. The gel was stained with Coomassie Brilliant Blue overnight and destained in a solution of 10% methanol and 10% acetic acid. Lane 1, protein markers; lane 2, uninduced total cell proteins; lane 3, induced total cell proteins.

yielding a matched base pair (C) and three mismatched base pairs (G, A, T) to the template (G). Construction of the Artificial Operon of Pol III Core—The dnaQ gene of pNS121 (Scheuermann et al., 1983) was amplified using two primers, the 5Ј-primer contains a 22-nucleotide sequence complementary to FIG.1.Construction of plasmids to overexpress ␣⑀ and pol III. dnaQ, a Shine-Dalgarno site (AGGAGG), and a BglII restriction en- Plasmids were constructed as described under “Experimental Proce- zyme site; the 3Ј-primer has a 16-nucleotide sequence complementary dures.” The backbone of the vectors was derived from pJC1, an HIV to dnaQ and three cloning sites (PstI, DraIII, and HindIII). The polym- nucleocapsid (NC)-overexpressing plasmid (You and McHenry, 1993), which has a tac promoter, a replication origin (Ori), lacIq gene, two erase chain reaction was conducted as described (Saiki et al., 1988). The transcriptional terminators (T1 and T2), (Brosius et al., 1981), and the polymerase chain reaction products of dnaQ were digested with BglII structural gene for ␤-lactamase (ampR). S/D indicates a Shine-Dalgarno and HindIII and ligated to the large fragment of pJC1 (You and site. McHenry, 1993) digested at the same restriction sites (Fig. 1). The resulting plasmid was named pHN1, an ⑀ overexpressing plasmid. The plasmid pHN3, an ␣⑀ overexpressing plasmid, was generated by liga- reconstituted and purified as described (Dallmann and McHenry, tion of the PstI-DraIII fragment of pHN1 and the same restriction 1995). Sequenase version 2.0 and Dnase I were obtained from United enzyme-digested fragment of pOPPA50–4a2, an ␣ overexpressing plas- States Biochemical Corp. Terminal deoxynucleotidyltransferase was mid (Tomasiewicz, 1991). Finally, the holE gene, encoding the ␪ sub- from International Biotechnologies, Inc. Rabbit anti-␤ IgG was pre- unit, from pHN100 (Carter et al., 1993) was inserted into pHN3 at the pared as described (Johanson and McHenry, 1980). PstI site between dnaQ and dnaE to generate plasmid pHN4, which Buffers—Buffer I is 50 mM imidazole (pH 6.5), 1 mM EDTA, 5 mM overexpressed pol III core (␣⑀␪) complex. Each gene of pol III in this DTT, and 20% glycerol; buffer A is 20 mM potassium phosphate (pH plasmid contains its own Shine-Dalgarno sequence in front of a start 6.5), 1 mM EDTA, 5 mM DTT, and 25% glycerol; 30% A.S. buffer is 50 mM codon (ATG) with an AT-rich, 9-nucleotide spacer. Tris-HCl (pH 7.5), 1 mM EDTA, 5 mM DTT, and 175 g of ammonium Determination of Molar Extinction Coefficients—The extinction coef- sulfate added to 1 liter (30% saturation at 4 °C); 15% A.S. buffer is 50 ficients of the ␣ subunit, ␣⑀ complex, and pol III core (␣⑀␪) complex at Ϫ1 Ϫ1 mM Tris-HCl (pH 7.5), 1 mM EDTA, 5 mM DTT, and 84 g of ammonium ⑀280 were 99,920, 112,370, and 123,000 liters mol cm , respectively, sulfate added to 1 liter (15% saturation at 4 °C); buffer B is 50 mM as determined by the method of Edelhoch (1967). Proteins were dia- HEPES (pH 7.5), 20% glycerol (v/v), 10 mM DTT, 0.1 mM EDTA, 100 mM lyzed against buffer E overnight, and their extinction coefficients were potassium glutamate, and 5 mM magnesium acetate; buffer C is 50 mM determined in buffer E, in the presence or absence of 6 M guanidine Tris-HCl (pH 7.5), 1 mM EDTA, 5 mM DTT, and 30% glycerol; buffer E hydrochloride. Spectra of the three polymerases were measured on a is 50 mM Tris-HCl (pH 7.5), 5% glycerol, and 1 mM EDTA; enzyme Hewlett-Packard 8450Z diode array spectrophotometer between 240 dilution buffer (EDB) is 50 mM HEPES (pH 7.5), 0.02% Nonidet P-40, and 340 nm. Extinction coefficients of denatured proteins were calcu- 200 ␮g/ml BSA, 20% glycerol (v/v), 10 mM DTT, and 100 mM potassium lated from the number of tryptophan and tyrosine residues in each glutamate; buffer TE is 50 mM Tris-HCl (pH 8.0) and 1 mM EDTA. protein (Edelhoch, 1967) and corrected by the ratio of the absorbances Oligonucleotides—Two primers used to amplify the dnaQ gene were of the native proteins to the absorbances of the proteins in 6 M guani- synthesized on a Biosearch 8600 DNA synthesizer. The 3Ј-primer is dine hydrochloride. 5Ј-GGG GGA GAT CTA GGA GGT TTA AAA TAA TGA GCA CTG CAA Gap-filling Polymerase Assay—This assay was performed by a mod- TTA CAC GCC-3Ј (48-mer), and the 5Ј-primer is 5Ј-CCC CCC CAA GCT ification of the method of McHenry and Crow (1979) and used to detect TCA CCC AGT GGC GGC CGC TGC AGT TAT GCT CGC CAG A-3Ј enzyme activity during protein purification. The reaction was initiated (50-mer). These two oligonucleotides were purified by DE52 column by the addition of enzyme to a 25-␮l solution containing four dNTPs 3 chromatography as described (Hagerman, 1985). Another four oligonu- (100 cpm of H/pmol dNTPs), 10 mM MgCl2, and 5 ␮g of activated calf cleotides (5Ј-AGG CGC ATA GGC TGG CTG ACC TT(N)-3Ј) comple- thymus DNA in 50 mM HEPES (pH 7.5), 10 mM DTT, 200 mg/ml BSA, mentary to the M13Gori DNA (nucleotides 993-1016) were purified by 0.02% Nonidet P-40, and 20% glycerol and incubated at 30 °C for 5 min. gel electrophoresis (Hagerman, 1985). They have the same DNA se- One unit is defined as the amount of enzyme catalyzing the incorpora- quence except for the nucleotide at the 3Ј end (either C, G, A, or T), tion of 1 pmol of dNTPs per min at 30 °C. In Vivo Assembly of Overproduced DNA Pol III 20683

Preparation of Activated Calf Thymus DNA—Calf thymus DNA (100 primase (87,500 units), four NTPs (each 0.5 mM), and magnesium mg) was dissolved in 50 ml of 20 mM KCl with stirring overnight at 4 °C. acetate (10 mM) in buffer B was incubated at 30 °C for 10 min and The dissolved DNA was treated with 0.4 ␮g of DNase I (1 mg/ml in 1 mM applied to a Bio-Gel A-5m column (1 ϫ 25 cm) equilibrated with buffer

CaCl2 and 50% glycerol) per 10 mg DNA in a reaction mix containing 50 B at 4 °C. Primed M13Gori DNA was eluted with 40 ml of buffer B (flow mM Tris-HCl (pH 7.5), 10 mM MgCl2, and 50 ␮g/ml BSA at 37 °C for 20 rate 125 ␮l/min) and detected by assaying with holoenzyme subunits min. DNase I was inactivated at 65 °C for 10 min, followed by phenol: and dNTPs (Dallmann et al., 1995). The peak fractions (total 1.8 ml) chloroform extraction, and an additional chloroform extraction. The that incorporate more than 40 pmol of nucleotides in a 25-␮l assay DNA was precipitated by addition of 2 volumes of ethanol and 1/10 using 1 ␮l of each fraction were combined (83% yield based on replica- volume of 3 M sodium acetate (pH 5.2) (Ϫ20 °C overnight). DNA samples tion assay). were centrifuged at 10,000 ϫ g for 35 min and rinsed with 70% ethanol Analysis of DNA Elongation Rate—The DNA synthesis rate of the twice. The DNA pellet was initially dissolved in 30 ml of buffer TE and holoenzyme-like activity reconstituted with three polymerases (␣, ␣⑀, dialyzed against 2 liters of buffer TE for 4 h with one buffer change. The ␣⑀␪) and ␶ complex and ␤ was determined by a modification of the DNA concentration was determined spectrophotometrically at 260 nm method of Fay et al. (1981). A reaction mix (50 ␮l) containing RNA-

(17 A260 ϭ 1 mg/ml) and adjusted to 5 mg/ml final concentration by primed, SSB-coated M13Gori DNA template (480 fmol as a circle), ␶ dilution with buffer TE. Determination of Steady-state Kinetic Parameters—Kinetic parameters for dNTPs and activated DNA of the three polymerases (␣, ␣⑀, ␣⑀␪) TABLE I were determined in the gap-filling polymerase assay described above in Purification of pol III the presence of 67 fmol of each polymerase at 30 °C for various times. Fraction Total protein Total units Specific activity Yield dNTPs were titrated in the presence of 605 ␮M activated calf thymus DNA (as nucleotide), or activated DNA was titrated in the presence of mg ϫ10Ϫ8 ϫ10Ϫ3 units/mg % 60 ␮M dNTPs to determine an initial velocity at each substrate concen- I. Lysate 15,600 2.9 19 100 tration. The Km and Vmax were calculated from Lineweaver-Burk plots II. Ammonium 945 1.9 201 65 or by nonlinear least squares curve fitting (Kaleidagraph 3.0.1 soft- sulfate ware) to the equation v ϭ V [S]/(K ϩ [S]), where v is initial III. BioRex-70 98 1.3 1,360 45 0 max m 0 a a velocity at a given substrate concentration, and S is substrate concen- IV. S-300 43 1.0 2,500 34 a tration. The kinetic parameters (Km and Vmax) determined by these two For purposes of convenient comparison, the protein concentrations different methods agree to within ϳ5%. reported were determined by the method of Bradford (1976). The actual Preparation of Primed M13Gori DNA—RNA-primed and SSB-coated yield of pol III from S-300 was 18 mg as determined using the extinction M13Gori DNA was prepared (Fay et al., 1981). A reaction mix (1 ml) coefficient. Thus, the true specific activity of pure pol III is 5.9 ϫ 106 containing M13Gori DNA (1 ␮mol as nucleotide), SSB (1.6 mg), DnaG units/mg.

FIG.3.Chromatographic purification of pol III. A, Bio-Rex 70 column profile. The first 20 fractions are flow-through, and the following 10 fractions result from the wash step. The gradient started at fraction 30. A total of 140 fractions (17 ml each) were collected. Polymerase activity (f) and protein (●) were assayed as described under “Experimental Procedures.” Conductivity (ࡗ) is reported relative to NaCl standards. B, SDS-polyacrylamide gel electrophoresis of Bio-Rex 70 fractions (70 ␮l) and Fr II (50 ␮g, load lane). Fractions were analyzed by 10–20% gradient SDS-polyacrylamide gel electrophoresis. The gel was stained with Coomassie Blue to visualize proteins. The protein content of applied peak fractions was about 20 ␮g. Numbers above the gel are column fractions. C, Sephacryl S-300 column profile. A total of 80 fractions (1.375 ml each) were collected. Polymerase activity (f) and protein (●) were assayed as described under “Experimental Procedures.” D, SDS-polyacrylamide gel electrophoresis of individual Sephacryl S-300 fractions (50 ␮l) and Fr III (20 ␮g, load lane). Fractions were analyzed on a 10–20% gradient SDS-polyacrylamide gel stained with Coomassie Blue. Fractions 48–52 contain about 175 ␮g total protein. Numbers above gel are column fractions. 20684 In Vivo Assembly of Overproduced DNA Pol III complex (1.64 pmol as ␶4␦␦Ј␹␺), ␤ subunit (2.26 pmol as dimer), and 200 Purification of Pol III (␣⑀␪) ␮M ATP in enzyme dilution buffer was incubated at 30 °C for 5 min with 1 pmol of ␣, ␣⑀,or␣⑀␪ to allow formation of an initiation complex. The E. coli HB101 containing pHN4 was used to express pol III. reaction was then placed in a 22 °C water bath for 5 min to permit The ␣, ⑀, and ␪ subunits were expressed at ϳ1, 13, and 6% of thermal equilibration and started by the addition of 6 ␮l of dNTPs (each total proteins, respectively, as determined by densitometric 0.8 mM) at 22 °C. All reaction mixtures were initially made in a batch, and a 50-␮l sample was removed at the indicated time and quenched in TABLE II 200 ␮l of ethanol and 5 ␮lof4MNaCl in a dry ice/ethanol bath. DNA Purification of the ␣⑀ complex samples were precipitated at Ϫ80 °C overnight, spun at 15,000 ϫ g for

45 min at 4 °C, and resuspended in 26 ␮lofH2O. The DNA was digested Fraction Total protein Total units Specific activity Yield with BbvIina30-␮l volume at 37 °C for 1 h, loaded on an 8% native mg ϫ10Ϫ8 ϫ10Ϫ3 units/mg % polyacrylamide gel (1.5 ϫ 25 ϫ 15 cm), and run at 100 V overnight. The gel was stained with ethidium bromide (25 ␮g/ml) solution for 30 min I. Lysate 40,390 18 44 100 II. Ammonium sulfate 2,418 9 370 50 and destained ina1mMMgSO solution for 10 min to visualize DNA 4 III. Bio-Rex 70 367 6.4 1,731 35 fragments with a UV illuminator. IV. S-300 122a 3.6 2,860b 20 Other Methods—Protein concentration during protein purification was determined by the method of Bradford (1976). Protein concentra- a The yield determined using the extinction coefficient was 55 mg. tion of all purified proteins was determined using the extinction coef- b Thus, the true specific activity of ␣⑀ is 6.3 ϫ 106 units/mg. ficient. SDS-polyacrylamide gel electrophoresis was performed by a modification of the method of Laemmli (1970). TABLE III Purification of the ␣ subunit RESULTS Fraction Total protein Total units Specific activity Yield We constructed artificial operons to overexpress pol III subunit complexes assembled in vivo. The dnaE, dnaQ, and holE mg ϫ10Ϫ8 ϫ10Ϫ3 units/mg % genes were inserted into a vector with a tac promoter to I. Lysate 23,904 6.4 26 100 produce the ␣⑀␪ complex, whereas dnaE and dnaQ were II. Ammonium sulfate 8,700 8.5 97 132 III. BioRex-70 217 3.7 1,687 58 inserted to obtain a plasmid expressing the ␣⑀ complex (Fig. 1). IV. Phenyl-650M 66 1.5 2,216 23 Three polymerases (␣, ␣⑀ or ␣⑀␪) were overexpressed and pu- V. S-300 32 0.8 2,546 12 rified to 99% homogeneity from overexpressing E. coli strains. VI. Heparin 17a 0.5 2,941b 8 All steps of preparation were performed at 0–4 °C unless noted a The yield determined using the extinction coefficient was 9.5 mg. otherwise. b Thus, the true specific activity of ␣ is 6.6 ϫ 106 units/mg.

FIG.4.Chromatographic purification of ␣. A, Toyopearl phenyl-650M column profile. A total of 80 fractions (7.5 ml each) were collected. Polymerase activity (f) and protein (●) were assayed as described under “Experimental Procedures.” Conductivity (ࡗ) is given as the saturation percentage of ammonium sulfate at 4 °C. B, SDS-polyacrylamide gel electrophoresis of Toyopearl phenyl-650M fractions (35 ␮l) and Fr III (40 ␮g, load lane). Fractions were analyzed by 10% SDS-polyacrylamide gel electrophoresis. The gel was stained with Coomassie Blue. Applied peak fractions in this gel contain about 30 ␮g of protein. Numbers above the gel are column fractions. C, heparin-Sepharose column profile. A total of 80 fractions (3 ml each) were collected. Polymerase activity (f) and protein (●) were assayed as described under “Experimental Procedures.” Conductivity (ࡗ) is given as mM NaCl. D, SDS-polyacrylamide gel electrophoresis of heparin-Sepharose fractions (30 ␮l) and Fr V (40 ␮g, load lane). Fractions were analyzed by 10% SDS-polyacrylamide gel electrophoresis and the gel was stained with Coomassie Blue. Applied peak fractions contain about 37 ␮g. Numbers above the gel are column fractions. In Vivo Assembly of Overproduced DNA Pol III 20685 scan (Fig. 2, lane 3). The presence of free ␣ or ␣⑀ would not be 490 ml, Table I) was precipitated by the addition of an equal expected since ␣ was the limiting subunit. The extra ⑀ and ␪ volume of saturated ammonium sulfate solution. The purity of subunits formed a soluble, separable complex (data not shown) pol III after this column was more than 80% based on densito- through a direct interaction; excess ⑀ itself was insoluble. metric scan of an SDS gel (Fig. 3B). All three subunits (␣, ⑀, and Cell Lysis and Ammonium Sulfate Precipitation—Frozen ␪) eluted in a constant ratio across the peak (Fig. 3B). cells (180 g) were thawed and lysed (2 mg of lysozyme per g of Sephacryl S-300 HR Gel Filtration Chromatography—The cells) to prepare cell lysates (Fr I) as described (Cull and protein pellet obtained from ammonium sulfate precipitation of McHenry, 1995). Initially, 0.226 g of ammonium sulfate (40% Fr III was dissolved in buffer A ϩ 100 mM KCl. The resulting saturation at 0 °C) for each ml of cell lysate was added, followed protein solution (2 ml) was loaded onto a 110-ml, equilibrated by two sequential backwashes with 0.200 and 0.170 g of am- Sephacryl S-300 column (1.5 ϫ 62.3 cm), and 80 fractions were monium sulfate added to each ml as described (Cull and collected at a flow rate of 0.1-column volume/h. Pol III eluted at McHenry, 1995). The final ammonium sulfate precipitate was fractions 44–54 (Fig. 3C), resulting in Fr IV (15.1 ml, Table I). resuspended in buffer I 25 mM NaCl to yield Fr II (44 ml, ϩ Individual fractions (50 ␮l) were analyzed by SDS-polyacryl- Table I). amide gel electrophoresis (Fig. 3D). The gel was overloaded Bio-Rex 70 Cation Exchange Chromatography—Fr II was (about 175 ␮g at fractions 46–52), so that trace contaminants dialyzed overnight against buffer I ϩ 25 mM NaCl, diluted to a could be detected. However, when 10–20 ␮g of this pool was conductivity equivalent to buffer I ϩ 25 mM NaCl by the addi- loaded and resolved on a gel, no contamination was detected tion of buffer I, and applied to a 190-ml pre-equilibrated Bio- (Fig. 5). The overall yield of pol III in the purification was 34% Rex 70 column (5.75 ϫ 7.32 cm). The column was washed with (Table I). 3-column volumes of buffer I ϩ 25 mM NaCl, and proteins were eluted with a 10-column volume gradient of buffer I ϩ 25 mM NaCl to buffer I ϩ 300 mM NaCl (flow rate ϭ 1-column volume Purification of ␣⑀ Complex per h). Pol III started eluting at a conductivity of buffer I ϩ 125 Cell Lysis and Ammonium Sulfate Precipitation—E. coli mM NaCl (Fig. 3A). The pool of peak fractions (72–98) (Fr III, HB101 containing pHN3 produced the ␣⑀ complex at a level of 2% ␣ and 20% ⑀ of total proteins (data not shown). Cell lysis (286 g of cells) and ammonium sulfate precipitation were performed under the same conditions as described for pol III purification except that the 0.170 backwash was skipped because it resulted in solubilization of a significant amount of ␣⑀. ␣ and ⑀ formed a tight complex at a 1:1 ratio, and the excess insoluble ⑀ subunits sedimented with cell debris (data not shown). Bio-Rex 70 and Sephacryl S-300 Chromatography—A 280-ml Bio-Rex 70 column (2.7 ϫ 49.5 cm) was prepared, and proteins were loaded onto the equilibrated column as described for pol III. The column was washed with buffer I ϩ 75 mM NaCl, and proteins were eluted with a 10-column volume buffer I ϩ 75 to buffer I ϩ 300 mM NaCl gradient. The ␣⑀ complex started to elute at a conductivity of buffer I ϩ 100 mM NaCl, but some proportion of ␣⑀ complex eluted in early fractions (data not shown). Like pol III, the ␣⑀ complex was purified to near- homogeneity from the cation exchange chromatographic step. The pool (Fr III, 965 ml, Table II) of fractions 37 to 78 was concentrated by ammonium sulfate precipitation. A 160-ml Sephacryl S-300 column (1.5 ϫ 90 cm) was used for the final purification. The protein pellet obtained from ammonium sul-

FIG.5.Homogeneity of three purified polymerase forms. Puri- fate precipitation of Fr III was dissolved in buffer A (2.0 ml) fied ␣, ␣⑀, and ␣⑀␪ (10 ␮g each) were loaded on a 12.5% SDS-polyacryl- and loaded onto the column. The activity eluted at fractions amide gel. After separation of proteins at a 65 V overnight, the gel was 42–49, resulting in Fr IV (16 ml). The overall yield of ␣⑀ stained with Coomassie Brilliant Blue overnight, destained in a solution of 10% methanol and 10% acetic acid, and subjected to a densito- complex was 20% (Table II), and the overall purification of the metric scan (Molecular Dynamics). Lane 1, purified pol III core; lane 2, ␣⑀ complex was very similar to pol III in terms of column profile purified ␣⑀ complex; lane 3, purified ␣ subunit. and purity.

TABLE IV Kinetics of three pol III polymerase forms: ␣, ␣⑀, and ␣⑀␪ Data were derived from a Lineweaver-Burk plot and nonlinear curve fitting program and represent the average of three separate experiments. DNA concentration is given as nucleotide; ϳ150 ␮M nucleotide is equivalent to 1 ␮M 3Ј-hydroxyl end of activated DNA.

Substrate pol III Km kcat kcat/Km

Ϫ1 Ϫ1 Ϫ1 ␮M s s (mol/liter) dNTP ␣ 22 Ϯ 1 12.1 Ϯ 1.2 5.5 ϫ 105 ␣⑀ 21 Ϯ 2 19.8 Ϯ 2.5 9.4 ϫ 105 ␣⑀␪ 24 Ϯ 1 26.9 Ϯ 2.7 1.1 ϫ 106

DNA ␣ 100 Ϯ 5 11.9 Ϯ 1.0 1.2 ϫ 105 ␣⑀ 85 Ϯ 3 21.0 Ϯ 3.3 2.5 ϫ 105 ␣⑀␪ 55 Ϯ 5 21.6 Ϯ 3.5 3.9 ϫ 105 20686 In Vivo Assembly of Overproduced DNA Pol III

V, 13.5 ml) of this column was nearly pure, but some contamination was detected when more than 75 ␮g of protein was loaded on a gel (data not shown). Heparin-Sepharose Chromatography—We conducted another chromatographic step to remove all trace contaminants. Fr V was dialyzed against buffer I ϩ 10 mM NaCl and loaded onto a 19-ml heparin-Sepharose column (0.75 ϫ 44 cm) equilibrated with buffer I ϩ 10 mM NaCl. The column was washed with 2-column volumes of buffer I ϩ10 mM NaCl and eluted with 10-column volumes of gradient (buffer I ϩ 10 3 300 mM NaCl) at a flow rate of 1-column volume per h. Fractions 58–62 (Fr VI, 15 ml, Table III) were combined. The ␣ subunit eluted at a conductivity of buffer I ϩ 150 mM NaCl (Fig. 4,Cand D). SDS-Polyacrylamide Gel Electrophoresis of Purified Polym- erase Forms—The three purified pol III polymerases (each 10 ␮g) were analyzed by SDS-gel electrophoresis (Fig. 5). Based on FIG.6.Determination of steady-state kinetic constants for ␣, densitometric scan, all pol III polymerases were purified to 99% ␣⑀, and ␣⑀␪. The initial velocity (v0)of␣(E), ␣⑀ (Ⅺ), and ␣⑀␪ (Ç) determined by gap-filling assay was plotted versus the concentration (as homogeneity. Stoichiometries of ␣⑀ and ␣⑀␪ were determined by nucleotide) of activated DNA (S). This plot was generated by a nonlin- laser densitometry of complexes subjected to SDS-polyacryl- ear curve fitting program as described under “Experimental Proce- amide gel electrophoresis, stained with Coomassie Blue, and dures.” The estimated Km and Vmax values from this plot are 87 and 2 ␮M/min for ␣, 108 and 3.3 ␮M/min for ␣⑀, and 76 and 3.5 ␮M/min for ␣⑀␪, corrected for molecular weight. The stoichiometry of the three respectively. pol III subunits was 1.0:1.1:0.9 (␣:⑀:␪). In the ␣⑀ preparation, the ratio was 1.0:1.1 (␣:⑀). Purification of the ␣ Subunit Steady-state Kinetics of Polymerases in Gap-filling As- say—To examine and compare the kinetic properties of the Cell Lysis and Ammonium Sulfate Precipitation—E. coli three polymerase forms, the K and k values for dNTPs or MC1061 containing pOPPA50–4a2 (Tomasiewicz, 1991) was m cat activated DNA substrates (Table IV) were calculated by an used to express the ␣ subunit to ϳ5% of total cell proteins (data iterative fit to the equation v ϭ V [S]/(K ϩ[S]) as described not shown). Cell lysis (239 g of cells) was performed as de- 0 max m under “Experimental Procedures” (Fig. 6) and from Lin- scribed for pol III purification. The ␣ subunit alone was much eweaver-Burk plots. The k was calculated from the equation more soluble in ammonium sulfate than the other two polym- cat k ϭ V /E , where E is total enzyme concentration. The K erase forms; it did not precipitate significantly under condi- cat max t t m of all three polymerase forms for dNTPs was in the range of tions described for pol III or ␣⑀ complex purification. Initially, Ϫ1 21–24 ␮M. The k value (ϳ12 s )of␣was 2-fold less than 0.164 g of ammonium sulfate (30% saturation at 0 °C) was cat that of the other two polymerase forms (Table IV). The K of all added to each ml of Fr I lysate. Insoluble protein was removed m three polymerase forms for activated calf thymus DNA was by centrifugation (23,300 ϫ g at 0 °C for 1 h). The supernatant, very similar, although K for pol III was slightly lower. Be- containing ␣, was adjusted to a final concentration 0.291 g/ml m ammonium sulfate (50% saturation at 0 °C). Precipitates (Fr cause activated calf thymus DNA is such a heterogeneous II) were collected by centrifugation as described above. template, Km values were defined in terms of total nucleotide Bio-Rex 70 Chromatography—A 450-ml Bio-Rex 70 column concentration, permitting a relative comparison. The kcat/Km of (5.75 ϫ 17.3 cm) was used in an identical manner as described pol III for both dNTP and DNA was about 2-fold higher than for pol III. The ␣ subunit eluted at the same conductivity as the that of ␣ alone, indicating that ⑀ and ␪ made a modest contri- ␣⑀ and ␣⑀␪ complexes (data not shown). Fractions 78–93 (Fr bution to the gap-filling polymerase activity of ␣. The 3Ј-OH III, 445 ml, Table III) were pooled and precipitated by the concentration of activated DNA was estimated from the aver- addition of an equal volume of saturated ammonium sulfate age size of DNA fragments determined by denaturing gel elec- solution. trophoresis and dNTPs incorporated between gaps, ϳ150 ␮M as Toyopearl Phenyl-650M Hydrophobic Chromatography—Al- nucleotide was equivalent to 1 ␮M of 3Ј-OH. though ␣ was purified to near-homogeneity from the Bio-Rex 70 Primer Extension from Mismatched 3Ј Ends by Holoenzymes column, it was less pure than pol III or ␣⑀. Thus, additional Reconstituted with ␣, ␣⑀ and ␣⑀␪—The ␣ subunit itself does not chromatographic steps were required. Ammonium sulfate-pre- have proofreading activity to remove misincorporated bases cipitated Fr III was dissolved in 30% A.S. buffer and loaded during replication. We asked whether ␣-reconstituted holoen- onto a pre-equilibrated hydrophobic Toyopearl phenyl-650M zyme extends nucleotides further from the mispaired 3Ј end or, column (60 ml, 1.5 ϫ 36 cm). The column was washed with instead, pauses in DNA elongation when an incorrect nucleo- 1-column volume of 30% A.S. buffer followed by 2-column vol- tide is incorporated. Using four oligonucleotides annealed to umes of 15% A.S. buffer. Proteins were eluted with a 10-column M13Gori DNA (Fig. 7A) and saturating levels of polymerases, volume 15–0% ammonium sulfate gradient at a flow rate of we carried out assays for various times to examine utilization 0.84-column volume per h. The ␣ subunit eluted at 8–6% of mispaired primer termini and found that ␣ elongated DNA saturating ammonium sulfate (Fig. 4, A and B). Fractions from the 3Ј ends of a paired C-G and mispaired T-G but could 42–60 were combined, resulting in Fr IV (150 ml, Table III). not overcome G-G and A-G mismatches (Fig. 7, B-E). Perhaps The pool of fractions was precipitated by the addition of an T was elongated by forming a transient base pair with G. Both equal volume of saturated ammonium sulfate. ␣⑀ and pol III utilized each 3Ј end at almost the same rate Sephacryl S-300 Chromatography—Gel filtration chroma- because of their 3Ј 3 5Ј proofreading activity which removes tography to remove aggregated proteins as well as to purify ␣ mispaired nucleotides (Fig. 7, B-E). further was carried out as for ␣⑀ and ␣⑀␪. Ammonium sulfate- DNA Elongation Rates of Holoenzymes Reconstituted with precipitated Fr IV was dissolved in 2 ml of buffer A ϩ 100 mM the Three Polymerase Forms—Earlier studies concluded that ⑀ KCl and loaded onto a Sephacryl S-300 gel filtration column is required for ␣ to achieve rapid and highly processive DNA (160 ml, 1.5 ϫ 90 cm). ␣ eluted at fractions 46–52. The pool (Fr synthesis (Studwell-Vaughan and O’Donnell, 1990). The avail- In Vivo Assembly of Overproduced DNA Pol III 20687

FIG.7.Extension of mispaired primers termini by ␣, ␣⑀, and ␣⑀␪. A, a schematic presentation of M13Gori DNA primed by oligonucleotides with different 3Ј-termini. The primers (24-mer) containing various nucleotides at the 3Ј end were annealed to the template (993–1016 region of M13Gori single-stranded DNA) at a 2:1 ratio, and primed templates were purified from a Bio-Gel A-5m column as described (Fay et al., 1981). B, primer extension from the G-C matched 3Ј end. The mix (25 ␮l) containing primed templates (58 fmol as a circle), ␶ complex (116 fmol as ␶4␦␦Ј␹␺), ␤ subunit (285 fmol as a dimer), SSB (1.6 ␮g), either ␣ (f), ␣⑀ (●), or ␣⑀␪ (å) (each 100 fmol), and dNTPs (100 cpm 3H/pmol of dNTPs) in enzyme dilution buffer was incubated at 30 °C for various times (0.25–5 min). Subsequent steps of this assay were performed as described (McHenry and Crow, 1979). C, primer extension from the G-G mismatched 3Ј end. D, primer extension from the G-A mismatched 3Ј end. E, primer extension from the G-T mismatched 3Ј end. ability of three highly purified polymerase forms allowed us to and ␣⑀␪-reconstituted holoenzymes, and fragment h appeared study the effect of ⑀ or ␪ on DNA synthesis rates by reconsti- at 5 min for ␣-reconstituted holoenzyme and at 1 min for ␣⑀- tuted holoenzyme. Initiation complexes were formed using sat- and ␣⑀␪-reconstituted holoenzymes (Fig. 8B). Based on the urating levels of either ␣, ␣⑀,or␣⑀␪ as described under “Ex- production of these fragments, the elongation rates of reconsti- perimental Procedures.” Reactions were started by the addition tuted holoenzymes at 22 °C were calculated as 28 Ϯ 2, 126 Ϯ 4, of dNTPs at 22 °C (Fig. 8A). The DNA fragments generated and 126 Ϯ 4 nucleotides/s for ␣-, ␣⑀,- and ␣⑀␪-reconstituted from BbvI digestion are a (900 bp), b (175 bp), c (690 bp), d holoenzymes, respectively. The elongation rate of ␣-reconsti- (1739 bp), e (611 bp), f (1154 bp), g (410 bp), h (2000 bp), i (688 tuted holoenzyme was approximately 5-fold slower than that of bp), and j (256 bp) in the order produced. Fragment d appeared ␣⑀- and ␣⑀␪-reconstituted holoenzymes. at 2 min for ␣-reconstituted holoenzyme and at 30 s for both ␣⑀- Processivity of the ␣-Reconstituted Holoenzyme Using ␶ Com- 20688 In Vivo Assembly of Overproduced DNA Pol III

FIG.9. Processivity of ␣-reconstituted holoenzyme. Initiation complexes were formed by addition of either ␶ (lanes 1-5)or␥(lanes

6-10) complex (1.2 pmol as ␶4␦␦Ј␹␺ or ␥4␦␦Ј␹␺), ␤ (565 fmol as dimer), and either 1 pmol of ␣ (lanes 2, 4, 6, and 8)or␣⑀␪ (lanes 3, 5, 7, and 9) to primed M13Gori DNA (500 fmol as circle) as prepared under “Exper- imental Procedures” in 50 ␮l of enzyme dilution buffer containing 10 mM magnesium acetate and 200 ␮M ATP by an incubation at 30 °C for 5 min. Elongation reaction was initiated by the addition of each 48 ␮M of dATP, dCTP, and dGTP and 18 ␮M of [3H]dTTP (100 cpm/pmol) at 30 °C for 5 min. To block reinitiation during polymerase cycling so that processivity could be measured, 20 ␮g of anti-␤ IgG was added to tubes (lanes 4, 5, 8, and 9) prior to dNTP addition. Anti-␤ IgG was omitted from lanes 2, 3, 6, and 7. Subsequent steps were performed as described in Fig. 8. Lanes 1 and 10 are controls in the absence of polymerases. As a control to demonstrate that anti-␤ IgG under these reaction condi- FIG.8.Determination of the elongation rate of ␣, ␣⑀, and ␣⑀␪ in tions can rapidly inhibit cycling polymerase, tubes containing all com- reconstituted holoenzyme. A, scheme of elongation assay. The initi- ponents except polymerase (either ␣ or ␣⑀␪), DnaX complex, and ␤ ation complex on RNA-primed, SSB-coated M13Gori DNA was formed either in the presence or absence of anti-␤ IgG (20 ␮g) were incubated at 30 °C for 5 min. The reaction was started at 22 °C by the addition of at 30 °C for 5 min, immediately added to a mix of polymerase, DnaX dNTPs. The numbers on replicated double-stranded DNA indicate the complex, and ␤ and incubated for an additional 15 s and 1 min for ␣⑀␪- distance of BbvI cleavage sites from the 3Ј-OH primer terminus. Frag- and ␣-reconstituted holoenzymes, respectively. Radioactivity of ments generated from the restriction enzyme digestion are given as a [3H]dTMP-incorporated DNA was measured as described (McHenry (900 bp), b (175 bp), c (690 bp), d (1, 739 bp), e (611 bp), f (1, 154 bp), g and Crow, 1979), and anti-␤ IgG inhibited 99% of holoenzyme activity. (410 bp), h (2000 bp), i (688 bp), and j (256 bp). Details are described Letters on the right side of the gel indicate the DNA fragments (a–j) under “Experimental Procedures.” The arrow in the initiation complex generated by restriction enzyme digestion as described in Fig. 8. indicates the direction of DNA synthesis. H, DNA polymerase III holoenzyme reconstituted using ␶ complex, ␤, and either ␣, ␣⑀,or␣⑀␪. B, restriction gel analysis. The ␣-reconstituted holoenzyme (lanes 1–6), complex, and ␤ followed by incubations for sufficient time for ␣⑀-reconstituted holoenzyme (lanes 7–13), and ␣⑀␪-reconstituted ho- replication of a full circle of M13Gori DNA (15 s for pol III and loenzyme (lanes 14–20) were incubated at 22 °C for various times: 0 1 min for ␣). The intensity of each fragment in the same lane (lane 1),1(lane 2),2(lane 3),5(lane 4), 10 (lane 5), and 20 min (lane 6) was determined by a densitometric scan. Comparison of ratios for ␣-reconstituted holoenzyme; 0 (lanes 7 and 14), 10 (lanes 8 and 15), 20 (lanes 9 and 16), 30 s (lanes 10 and 17), 1 min (lanes 11 and 18), 2 min of early fragments (a, b,ord) and late fragments (f or g)inthe (lanes 12 and 19), and 5 min (lanes 13 and 20) for ␣⑀ and ␣⑀␪-reconsti- presence or absence of anti-␤ IgG showed that once holoenzyme tuted holoenzymes. The DNA fragments (a–j) were separated by 8% is reconstituted with either ␣ or ␣⑀␪ using ␶ complex to initiate native polyacrylamide gel electrophoresis and stained with ethidium DNA synthesis, it processively replicates M13Gori DNA (until bromide. fragment g). However, the ratio of early fragments and one particular late fragment (h)of␣-reconstituted holoenzyme us- plex—In previous studies, the ␣-reconstituted holoenzyme ing ␶ complex was 3–4-fold higher in the absence of anti-␤ IgG showed a processivity of 1–3 kb when ␥ complex was used as a (lane 2) than in its presence (lane 4), indicating that only clamp loader, whereas both ␣⑀- and ␣⑀␪-reconstituted holoen- 25–30% of initiation complexes completed the synthesis of frag- zymes were fully processive for a full cycle of replication on an ment h in the presence of anti-␤ IgG during the time course of M13 DNA template (Studwell-Vaughan and O’Donnell, 1990). the reaction. Perhaps the movement of holoenzyme is impeded We determined the processivity of the ␣-reconstituted holoen- by a strong hairpin in the bacteriophage M13 replication origin zyme using anti-␤ IgG to prevent reinitiation onto the DNA (van Wezenbeek et al., 1980) located in fragment h. template once an initiation complex was formed (Fay et al., 1981). Holoenzymes reconstituted using either the ␣ subunit or DISCUSSION pol III in the absence of anti-␤ IgG synthesized a full circle of Three subunits of pol III are tightly associated and can be M13Gori DNA regardless of which DnaX complex (␶ or ␥ com- isolated from wild-type cell lysates (McHenry and Crow, 1979). plex) was used (Fig. 9, lanes 2, 3, 6, and 7). When anti-␤ IgG Since a cell contains only 10–20 molecules of pol III (Wu et al., was added after initiation complex formation, the ␣- and ␣⑀␪- 1984), purification of pol III from wild-type E. coli required a reconstituted holoenzyme using ␶ complex was fully processive 30,000-fold purification and enormous quantities of cells (compare lanes 4 and 5 of Fig. 9), while the ␣-reconstituted (McHenry and Crow, 1979). We have constructed an artificial holoenzyme using ␥ complex showed a processivity of 3.6 kb operon containing combinations of the three pol III genes based on the last fragment (d) synthesized from this reaction (dnaE, dnaQ, and holE) which permitted overexpression of (Fig. 9, lane 8). Anti-␤ IgG inhibited 99% of holoenzyme activity three pol III subunits from a single promoter and the assembly if it was added to a reaction mix before pol III (or ␣), DnaX of complexes in vivo. From 180 g of pol III overexpressing cells, In Vivo Assembly of Overproduced DNA Pol III 20689

18 mg of pol III was purified to 99% homogeneity in two action is required for replicase-primosome coupling (Kim et al., chromatographic steps. It is interesting that ⑀, normally insol- 1996b) but that does not preclude additional primosome-ho- uble when overproduced alone (Sheuermann and Echols, 1984), loenzyme interactions. Finally, ␪ might somehow mediate the forms a defined 1:1 complex with ␣ when coexpressed in vivo, conformational change in pol III that is probably necessary in resulting in a soluble complex. Excess ⑀ is found in inclusion switching between the polymerase and exonuclease activity of bodies. pol III during replication. Firm conclusions about the function Gap-filling polymerase assays were used to determine the of ␪ await further genetic, structural, and functional studies. contribution of ⑀ or ␪ to the kinetic properties of ␣. All three REFERENCES polymerase forms, ␣, ␣⑀, and ␣⑀␪, had identical affinities for Blanco, L., Bernard, A., Blasco, M. A., and Salas, M. (1991) Gene (Amst.) 100, dNTP, suggesting that ⑀ and ␪ are not involved in dNTP bind- 27–38 Bradford, M. M. (1976) Anal. Biochem. 72, 248–254 ing by ␣. The Km of ␣ for DNA was about 2-fold higher than ␣⑀␪. Brosius, J., Dull, T. J., Sleeter, D. D., and Noller, H. F. (1981) J. Mol. Biol. 148, The kcat of ␣ was 2-fold lower than that of ␣⑀ and ␣⑀␪, indicating 107–129 a modest contribution from ⑀, perhaps by stabilizing a more Carter, J. R., Franden, M. A., Asbersold, R., Kim, D. R., and McHenry, C. S. (1993) active conformation of ␣; ␪ made no detectable contributions, Nucleic Acids Res. 21, 3281–3286 Cull, M. A., and McHenry, C. S. (1995) Methods Enzymol. 262, 22–35 consistent with other biochemical (Studwell-Vaughan and Dallmann, G. H., and McHenry, C. S. (1995) J. Biol. Chem. 270, 29563–29569 O’Donnell, 1990) and genetic (Slater et al., 1994) studies. Dallmann, G. H., Thimmig, R., and McHenry, C. S. (1995) J. Biol. Chem. 270, 29555–29562 To complete the synthesis of 4.4-megabase pairs of the E. coli DiFrancesco, R., Bhatnagar, S. K., Brown, A., and Bessman, M. J. (1984) J. Biol. genome within 40 min, holoenzyme must synthesize DNA at a Chem. 259, 5567–5573 rate of about 1 kb per s. In vitro replication assays have shown Edelhoch, H. (1967) Biochemistry 7, 1948–1954 Fay, P. J., Johanson, K. O., McHenry, C. S., and Bambara, R. A. (1981) J. Biol. that naturally purified as well as reconstituted holoenzyme Chem. 256, 976–983 synthesize DNA at a rate of about 500 nucleotides/s at 30 °C Gefter, M. L., Hirotoa, T., Kornberg, T., Weshsler, J. A., and Barnoux, C. (1971) Proc. Natl. Acad. Sci. U. S. A. 68, 3150–3153 (Johanson and McHenry, 1981). Our gel analysis of restriction Hagerman, P. J. (1985) Biochemistry 24, 7033–7037 fragments indicated a DNA synthesis rate of holoenzyme re- Horiuchi, T., Maki, H., Maruyama, M., and Sekiguchi, M. (1981) Proc. Natl. Acad. constituted with pol III, ␤, and ␶ complex of about 130 nucleo- Sci. U. S. A. 78, 3770–3774 Johanson, O. K., and McHenry, C. S. (1980) J. Biol. Chem. 255, 10984–10990 tides per s at 22 °C. The ␣⑀-reconstituted holoenzyme synthe- Johanson, O. K., and McHenry, C. S. (1981) in Structure and DNA-Protein Inter- sized DNA at the same rate as ␣⑀␪-reconstituted holoenzyme, actions of Replication Origins (Ray, D., ed) pp. 425–436, Academic Press, New York but the ␣-reconstituted holoenzyme elongated DNA at a rate Kim, D. R., and McHenry, C. S. (1996) J. Biol. Chem. 271, 20690–20698 ϳ5-fold slower. Therefore, rapid DNA synthesis by holoenzyme Kim, S., Dallmann, G. H., McHenry, C. S., and Marians, K. J. (1996a) J. Biol. is dependent on ⑀. By contrast, ␪ appears to have no influence Chem. 271, 4315–4318 Kim, S., Dallmann, G. H., McHenry, C. S., and Marians, K. J. (1996b) Cell 84, on DNA elongation at 22 °C. Although the ␣-reconstituted ho- 643–650 loenzyme using ␶ complex showed a 5-fold slower elongation LaDuca, R. J., Crute, J. J., McHenry, C. S., and Bambara, R. A. (1986) J. Biol. Chem. 261, 7550–7557 rate than holoenzyme reconstituted using ␣⑀ or ␣⑀␪, its proces- Laemmli, U. K. (1970) Nature 227, 680–685 sivity was very similar to that of the ␣⑀␪-reconstituted holoen- Maki, H., and Kornberg, A. (1985) J. Biol. Chem. 260, 12987–12992 zyme. When ␥ complex replaced ␶ complex, the processivity of Maki, H., and Kornberg, A. (1987) Proc. Natl. Acad. Sci. U. S. A. 84, 4389–4392 McHenry, C. S. (1982) J. Biol. Chem. 257, 2657–2663 ␣-reconstituted holoenzyme decreased (Fig. 9). ␶ protects ␤ McHenry C. S., and Crow, W. (1979) J. Biol. Chem. 254, 1748–1753 from removal by ␥ complex (Kim et al. 1996a). Presumably, in Onrust, R., Finkelstein, J., Naktinis, V., Turner, J., Fang, L., and O’Donnell, M. (1995) J. Biol. Chem. 270, 13348–13357 the absence of ␶, the slower moving ␣ provides more time for ␤ Saiki, R. K., Gelfand, D. H., Stoffel, S., Scharf, S. J., Higuchi, R., Horn, G. T., removal, resulting in a lower apparent processivity. Mullis, K. B., and Erlich, H. A. (1988) Science 239, 487–491 The function of ␪ in DNA replication is not clear. Analysis of Scheuermann, R. H., and Echols, H. (1984) Proc. Natl. Acad. Sci. U. S. A. 81, 7747–7751 a null mutation in holE has shown that ␪ is dispensable for E. Scheuermann, R. H., Tam, S., Burgers, P. M., Lu, C., and Echols, H. (1983) Proc. coli growth (Slater et al., 1994). Our kinetic and functional Natl. Acad. Sci. U. S. A. 80, 7085–7089 Slater, S. C., Lifsics, M. R., O’Donnell, M., and Maurer, R. (1994) J. Bacteriol. 176, studies of three polymerases did not show any significant effect 815–821 of ␪ in DNA replication, although it slightly increased ␣’s bind- Studwell-Vaughan, P., and O’Donnell, M. (1990) J. Biol. Chem. 265, 1171–1178 ing to DNA substrates. The ␪ subunit has also been shown to Studwell-Vaughan, P., and O’Donnell, M. (1991) J. Biol. Chem. 266, 19833–19841 Studwell-Vaughan, P., and O’Donnell, M. (1993) J. Biol. Chem. 268, 11785–11791 slightly stimulate the 3Ј 3 5Ј proofreading exonuclease activity Stukenberg, P. T., Studwell-Vaughan, P., and O’Donnell, M. (1991) J. Biol Chem. of the ⑀ subunit at a mismatched base pair (Studwell-Vaughan 266, 11328–11334 Tomasiewicz, H. (1991) Ph.D. thesis, University of Colorado Health Sciences and O’Donnell, 1993). Another possible role of ␪ is communica- Center tion with other replication proteins such as primase or helicase van Wezenbeek, P. M., Hulsebos, T. J., and Schoenmarkers, J. G. (1980) Gene at the replication fork. One of two holoenzymes at the replica- (Amst.) 11, 129–148 Welch, M. M., and McHenry, C. S. (1982) J. Biol. Chem. 152, 351–356 tion fork continuously recycles onto newly synthesized primer Wu, Y. H., Franden, M. A., Hawker, J. R., and McHenry, C. S. (1984) J. Biol. Chem. after completion of the synthesis of a preceding Okazaki frag- 259, 12117–12122 Wu, C. A., Zechmer, E. L., Reems, J. A., McHenry, C. S., and Marians, K. J. (1992) ment, which requires a signal between a new primer and sub- J. Biol. Chem. 267, 4074–4082 units of holoenzyme (Wu et al., 1992). Clearly a ␶-DnaB inter- You, J. C., and McHenry, C. S. (1993) J. Biol. Chem. 268, 16519–16527