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 a, a-e, AND a-e-u 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 (aeu) of the DNA 1984). The function of the u 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 t and a 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 a, ae and aeu were O’Donnell, 1991). The C-terminal region of a binds to a t dimer overexpressed and assembled in E. coli and purified to with a high affinity (KD 5 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 3 polymerase on a primed template with other accessory sub- gap-filling assay. Kinetics studies showed that neither e units (Fay et al., 1981). The DnaX complex (t dd9xc or g dd9xc) nor u influenced the K of a for deoxynucleotide triphos- 4 4 m loads the b sliding clamp onto the primed template by coupling phate and only slightly decreased the K of a for DNA, m ATP hydrolysis (Dallmann and McHenry, 1995; Onrust et al., although e was absolutely required for maximal DNA synthesis. The rate of DNA synthesis by a-reconstituted 1995). The b sliding clamp provides pol III with high proces- holoenzyme using t complex was about 5-fold less than sivity by tethering it to the template (LaDuca et al., 1986; that of ae or aeu-reconstituted holoenzyme as deter- Stukenberg et al., 1991). mined by a gel analysis. The processivity of a-reconsti- Each subunit of pol III works cooperatively and stimulates tuted holoenzyme was very similar to that of aeu-recon- the activity of other subunits. For instance, the a subunit can stituted holoenzyme when t complex was used as a stimulate the exonuclease activity of the e subunit 10–80-fold clamp loader. by increasing the affinity of e for the 39-hydroxyl terminus (Maki and Kornberg, 1987). The e exonuclease activity is also slightly stimulated by the u subunit (Studwell-Vaughan and The DNA polymerase III core (pol III)1 of the DNA polymer- O’Donnell, 1993). Additionally, e induces a 3-fold increase in ase III holoenzyme is a heterotrimer, composed of a, e, and u the polymerase activity of the a 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; a is encoded by dnaE (Gefter et al., 1971; tide, suggesting that the two activities are interactive (Blanco Welch and McHenry, 1982), e by dnaQ (Horiuchi et al., 1981; et al., 1991). Sheuermann et al., 1983), and u 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 a, ae,oraeu complexes assembled in vivo form a very stable complex at a ratio of 1:1:1. a binds e which and purified them to homogeneity without the denaturation- binds u, but a direct a-u contact has not been observed renaturation step required for the purification of e 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 a subunit contains catalytic po- compared. lymerase activity and synthesizes DNA at a rate of approxi- mately 10 nucleotides/s (Maki and Kornberg, 1987; Maki and EXPERIMENTAL PROCEDURES Kornberg, 1985). The e subunit (the dnaQ product) contains 39 Strains—E. coli strains HB101 (F2, recA13, ara 14, proA2, lacY1, 3 59 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(2), 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 overexpress- ing 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 mg/ml ican Cancer Society and facilities support from the Lucille P. Markey 2 4 4 ampicillin at 37 °C. Cells were induced by isopropyl-b-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 a in part by the payment of page charges. This article must therefore be 600 5 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 t complex (t4dd9xc) and g complex (g4dd9xc) were 20681 20682 In Vivo Assembly of Overproduced DNA Pol III FIG.2. Overexpression of a, e, and u 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 ml 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 59-primer contains a 22-nucleotide sequence complementary to FIG.1.Construction of plasmids to overexpress ae 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 39-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 b-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 e overexpressing plasmid. The plasmid pHN3, an ae 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 a overexpressing plas- States Biochemical Corp. Terminal deoxynucleotidyltransferase was mid (Tomasiewicz, 1991). Finally, the holE gene, encoding the u sub- from International Biotechnologies, Inc. Rabbit anti-b 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 (aeu) 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.