A Covalent Linkage between the Gene 5 DNA Polymerase of Bacteriophage T7 and Escherichia coli Thioredoxin, the Processivity Factor
2003; Elsevier BV; Volume: 278; Issue: 26 Linguagem: Inglês
10.1074/jbc.m301366200
ISSN1083-351X
AutoresDonald E. Johnson, Charles C. Richardson,
Tópico(s)Bacteriophages and microbial interactions
ResumoGene 5 protein (gp5) of bacteriophage T7 is a non-processive DNA polymerase, which acquires high processivity by binding to Escherichia coli thioredoxin. The gene 5 protein-thioredoxin complex (gp5/trx) polymerizes thousands of nucleotides before dissociating from a primer-template. We have engineered a disulfide linkage between the gene 5 protein and thioredoxin within the binding surface of the two proteins. The polymerase activity of the covalently linked complex (gp5-S-S-trx) is similar to that of gp5/trx on poly(dA)/oligo(dT). However, gp5-S-S-trx has only one third the polymerase activity of gp5/trx on single-stranded M13 DNA. gp5-S-S-trx has difficulty polymerizing nucleotides through sites of secondary structure on M13 DNA and stalls at these sites, resulting in lower processivity. However, gp5-S-S-trx has an identical processivity and rate of elongation when E. coli single-stranded DNA-binding protein (SSB protein) is used to remove secondary structure from M13 DNA. Upon completing synthesis on a DNA template lacking secondary structure, both complexes recycle intact, without dissociation of the processivity factor, to initiate synthesis on a new DNA template. However, a complex stalled at secondary structure becomes unstable, and both subunits dissociate from each other as the polymerase prematurely releases from M13 DNA. Gene 5 protein (gp5) of bacteriophage T7 is a non-processive DNA polymerase, which acquires high processivity by binding to Escherichia coli thioredoxin. The gene 5 protein-thioredoxin complex (gp5/trx) polymerizes thousands of nucleotides before dissociating from a primer-template. We have engineered a disulfide linkage between the gene 5 protein and thioredoxin within the binding surface of the two proteins. The polymerase activity of the covalently linked complex (gp5-S-S-trx) is similar to that of gp5/trx on poly(dA)/oligo(dT). However, gp5-S-S-trx has only one third the polymerase activity of gp5/trx on single-stranded M13 DNA. gp5-S-S-trx has difficulty polymerizing nucleotides through sites of secondary structure on M13 DNA and stalls at these sites, resulting in lower processivity. However, gp5-S-S-trx has an identical processivity and rate of elongation when E. coli single-stranded DNA-binding protein (SSB protein) is used to remove secondary structure from M13 DNA. Upon completing synthesis on a DNA template lacking secondary structure, both complexes recycle intact, without dissociation of the processivity factor, to initiate synthesis on a new DNA template. However, a complex stalled at secondary structure becomes unstable, and both subunits dissociate from each other as the polymerase prematurely releases from M13 DNA. DNA polymerases responsible for copying genomic DNA require high processivity to incorporate thousands of nucleotides without dissociating from the DNA (1Kornberg A. Baker T.A. DNA Replication. 2nd Ed. W. H. Freeman, New York1992: 494-496Google Scholar, 2Onrust R. Stukenberg P.T. O'Donnell M. J. Biol. Chem. 1991; 266: 21681-21686Google Scholar, 3Kelman Z. Hurwitz J. O'Donnell M. Structure. 1998; 6: 121-125Google Scholar). In most cases, a DNA polymerase achieves high processivity by utilizing accessory proteins that act as a sliding clamp that encirles the DNA to tether the polymerase to a primed DNA template (4Stukenberg P.T. Studwell-Vaughan P.S. O'Donnell M. J. Biol. Chem. 1991; 266: 11328-11334Google Scholar, 5Kuriyan J. O'Donnell M. J. Mol. Biol. 1991; 234: 915-925Google Scholar, 6Kelman Z. O'Donnell M. Curr. Opin. Genet. Dev. 1994; 4: 185-195Google Scholar). For example, Escherichia coli DNA polymerase III must have its processivity factor, the β-clamp, pre-assembled on the primer-template before the polymerase can productively bind to the DNA to initiate DNA synthesis. In contrast, several viruses utilize a different mechanism to achieve high processivity. For example, the DNA polymerase of bacteriophage T7 adopts the host protein E. coli thioredoxin (trx) 1The abbreviations used are: trx, thioredoxin; ssDNA, single-stranded DNA; dsDNA, double-stranded DNA; IPTG, isopropyl-β-d-thiogalactopyranoside; DTT, dithiothreitol; NEM, N-ethylmaleimide; nt, nucleotide; SSB, single-stranded DNA-binding; wt, wild type. 1The abbreviations used are: trx, thioredoxin; ssDNA, single-stranded DNA; dsDNA, double-stranded DNA; IPTG, isopropyl-β-d-thiogalactopyranoside; DTT, dithiothreitol; NEM, N-ethylmaleimide; nt, nucleotide; SSB, single-stranded DNA-binding; wt, wild type. as a processivity factor.After infecting its host, E. coli, bacteriophage T7 induces the synthesis of a replicative DNA polymerase, the product of gene 5 (7Grippo P. Richardson C.C. J. Biol. Chem. 1971; 246: 6867-6873Google Scholar). Gene 5 protein alone is a distributive enzyme, dissociating from a primed DNA template after incorporation of only a few nucleotides (8Tabor S. Huber H.E. Richardson C.C. J. Biol. Chem. 1987; 262: 16212-16223Google Scholar). The gene 5 protein achieves high processivity by forming a 1:1 complex (Kd, ∼5 nm) with E. coli thioredoxin (8Tabor S. Huber H.E. Richardson C.C. J. Biol. Chem. 1987; 262: 16212-16223Google Scholar, 9Huber H.E. Tabor S. Richardson C.C. J. Biol. Chem. 1987; 262: 16224-16232Google Scholar, 10Modrich P. Richardson C.C. J. Biol. Chem. 1975; 250: 5515-5522Google Scholar, 11Mark D. Richardson C.C. Proc. Natl. Acad. Sci. U. S. A. 1976; 73: 780-784Google Scholar). The complex of T7 gene 5 DNA polymerase and thioredoxin is designated as gp5/trx, also known as T7 DNA polymerase. Thioredoxin allows the gene 5 protein to incorporate thousands of nucleotides per polymerization cycle, a result of an 80-fold increase in the affinity of gp5/trx for the 3′-terminus of the primer-template (8Tabor S. Huber H.E. Richardson C.C. J. Biol. Chem. 1987; 262: 16212-16223Google Scholar, 9Huber H.E. Tabor S. Richardson C.C. J. Biol. Chem. 1987; 262: 16224-16232Google Scholar). Thioredoxin also markedly increases the 3′-5′ double-stranded DNA exonuclease activity of the polymerase, but does not affect the single-stranded DNA exonuclease activity (8Tabor S. Huber H.E. Richardson C.C. J. Biol. Chem. 1987; 262: 16212-16223Google Scholar, 12Adler S. Modrich P. J. Biol. Chem. 1979; 254: 11605-11614Google Scholar, 13Hori K. Mark D. Richardson C.C. J. Biol. Chem. 1979; 254: 11598-11604Google Scholar).The crystal structure of gp5/trx has been determined at 2.2 Å resolution with the polymerase captured in a polymerization mode (Ref. 17Doublie S. Tabor S. Long A.M. Richardson C.C. Ellenberger T. Nature. 1998; 391: 157-166Google Scholar and Fig. 1). T7 gene 5 protein is a member of the pol I family of DNA polymerases with three subdomains: palm, fingers, and thumb. The three subdomains together form a DNA binding groove with the palm forming the base of a cleft, and the fingers and thumb creating a wall on each side. In this structure thioredoxin is bound to the polymerase at a flexible loop extending from the thumb and is rotated slightly up and away from the cleft in which the primer-template lies. Previous biochemical studies have characterized the domain in gene 5 protein that is responsible for binding thioredoxin. An amino acid alignment of gene 5 protein with homologous regions of the Klenow fragment of E. coli DNA polymerase I revealed a 71 amino acid extension between α-helices H and H1 of the thumb that is absent from the Klenow fragment (18Braithwaite D.K. Ito J. Nucleic Acids Res. 1993; 21: 787-802Google Scholar). Mutations within this domain affect the ability of the polymerase to bind thioredoxin (19Yang X.-M. Richardson C.C. J. Biol. Chem. 1997; 272: 6599-6606Google Scholar). Furthermore, insertion of this domain into the corresponding region of the thumb in the Klenow fragment results in a chimeric DNA polymerase that can bind thioredoxin and achieve higher processivity (20Bedford E. Tabor S. Richardson C.C. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 479-484Google Scholar).The precise molecular mechanism by which thioredoxin increases the processivity of gp5 is not known. However, unlike the processivity factor of E. coli DNA polymerase III, thioredoxin does not appear to encircle the DNA as a clamp. It is likely that in the crystal structure the polymerase-DNA complex has been captured in a non-processive mode. In a processive mode the thumb and bound thioredoxin is postulated to swing down onto the duplex portion of the primer-template to prevent the DNA from dissociation prior to the next polymerization cycle. Suppressor analysis of a genetically altered thioredoxin supports this scenario (21Himawan J.S. Richardson C.C. J. Biol. Chem. 1996; 271: 19999-20008Google Scholar). Amino acids in gp5 that restore the ability of the altered thioredoxin to confer processivity on the polymerase reside within the thioredoxin binding segment while another is located within the exonuclease domain. The latter site is interesting since it raises the possibility that the extended loop of the thioredoxin binding segment might swing down and dock on the lip of the crevice located within the exonuclease domain, thus encircling gp5/trx around the DNA within a structure similar to a sliding clamp. Alternatively, thioredoxin could be increasing the electrostatic interactions between the polymerase and the DNA template.At a replication fork, gp5/trx interacts with the T7 gene 4 helicase-primase (14Notarnicola S.M. Mulcahy H.L. Lee J. Richardson C.C. J. Biol. Chem. 1997; 272: 18425-18433Google Scholar) and the T7 gene 2.5 single-stranded DNA-binding protein (40Kim Y.T. Tabor S. Churchich J.E. Richardson C.C. J. Biol. Chem. 1992; 267: 15032-15040Google Scholar) to mediate coordinated leading and lagging strand DNA synthesis (15Debyser Z. Tabor S. Richardson C.C. Cell. 1994; 77: 157-166Google Scholar, 16Lee J. Chastain II, P.D. Kuskabe T. Griffith J.D. Richardson C.C. Mol. Cell. 1998; 1: 1001-1010Google Scholar). Like E. coli DNA polymerase III, gp5/trx synthesizes both strands processively (15Debyser Z. Tabor S. Richardson C.C. Cell. 1994; 77: 157-166Google Scholar, 16Lee J. Chastain II, P.D. Kuskabe T. Griffith J.D. Richardson C.C. Mol. Cell. 1998; 1: 1001-1010Google Scholar). The leading strand polymerase synthesizes DNA at the replication fork in a continuous manner, while the lagging strand polymerase replicates Okazaki fragments in a discontinuous manner. It is postulated that DNA polymerase III must rapidly recycle from the DNA and β-clamp upon completion of an Okazaki fragment to associate with another pre-assembled β-clamp for processive synthesis of the next Okazaki fragment (3Kelman Z. Hurwitz J. O'Donnell M. Structure. 1998; 6: 121-125Google Scholar). During coordinated DNA synthesis by the T7 replisome, the lagging strand gp5/trx also recycles from a completed Okazaki fragment to a new primer (15Debyser Z. Tabor S. Richardson C.C. Cell. 1994; 77: 157-166Google Scholar, 16Lee J. Chastain II, P.D. Kuskabe T. Griffith J.D. Richardson C.C. Mol. Cell. 1998; 1: 1001-1010Google Scholar). However, it is unclear whether gp5 dissociates from thioredoxin as the polymerase recycles. In this study, we have examined the fate of thioredoxin during recycling by forming a covalent linkage between the polymerase and thioredoxin.Thioredoxin is a versatile protein found in all species, serving as a cofactor to reduce disulfide bonds in many proteins (22Holmgren A. Annu. Rev. Biochem. 1985; 54: 237-271Google Scholar, 23Holmgren A. Structure. 1995; 3: 239-243Google Scholar). Among its many functions it acts as a hydrogen donor for the enzyme ribonucleotide reductase. The activities of thioredoxin have been attributed to two active site cysteines that can form a disulfide linkage between their sulfhydryl groups or can participate in reversible oxidation-reductions with other proteins. The thioredoxin active site cysteines are part of a conserved sequence, Cys-Gly-Pro-Cys (residues 32–35) located in a loop that is partially exposed to the surface of the protein (24Holmgren A. Soderberg B.-O. Eklund H. Branden C.-I. Proc. Natl. Acad. Sci. U. S. A. 1975; 72: 2305-2309Google Scholar, 25Katti S.K. LeMaster D.M. Eklund H. J. Mol. Biol. 1990; 212: 167-184Google Scholar). This loop participates in a hydrophobic surface that is responsible for binding to protein substrate (26Eklund H. Gleason F.K. Holmgren A. Proteins. 1991; 11: 13-28Google Scholar). Once bound to a protein having a disulfide bond, residues Cys-32 and Cys-35 of thioredoxin act together to reduce their target substrate. Cys-32 acts as a nucleophile to form a covalently mixed disulfide with the target protein in the transition state (22Holmgren A. Annu. Rev. Biochem. 1985; 54: 237-271Google Scholar, 23Holmgren A. Structure. 1995; 3: 239-243Google Scholar, 27Kallis G.B. Holmgren A. J. Biol. Chem. 1980; 255: 10261-10265Google Scholar). Cys-35 then resolves this intermediate mixed disulfide to yield the reduced target protein (27Kallis G.B. Holmgren A. J. Biol. Chem. 1980; 255: 10261-10265Google Scholar).The structure of reduced thioredoxin in gp5/trx is very similar to that of oxidized thioredoxin (17Doublie S. Tabor S. Long A.M. Richardson C.C. Ellenberger T. Nature. 1998; 391: 157-166Google Scholar), yet only reduced thioredoxin binds to gene 5 protein (12Adler S. Modrich P. J. Biol. Chem. 1979; 254: 11605-11614Google Scholar). In the crystal structure of gp5/trx the thioredoxin binding loop of gp5 wraps around the base of thioredoxin, burying the active site cysteines (17Doublie S. Tabor S. Long A.M. Richardson C.C. Ellenberger T. Nature. 1998; 391: 157-166Google Scholar). Thus, it is not surprising that the active site cysteines are not required for their reducing power when thioredoxin binds gp5 (29Huber H.E. Russel M. Model P. Richardson C.C. J. Biol. Chem. 1986; 261: 15006-15012Google Scholar). Both Cys-32 and Cys-35 can be replaced with residues that abolish the ability of thioredoxin to undergo oxidation-reduction reactions, but these altered forms of thioredoxin can form functional polymerase-thioredoxin complexes in vitro, albeit with a reduced binding affinity. These results show that the active site residues of thioredoxin only function in binding thioredoxin to the polymerase. The three-dimensional structure of gp5/trx supports these findings, revealing that thioredoxin Cys-32 is exposed to the protein-protein interface and hydrogen bonds with Thr-327 of the polymerase thumb (17Doublie S. Tabor S. Long A.M. Richardson C.C. Ellenberger T. Nature. 1998; 391: 157-166Google Scholar). Thr-327 of the polymerase selects for reduced thioredoxin through its hydrogen bond with the sulfhydryl group of Cys-32. 2D. Johnson, S. Tabor, T. Ellenberger, and C. C. Richardson, unpublished results. 2D. Johnson, S. Tabor, T. Ellenberger, and C. C. Richardson, unpublished results. This interaction effectively decreases the polarity of Thr-327 within the hydrophobic subunit interface and thus explains the requirement for reduced thioredoxin for binding. Cys-32 of oxidized thioredoxin cannot participate in a hydrogen bond with Thr-327 of gp5 because it forms a disulfide linkage with Cys-35. In the present study we have substituted Thr-327 of the polymerase thumb with cysteine (gp5(T327C)) so that it can react with Cys-32 of thioredoxin to facilitate a mixed disulfide between the two proteins. We have used the covalently linked complex (gp5-S-S-trx) to examine processivity and to determine if there is a requirement for thioredoxin to dissociate when the polymerase recycles from one template to another.EXPERIMENTAL PROCEDURESMaterialsBacterial Strains and DNA—Bacterial strain BL21(DE3), used to express wild-type thioredoxin, was purchased from Invitrogen. E. coli A307 (HrfC, ΔtrxA307) was a gift from Stan Tabor (Harvard Medical School). E. coli A307(DE3) was constructed from E. coli A307 using a DE3 lysogenization kit from Novagen. Using this kit, E. coli A307(DE3) was infected with a λDE3 prophage carrying the gene for T7 RNA polymerase under lacUV5 control so that expression of cloned genes having a T7 promoter could be induced in the presence of IPTG. T7Δ5 phage, lacking gene 5, were a gift from Stan Tabor. M13 mp18 bacteriophage were a gift from Kajal Chowdhury (Harvard Medical School). M13 phage were grown and purified as described (33Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual. 2nd Ed. Cold Spring Harbor, NY1989: 6.20-6.21Google Scholar). Single-stranded M13 DNA was purified using a Lambda Maxi kit purchased from Qiagen, Inc. Poly(dA)350-oligo(dT)25 and oligonucleotide primers for M13 DNA were obtained from Midland Certified Reagent Co. Plasmids pGP5-3, pTrx-3, and pGP1-2, vectors having wild-type T7 gene 5, E. coli trxA, and T7 gene 1, respectively, were gifts from Stan Tabor. Plasmid pT7-7, the parent vector of pGP5-3 and pTrx(C35S)-1, was a gift from Stan Tabor. Plasmid pET-24a, the parent vector of pTrxA and pTrx(C35S)-2, was purchased from Novagen.Mutagenesis of T7 Gene 5—Plasmid pGP5(T327C) was constructed by mutagenesis of T7 gene 5 within pGP5-3 using an "overlap extension" method (34Ho S.N. Hunt H.D. Horton R.M. Pullen J.K. Pease L.R. Gene (Amst.). 1989; 77: 51-59Google Scholar). The mutagenesis required three separate PCR reactions using using PfuTurbo DNA polymerase (Stratagene). The first PCR reaction used the primers DJ4 (5′-GAAGGGTTAAACACAACATGTTCAACTGGGCAGTAAGGAGCACCAGCAACGTACT-3′) and BCMP97 (5′-GCATTGACCAAACTGGCAAAG-3′) to generate a 5′ fragment of T7 gene 5 that contains a codon that corresponds to a Thr-327 to Cys alteration. The altered codon of primer DJ4 is underlined. A 3′ fragment containing the same codon was generated in a second PCR reaction using primers DJ3 (5′-AGTACGTTGCTGGTGCTCCTTACTGCCCAGTTGAACATGTTGTGTTTAACCC-3′) and JH8 (5′-GCGAGCCATGAAGTGAGCC-3′). The 5′ and 3′ fragments were purified by agarose gel electrophoresis and then used in a final PCR reaction. These fragments overlap and generate a longer fragment when amplified with primers BCMP97 and JH8. The final PCR product was purified on an agarose gel, digested with StyI and MfeI, and ligated into corresponding sites on plasmid pGP5-3 to create pGP5(T327C). The desired clone was confirmed by DNA sequencing.Mutagenesis of E. coli trxA—Plasmid pTrx(C35S) was constructed by mutagenesis of E. coli trxA of plasmid pTrx-3 using a "Megaprimer" method (35Sarker G. Sommer S. BioTechniques. 1990; 8: 404-407Google Scholar). Plasmid pTrx-3 contains a copy of wild-type E. coli trxA. The mutagenesis required two separate PCR reactions using PfuTurbo DNA polymerase (Stratagene). The first PCR reaction used primer A (5′-GAGTGGTGCGGTCCGTCCAAAATGATCGCCCCGATT-3′) and primer B (5′-GCTTCTAAGCTTCCCTTACGCCAGGTT-3′) to generate a 5′ fragment of the trxA gene having a codon that corresponds to a Cys-35 to Ser alteration. The altered codon of primer A is underlined. This 5′-fragment was purified by agarose gel electrophoresis and then used in a final PCR reaction with the primer C (5′-GTTGGTAGCGGCCATATGAGCGATAAAATTATTCAC-3′) to generate a full-length copy of the trxA gene with the desired mutation. The final PCR product was purified on an agarose gel, digested with NdeI and HindIII, and ligated into corresponding sites on plasmid pT7-7 to create pTrx (C35S)-1, the plasmid used to express trx(C35S) in the presence of ampicillin for purification. The restricted fragment containing the mutated trxA gene was also ligated into corresponding sites on plasmid pET-24a to create pTrx(C35S)-2, the plasmid used for determining plating efficiencies in the presence of kanamycin. Plasmid pTrxA was used to express wild-type thioredoxin in the presence of kanamycin. It was created by PCR of trxA of plasmid pTrx-3 using primer A and primer C, followed by digestion with NdeI and HindIII, and then ligation into corresponding sites on plasmid pET-24a. The desired clones were confirmed by DNA sequencing.Other Materials—PfuTurbo DNA polymerase or AmpliTaq DNA polymerase (Applied Biosystems) were used for standard PCR reactions. Unlabeled nucleotides (HPLC grade) and [methyl-3H]TTP (3000 Ci/mmol) were obtained from Amersham Biosciences. Restriction enzymes NdeI, HindIII, StyI, and MfeI were purchased from New England Biolabs. T4 DNA ligase, T4 polynucleotide kinase, and ThermoSequenase were from Amersham Biosciences. IPTG, DTT, kanamycin, and ampicillin were obtained from American Bioanalytical. DEAE-cellulose (DE52), phosphocellulose (P11), and DE81 filter papers were obtained from Whatman Paper Ltd. Sephadex G-50, HiTrap heparin columns (5-ml bed volume), HiTrap Q Sepharose HP columns (5-ml bed volume), and agarose-HE were from Amersham Biosciences. Ceramic hydroxyapatite columns (5-ml Econo-PacCHT-II cartridges) and bovine serum albumin were from Bio-Rad.MethodsPlating Efficiencies—Plating efficiencies of T7Δ5 phage were measured on E. coli A307(ΔtrxA) harboring either plasmid pT7-7, pGP5-3, pGP5(T327C), pET-24a, pTrxA, pTrx(C35S)-2, or a combination of two of these plasmids (Table II). Cells having plasmids pT7-7, pGP5-3, and pGP5(T327C) were selected for ampicillin resistance. Cells harboring plasmids pET-24a, pTrxA, and pTrx(C35S)-2 were selected for kanamycin resistance. 10-fold serial dilutions of T7Δ5 phage (100 μl) were mixed with a 100-μl plating culture and 3 ml of top agar (1% tryptone, 0.5% yeast extract, 0.5% NaCl, 0.7% agar, pH 7.0) that was preincubated at 48 °C. Mixtures were plated on TB plates at room temperature (1% tryptone, 0.5% yeast extract, 0.5% NaCl, 1.5% agar, pH 7.0) having appropriate antibiotics. The plates were incubated at 37 °C for 5 h. The efficiency of plating was determined by dividing the number of plaque-forming units by the amount of plaque-forming units observed for cells having pTrxA and pGP5-3 and represents an average of at least two experiments.Table IIAbility of gene 5 and trxA mutants to complement phage growthPlasmidMutationEfficiency of platingaPlating efficiencies for wild-type T7Δ5 phage were measured as described under "Experimental Procedures." T7Δ5 refers to T7Δ5 phage. T7Δ5pET-24aNo trxA<10-6pTrxWt trxA<10-6pTrx(C35S)trx(C35S)<10-6pT7-7No gene 5<10-6pGP5-3Wt gene 5<10-6pGP5(T327C)gp5 (T327C)<10-6pT7-7/pET-24aNo gene 5/no trxA<10-6pGP5-3/pTrxWt gene 5/wt trxA1.0pGP5-3/pTrx(C35S)Wt gene 5/trx(C35S)0.9pGP5(T327C)/pTrxgp5(T327C)/wt trxA0.1pGP5(T327C)/pTrx(C35S)gp5(T327C)/trx(C35S)0.07a Plating efficiencies for wild-type T7Δ5 phage were measured as described under "Experimental Procedures." T7Δ5 refers to T7Δ5 phage. Open table in a new tab Protein Purification—gp5/trx, gene 5 protein, and gp5(T327C) were overexpressed in E. coli A307(DE3) using plasmids pGP5-3/pTrxA, pGP5-3, and pGP5(T327C), respectively and then purified using procedures described previously (3Kelman Z. Hurwitz J. O'Donnell M. Structure. 1998; 6: 121-125Google Scholar). However, hydroxyapatite chromatography was omitted from the purification procedures of gene 5 protein and gp5(T327C). Wild-type thioredoxin was overexpressed in E. coli BL21(DE3) using plasmid pTrxA and purified as described (20Bedford E. Tabor S. Richardson C.C. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 479-484Google Scholar). trx(C35S) is toxic to growth of E. coli BL21(DE3) (data not shown), so this protein was overexpressed in E. coli A307 using a heat shock system as described (36Tabor S. Richardson C.C. J. Biol. Chem. 1985; 82: 1074-1078Google Scholar). The heat shock system is a two-plasmid system that maintains toxic proteins under tight transcriptional control. One plasmid, pGP1-2, has the gene for T7 RNA polymerase controlled by the λ PL promoter, the gene for the temperature-sensitive λ repressor cI857, and the gene for kanamycin resistance. The second plasmid is pTrx(C35S)-1, which has the gene for trx(C35S) under control of a T7 promoter and carrying the gene for ampicillin resistance. The λ repressor cI857 tightly represses expression of T7 RNA polymerase at 25 °C, but exclusive expression of trx(C35S) is achieved after heat induction of T7 RNA polymerase at 42 °C. Thus, E. coli A307 was transformed with both plasmids pGP1-2 and pTrx(C35S)-1 in the presence of kanamycin and ampicillin, and then initially grown at 25 °C until an OD600 of 1. The cells were heat-induced at 42 °C for 1 h followed by protein synthesis for 3 h at 37 °C. trx(C35S) was then purified from cells using procedures previously described for wild-type thioredoxin (36Tabor S. Richardson C.C. J. Biol. Chem. 1985; 82: 1074-1078Google Scholar).The covalently linked gp5-S-S-trx was formed by mixing 20 μm purified gp5(T327C) and 150 μm trx(C35S) overnight at 0 °C in 4 ml of buffer containing 40 mm potassium phosphate (pH 7.4), 25 mm NaCl, 1 mm EDTA, 1 mm DTT, 50% glycerol. gp5-S-S-trx was purified from both free gp5(T327C) and trx(C35S) on a HiTrap heparin column (5-ml bed volume) at 4 °C after diluting the mixture 10-fold with Buffer H (50 mm Tris-HCl, pH 7.5, 1 mm EDTA, 0.1 mm DTT, and 10% glycerol). After loading the diluted mixture on the HiTrap heparin column using a BioCad Sprint System (Perseptive BioSystems Inc.), the column was washed with 200 mm NaCl in Buffer H to remove free trx(C35S). gp5-S-S-trx was separated from free gp5(T327C) using a 200–800 mm NaCl continuous gradient in Buffer H over 60 min. Under these conditions, gp5-S-S-trx eluted at 610 mm NaCl and was determined to be greater than 95% pure by analysis on a 4–20% SDS-PAGE gel. Purified gp5-S-S-trx was stored at –12 °C in a buffer containing 40 mm potassium phosphate (pH 7.4), 0.1 mm DTT, 1 mm EDTA, and 50% glycerol. Protein concentrations were determined by the Bradford method using a Bio-Rad protein assay kit and bovine serum albumin as a standard.DNA Polymerase Assays—DNA polymerase activity was measured by procedures modified from those previously described (3Kelman Z. Hurwitz J. O'Donnell M. Structure. 1998; 6: 121-125Google Scholar, 37Kumar J.K. Kremsdorf R. Tabor S. Richardson C.C. J. Biol. Chem. 2001; 82: 46151-46159Google Scholar, 38Bryant F.R. Johnson K.A. Benkovic S.J. Biochemistry. 1983; 22: 3537-3546Google Scholar). The DNA polymerase assay (300 μl) for M13 DNA contained 50 mm Tris-Cl (pH 7.5), 10 mm MgCl2, 0.1 mm DTT, 50 mm NaCl, 250 μm each dGTP, dATP, dCTP, and [3H]TTP (10 cpm/pmol). Reactions also had 14 nm single-stranded M13 mp8 DNA primed with a 17-nt oligonucleotide (–40 primer) and 4 nm of either gp5/trx or gp5-S-S-trx. The polymerase assays were carried out at 37 °C. Aliquots (20 μl) were removed at the times indicated and stopped by addition of EDTA to a final concentration of 25 mm. Aliquots were spotted on DE81 ion exchange filters, and unincorporated radiolabeled nucleotides were washed away with three successive 10-min washes in 300 mm ammonium formate (pH 8.0). Filters were dried under a heat lamp, and the amount of [3H]dTMP incorporated was measured by liquid scintillation counting. Assays to study the effect of E. coli SSB protein on polymerase activity contained 60 μg of SSB protein per 300 μl of reaction mixture. Reactions to monitor polymerase activity on poly(dA)350-oligo(dT)25 were similar for those with single-stranded M13 DNA except poly(dA)350-oligo(dT)25 was added at a concentration of 200 nm, resulting in a 50-fold molar excess of DNA over polymerase. Reactions involving poly(dA)350-oligo(dT)25 were incubated at 25 °C.M13 DNA (100 nm) was primed for polymerase assays by annealing with a 17-nt primer (–40 primer) in 50 mm Tris-Cl (pH 7.5), 50 mm NaCl. Annealing reactions were incubated at 75 °C for 5 min followed by 30 min at room temperature. For polymerase assays using linear DNA templates, poly(dA)350 (2 μm) was annealed to an oligo(dT)25 primer in a 1:1 molar ratio at 0 °C for 30 min.Exonuclease Activity—The 3′ to 5′ single- and double-stranded DNA exonuclease activity of gp5/trx and gp5-S-S-trx were assayed using procedures modified from those previously described (37Kumar J.K. Kremsdorf R. Tabor S. Richardson C.C. J. Biol. Chem. 2001; 82: 46151-46159Google Scholar, 39Chowdhury K. Tabor S. Richardson C.C. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 12469-12474Google Scholar). Uniformly 3H-labeled double-stranded M13 DNA was prepared by annealing a 17-nt primer (–40 primer) to M13 mp8 DNA. The primer was partially extended by 8 nm gp5/trx in a DNA polymerase reaction containing 10 mm MgCl2, 0.1 mm DTT, 20 μm each dGTP, dATP, dCTP, and [3H]TTP (10 cpm/pmol). The reaction was incubated at 37 °C for 5 min and stopped by heating at 75 °C for 5 min, followed by re-annealing at room temperature for 30 min. The labeled DNA was extracted with phenol-chloroform and then purified by passing through Biospin 6 columns (Bio-Rad) to remove free nucleotides. Radiolabeled single-stranded M13 DNA was prepared by alkali denaturation of 3H-labeled double-stranded M13 DNA by treatment with 50 mm NaOH at room temperature for 15 min followed by neutralization with HCl.The double-stranded DNA exonuclease assays (200 μl) contained 50 mm Tris-Cl (pH 7.5), 10 mm MgCl2, 0.1 mm DTT, 50 mm NaCl, 16 nm gp5/trx or gp5-S-S-trx, and 8 nm3H-labeled, double-stranded M13 mp8 DNA. Reaction mixtures were incubated at 37 °C, and 20 μl aliquots were removed at 1-min intervals from 1 to 10 min and stopped by addition of EDTA to a final concentration of 25 mm. Aliquots were spotted on DE81 ion exchange filters and washed with three successive 10-min washes of 300 mm ammonium formate (pH 8.0). Filters were dried under a heat lamp, and the amount of [3H]DNA remaining was measured by liquid scintillation counting to calculate the amount of DNA hydrolyzed.Reaction mixtures for single-stranded DNA exonuclease assays were similar as for the reactions for double-stranded M13 DNA except reactions contained 8 nm gp5/trx or gp5-S-S-trx and ∼8 nm3H-labeled, single-stranded M13 mp8 DNA. Assays to study the effect of SSB protein on polymerase activity contained 40 μg of SSB protein per 200-μl volume reactio
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