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• W2000554826 abstract "The crystal structure of the DNA polymerase encoded by gene 5 of bacteriophage T7, in a complex with its processivity factor, Escherichia coli thioredoxin, a primer-template, and an incoming deoxynucleoside triphosphate reveals a putative hydrogen bond between the C-terminal residue, histidine 704 of gene 5 protein, and an oxygen atom on the penultimate phosphate diester of the primer strand. Elimination of this electrostatic interaction by replacing His704 with alanine renders the phage nonviable, and no DNA synthesis is observed in vivo. Polymerase activity of the genetically altered enzyme on primed M13 DNA is only 12% of the wild-type enzyme, and its processivity is drastically reduced. Kinetic parameters for binding a primer-template (K Dapp ), nucleotide binding (Km), andkoff for dissociation of the altered polymerase from a primer-template are not significantly different from that of wild-type T7 DNA polymerase. However, the decrease in polymerase activity is concomitant with increased hydrolytic activity, judging from the turnover of nucleoside triphosphate into the corresponding nucleoside monophosphate (percentage of turnover, 65%) during DNA synthesis. Biochemical data along with structural observations imply that the terminal amino acid residue of T7 DNA polymerase plays a critical role in partitioning DNA between the polymerase and exonuclease sites. The crystal structure of the DNA polymerase encoded by gene 5 of bacteriophage T7, in a complex with its processivity factor, Escherichia coli thioredoxin, a primer-template, and an incoming deoxynucleoside triphosphate reveals a putative hydrogen bond between the C-terminal residue, histidine 704 of gene 5 protein, and an oxygen atom on the penultimate phosphate diester of the primer strand. Elimination of this electrostatic interaction by replacing His704 with alanine renders the phage nonviable, and no DNA synthesis is observed in vivo. Polymerase activity of the genetically altered enzyme on primed M13 DNA is only 12% of the wild-type enzyme, and its processivity is drastically reduced. Kinetic parameters for binding a primer-template (K Dapp ), nucleotide binding (Km), andkoff for dissociation of the altered polymerase from a primer-template are not significantly different from that of wild-type T7 DNA polymerase. However, the decrease in polymerase activity is concomitant with increased hydrolytic activity, judging from the turnover of nucleoside triphosphate into the corresponding nucleoside monophosphate (percentage of turnover, 65%) during DNA synthesis. Biochemical data along with structural observations imply that the terminal amino acid residue of T7 DNA polymerase plays a critical role in partitioning DNA between the polymerase and exonuclease sites. single-stranded DNA double-stranded DNA nucleotide bovine serum albumin dithiothreitol Gene 5 protein encoded by bacteriophage T7 is a replicative DNA polymerase with an associated 3′-5′ exonuclease activity (1Grippo P. Richardson C.C. J. Biol. Chem. 1971; 246: 6867-6873Abstract Full Text PDF PubMed Google Scholar). This 80-kDa enzyme, by itself, is distributive for DNA synthesis and can only incorporate less than 15 nucleotides before dissociating from a primer terminus (2Tabor S. Huber H.E. Richardson C.C. J. Biol. Chem. 1987; 262: 16212-16223Abstract Full Text PDF PubMed Google Scholar). However, in a 1:1 complex with the hostEscherichia coli protein, thioredoxin (KD= 5 nm), gene 5 protein processively catalyzes the addition of thousands of nucleotides at rates approaching 300 nucleotides/s (2Tabor S. Huber H.E. Richardson C.C. J. Biol. Chem. 1987; 262: 16212-16223Abstract Full Text PDF PubMed Google Scholar, 3Modrich P. Richardson C.C. J. Biol. Chem. 1975; 250: 5508-5514Abstract Full Text PDF PubMed Google Scholar, 4Huber H.E. Russel M. Model P. Richardson C.C. J. Biol. Chem. 1986; 261: 5006-5012Google Scholar, 5Tabor S. Huber H.E. Richardson C.C. Holmgren A. Branden C.-I. Jornvall H. Sjoberg B.-M. Thioredoxin and Glutaredoxin Systems: Structure and Function. Raven Press, New York1986Google Scholar, 6Huber H.E. Tabor S. Richardson C.C. J. Biol. Chem. 1987; 262: 16224-16232Abstract Full Text PDF PubMed Google Scholar). The gene 5 protein-thioredoxin complex will be referred to as T7 DNA polymerase in this manuscript. A recent crystal structure of T7 DNA polymerase in complex with a primer-template and an incoming dideoxynucleoside triphosphate solved at 2.2 Å resolution (7Doublie S. Tabor S. Long A.M. Richardson C.C. Ellenberger T. Nature. 1998; 391: 251-258Crossref PubMed Scopus (1108) Google Scholar) reveals a bipartite structure with distinct C-terminal polymerase and N-terminal exonuclease domains (Fig.1). Most polymerases of the polymerase I family have a similar bipartite architecture (8Ollis D.L. Brick P. Hamlin R. Xuong N.G. Steitz T.A. Nature. 1985; 313: 762-766Crossref PubMed Scopus (739) Google Scholar, 9Beese L.S. Derbyshire V. Steitz T.A. Science. 1993; 260: 352-355Crossref PubMed Scopus (452) Google Scholar, 10Joyce C.M. Steitz T.A. Annu. Rev. Biochem. 1994; 63: 777-822Crossref PubMed Scopus (571) Google Scholar, 11Brautigam C.A. Steitz T.A. Curr. Opin. Struct. Biol. 1998; 8: 54-63Crossref PubMed Scopus (338) Google Scholar, 12Doublie S. Sawaya M.R. Ellenberger T. Structure Folding Design. 1999; 7: R31-R35Abstract Full Text Full Text PDF Scopus (290) Google Scholar). A “right hand” with distinct fingers, palm, and thumb subdomains forms a distinct DNA-binding groove that leads to the polymerase active site (13Steitz T.A. Curr. Opin. Struct. Biol. 1993; 3: 31-38Crossref Scopus (201) Google Scholar). The fingers, palm, and thumb grip the primer-template by a number of direct and water-mediated contacts mainly to the phosphodiester backbone of DNA such that the 3′-end of the primer strand is positioned next to the nucleotide-binding site. The 3′-5′ exonuclease activity on ssDNA1 and dsDNA serves to excise mismatches during processive DNA synthesis (14Jovin T.M. Englund P.T. Bertsch L.L. J. Biol. Chem. 1969; 244: 2996-3008Abstract Full Text PDF PubMed Google Scholar, 15Hori K. Mark D.F. Richardson C.C. J. Biol. Chem. 1979; 254: 11598-11604Abstract Full Text PDF PubMed Google Scholar). The polymerase and the exonuclease active sites are ∼35 Å apart. Although the error frequency during base selection by the polymerase activity occurs at the low rate of one misincorporation per 106 turnovers, the 3′-5′ proofreading activity further reduces the error frequency by 10–200-fold depending on the method of measurement (16Kunkel T.A. Patel S.S. Johnson K.A. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 6830-6834Crossref PubMed Scopus (92) Google Scholar, 17Wong I. Patel S.S. Johnson K.A. Biochemistry. 1991; 30: 526-537Crossref PubMed Scopus (346) Google Scholar). It is believed that the 3′-5′ exonuclease activity maximizes its contribution to replication fidelity by minimal hydrolysis of correctly base paired DNA (18Donlin M.J. Patel S.S. Johnson K.A. Biochemistry. 1991; 30: 538-546Crossref PubMed Scopus (182) Google Scholar). The pre-steady-state kinetic pathways for the polymerization and the exonuclease reactions have been described in detail for T7 DNA polymerase (17Wong I. Patel S.S. Johnson K.A. Biochemistry. 1991; 30: 526-537Crossref PubMed Scopus (346) Google Scholar, 18Donlin M.J. Patel S.S. Johnson K.A. Biochemistry. 1991; 30: 538-546Crossref PubMed Scopus (182) Google Scholar, 19Patel S.S. Wong I. Johnson K.A. Biochemistry. 1991; 30: 511-525Crossref PubMed Scopus (473) Google Scholar) and the Klenow fragment of E. coli DNA polymerase I (20Bryant F.R. Johnson K.A. Benkovic S.J. Biochemistry. 1983; 22: 3537-3546Crossref PubMed Scopus (150) Google Scholar, 21Eger B.T. Kuchta R.D. Carroll S.S. Benkovic P.A. Dahlberg M.E. Joyce C.M. Benkovic S.J. Biochemistry. 1991; 30: 1441-1448Crossref PubMed Scopus (90) Google Scholar, 22Guest C.R. Hochstrasser R.A. Dupuy C.G. Allen D.J. Benkovic S.J. Millar D.P. Biochemistry. 1991; 30: 8759-8770Crossref PubMed Scopus (93) Google Scholar, 23Eger B.T. Benkovic S.J. Biochemistry. 1992; 31: 9227-9236Crossref PubMed Scopus (73) Google Scholar, 24Capson T.L. Peliska J.A. Kaboord B.F. Frey M.W. Lively C. Dahlberg M. Benkovic S.J. Biochemistry. 1992; 31: 10984-10994Crossref PubMed Scopus (227) Google Scholar, 25Carver T.E. Hochstrasser R.A. Millar D.P. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 10670-10674Crossref PubMed Scopus (73) Google Scholar). An equilibrium exists between DNA binding to the polymerase or exonuclease active site, with binding to the polymerase site being thermodynamically more favored (18Donlin M.J. Patel S.S. Johnson K.A. Biochemistry. 1991; 30: 538-546Crossref PubMed Scopus (182) Google Scholar). Any process that slows the rate of the elongation reaction, for example a mismatch, allows time for the transfer of DNA to the exonuclease active site (17Wong I. Patel S.S. Johnson K.A. Biochemistry. 1991; 30: 526-537Crossref PubMed Scopus (346) Google Scholar, 25Carver T.E. Hochstrasser R.A. Millar D.P. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 10670-10674Crossref PubMed Scopus (73) Google Scholar). The data also suggest that DNA is transferred intramolecularly in both directions between the polymerase and exonuclease sites without dissociating from the enzyme. However, the mechanisms that underlie the occupancy of DNA in the polymerase versus the exonuclease site are not well understood. Residues that contact DNA may be expected to govern the partitioning of DNA between the two active sites. The crystal structure of T7 DNA polymerase reveals a putative hydrogen bond between the C-terminal residue His704 of gene 5 protein and an oxygen atom on the penultimate phosphate diester of the primer strand (Fig. 1). This C-terminal residue is conserved in T7 DNA polymerase (His704) and in E. coli DNA polymerase I (His928). In the DNA polymerases from Bacillus stearothermophilus (26Kiefer J.R. Mao C. Hansen C.J. Basehore S.L. Hogrefe H.H. Braman J.C. Beese L.S. Structure. 1997; 5: 95-108Abstract Full Text Full Text PDF PubMed Scopus (136) Google Scholar, 27Kiefer J.R. Mao C. Braman J.C. Beese L.S. Nature. 1998; 391: 304-307Crossref PubMed Scopus (485) Google Scholar) and Thermus aquaticus(28Korolev S. Nayal M. Barnes W.M. Dicera E. Waksman G. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 9264-9268Crossref PubMed Scopus (170) Google Scholar, 29Kim Y. Eom S.H. Wang J.M. Lee D.S. Suh S.W. Steitz T.A. Nature. 1995; 376: 612-616Crossref PubMed Scopus (329) Google Scholar, 30Eom S.H. Wang J.M. Steitz T.A. Nature. 1996; 382: 278-281Crossref PubMed Scopus (309) Google Scholar), the homologous residues are Lys876 and Lys832, respectively. To probe the role of this interaction with the 3′-OH primer terminus, His704 of T7 DNA polymerase was substituted with alanine. The loss of this interaction could result either in reduced affinity of the polymerase for the primer-template or in reduced affinity for just the primer strand. In this report, we show that the C-terminal histidine of T7 DNA polymerase plays a critical role in orienting the primer terminus in position for catalysis and possibly in the controlled shuttling of DNA between the polymerase and exonuclease sites. M13 mGP1-2 is a 9950-nt derivative of vector M13 mp8 containing an insert of phage T7 DNA (31Tabor S. Richardson C.C. Proc. Natl. Acad. Sci. U. S. A. 1987; 84: 4767-4771Crossref PubMed Scopus (1687) Google Scholar). The 24-nt M13 sequencing primer (−47) (5′-CGCCAGGGTTTTCCCAGTCACGAC-3′) and oligonucleotides forin vitro mutagenesis were from Oligos Etc. Activated calf thymus DNA (Type XV) was obtained from Sigma. Poly(dA)280was obtained from Amersham Pharmacia Biotech, and oligo(dT)22 was obtained from Integrated DNA Technologies. Poly(dA)280 and oligo(dT)22 were dissolved in a 1:1 molar ratio (20 μm) in 40 mm Tris-Cl, pH 7.5, and 50 mm NaCl and annealed by heating to 95 °C for 5 min, followed by slowly cooling to room temperature. The 24-nt M13 sequencing primer and M13 mGP1-2 DNA were mixed in a 1:1 molar ratio (100 nm) and annealed using the same protocol. Oligonucleotide concentrations were determined spectrophotometrically. DNA concentrations are expressed in terms of primer 3′-ends. E. coli strains C600 and HMS174 are from the laboratory collection.E. coli HMS174 (DE3)/pLysS cells are from Novagen. Wild-type bacteriophage T7 and mutant T7Δ5 (gene 5 deletion) phage are from the laboratory collection. Plasmid pGP5-3 contains T7 gene 5 under the control of the T7 RNA polymerase promotor Φ10 (2Tabor S. Huber H.E. Richardson C.C. J. Biol. Chem. 1987; 262: 16212-16223Abstract Full Text PDF PubMed Google Scholar). Plasmid pGP5-5,7A contains T7 gene 5 in which two exonuclease active site residues of gene 5 (Asp5 and Glu7) have been substituted with alanine. Plasmid pT7-7 is the parent vector of pGP5-3 missing the GP5 insert. Growth and manipulation of bacteriophage T7 and E. coli were performed as described (32Tabor S. Richardson C.C. J. Biol. Chem. 1989; 264: 6447-6458Abstract Full Text PDF PubMed Google Scholar, 33Studier F.W. J. Mol. Biol. 1975; 94: 283-295Crossref PubMed Scopus (134) Google Scholar). Plasmid pGP5-H704A was constructed using standard polymerase chain reaction and cloning techniques. Two oligonucleotide primers, (5′-CGGGATCCTCAGCCGCAAATCGCCCAATTA-3′) and (5′-CCGTTGGTGCCGGCAAAGAGCGCGG-3′) were used to amplify 340 base pairs of the T7 DNA sequence containing His704. The codon in bold type corresponds to the amino acid alteration. Polymerase chain reaction-generated fragments were digested with BamHI andPshAI and then cloned into the PshAI andBglII sites of plasmid pGP5-3. Plasmid pGP5-H704A contains the genetically altered gene 5 that encodes gp5-H704A under control of the T7 Φ10 promotor. Plasmid pGP5-H704A was digested with restriction enzymes HindIII and StyI, and the resulting fragment was ligated into the HindIII and StyI site of pGP5-5,7A giving rise to pGP5-H704A-5,7 A. The identity of the clones was confirmed by DNA sequencing. Gene 5 proteins were overproduced from E. coli HMS174 (DE3)/pLysS cells carrying plasmids. The 1:1 complex of polymerase and thioredoxin was purified to apparent homogeneity as described (2Tabor S. Huber H.E. Richardson C.C. J. Biol. Chem. 1987; 262: 16212-16223Abstract Full Text PDF PubMed Google Scholar). Protein concentrations were determined by the method of Bradford (34Bradford M.M. Anal. Biochem. 1976; 72: 248-254Crossref PubMed Scopus (217544) Google Scholar) and were confirmed by amino acid analysis. Restriction enzymes were from New England Biolabs. Unlabeled nucleotides (high pressure liquid chromatography grade) were obtained from Amersham Pharmacia Biotech. [3H]dTTP (3000 Ci/mmol) was obtained fromAmersham Pharmacia Biotech and diluted with dTTP to ∼2 cpm/pmol. Methyl-[3H]thymidine (25 Ci/mmol) and [α-32P]dTTP (3000 Ci/mmol) were obtained from Amersham Pharmacia Biotech. Plating efficiencies of wild-type and Δ5 T7 phage were measured on E. coli C600 cells harboring either the plasmid pT7-7, pGP5-3, or pGP5-H704A. The cells were grown to a density of 2 × 108 cells/ml. Dilutions of phage solutions were mixed with 0.5 ml of cells, 3 ml of top agar (1% tryptone, 0.5% yeast, 0.5% NaCl, 0.7% agar, pH 7.0) and ampicillin (200 μg) and plated on TB (1% tryptone, 0.5% yeast, 0.5% NaCl, 1.5% agar, pH 7.0) plates. The plates were incubated at 37 °C for 5 h before being analyzed for plaques. Thymidine incorporation assays were carried out at 30 °C (35Saito H. Richardson C.C. J. Virol. 1981; 37: 343-351Crossref PubMed Google Scholar). E. coli C600 cells harboring plasmid pGP5-3 or pGP5-H704A were grown to a density of 3 × 108 cells/ml in Davis medium (0.7% potassium diphosphate, 2% potassium monophosphate, 0.05% sodium citrate, 0.01% magnesium sulfate, 0.1% ammonium sulfate) supplemented with glucose, thiamine, casamino acids, and ampicillin (80 μg/ml). The cells were infected with either wild-type T7 phage or T7 phage containing a deletion of gene 5 (Δ5 phage) at a multiplicity of infection of ∼5 (35Saito H. Richardson C.C. J. Virol. 1981; 37: 343-351Crossref PubMed Google Scholar). At indicated time intervals, 200 μl of the samples were removed, and [3H]thymidine was added to a final concentration of 50 μCi/ml. Radioactive labeling was terminated after 90 s by the addition of 3 ml of ice-cold 0.3 ntrichloroacetic acid. Acid-insoluble radioactivity was collected via filtration on glass microfiber filters and washed three times with 1m HCl (3 ml) and twice with ethanol (3 ml). The acid insoluble radioactivity was measured using a scintillation counter. The reaction mixtures (50 μl) with primed M13 mGP1-2 DNA contained 40 mm Tris-Cl, pH 7.5, 10 mm MgCl2, 5 mm DTT, 50 mm NaCl, 20 nm M13 DNA annealed to a 24-nt oligonucleotide, 500 μm each of dATP, dCTP, dGTP, and [3H]dTTP (2 cpm/pmol), 50 μg/ml BSA, and 0.3 nm DNA polymerase. The reaction mixtures were incubated at 37 °C for the indicated periods of time. The reactions were stopped by the addition of 10 μl of 0.5 m EDTA, pH 7.5. The incorporation of [3H]dTMP was measured on DE81 filter discs as described (20Bryant F.R. Johnson K.A. Benkovic S.J. Biochemistry. 1983; 22: 3537-3546Crossref PubMed Scopus (150) Google Scholar). To measure a single round of DNA synthesis, (dA)280·(dT)22 was used as a primer-template, and the reactions were carried out at 22 °C as described (6Huber H.E. Tabor S. Richardson C.C. J. Biol. Chem. 1987; 262: 16224-16232Abstract Full Text PDF PubMed Google Scholar). The DNA polymerase was preincubated with the primer-template in the absence of Mg2+ and dTTP for 5 min. The reactions were initiated by the addition of Mg2+, dTTP, and challenger DNA to trap any free polymerase. The aliquots were withdrawn at the indicated times and quenched with a final concentration of 100 mm EDTA. The control reactions to measure background incorporation by polymerase not trapped by challenger DNA were carried out by adding challenger DNA to the preincubation mix. This background reaction was subtracted wherever applicable. The preincubation mixture (30 μl) contained 270 nm poly(dA)280·oligo(dT)22 and 40 nm DNA polymerase. The reaction was initiated by the addition of 25 mm MgCl2, 0.75 mm[3H]dTTP (2 cpm/pmol), and 10 μg calf thymus DNA in 20 μl. Final concentrations were 160 nmpoly(dA)280·oligo(dT)22, 0.3 mm[3H]dTTP, 25 nm DNA polymerase, 10 mm MgCl2, and 200 μg/ml calf thymus DNA. All reaction mixtures also contained 40 mm Tris-Cl, pH 7.5, 5 mm DTT, 50 mm NaCl, and 50 μg/ml BSA. The 3′-5′ exonuclease activity was measured using uniformly 3H-labeled M13 ssDNA or dsDNA. This substrate was prepared by annealing the 24-nt oligonucleotide to M13 mGP1-2 DNA and then extending the primer in a reaction mixture (300 μl) that contained 30 mm Tris-Cl, pH 7.5, 10 mm MgCl2, 5 mm DTT, 50 mm NaCl, 50 μm each of dATP, dCTP, dGTP, and [3H]dTTP (3000 Ci/mmol), and 200 nm T7 DNA polymerase. After incubation at 37 °C for 8 min, the DNA was extracted with an equal volume of buffer-saturated phenol, pH 8.0:chloroform:isoamyl alcohol (24:24:1), and the labeled DNA was purified through a Sepharose CL-6B (Amersham Pharmacia Biotech) column.3H-Labeled M13 mGP1-2 ssDNA was prepared by alkali denaturation of 3H-labeled dsDNA by treatment with 50 mm NaOH at 20 °C for 15 min, followed by neutralization with HCl. M13 3H-labeled ssDNA was used immediately.3H-labeled M13 dsDNA was stored at 4 °C. The reaction mixtures for exonuclease assays (100 μl) contained 40 mm Tris-Cl, pH 7.5, 10 mm MgCl2, 5 mm DTT, 50 mm NaCl, 1 nmol (in terms of total nucleotides) [3H]-labeled M13 mGP1-2 ssDNA or dsDNA, and (0.06–6 nm) DNA polymerase. After incubation at 37 °C for 10 min, the reaction was quenched by the addition of 30 μl of BSA (10 mg/ml) and 30 μl of trichloroacetic acid (100% w/v). After incubation at 0 °C for 15 min, precipitated DNA was collected by centrifugation at 12,000 × g for 30 min. The acid-soluble radioactivity was measured by scintillation counting in Ultra fluor (Packard). One unit of exonuclease activity catalyzes the release of 1 pmol of total nucleotides into an acid-soluble form in 1 min. The DNA used for processivity assays was a 5′ 32P-labeled 24-nt primer annealed to M13 mGP1-2 ssDNA as described (2Tabor S. Huber H.E. Richardson C.C. J. Biol. Chem. 1987; 262: 16212-16223Abstract Full Text PDF PubMed Google Scholar). The reaction mixture (22.5 μl) contained 40 mm Tris-Cl, pH 7.5, 10 mm MgCl2, 5 mm DTT, 50 mm NaCl, 500 μm each of dTTP, dATP, dGTP, and dCTP, and 8 nm primer-template. This mixture was preincubated at 37 °C for 3 min, and the reaction was started by the addition of 2.5 μl of T7 DNA polymerase. Final concentration of the polymerase was 0.3 nm. After the indicated times, the reaction was stopped by the addition of 5 μl of stop solution that contained 40% sucrose, 20 mm EDTA, and 0.05% bromphenol blue. The reaction mixtures were subjected to electrophoresis on a 0.6% agarose gel in a buffer containing 100 mm Tris borate, pH 8.3, 1 mm EDTA, and 0.06 μg/ml ethidium bromide. The gels were dried and autoradiographed. At subsaturating concentrations of ethidium bromide, nicked M13 dsDNA has lower electrophoretic mobility than M13 ssDNA annealed to a 24-nt oligonucleotide. The reaction mixture for turnover assays was essentially the same as for polymerase assays with primed M13 mGP1-2 DNA. The concentrations of wild-type T7 DNA polymerase and gp5-H704A were 2 and 4 nm, respectively. The incorporation of [α-32P]dTMP into the primer strand was measured as described for polymerase assays. The amount of [α-32P]dTMP obtained upon exonucleolytic hydrolysis by the enzyme was quantified by TLC. An aliquot of the reaction mixture was applied to a polyethyleneimine plate (JT Baker) prewashed with distilled water. The thin layer plate was developed with 0.6m LiCl in 1 m formic acid. The amount of [α-32P]dTMP formed was measured using phosphorus imaging analysis with a Fuji BAS 1000 bio-imaging analyzer. The half-life of a preformed polymerase-DNA complex was determined by preincubating the polymerase with poly(dA)280·oligo(dT)22 and adding challenger DNA at time 0. After varying periods of time (5–60 s), the polymerase reaction was started by the addition of Mg2+ and dTTP and stopped after 30 s by the addition of EDTA to a final concentration of 100 mm. Final concentrations of all of the reagents were identical to those used in the polymerase assay with poly(dA)280·oligo(dT)22. Assays to determine Km were carried out at 22 °C with poly(dA)280·oligo(dT)22. A series of 10 concentrations of dTTP were used to bracket the Kmvalue. All reaction mixtures contained 40 mm Tris-Cl, pH 7.5, 10 mm MgCl2, 5 mm DTT, 50 mm NaCl, 160 nmpoly(dA)280·oligo(dT)22 and varying amounts (5–800 μm) of [3H]dTTP (2 cpm/pmol). The reactions were initiated by the addition of polymerase diluted with BSA (final concentrations of 5 nm DNA polymerase and 50 μg/ml BSA) and stopped after 30 s by the addition of EDTA to a final concentration of 100 mm. Dissociation constants for polymerase-DNA complexes were determined for a single processive cycle of DNA synthesis using calf thymus DNA as a trap (6Huber H.E. Tabor S. Richardson C.C. J. Biol. Chem. 1987; 262: 16224-16232Abstract Full Text PDF PubMed Google Scholar). The preincubation mixture (30 μl) contained 42–660 nm poly(dA)280·oligo(dT)22 and 40 nm DNA polymerase. The reaction was initiated by the addition of 20 μl of 25 mm MgCl2, 0.75 mm [3H]dTTP (2 cpm/pmol), and 10 μg calf thymus DNA. Final concentrations were 25–400 nmpoly(dA)280·oligo(dT)22, 0.3 mm[3H]dTTP, 25 nm DNA polymerase, 10 mm MgCl2, and 200 μg/ml calf thymus DNA. All reaction mixtures also contained 40 mm Tris-Cl, pH 7.5, 5 mm DTT, 50 mm NaCl, and 50 μg/ml BSA. The data were fit by nonlinear regression (Enzyme Kinetics, version 1.4). To ascertain the role of the C-terminal histidine in T7 DNA polymerase, a gene 5 mutant was constructed in which histidine 704 was replaced with alanine (gp5-H704A). The effect of the genetically altered gene 5 protein on the growth of T7 phage was tested (TableI). When gp5-H704A is produced from a plasmid, it is unable to support the growth of T7 phage in which the wild-type gene 5 has been deleted (T7Δ5). T7Δ5 phage are dependent on the expression of plasmid encoded gene 5 protein for viability. Furthermore, gp5-H704A inhibits the growth of wild-type T7 phage that is expressing the wild-type gene 5. In contrast, production of wild-type gene 5 protein from a plasmid supports the growth of T7Δ5 phage and has no effect on the growth of on wild-type T7 phage. Thus, the histidine to alanine substitution is dominant lethal for T7 phage growth.Table IAbility of gene 5 plasmids to complement T7 phage growthPlasmidaPlasmid pGP5–3 contains T7 gene 5 under the control of the T7 RNA polymerase promotor φ10, pGP5-H704A contains T7 gene 5 with the H704A codon substitution, and pT7-7 is the parent vector of pGP5-3 missing the gene 5 insert.MutationEfficiency of platingT7Δ5T7 wtNo Plasmid<10−61pT7-7No gene 5<10−61pGP5-3Wild type11pGP5-H704AH704A<10−6<10−6Efficiency of plating of wild-type and Δ5 T7 phage on E. coli C600 was measured as described under “Experimental Procedures.” E. coli C600 cells harboring the indicated plasmids were infected with wild-type or T7 Δ5 phage. The efficiency of plating is calculated by dividing the number of plaque forming units observed when plated on cells containing the indicated plasmid by the number of plaque forming units on pGP5–3 and represents an average of three experiments.a Plasmid pGP5–3 contains T7 gene 5 under the control of the T7 RNA polymerase promotor φ10, pGP5-H704A contains T7 gene 5 with the H704A codon substitution, and pT7-7 is the parent vector of pGP5-3 missing the gene 5 insert. Open table in a new tab Efficiency of plating of wild-type and Δ5 T7 phage on E. coli C600 was measured as described under “Experimental Procedures.” E. coli C600 cells harboring the indicated plasmids were infected with wild-type or T7 Δ5 phage. The efficiency of plating is calculated by dividing the number of plaque forming units observed when plated on cells containing the indicated plasmid by the number of plaque forming units on pGP5–3 and represents an average of three experiments. The rates of in vivo DNA synthesis were measured by monitoring the incorporation of [3H]thymidine into DNA in phage-infected cells (Fig.2). E. coli C600 cells harboring a plasmid (pGP5-3 or pGP5-H704A) were infected with either T7Δ5 phage (Fig. 2 A) or wild-type T7 phage (Fig.2 B). T7 DNA synthesis in infected cells starts ∼10 min after infection, presumably after the shut down of host DNA synthesis (33Studier F.W. J. Mol. Biol. 1975; 94: 283-295Crossref PubMed Scopus (134) Google Scholar). Fig. 2 A shows a plot of the incorporation of [3H]thymidine as a function of time upon infection with T7Δ5 phage. When wild-type gene 5 protein is produced from the plasmid, DNA synthesis starts to increase 10 min after infection and continues to increase up to 30 min after infection. In contrast, when gp5-H704A is produced from the plasmid, DNA synthesis is strongly inhibited 10 min after infection with T7Δ5 phage. Even more striking is the kinetics of DNA synthesis upon infection with wild-type T7 phage (Fig. 2 B). With wild-type gene 5 protein, T7 DNA synthesis starts 10 min after infection, reaching a maximum at 40 min after infection. In contrast, with gp5-H704A, DNA synthesis starts to decrease immediately upon infection. These data indicate that the altered protein, gp5-H704A, cannot restore DNA synthesis in T7Δ5 phage-infected cells, and furthermore, gp5-H704A inhibits DNA synthesis in wild-type T7 phage-infected cells. To determine how the mutation in gene 5 leads to decreased DNA synthesis in vivo, gp5-H704A was overproduced, purified, and characterized biochemically. Initially, gp5-H704A was compared with wild-type T7 DNA polymerase by measuring the rate of polymerization of nucleotides. Initial rates of DNA synthesis with purified enzymes were measured by following the incorporation of [3H]dTMP into DNA using primed M13 ssDNA (Fig. 3) or poly(dA)280·oligo(dT)22 (Fig.4) as primer-templates. The reactions were carried out under conditions of excess nucleotides and DNA. The steady-state rate constant for polymerization (kpol) was determined by dividing the rate of incorporation of nucleotides by the polymerase concentration. The polymerase activity has been expressed in terms ofkpol. Gp5-H704A has 8-fold lower polymerase activity (kpol = 30 s−1) than the wild-type polymerase (kpol = 240 s−1) on M13 ssDNA (Fig. 3). With poly(dA)280·oligo(dT)22, the polymerase activity of gp5-H704A (kpol = 60 s−1) is 3-fold lower than of the wild-type polymerase (kpol = ∼200 s−1) (Fig.4 A). One possible explanation of the lower rate of DNA synthesis on the long M13 DNA template relative to that observed on the shorter poly(dA)280·oligo(dT)22 template is that gp5-H704A is less processive.Figure 4Polymerase assays with preformed polymerase-poly(dA)280·oligo(dT)22 complexes in the presence of challenger DNA. Wild-type (○) or gp5-H704A (●) polymerase (40 nm) was preincubated with 270 nm poly(dA)280·oligo(dT)22 for 5 min to form a polymerase-poly(dA)280·oligo(dT)22 complex. Time course of [3H]dTMP incorporation when the reaction was initiated by the addition of Mg·dTTP alone (A), the reaction was initiated by the addition of Mg·dTTP and challenger DNA (B), or the polymerase was preincubated with challenger DNA and poly(dA)280·oligo(dT)22 before the addition of Mg·dTTP (C). The reactions were carried out at 22 °C and stopped by the addition of 100 mm EDTA.View Large Image Figure ViewerDownload Hi-res image Download (PPT) DNA synthesi" @default.
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• W2000554826 title "Role of the C-terminal Residue of the DNA Polymerase of Bacteriophage T7" @default.
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