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• W2066857980 abstract "T7 gene 5 DNA polymerase forms a complex with Escherichia coli thioredoxin (its processivity factor), and a 76-amino acid sequence (residues 258-334), unique to gene 5 protein, has been implicated in this interaction. We have examined the effect of amino acid substitution(s) in this region on T7 phage growth and on the interaction of the polymerase with thioredoxin. Among the mutations in gene 5, we found that a substitution of either Glu or Ala for Lys-302 yielded a protein that could not complement T7 phage lacking gene 5 (T7Δ5) to grow on E. coli having reduced thioredoxin levels. One triple mutant (K300E,K302E,K304E) could not support the growth of T7Δ5 even in wild type cells. This altered polymerase is stimulated 4-fold less by thioredoxin than is the wild type enzyme and the polymerase-thioredoxin complex has reduced processivity. The exonuclease activity of the altered polymerase is not stimulated to the same extent as that of the wild type enzyme by thioredoxin. The observed dissociation constant of the gene 5 protein K(300,302,304)E-thioredoxin complex is 7-fold higher than that of the wild type complex. The altered polymerase also has a lower binding affinity for double-stranded DNA. T7 gene 5 DNA polymerase forms a complex with Escherichia coli thioredoxin (its processivity factor), and a 76-amino acid sequence (residues 258-334), unique to gene 5 protein, has been implicated in this interaction. We have examined the effect of amino acid substitution(s) in this region on T7 phage growth and on the interaction of the polymerase with thioredoxin. Among the mutations in gene 5, we found that a substitution of either Glu or Ala for Lys-302 yielded a protein that could not complement T7 phage lacking gene 5 (T7Δ5) to grow on E. coli having reduced thioredoxin levels. One triple mutant (K300E,K302E,K304E) could not support the growth of T7Δ5 even in wild type cells. This altered polymerase is stimulated 4-fold less by thioredoxin than is the wild type enzyme and the polymerase-thioredoxin complex has reduced processivity. The exonuclease activity of the altered polymerase is not stimulated to the same extent as that of the wild type enzyme by thioredoxin. The observed dissociation constant of the gene 5 protein K(300,302,304)E-thioredoxin complex is 7-fold higher than that of the wild type complex. The altered polymerase also has a lower binding affinity for double-stranded DNA. INTRODUCTIONProtein-protein interactions are essential for the coordination of the multiple reactions that occur at a replication fork. Although the replication machinery of bacteriophage T7 is simple in comparison to that of its host, Escherichia coli, specific interactions among the relatively few proteins are important (1Richardson C.C. Cell. 1983; 33: 315-317Abstract Full Text PDF PubMed Scopus (98) Google Scholar, 2Debyser Z. Tabor S. Richardson C.C. Cell. 1994; 77: 157-166Abstract Full Text PDF PubMed Scopus (79) Google Scholar, 3Kim Y.T. Richardson C.C. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 10173-10177Crossref PubMed Scopus (58) Google Scholar). In fact, the economy of proteins involved in T7 DNA replication has made it an attractive model for dissecting their interactions. An essential component of the T7 replisome is the T7 DNA polymerase, the 80-kDa product of gene 5 of the phage. Gene 5 protein physically interacts with the hexameric gene 4 protein of the phage, a protein that provides both helicase and primase activity at the replication fork to coordinate both leading and lagging strand synthesis (2Debyser Z. Tabor S. Richardson C.C. Cell. 1994; 77: 157-166Abstract Full Text PDF PubMed Scopus (79) Google Scholar, 4Nakai H. Richardson C.C. J. Biol. Chem. 1988; 263: 9831-9839Abstract Full Text PDF PubMed Google Scholar, 5Romano L.J. Richardson C.C. J. Biol. Chem. 1979; 254: 10483-10489Abstract Full Text PDF PubMed Google Scholar, 6Notarnicola S.M. Park K. Griffith J.D. Richardson C.C. J. Biol. Chem. 1995; 270: 20215-20224Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar). Both the gene 5 and gene 4 proteins in turn interact with the T7 gene 2.5 protein, a single-stranded DNA (ssDNA) 1The abbreviations used are:sssingle-strandeddsdouble-strandedPCRpolymerase chain reactionPAGEpolyacrylamide gel electrophoresisDTT1,4-dithiothreitolBSAbovine serum albuminbpbase pair(s).-binding protein that stimulates both polymerase and primase activities (4Nakai H. Richardson C.C. J. Biol. Chem. 1988; 263: 9831-9839Abstract Full Text PDF PubMed Google Scholar, 7Scheringer E. Liftin G. Jost E. Mol. Gen. Genet. 1973; 123: 247-262Crossref PubMed Scopus (40) Google Scholar, 8Kim Y.T. Tabor S. Churchich J.E. Richardson C.C. J. Biol. Chem. 1992; 267: 15032-15040Abstract Full Text PDF PubMed Google Scholar). Thus, all three of these phage-encoded proteins physically interact with one another.In addition to the interactions of the T7 DNA polymerase with the other two phage-encoded proteins, the gene 5 protein also physically interacts with one host protein, E. coli thioredoxin (9Tabor S. Huber H.E. Richardson C.C. J. Biol. Chem. 1987; 262: 16212-16223Abstract Full Text PDF PubMed Google Scholar, 10Modrich P. Richardson C.C. J. Biol. Chem. 1975; 250: 5508-5514Abstract Full Text PDF PubMed Google Scholar, 11Modrich P. Richardson C.C. J. Biol. Chem. 1975; 250: 5515-5522Abstract Full Text PDF PubMed Google Scholar, 12Mark D.F. Richardson C.C. Proc. Natl. Acad. Sci. U. S. A. 1976; 73: 780-781Crossref PubMed Scopus (175) Google Scholar). Thioredoxin forms a stable one-to-one complex with T7 gene 5 protein with an apparent dissociation constant of 5 nM (13Huber H.E. Russel M. Model P. Richardson C.C. J. Biol. Chem. 1986; 261: 15006-15012Abstract Full Text PDF PubMed Google Scholar). The consequence of the interaction is to convert gene 5 protein from a polymerase with low processivity of polymerization of nucleotides to one of high processivity (9Tabor S. Huber H.E. Richardson C.C. J. Biol. Chem. 1987; 262: 16212-16223Abstract Full Text PDF PubMed Google Scholar, 10Modrich P. Richardson C.C. J. Biol. Chem. 1975; 250: 5508-5514Abstract Full Text PDF PubMed Google Scholar, 11Modrich P. Richardson C.C. J. Biol. Chem. 1975; 250: 5515-5522Abstract Full Text PDF PubMed Google Scholar, 12Mark D.F. Richardson C.C. Proc. Natl. Acad. Sci. U. S. A. 1976; 73: 780-781Crossref PubMed Scopus (175) Google Scholar). The increased processivity arises as a result of the 80-fold greater affinity of the DNA polymerase-thioredoxin complex for a primer-template (13Huber H.E. Russel M. Model P. Richardson C.C. J. Biol. Chem. 1986; 261: 15006-15012Abstract Full Text PDF PubMed Google Scholar, 14Huber H.E. Tabor S. Richardson C.C. J. Biol. Chem. 1987; 262: 16224-16232Abstract Full Text PDF PubMed Google Scholar). T7 gene 5 protein also has a 3′ to 5′ exonuclease activity that is active on both ssDNA and double-stranded DNA (dsDNA) (9Tabor S. Huber H.E. Richardson C.C. J. Biol. Chem. 1987; 262: 16212-16223Abstract 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). Thioredoxin stimulates activity on dsDNA but the activity on ssDNA is not affected. The enhanced activity on dsDNA is most likely due to a higher affinity of the polymerase-thioredoxin complex to the 3′-termini, resulting in increased processivity of hydrolysis (14Huber H.E. Tabor S. Richardson C.C. J. Biol. Chem. 1987; 262: 16224-16232Abstract Full Text PDF PubMed Google Scholar).Studies on both E. coli thioredoxin and T7 gene 5 protein have provided information on the domains in each protein that are important for the interactions of the two proteins. Thioredoxin, a 12-kDa protein that contains an active center consisting of two cysteine residues (Cys-32 and Cys-35) that can form a disulfide bridge, provides the reducing power for a number of reactions in E. coli (16Holmgren A. Curr. Top. Cell. Regul. 1981; 19: 47-76Crossref PubMed Scopus (75) Google Scholar, 17Holmgren A. Annu. Rev. Biochem. 1985; 54: 237-271Crossref PubMed Google Scholar, 18Holmgren A. Bjornstedt M. Methods Enzymol. 1995; 252: 199-208Crossref PubMed Scopus (813) Google Scholar). However, the active-center cysteines are not essential for the interaction of thioredoxin and gene 5 protein, since genetically altered thioredoxins in which either one or both Cys have been replaced with Ser, form a functional complex with gene 5 protein and support T7 phage growth (13Huber H.E. Russel M. Model P. Richardson C.C. J. Biol. Chem. 1986; 261: 15006-15012Abstract Full Text PDF PubMed Google Scholar). Other studies have implicated a group of residues (Gly-33, Pro-34, Ile-75, Pro-76, Gly-92, and Ala-93) that form a hydrophobic surface as being involved in interactions with a number of other proteins (17Holmgren A. Annu. Rev. Biochem. 1985; 54: 237-271Crossref PubMed Google Scholar, 19Eklund H. Cambillau C. Sjöberg B.-M. Holmgren A. Jörnvall H. Höög J.-O. Brändén C.I. EMBO J. 1984; 3: 1443-1449Crossref PubMed Scopus (158) Google Scholar). The three-dimensional structures of both oxidized (20LeMaster D.M. Katti S.K. J. Mol. Biol. 1990; 212: 167-184Crossref PubMed Scopus (532) Google Scholar) and reduced thioredoxin (21Dyson H.J. Gippert G.P. Case D.A. Holmgren A. Wright P.E. Biochemistry. 1990; 29: 4129-4136Crossref PubMed Scopus (161) Google Scholar) are known; these residues are located in three loops formed between β-sheet 2 (β2)-α-helix 2 (α2), α3-β4, and β5-α4, and all face the same side of the thioredoxin molecule. A comparison of the structures of oxidized and reduced thioredoxin reveals that the transition of Cys-32 and Cys-35 between reduced and oxidized forms introduces a significant change in the position of Pro-34, resulting in a local conformational change around Cys-35. This conformational change could explain why only reduced thioredoxin binds to gene 5 protein (20LeMaster D.M. Katti S.K. J. Mol. Biol. 1990; 212: 167-184Crossref PubMed Scopus (532) Google Scholar). Genetic (22Himawan J.S. Richardson C.C. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 9774-9778Crossref PubMed Scopus (21) Google Scholar) and biochemical analyses (13Huber H.E. Russel M. Model P. Richardson C.C. J. Biol. Chem. 1986; 261: 15006-15012Abstract Full Text PDF PubMed Google Scholar, 23Himawan J.S. Richardson C.C. J. Biol. Chem. 1996; 271: 19999-20008Abstract Full Text Full Text PDF PubMed Scopus (23) Google Scholar) of thioredoxin mutants that are defective in supporting T7 phage growth have shown that mutations at Cys-32, Cys-34, Gly-74, and Gly-92 affect the binding of thioredoxin to gene 5 protein, suggesting that the same hydrophobic surface of thioredoxin is also involved in its interaction with T7 gene 5 protein. Several other residues in thioredoxin have also been postulated to be involved in the interaction with gene 5 protein: Pro-34, Gly-74, and Gly-92 (13Huber H.E. Russel M. Model P. Richardson C.C. J. Biol. Chem. 1986; 261: 15006-15012Abstract Full Text PDF PubMed Google Scholar, 22Himawan J.S. Richardson C.C. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 9774-9778Crossref PubMed Scopus (21) Google Scholar); recently, it has been proposed that the active-site cysteines, Gly-74, and Gly-92 define part of the thioredoxin surface that contacts gene 5 protein (23Himawan J.S. Richardson C.C. J. Biol. Chem. 1996; 271: 19999-20008Abstract Full Text Full Text PDF PubMed Scopus (23) Google Scholar, 24Yang X. Richardson C.C. J. Biol. Chem. 1996; 271: 24207-24212Abstract Full Text Full Text PDF PubMed Scopus (12) Google Scholar).Although the three-dimensional structure of T7 DNA polymerase is not known, it shares a high degree of homology to the large fragment of E. coli DNA polymerase I (25Ollis D.S. Kline C. Steitz T.A. Nature. 1985; 313: 818-819Crossref PubMed Scopus (88) Google Scholar), whose crystal structure is known (26Ollis D.L. Brick P. Hamlin R. Xuong N.G. Steitz T.A. Nature. 1985; 313: 726-766Google Scholar, 27Beese L.S. Derbyshire V. Steitz T.A. Science. 1993; 260: 352-355Crossref PubMed Scopus (450) Google Scholar). Furthermore, a number of studies indicate that the active sites of the enzyme are similar and that the two proteins share some structural similarities. For example the polymerase activity site of T7 DNA polymerase, like that of E. coli DNA polymerase I, resides within the carboxyl-terminal half of the molecule, while the 3′ to 5′ proofreading domain is located within the amino-terminal half of the molecule (24Yang X. Richardson C.C. J. Biol. Chem. 1996; 271: 24207-24212Abstract Full Text Full Text PDF PubMed Scopus (12) Google Scholar, 28Tabor S. Richardson C.C. J. Biol. Chem. 1989; 264: 6447-6458Abstract Full Text PDF PubMed Google Scholar). Furthermore, relatively large segments of the polypeptide chain that constitutes the polymerase active site of E. coli DNA polymerase I can be exchanged with the homologous region of T7 DNA polymerase with retention of polymerase activity (29Tabor S. Richardson C.C. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 6339-6343Crossref PubMed Scopus (300) Google Scholar).Two lines of evidence suggest that at least a portion of the thioredoxin binding domain of gene 5 protein resides within the polymerase domain. T7 DNA polymerase purified in the absence of thioredoxin is subject to proteolytic attack at three clustered sites that lie within the COOH-terminal region of the protein, and this proteolysis is inhibited by thioredoxin (9Tabor S. Huber H.E. Richardson C.C. J. Biol. Chem. 1987; 262: 16212-16223Abstract Full Text PDF PubMed Google Scholar). Mapping of these sites showed the proteolytic cleavage to reside at positions Ile-289, Lys-299, and Ala-323 of the T7 gene 5 protein (24Yang X. Richardson C.C. J. Biol. Chem. 1996; 271: 24207-24212Abstract Full Text Full Text PDF PubMed Scopus (12) Google Scholar). These results implicated the sequence Ile-289 to Ala-323 as a region that physically interacts with thioredoxin. This sequence, which does have a homologous counterpart in E. coli DNA polymerase I (30Bernad A. Blasco L. Salas M. Gene (Amst.). 1991; 100: 27-38Crossref PubMed Scopus (192) Google Scholar, 31Scarlato V. Gargano S. Gene (Amst.). 1992; 118: 109-113Crossref PubMed Scopus (15) Google Scholar), has an unusually high content of hydrophilic residues. In the structure of the large fragment of E. coli DNA polymerase I, this 76-amino acid sequence is located in a region referred to as the “thumb” that partially covers the crevice through which the DNA passes, a region that has been proposed to be involved in the interaction of the polymerase with duplex DNA (27Beese L.S. Derbyshire V. Steitz T.A. Science. 1993; 260: 352-355Crossref PubMed Scopus (450) Google Scholar). Further evidence suggesting that this unique amino acid sequence interacts with thioredoxin comes from studies on suppressor mutations that allow T7 phage to use a genetically altered thioredoxin (22Himawan J.S. Richardson C.C. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 9774-9778Crossref PubMed Scopus (21) Google Scholar). One suppressor mutation (Glu-319 → Lys) resides within this region and restores the ability of T7 DNA polymerase to interact with this particular mutant thioredoxin (23Himawan J.S. Richardson C.C. J. Biol. Chem. 1996; 271: 19999-20008Abstract Full Text Full Text PDF PubMed Scopus (23) Google Scholar).In this report we have introduced specific amino acid changes into this unique hydrophilic region of T7 gene 5 protein. We show that alteration of amino acid within this sequence results in the failure to support the growth of T7 phage due to decreased binding of the polymerase to thioredoxin and reduced processivity of the polymerase-thioredoxin complex.EXPERIMENTAL PROCEDURESMaterialsE. coli strains, Bacteriophage, and Plasmids—E. coli strains A307OmpT (HrfC, ΔtrxA307, ompt−), HMS249 (F−, opt Al, dapD4), C600, C600trxA− (trxA gene deletion), HMS157 (F−, recC22, sbcA5, endA−, gal −, thi −, sup−), HMS157trxA−, and HB101 are from the laboratory collection. Wild type bacteriophage T7 is from the laboratory collection, and mutant phages T7Δ5 (gene 5 deletion), T7trx5 (E. coli trxA gene inserted into T7 phage genome between genes 1 and 1.1) were from S. Tabor (Harvard Medical School). Plasmid pGP5-3 containing wild type T7 gene 5 under control of T7 RNA polymerase promoter was obtained from S. Tabor (Harvard Medical School) (9Tabor S. Huber H.E. Richardson C.C. J. Biol. Chem. 1987; 262: 16212-16223Abstract Full Text PDF PubMed Google Scholar). M13mGP1-2 is a 9950-base pair derivative of phage M13 containing an insert of phage T7 DNA, which expresses T7 RNA polymerase upon induction by isopropyl-1-thio-β-D-galactopyranoside (9Tabor S. Huber H.E. Richardson C.C. J. Biol. Chem. 1987; 262: 16212-16223Abstract Full Text PDF PubMed Google Scholar). Growth and manipulation of bacteriophage T7 and E. coli were performed as described (28Tabor S. Richardson C.C. J. Biol. Chem. 1989; 264: 6447-6458Abstract Full Text PDF PubMed Google Scholar, 32Studier F.W. Virology. 1969; 39: 562-574Crossref PubMed Scopus (314) Google Scholar, 33Studier F.W. J. Mol. Biol. 1975; 94: 283-295Crossref PubMed Scopus (132) Google Scholar).Nucleotides, Oligonucleotides, and DNAM13mp18 DNA and the 23-mer universal cycle sequencing primer were obtained from Amersham Life Sciences, Inc. Calf thymus DNA was from Sigma. Oligonucleotides of the xy52 series used for mutagenesis (each harbors one of the mutated amino acid codons), JH10 (5′-CCTTTAATCCTGCGGCG-3′) complementary to T7 gene 5 and Liu12 (5′-TACGACTCACTATCAGGGAG-3′) complementary to T7 RNA polymerase promoter of plasmid pGP5-3, are from the laboratory collection. Nucleoside 5′-triphosphates (dNTPs) were from Pharmacia Biotech Inc. [α-32P]dTTP (800 Ci/mmol, 1 Ci = 37 Gbq), [γ-32P]ATP (3000 Ci/mmol), [α-32P]dATP (3000 Ci/mmol), and [3H]dTTP (15 Ci/mmol) were products of DuPont NEN or Amersham Life Science, Inc.ProteinsPurified bacteriophage T7 gene 5 protein and gene 2.5 protein were obtained from S. Tabor (Harvard Medical School). T7 gene 4 proteins were provided by S. Notarnicola and B. B. Beauchamp (Harvard Medical School). E. coli thioredoxin and endonuclease AvaI are products of Amersham Life Science, Inc. Bovine serum albumin was from Miles Laboratories. Ampli-Taq® DNA polymerase was from Perkin Elmer. Polyclonal antiserum specific to gene 5 protein and to E. coli thioredoxin was from Hazelton Research Products, Inc.Other MaterialsdsDNA-cellulose was obtained from B. B. Beauchamp (Harvard Medical School). DEAE-Sephadex A-50, Sepharose CL-2B cellulose (P-11), and DE-81 filter discs were obtained from Whatman. Microspin Columns were products of Pharmacia Biotech Inc. Bio-Spin Chromatography columns, Mini-Protein ready gels, and protein silver stain kits were purchased from Bio-Rad. Protein electrophoresis standards were from Amersham Life Science, Inc. Premixed polyacrylamide solutions were from Boehringer Mannheim or National Diagnostics. 1,4-Dithiothreitol (DTT) is the product of ICN Biochemicals Inc.Methods DNA ManipulationIf not indicated specifically, DNA manipulation were performed according to the protocols described (34Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1989Google Scholar). DNA sequence analysis was performed using the method of dideoxynucleotide chain-termination (35Sanger F. Nicklen S. Coulson A.R. Proc. Natl. Acad. Sci. U. S. A. 1977; 74: 5463-5467Crossref PubMed Scopus (52347) Google Scholar) with Sequenase 2.0 (Amersham Life Science, Inc.).Site-directed MutagenesisIn vitro mutagenesis of bacteriophage T7 gene 5 was carried out by using a modified “overlap extension” method as described elsewhere (36Ho S.N. Hunt H.D. Horton R.M. Pullen J.K. Pease L.R. Gene (Amst.). 1989; 77: 51-59Crossref PubMed Scopus (6795) Google Scholar). Two partially overlapping oligonucleotide primers (xy52 series), each bearing appropriate codons corresponding to the desired amino acid residue substitution in the overlapping region, were used in the polymerase chain reaction (PCR) (37Saiki R. Scharf S.J. Faloona F. Mullis K.B. Horn H.A. Arnheim N. Science. 1985; 242: 1350-1354Crossref Scopus (6691) Google Scholar) to generate each mutant T7 gene 5 Each mutagenesis involved three separate PCR reactions. For example, two oligonucleotide primers, xy52-5 (5′-TCTCGCTGTGCCTTCTCGTTAGGCTTTTTAAAGATACC-3′) and xy52-5c (5′-GGTATCTTTAAAAAGCCTGAGAACAAGGCACAGCG-3′), were used to replace the Lys-302 (AAG) to a Glu (GAG) in gene 5 protein. The underlined codon corresponds to the amino acid residue alteration. In the first PCR, the oligonucleotide primer (xy52-5) and one upstream primer (Liu12) were used to generate a 980-base pair (bp) fragment. Another oligonucleotide primer (xy52-5c) and a downstream primer (JH10) were used in the second PCR to generate a fragment of 900 bp. The 980- and 900-bp fragments from the first two PCR were mixed and used as a template in the third PCR in the presence of the upstream (Liu12) and downstream (JH10) primers. The product from the third PCR (1800 bp) was digested with restriction enzymes MunI and HpaI, and the resulting fragment was ligated into the MunI and HpaI site of pGP5-3 giving rise to pGP5-3K302E. All the mutants were prepared in a similar way as described above. All clones were confirmed by DNA sequencing.Preparation of DNA SubstratesCircular M13 DNA to which a 25-nucleotide oligonucleotide primer was annealed was used in the polymerase and processivity assays. In the latter assay, the oligonucleotide was radioactively labeled at its 5′-terminus using polynucleotide kinase and [γ-32P]ATP (34Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1989Google Scholar). After incubation at 37°C for 30 min followed by 15 min at 70°C, the labeled oligonucleotide was annealed to M13mp18 DNA in the presence of MgCl2 (10 mM) and NaCl (100 mM). After purification using an S300 Spin-Column, the DNA was extracted with an equal volume of phenol/chloroform. The DNA was precipitated with ethanol (34Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1989Google Scholar) and dissolved in TE buffer.DNA for use in the exonuclease assay was uniformly labeled [3H]dsDNA and ssDNA. dsDNA (1,800 base pairs) was amplified by PCR using primers JH10 and Liu12 in the presence of [3H]dTTP and three other nucleoside triphosphates. The PCR products were isolated by S400 Spin-Columns and digested with endonuclease AvaI, generating a fragment of 1600 base pairs with protruding cohesive 5′ termini. After separation on an agarose gel, the desired DNA was purified by GeneClean (Bio 101, Inc.) and stored in TE buffer (specific activity 15-20 cpm/pmol). ssDNA was prepared by incubating the [3H]dsDNA with 1 M NaOH for 5 min at room temperature. The solution was neutralized by the addition of 1 M HCl and 1 mM EDTA.Generation of Phage T7Δ5trxADNA isolated from phage T7trx5 and T7Δ5 were digested with endonuclease BglII, which cleaves the T7 DNA once at position 11,515, giving rise to two DNA fragments. The shorter fragment contains T7 DNA from position 1 to 11,515, and the longer fragment contains T7 DNA from position 11,516 to the 3′-end of the molecule. The shorter fragment from T7trx5 and the longer fragment from T7Δ5 were isolated and ligated, and the ligated DNA was transfected into E. coli HMS157 harboring plasmid pGP5-3 (HMS157/pGP5-3). The transformed cells were plated on E. coli C600 harboring plasmid pGP5-3 (C600/pGP5-3). Individual phage plaques were picked, and the phages were screened for their ability to grow on HMS157trxA−/pGP5-3.Determination of Thioredoxin Production in Cells Infected with Phage T7Δ5trxAIn order to measure the production of thioredoxin in E. coli infected with phage T7Δ5trxA, E. coli C600 and C600trxA−, each harboring the plasmid pGP5-3 (C600/pGP5-3, C600trxA−/pGP5-3), were grown in LB medium (33Studier F.W. J. Mol. Biol. 1975; 94: 283-295Crossref PubMed Scopus (132) Google Scholar) at 37°C. At an A590 of 1.0, the cells were infected with wild type T7 and with T7Δ5trxA phage, at a multiplicity of infection of 5 phage. Aliquots of the culture were transferred before and after infection, and the cells were lysed in buffer containing 1.25% sodium dodecyl sulfate, 60 mM Tris-HCl, pH 6.8, 12.5% glycerol, 30 mM 2-mercaptoethanol, and 0.014% bromphenol blue. After separation by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, proteins were electroblotted onto polyvinylidene difluoride membranes and the proteins detected with polyclonal antibodies specific to gene 5 protein and E. coli thioredoxin. The results were analyzed by a densitometer.Phage Complementation AssaysPlating efficiencies of T7Δ5 and T7Δ5trxA phage were measured as follows. E. coli strains C600 or C600trxA− harboring plasmids containing wild type or one of the mutated T7 gene 5 were grown to 2 × 108 cells/ml. Various phage (either T7Δ5 or T7Δ5trxA) diluted in LB medium (0.1 ml) were mixed with 0.1 ml of E. coli cell culture and 3 ml of top agar. The mixtures were plated on LB or LB/ampicillin plates and incubated at 37°C.Overproduction and Purification of Mutant Gene 5 ProteinThe plasmid pGP5-3KE containing the mutated gene 5 was used to produce the altered gene 5 protein designated gp5K(300,302,304)E, which has three amino acid substitutions: K300E, K302E, and K304E. E. coli A307OmpT harboring the plasmid pGP5-3KE were grown at 37°C, and at an A590 of 1, gene 5 expression was induced by infection with M13mGP1-2 and the addition of isopropyl-1-thio-β-D-galactopyranoside to a concentration of 0.3 mM (9Tabor S. Huber H.E. Richardson C.C. J. Biol. Chem. 1987; 262: 16212-16223Abstract Full Text PDF PubMed Google Scholar). Three hours later, the cells were harvested by centrifugation at 10,000 × g for 10 min at 4°C. Cell pellets were washed twice with ice-cold 2-fold volumes of High EDTA Buffer (50 mM Tris-HCl, pH 7.5, 10% sucrose, 25 mM EDTA), and twice with Low EDTA Buffer (50 mM Tris-HCl, pH 7.5, 10% sucrose, 5 mM EDTA). Purification of altered gene 5 protein was performed as described (9Tabor S. Huber H.E. Richardson C.C. J. Biol. Chem. 1987; 262: 16212-16223Abstract Full Text PDF PubMed Google Scholar).Reconstitution of Gene 5 Protein-Thioredoxin ComplexThe gene 5 protein-thioredoxin complex found in T7-infected E. coli cells can be reconstituted from purified T7 gene 5 protein and E. coli thioredoxin (9Tabor S. Huber H.E. Richardson C.C. J. Biol. Chem. 1987; 262: 16212-16223Abstract Full Text PDF PubMed Google Scholar). The gene 5 protein-thioredoxin complex was reconstituted by incubation of gene 5 protein and thioredoxin in ice for 5 min at a 1:1000 molar ratio (0.1 μg of gene 5 protein:15 μg of thioredoxin). The mixture was then diluted into 40 mM Tris-HCl, pH 7.5, 5 mM DTT, 0.5 mg/ml BSA (19Eklund H. Cambillau C. Sjöberg B.-M. Holmgren A. Jörnvall H. Höög J.-O. Brändén C.I. EMBO J. 1984; 3: 1443-1449Crossref PubMed Scopus (158) Google Scholar) before use.DNA Polymerase AssayDNA polymerase activity was measured in an assay based on previously described procedures (23Himawan J.S. Richardson C.C. J. Biol. Chem. 1996; 271: 19999-20008Abstract Full Text Full Text PDF PubMed Scopus (23) Google Scholar). The primer-template used was either heat-denatured calf thymus DNA (0.1 mg/ml) or single-stranded M13 DNA annealed to a 23-mer oligonucleotide (17 nM). The reaction mixture (25-50 μl) contained, in addition to the primer-template, 40 mM Tris-HCl, pH 7.5, 10 mM MgCl2, 50 mM NaCl, 1 mM DTT, 0.1 mg/ml BSA, 0.3 mM each dATP, dGTP, dCTP, and [3H]dTTP (final specific activity is 3-4 cpm/pmol). Reactions were initiated by the addition of enzyme. After 15 min at 37°C, reactions were terminated by the addition of EDTA to 50 mM. The 3H-labeled DNA product was adsorbed onto DEAE-81 filters, and the radioactivity was measured as described (23Himawan J.S. Richardson C.C. J. Biol. Chem. 1996; 271: 19999-20008Abstract Full Text Full Text PDF PubMed Scopus (23) Google Scholar).3′ to 5′ Exonuclease Assay3′ to 5′ exonuclease activity of DNA polymerase was determined as described previously (23Himawan J.S. Richardson C.C. J. Biol. Chem. 1996; 271: 19999-20008Abstract Full Text Full Text PDF PubMed Scopus (23) Google Scholar) using uniformly labeled dsDNA or ssDNA. The reaction (50 μl) contained 40 mM Tris-HCl, pH 7.5, 10 mM MgCl2, 50 mM NaCl, 5 mM DTT, and 1200 pmol of [3H]DNA (in nucleotide equivalent). The reactions were initiated by the addition of enzyme (2 nM). After 15 min at 37°C, the reactions were terminated by the addition of EDTA to 50 mM. DNA was precipitated by the addition of 15 μl of an ice-cold solution of bovine serum albumin (10 mg/ml) and 15 μl of an ice-cold solution of trichloroacetic acid (100%). After 15 min on ice, the mixture was centrifuged for 30 min at 13,000 × g at 4°C and the acid-soluble radioactivity in the supernatant was measured by liquid scintillation counting.Processivity AssayProcessivity of nucleotide polymerization in the gene 5 protein polymerase reaction was analyzed by a method previously described (23Himawan J.S. Richardson C.C. J. Biol. Chem. 1996; 271: 19999-20008Abstract Full Text Full Text PDF PubMed Scopus (23) Google Scholar) using a circular M13 ssDNA annealed to a 5′-32P-labeled oligonucleotide primer (BCMP-57, 5′- TTTTCCCAGTCACGACGTTGTAAAACGACGGCCAGTGCCA-3′). After labeling at the 5′-end with T4 polynucleotide kinase, the oligonucleotide was annealed to M13mp18 DNA, and the primer-template was purified using a GeneClean kit (Bio 101, Inc.). The reaction mixture (25 μl) containing 20 nM primer-template and the same components described for the polymerase assay was preincubated at 37°C for 1 min, and then the reaction was initiated" @default.
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• W2066857980 title "Amino Acid Changes in a Unique Sequence of Bacteriophage T7 DNA Polymerase Alter the Processivity of Nucleotide Polymerization" @default.
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• W2066857980 cites W1486075102 @default.
• W2066857980 cites W1494109455 @default.
• W2066857980 cites W1496831544 @default.
• W2066857980 cites W1530429246 @default.
• W2066857980 cites W1532259512 @default.
• W2066857980 cites W1535710222 @default.
• W2066857980 cites W1537955742 @default.
• W2066857980 cites W1546917481 @default.
• W2066857980 cites W1551501575 @default.
• W2066857980 cites W1575228817 @default.
• W2066857980 cites W1587695639 @default.
• W2066857980 cites W1593932996 @default.
• W2066857980 cites W180833288 @default.
• W2066857980 cites W1968746934 @default.
• W2066857980 cites W1973357632 @default.
• W2066857980 cites W1976799117 @default.
• W2066857980 cites W1979699887 @default.
• W2066857980 cites W2002841189 @default.
• W2066857980 cites W2009412786 @default.
• W2066857980 cites W2012961354 @default.
• W2066857980 cites W2014976263 @default.
• W2066857980 cites W2018989186 @default.
• W2066857980 cites W2021014340 @default.
• W2066857980 cites W2021914960 @default.
• W2066857980 cites W2025735321 @default.
• W2066857980 cites W2034655946 @default.
• W2066857980 cites W2035674607 @default.
• W2066857980 cites W2041249795 @default.
• W2066857980 cites W2050551172 @default.
• W2066857980 cites W2050717506 @default.
• W2066857980 cites W2056455068 @default.
• W2066857980 cites W2060913542 @default.
• W2066857980 cites W2065014822 @default.
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• W2066857980 cites W2091172849 @default.
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• W2066857980 doi "https://doi.org/10.1074/jbc.272.10.6599" @default.
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