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• W2161569491 abstract "The lagging strand of the replication fork is initially copied as short Okazaki fragments produced by the coupled activities of two template-dependent enzymes, a primase that synthesizes RNA primers and a DNA polymerase that elongates them.Gene 4 of bacteriophage T7 encodes a bifunctional primase-helicase that assembles into a ring-shaped hexamer with both DNA unwinding and primer synthesis activities. The primase is also required for the utilization of RNA primers by T7 DNA polymerase. It is not known how many subunits of the primase-helicase hexamer participate directly in the priming of DNA synthesis. In order to determine the minimal requirements for RNA primer utilization by T7 DNA polymerase, we created an altered gene 4 protein that does not form functional hexamers and consequently lacks detectable DNA unwinding activity. Remarkably, this monomeric primase readily primes DNA synthesis by T7 DNA polymerase on single-stranded templates. The monomeric gene 4 protein forms a specific and stable complex with T7 DNA polymerase and thereby delivers the RNA primer to the polymerase for the onset of DNA synthesis. These results show that a single subunit of the primase-helicase hexamer contains all of the residues required for primer synthesis and for utilization of primers by T7 DNA polymerase. The lagging strand of the replication fork is initially copied as short Okazaki fragments produced by the coupled activities of two template-dependent enzymes, a primase that synthesizes RNA primers and a DNA polymerase that elongates them.Gene 4 of bacteriophage T7 encodes a bifunctional primase-helicase that assembles into a ring-shaped hexamer with both DNA unwinding and primer synthesis activities. The primase is also required for the utilization of RNA primers by T7 DNA polymerase. It is not known how many subunits of the primase-helicase hexamer participate directly in the priming of DNA synthesis. In order to determine the minimal requirements for RNA primer utilization by T7 DNA polymerase, we created an altered gene 4 protein that does not form functional hexamers and consequently lacks detectable DNA unwinding activity. Remarkably, this monomeric primase readily primes DNA synthesis by T7 DNA polymerase on single-stranded templates. The monomeric gene 4 protein forms a specific and stable complex with T7 DNA polymerase and thereby delivers the RNA primer to the polymerase for the onset of DNA synthesis. These results show that a single subunit of the primase-helicase hexamer contains all of the residues required for primer synthesis and for utilization of primers by T7 DNA polymerase. single-stranded DNA dithiothreitol adenosine 5′-(β,γ-methylene triphosphate) polyacrylamide gel electrophoresis bovine serum albumin 2′,3′-dideoxythymidine DNA replication is mediated by a complex of proteins that assembles at the replication fork and directs the coordinated synthesis of two DNA strands. Four proteins account for the major reactions occurring at the replication fork of bacteriophage T7 as follows: the gene 5 DNA polymerase and its processivity factor,Escherichia coli thioredoxin, the gene 2.5 single-stranded DNA-binding protein, and the gene 4 primase-helicase (1Richardson C.C. Cell. 1983; 33: 315-317Abstract Full Text PDF PubMed Scopus (98) Google Scholar). The activities of these replication proteins are coordinated by their physical interactions during replication, which serve to couple the synthesis of the leading strand with that of the lagging strand (2Lee J. Chastain P.D. Kusakabe T. Griffith J.D. Richardson C.C. Mol. Cell. 1998; 1: 1001-1010Abstract Full Text Full Text PDF PubMed Scopus (124) Google Scholar). The primase-helicase is a fixture of the T7 replisome that directly contacts both the DNA polymerase and the single-stranded DNA-binding protein (2Lee J. Chastain P.D. Kusakabe T. Griffith J.D. Richardson C.C. Mol. Cell. 1998; 1: 1001-1010Abstract Full Text Full Text PDF PubMed Scopus (124) Google Scholar, 3Nakai H. Richardson C.C. J. Biol. Chem. 1986; 261: 15208-15216Abstract Full Text PDF PubMed Google Scholar, 4Notarnicola S.M. Mulcahy H.L. Lee J. Richardson C.C. J. Biol. Chem. 1997; 272: 18425-18433Abstract Full Text Full Text PDF PubMed Scopus (80) Google Scholar, 5Kim Y.T. Richardson C.C. J. Biol. Chem. 1994; 269: 5270-5278Abstract Full Text PDF PubMed Google Scholar, 6Kim Y.T. Tabor S. Churchich J.E. Richardson C.C. J. Biol. Chem. 1992; 267: 15032-15040Abstract Full Text PDF PubMed Google Scholar). A C-terminal acidic segment of the primase-helicase is required for its interaction with T7 DNA polymerase (4Notarnicola S.M. Mulcahy H.L. Lee J. Richardson C.C. J. Biol. Chem. 1997; 272: 18425-18433Abstract Full Text Full Text PDF PubMed Scopus (80) Google Scholar). This stable protein-protein interaction could correspond to an interaction between the helicase bound to the lagging strand of the replication fork and the polymerase on the leading strand. As the replication fork moves along a DNA duplex, DNA primase periodically deposits short RNA primers at specific priming sequences on the lagging strand, triggering the synthesis of Okazaki fragments that are subsequently processed to form a continuous DNA strand (7Fuller C.W. Beauchamp B.B. Engler M.J. Lechner R.L. Matson S.W. Tabor S. White J.H. Richardson C.C. Cold Spring Harb. Symp. Quant. Biol. 1983; 47: 669-679Crossref PubMed Google Scholar, 8Baker T.A. Kornberg A. DNA Replication. 2nd Ed. W. H. Freeman & Co., New York1992: 471-510Google Scholar). In most DNA replication systems, a separate primase protein transiently interacts with the DNA helicase to initiate primer synthesis on the lagging strand. In E. coli, the strength of this interaction affects the frequency of priming and thereby sets the average length of Okazaki fragments (9Tougu K. Marians K.J. J. Biol. Chem. 1996; 271: 21398-21405Abstract Full Text Full Text PDF PubMed Scopus (116) Google Scholar). The primase and helicase activities of bacteriophage T7 are fused in a single polypeptide that assembles into a ring-shaped hexamer (10Patel S.S. Hingorani M.M. J. Biol. Chem. 1993; 268: 10668-10675Abstract Full Text PDF PubMed Google Scholar, 11Notarnicola 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, 12Egelman H.H., Yu, X. Wild R. Hingorani M.M. Patel S.S. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 3869-3873Crossref PubMed Scopus (253) Google Scholar). The bifunctional primase-helicase unwinds DNA ahead of the replication fork, and it primes the discontinuous synthesis of the lagging strand of the replication fork. The short tetranucleotides synthesized by the primase domain of the primase-helicase are not extended by T7 DNA polymerase alone (13Nakai H. Richardson C.C. J. Biol. Chem. 1986; 261: 15217-15224Abstract Full Text PDF PubMed Google Scholar, 14Kusakabe T. Richardson C.C. J. Biol. Chem. 1997; 272: 12446-12453Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar, 15Scherzinger E. Lanka E. Morelli G. Seiffert D. Yuki A. Eur. J. Biochem. 1977; 72: 543-558Crossref PubMed Scopus (57) Google Scholar); they are elongated by the polymerase only if the primase-helicase is also present during primer extension. It is not known how many subunits of the hexameric primase-helicase directly participate in the priming of DNA synthesis, nor is it known how the primase-helicase stimulates primer utilization by T7 DNA polymerase. The primase-helicase protein consists of an N-terminal primase domain and C-terminal helicase domain (12Egelman H.H., Yu, X. Wild R. Hingorani M.M. Patel S.S. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 3869-3873Crossref PubMed Scopus (253) Google Scholar, 16Bird L.E. Hakansson K. Pan H. Wigley D.B. Nucleic Acids Res. 1997; 25: 2620-2626Crossref PubMed Scopus (53) Google Scholar, 17Patel S.S. Rosenberg A.H. Studier F.W. Johnson K.A. J. Biol. Chem. 1992; 267: 15013-15021Abstract Full Text PDF PubMed Google Scholar) that will separately catalyze tetraribonucleotide synthesis and DNA unwinding, respectively (16Bird L.E. Hakansson K. Pan H. Wigley D.B. Nucleic Acids Res. 1997; 25: 2620-2626Crossref PubMed Scopus (53) Google Scholar, 18Frick D.N. Baradaran K. Richardson C.C. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 7957-7962Crossref PubMed Scopus (66) Google Scholar, 19Guo S. Tabor S. Richardson C.C. J. Biol. Chem. 1999; 274: 30303-30309Abstract Full Text Full Text PDF PubMed Scopus (81) Google Scholar). However, the primase domain alone does not support the extension of primers by T7 DNA polymerase (18Frick D.N. Baradaran K. Richardson C.C. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 7957-7962Crossref PubMed Scopus (66) Google Scholar). The dual requirement for the primase-helicase and the T7 DNA polymerase during RNA-primed synthesis of DNA suggests that these proteins associate in a complex that initiates the elongation of RNA primers synthesized by the primase (20Chowdhury K. Tabor S. Richardson C.C. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 12469-12474Crossref PubMed Scopus (17) Google Scholar). The ring-shaped T7 primase-helicase catalyzes DNA unwinding by encircling one strand of DNA (12Egelman H.H., Yu, X. Wild R. Hingorani M.M. Patel S.S. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 3869-3873Crossref PubMed Scopus (253) Google Scholar, 21Yu X. Hingorani M.M. Patel S.S. Egelman E.H. Nat. Struct. Biol. 1996; 3: 740-743Crossref PubMed Scopus (116) Google Scholar) and moving in a 5′ to 3′ direction along one DNA strand while displacing the complementary strand (22Hacker K.J. Johnson K.A. Biochemistry. 1997; 36: 14080-14087Crossref PubMed Scopus (107) Google Scholar, 23Brown W.C. Romano L.J. J. Biol. Chem. 1989; 264: 6748-6754Abstract Full Text PDF PubMed Google Scholar, 24Ahnert P. Patel S.S. J. Biol. Chem. 1997; 272: 32267-32273Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar, 25Matson S.W. Richardson C.C. J. Biol. Chem. 1983; 258: 14009-14016Abstract Full Text PDF PubMed Google Scholar, 26Tabor S. Richardson C.C. Proc. Natl. Acad. Sci. U. S. A. 1981; 78: 205-209Crossref PubMed Scopus (107) Google Scholar). The vectorial movement of the protein on DNA is coupled to the hydrolysis of 2′-deoxythymidine triphosphate (dTTP) (25Matson S.W. Richardson C.C. J. Biol. Chem. 1983; 258: 14009-14016Abstract Full Text PDF PubMed Google Scholar,27Matson S.W. Tabor S. Richardson C.C. J. Biol. Chem. 1983; 258: 14017-14024Abstract Full Text PDF PubMed Google Scholar, 28Romano L.J. Richardson C.C. J. Biol. Chem. 1979; 254: 10483-10489Abstract Full Text PDF PubMed Google Scholar), and thus, the helicase is a type of molecular motor. Crystal structures of the helicase domain of the primase-helicase (29Sawaya M.R. Guo S. Tabor S. Richardson C.C. Ellenberger T. Cell. 1999; 99: 167-177Abstract Full Text Full Text PDF PubMed Scopus (245) Google Scholar, 30Singleton M.R. Sawaya M.R. Ellenberger T. Wigley D.B. Cell. 2000; 101: 589-600Abstract Full Text Full Text PDF PubMed Scopus (424) Google Scholar) revealed that the nucleotide-binding sites are located at the interfaces between subunits of the hexamer, where changes in the relative orientations of the subunits could influence the catalytic activities of the six potential active sites within the helicase. Three different relative orientations of adjacent subunits are observed in the hexameric helicase that was crystallized, and these different orientations affect the nucleotide binding properties of individual subunits (30Singleton M.R. Sawaya M.R. Ellenberger T. Wigley D.B. Cell. 2000; 101: 589-600Abstract Full Text Full Text PDF PubMed Scopus (424) Google Scholar). This conformational flexibility, together with previous biochemical and genetic data revealing the identities of functionally important residues and the cooperative behaviors of nucleotide binding and hydrolysis by the hexameric helicase, are the basis for several proposed mechanisms of DNA unwinding (30Singleton M.R. Sawaya M.R. Ellenberger T. Wigley D.B. Cell. 2000; 101: 589-600Abstract Full Text Full Text PDF PubMed Scopus (424) Google Scholar, 31Hingorani M.M. Washington M.T. Moore K.C. Patel S.S. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 5012-5017Crossref PubMed Scopus (90) Google Scholar). In these models, nucleotide hydrolysis is coupled to changes in protein conformation and DNA binding affinity that allow the protein to step along DNA, not unlike other motor proteins that travel along protein filaments in response to nucleotide hydrolysis (32Waksman G. Lanka E. Carazo J.M. Nat. Struct. Biol. 2000; 7: 20-22Crossref PubMed Scopus (22) Google Scholar, 33Lohman T.M. Thorn K. Vale R.D. Cell. 1998; 93: 9-12Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar). Some aspects of the proposed mechanisms of DNA unwinding resemble those of the bind-change mechanism of rotary catalysis proposed for the mitochondrial F1-ATPase (34Boyer P.D. Biochim. Biophys. Acta. 2000; 1458: 252-262Crossref PubMed Scopus (97) Google Scholar, 35Leslie A.G. Walker J.E. Philos. Trans. R. Soc. Lond-Biol. Sci. 2000; 355: 465-471Crossref PubMed Scopus (65) Google Scholar). There are currently few high resolution structures of primases, and none with substrates bound (36Keck J.L. Roche D.D. Lynch A.S. Berger J.M. Science. 2000; 287: 2482-2486Crossref PubMed Scopus (126) Google Scholar, 37Pan H. Wigley D.B. Struct. Fold Des. 2000; 8: 231-239Abstract Full Text Full Text PDF Scopus (63) Google Scholar, 38Huber R. Kaiser J.T. Augustin M.A. Nat. Struct. Biol. 2001; 8: 57-61Crossref PubMed Scopus (86) Google Scholar). The DNA binding and catalytic properties of the T7 primase are well characterized, making it an attractive candidate for structural analysis. Like the intact primase-helicase, a primase fragment (residues 1–271) of the T7 primase-helicase catalyzes the template-dependent synthesis of RNA oligomers at specific priming sites as follows: 5′-(G/T)(G/T)GTC-3′ (18Frick D.N. Baradaran K. Richardson C.C. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 7957-7962Crossref PubMed Scopus (66) Google Scholar, 39Frick D.N. Kumar S. Richardson C.C. J. Biol. Chem. 1999; 274: 35899-35907Abstract Full Text Full Text PDF PubMed Scopus (32) Google Scholar, 40Frick D.N. Richardson C.C. J. Biol. Chem. 1999; 274: 35889-35898Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar), making predominantly pppAC, pppACC(C/A), and pppACAC. The conserved 3′-C of the priming sites is required for primer synthesis, but it is not copied into the RNA products. The tetraribonucleotides synthesized by an isolated primase fragment are bona fide primers that can be extended by T7 DNA polymerase, provided the intact primase-helicase protein is added during primer extension (18Frick D.N. Baradaran K. Richardson C.C. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 7957-7962Crossref PubMed Scopus (66) Google Scholar). The primase domain fragment of the T7 primase-helicase is monomeric even at very high protein concentrations, and its failure to support primer utilization by T7 DNA polymerase suggested that several subunits of the hexameric primase-helicase might cooperate to deliver tetranucleotide primers to the polymerase. Such cooperation could occur either by recruiting the polymerase through protein-protein interactions or by preventing dissociation of the RNA primer from the DNA template by sequestering the primer in a stable protein-DNA complex. A long lived complex of the primase-helicase and M13 single-stranded DNA forms in the presence of ribonucleotide substrates for primer synthesis (13Nakai H. Richardson C.C. J. Biol. Chem. 1986; 261: 15217-15224Abstract Full Text PDF PubMed Google Scholar). This primase-helicase-DNA complex most likely contains the ribonucleotide product of the primase annealed to DNA and ready for elongation by T7 DNA polymerase (14Kusakabe T. Richardson C.C. J. Biol. Chem. 1997; 272: 12446-12453Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar). We wished to determine if a single subunit of the primase-helicase could provide all of the necessary DNA binding and protein contacts for priming synthesis of DNA by T7 DNA polymerase. In order to define the minimal requirements for primer utilization by T7 DNA polymerase, we have genetically engineered an altered primase-helicase that does not assemble into functional hexamers and therefore lacks DNA unwinding activity. We show that the monomeric primase physically associates with T7 DNA polymerase in a protein-DNA complex that initiates RNA-primed synthesis of DNA. The oligonucleotides used in these studies were synthesized on an Applied Biosystems model 394 DNA synthesizer and purified by high performance liquid chromatography using an HQ20 anion exchange column (Applied Biosystems) or by gel electrophoresis using 20% acrylamide, 7 m urea gels. The T7 primase-helicase and T7 DNA polymerase (a 1:1 complex of T7 gene 5 protein and E. coli thioredoxin) were purified by published procedures (41Notarnicola S.M. Richardson C.C. J. Biol. Chem. 1993; 268: 27198-27207Abstract Full Text PDF PubMed Google Scholar, 42Tabor S. Huber H.E. Richardson C.C. J. Biol. Chem. 1987; 262: 16212-16223Abstract Full Text PDF PubMed Google Scholar). A variant of T7 DNA polymerase with two amino acid substitutions in the active site of the 3′–5′-exonuclease (Asp-5 → Ala and Glu-7 → Ala; constructed by Stanley Tabor) was used for the assembly of stable protein-DNA complexes. This altered DNA polymerase has a wild-type level of DNA polymerase activity, but the exonuclease activity of the altered polymerase is reduced by a factor of 106 (43Patel S.S. Wong I. Johnson K.A. Biochemistry. 1991; 30: 511-525Crossref PubMed Scopus (472) Google Scholar). We therefore refer to this modified polymerase as exo− T7 DNA polymerase. The primase fragment (residues 1–271) of the T7 primase-helicase was purified as described previously (18Frick D.N. Baradaran K. Richardson C.C. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 7957-7962Crossref PubMed Scopus (66) Google Scholar). E. coli DH5α was obtained from Life Technologies, Inc., and E. coli BL21(DE3) was obtained from Novagen. M13 single-stranded DNA (ssDNA)1and restriction enzymes were obtained from New England Biolabs. dNTPs were purchased from Promega Corp. ATP, CTP, and all radiolabeled materials were from Amersham Pharmacia Biotech. The diribonucleotide ApC was obtained from Sigmao. The ribonucleoside triphosphate pppApC was prepared as described under “Exonuclease III Protection Assay“ below. The mutant T7 gene 4 protein, gp4ΔD2D3, has the sequence SASASG substituted for residues 368–382 of the primase-helicase, and consequently, it is nine residues shorter than the wild-type protein (Fig. 1). This amino acid substitution eliminates two α-helices (helices D2 and D3) located at the subunit interface of the helicase domain of the primase-helicase (29Sawaya M.R. Guo S. Tabor S. Richardson C.C. Ellenberger T. Cell. 1999; 99: 167-177Abstract Full Text Full Text PDF PubMed Scopus (245) Google Scholar, 30Singleton M.R. Sawaya M.R. Ellenberger T. Wigley D.B. Cell. 2000; 101: 589-600Abstract Full Text Full Text PDF PubMed Scopus (424) Google Scholar) and thus should prevent the formation of the hexamer. An expression plasmid for gp4ΔD2D3 was constructed by cloning aBsaI-AflII fragment containing the desired modifications from the plasmid m4D1 (encoding a C-terminal fragment of the gene 4 protein, provided by Leo Guo, Harvard Medical School) into an expression plasmid for the full-length primase-helicase, pETgp4A′-A (obtained from David Frick, Harvard Medical School). The resulting plasmid, pGP4ΔD2D3, is based on the pET24a vector (Novagen Inc.), and it places the gene encoding the modified primase-helicase under control of a T7 promoter with a binding site for the lac repressor, which in turn suppresses background expression of protein prior to induction. Codon 64 of the gp4ΔD2D3 coding sequence has been changed from methionine to a glycine codon in order to prevent internal initiation of translation at codon 64 (44). A 5-ml overnight culture ofE. coli BL21(DE3) cells transformed with the pGP4ΔD2D3 expression plasmid was inoculated into 1 liter of Luria-Bertani (LB) medium containing 60 μg/ml kanamycin and was shaken at 37 °C for about 6 h until the culture reached anA 600 of ∼2.0. The culture flasks were then chilled on ice for 15 min; isopropyl-β-d-thiogalactopyranoside was added to a final concentration of 0.5 mm, and the induced cultures were incubated with shaking at 10 °C for an additional 18 h. The cells did not grow during incubation at 10 °C, yet the gp4ΔD2D3 protein was expressed as a mixture of soluble and insoluble protein. The soluble protein accounted for ∼10% of the total gp4ΔD2D3 produced. Protein expression at 22 or 37 °C resulted in higher levels of gp4ΔD2D3 expression, but all of the protein was insoluble in native buffers. After overnight induction at 10 °C, the cells (25 g wet weight) were collected by centrifugation and resuspended in 120 ml of lysis buffer (100 mm Tris-HCl (pH 8.0), 500 mm NaCl, 1 mm EDTA, 1 mm DTT, and 0.1 mm phenylmethylsulfonyl fluoride). The suspended cells were lysed by sonication. The cell lysate was centrifuged at 49,000 × g for 40 min at 4 °C, and the supernatant (fraction I) was collected for purification of gp4ΔD2D3. About 90% of the gp4ΔD2D3 precipitated in the cell lysate, and no attempt was made to resuspend this insoluble material. 7.7 g of ammonium sulfate was added to 145 ml of fraction I (10% saturation), and the solution was centrifuged at 40,000 × g for 40 min at 4 °C. To the supernatant, ammonium sulfate (24.5 g, 40% saturation) was again added with stirring, and the solution was centrifuged as before. The resulting pellet containing the gp4ΔD2D3 protein (fraction II) was resuspended in 100 ml of Buffer A (20 mmTris-HCl (pH 8.0), 0.5 mm EDTA, and 0.5 mm DTT) and again centrifuged at 49,000 × g for 20 min at 4 °C to remove insoluble material. The clarified fraction II was loaded on a DEAE-Sepharose column (4.9 cm2 × 15 cm) and washed with 100 ml of Buffer A. The DEAE column was then eluted with a 400-ml gradient of NaCl (0–400 mm) in Buffer A. The fractions containing gp4ΔD2D3 were identified by SDS-PAGE and Coomassie Blue G-250 staining and then pooled (72 ml, fraction III). Fraction III was diluted to 180 ml with Buffer A to decrease the salt concentration, and then it was applied in 2 equal aliquots to a Mono Q column (0.79 cm2 × 10 cm, Amersham Pharmacia Biotech). The Mono Q column was washed with 20 ml of Buffer A and then eluted with a 120-ml gradient of NaCl (0–400 mm NaCl) in Buffer A. The fractions containing gp4ΔD2D3 were combined from both Mono Q separations (50 ml, fraction IV), diluted to 200 ml with Buffer A, and loaded onto two 5-ml Hi-Trap heparin-Sepharose columns (2 cm2 × 2.5 cm; Amersham Pharmacia Biotech) connected in series. After washing the heparin-Sepharose with 50 ml of Buffer A, the proteins were eluted with a 120-ml gradient of NaCl (0–400 mm) in Buffer A. The fractions containing gp4ΔD2D3 were combined, and the protein was precipitated by adding ammonium sulfate to 60% saturation and then resuspended in 2 ml of Buffer A (fraction V). Fraction V was loaded onto a Superdex 200 gel filtration column (5.3 cm2 × 60 cm; Amersham Pharmacia Biotech) that had been equilibrated with Buffer A containing 100 mm NaCl. gp4ΔD2D3 eluted from the gel filtration column at the position of a 66-kDa protein standard, consistent with the monomeric protein. The purified fractions containing gp4ΔD2D3 were combined (45 ml, fraction VI) and concentrated by ultrafiltration (Centriprep; Amicon Inc.) to a protein concentration of ∼20 mg/ml in Buffer A plus 100 mm NaCl. gp4ΔD2D3 purified from the soluble fraction of the cell lysate shows no signs of aggregation. The concentrated protein was used immediately or diluted 2-fold with glycerol (50% v/v final concentration) and stored at −20 °C. gp4ΔD2D3 prepared by this procedure is typically more than 95% pure, as judged by Coomassie Blue G-250 staining of the protein sample after SDS-PAGE. The purification procedure typically results in a yield of 2 mg of pure gp4ΔD2D3 per liter of culture. Native PAGE was performed in a 15% acrylamide Tris-glycine gel (Bio-Rad) with a Mini-PROTEAN II electrophoresis system (Bio-Rad). The electrophoresis buffer was 25 mm Tris-HCl, 190 mm glycine, 5 mmMgCl2, with 1 mm ATP added to stabilize the hexameric form of the primase-helicase (10Patel S.S. Hingorani M.M. J. Biol. Chem. 1993; 268: 10668-10675Abstract Full Text PDF PubMed Google Scholar, 19Guo S. Tabor S. Richardson C.C. J. Biol. Chem. 1999; 274: 30303-30309Abstract Full Text Full Text PDF PubMed Scopus (81) Google Scholar). Protein samples (10 μm monomer) in 20 mm Tris-HCl (pH 7.5), 10 mm DTT, 5 mm MgCl2, 5 mm β, γ-methylene ATP (AMP-PCP, to stabilize the hexamer), and 40% glycerol were incubated on ice for 30 min before loading on the gel and electrophoresing the samples for 4 h at 150 V at an ambient temperature of 4 °C. The proteins were visualized after electrophoresis by staining the gel with Coomassie Blue G-250. The dTTPase activity of the helicase domain of the primase-helicase is a sensitive indicator of hexamer formation (19Guo S. Tabor S. Richardson C.C. J. Biol. Chem. 1999; 274: 30303-30309Abstract Full Text Full Text PDF PubMed Scopus (81) Google Scholar) because the active site of the helicase is located at the interface between subunits of the hexamer (29Sawaya M.R. Guo S. Tabor S. Richardson C.C. Ellenberger T. Cell. 1999; 99: 167-177Abstract Full Text Full Text PDF PubMed Scopus (245) Google Scholar). Hydrolysis of dTTP was monitored using thin layer chromatography as described previously (11Notarnicola 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). The 10-μl reaction mixture contained 30 mm Tris-HCl (pH 7.5), 10 mm MgCl2, 10 mm DTT, 5 mm dTTP (including 5 μCi of [3H] dTTP), and the indicated concentrations of the wild-type primase-helicase or of gp4ΔD2D3. The hydrolysis reactions were incubated for 15 min at 30 °C and stopped by adding 5 μl of 500 mm EDTA. One microliter of the reaction was spotted on a cellulose polyethyleneimine plate (J. T. Baker Inc.), which was subsequently developed with 1m formic acid and 0.8 m LiCl for 1 h. After drying the gel, the separated substrate and products were visualized by autoradiography and quantitated by densitometry. The helicase activity of the T7 primase-helicase was measured with two different assays, either by monitoring the enzymatic separation of two DNA strands (27Matson S.W. Tabor S. Richardson C.C. J. Biol. Chem. 1983; 258: 14017-14024Abstract Full Text PDF PubMed Google Scholar) or by its ability to support DNA replication in a rolling circle DNA replication reaction (2Lee J. Chastain P.D. Kusakabe T. Griffith J.D. Richardson C.C. Mol. Cell. 1998; 1: 1001-1010Abstract Full Text Full Text PDF PubMed Scopus (124) Google Scholar). DNA strand separation activity was monitored as the dissociation of a 5′-32P-labeled 37-mer oligonucleotide (5′-TCACGACGTTGTAAAACGACGGCCAGTTTTTTTTTTT-3′) annealed to M13 ssDNA (11Notarnicola 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). The oligo(dT) sequence at the 3′ end of the oligonucleotide is not complementary to M13 ssDNA, which allows the primase-helicase to load onto the M13 DNA at the junction between single-stranded and double-stranded regions. The helicase catalyzed strand separation in a 5′–3′ direction along the circular M13 DNA template. The 10-μl reaction mixture contained 40 mmTris-HCl (pH 7.5), 10 mm MgCl2, 10 mm DTT, 50 μg/ml BSA, 5 mm dTTP, 20 ng/μl DNA substrate (described above), and the indicated concentrations of the primase-helicase. The mixture was incubated for 15 min at 23 °C, and then the reaction was stopped by adding 2 μl of 500 mm EDTA. The samples were loaded on the 15% nondenaturing polyacrylamide gel with TBE buffer (90 mm Tris-HCl, 90 mm borate, 2 mm EDTA (pH 8.0)) and electrophoresed at 300 V for 2 h at an ambient temperature of 4 °C. The amounts of radiolabeled oligonucleotide remaining annealed to the substrate or in the dissociated product strand were visualized by autoradiography. We also examined the ability of the primase-helicase to stimulate DNA synthesis by T7 DNA polymerase on a duplex DNA template in a reaction that requires DNA strand separation by the helicase (2Lee J. Chastain P.D. Kusakabe T. Griffith J.D. Richardson C.C. Mol. Cell. 1998; 1: 1001-1010Abstract Full Text Full Text PDF PubMed Scopus (124) Google Scholar). The DNA template for this assay was a circular 70-nucleotide DNA template annealed to an oligonucleotide with a 3′-hydroxyl annealed to the template and an unpaired 5′ end that facilitates the loading of the primase-helicase onto DNA (Fig. 4, inset). The sequence of this substrate was designed to identify and quantitate specifically the leading strand synthesis that is linked to the DNA unwinding by measuring the incorporation of [α-32P]dGMP into DNA. The DNA replication reaction (25 μl) contained 40 mmTris-HCl (pH 7.5), 10 mm MgCl2, 10 mm DTT, 100 μg/ml BSA, 50 mm potassium glutamate, 0.6 mm each of dATP, dCTP, dGTP, and dTTP, with 333 mCi/mmol [α-32P]dGTP, 100 nm DNA template, 80 nm T7 DNA polymerase, and the indicated concentrations of the T7 primase-helicase or gp4ΔD2D3. The reaction mixture was incubated at 30 °C, and 4-μl aliquots were removed at 1-min intervals and quenched immediately by the addition of EDTA. The amounts of DNA synthesized were measured by spotting the reaction aliquots onto DE81 filters, washing the filters 3 times with 0.3m ammonium formate (pH 8.0), and measuring the bound radioactivity by scintillation counting. Oligoribonucleotides s" @default.
• W2161569491 created "2016-06-24" @default.
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• W2161569491 date "2001-06-01" @default.
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• W2161569491 title "A Complex of the Bacteriophage T7 Primase-Helicase and DNA Polymerase Directs Primer Utilization" @default.
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• W2161569491 doi "https://doi.org/10.1074/jbc.m101470200" @default.
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