τ Binds and Organizes Escherichia coli Replication Proteins through Distinct Domains
2001; Elsevier BV; Volume: 276; Issue: 6 Linguagem: Inglês
10.1074/jbc.m009828200
ISSN1083-351X
AutoresDexiang Gao, Charles S. McHenry,
Tópico(s)Bacterial Genetics and Biotechnology
ResumoThe τ subunit dimerizes Escherichia coli DNA polymerase III core through interactions with the α subunit. In addition to playing critical roles in the structural organization of the holoenzyme, τ mediates intersubunit communications required for efficient replication fork function. We identified potential structural domains of this multifunctional subunit by limited proteolysis of C-terminal biotin-tagged τ proteins. The cleavage sites of each of eight different proteases were found to be clustered within four regions of the τ subunit. The second susceptible region corresponds to the hinge between domain II and III of the highly homologous δ′ subunit, and the third region is near the C-terminal end of the τ-δ′ alignment (Guenther, B., Onrust, R., Sali, A., O'Donnell, M., and Kuriyan, J. (1997) Cell 91, 335–345). We propose a five-domain structure for the τ protein. Domains I and II are based on the crystallographic structure of δ′ by Guenther and colleagues. Domains III–V are based on our protease cleavage results. Using this information, we expressed biotin-tagged τ proteins lacking specific protease-resistant domains and analyzed their binding to the α subunit by surface plasmon resonance. Results from these studies indicated that the α binding site of τ lies within its C-terminal 147 residues (domain V). The τ subunit dimerizes Escherichia coli DNA polymerase III core through interactions with the α subunit. In addition to playing critical roles in the structural organization of the holoenzyme, τ mediates intersubunit communications required for efficient replication fork function. We identified potential structural domains of this multifunctional subunit by limited proteolysis of C-terminal biotin-tagged τ proteins. The cleavage sites of each of eight different proteases were found to be clustered within four regions of the τ subunit. The second susceptible region corresponds to the hinge between domain II and III of the highly homologous δ′ subunit, and the third region is near the C-terminal end of the τ-δ′ alignment (Guenther, B., Onrust, R., Sali, A., O'Donnell, M., and Kuriyan, J. (1997) Cell 91, 335–345). We propose a five-domain structure for the τ protein. Domains I and II are based on the crystallographic structure of δ′ by Guenther and colleagues. Domains III–V are based on our protease cleavage results. Using this information, we expressed biotin-tagged τ proteins lacking specific protease-resistant domains and analyzed their binding to the α subunit by surface plasmon resonance. Results from these studies indicated that the α binding site of τ lies within its C-terminal 147 residues (domain V). nitrilotriacetic acid 3-cyclohexylamino-1-propanesulfonic acid polymerase chain reaction N-hydroxysuccinimide 1-ethyl-3-[(3-dimethylamino)propyl]-carbodiimide phenylmethylsulfonyl fluoride response units The structurally complex DNA polymerase III holoenzyme is responsible for replication of the majority of the chromosome inEscherichia coli. The polymerase of the enzyme and 3′ → 5′ exonuclease proofreading activities are contained within the heterotrimeric DNA polymerase III core (αεθ) subassembly. The holoenzyme contains seven different auxiliary subunits (β, γ, δ, δ′, τ, χ, and ψ) that confer a number of special properties requisite for replicative polymerase function (1McHenry C.S. Annu. Rev. Biochem. 1988; 57: 519-550Crossref PubMed Scopus (110) Google Scholar, 2McHenry C.S. J. Biol. Chem. 1991; 266: 19127-19130Abstract Full Text PDF PubMed Google Scholar, 3Kornberg A. J. Biol. Chem. 1988; 263: 1-4Abstract Full Text PDF PubMed Google Scholar, 4Kelman Z. O'Donnell M. Annu. Rev. Biochem. 1995; 64: 171-200Crossref PubMed Scopus (360) Google Scholar). These properties include a rapid elongation rate, high processivity, and the ability to communicate with primosomal proteins at the replication fork (5Fay P.J. Johanson K.O. McHenry C.S. Bambara R.A. J. Biol. Chem. 1981; 256: 976-983Abstract Full Text PDF PubMed Google Scholar, 6Wu C.A. Zechner E.L. Reems J.A. McHenry C.S. Marians K.J. J. Biol. Chem. 1992; 267: 4074-4083Abstract Full Text PDF PubMed Google Scholar, 7Wu C.A. Zechner E.L. Marians K.J. J. Biol. Chem. 1992; 267: 4030-4044Abstract Full Text PDF PubMed Google Scholar). The auxiliary subunits are divided between two functional assemblies: a β2 sliding clamp processivity factor, and the DnaX complex, a multiprotein ATPase that assembles the β2processivity factor onto the primer-template. Both the τ and γ subunits of the holoenzyme are expressed fromdnaX. Translation of dnaX gene yields the full-length τ subunit (71 kDa) as well as the γ subunit (47 kDa), which corresponds to the N-terminal two-thirds of the τ sequence (8McHenry C.S. Griep M.A. Tomasiewicz H. Bradley M. Molecular Mechanism in DNA Replication and Recombination. Alan R. Liss, Inc., New York1989: 115-126Google Scholar, 9Tsuchihashi Z. Kornberg A. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 2516-2520Crossref PubMed Scopus (224) Google Scholar, 10Blinkowa A.L. Walker J.R. Nucleic Acids Res. 1990; 18: 1725-1729Crossref PubMed Scopus (179) Google Scholar, 11Flower A.M. McHenry C.S. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 3713-3717Crossref PubMed Scopus (202) Google Scholar). The γ subunit results from −1 translational frameshifting to a frame with an early stop codon. The τ subunit plays central roles in the structure and function of the holoenzyme. Interactions between the τ and α subunits result in the formation of a dimeric DNA polymerase III′ (αεθ)2τ2(12McHenry C.S. J. Biol. Chem. 1982; 257: 2657-2663Abstract Full Text PDF PubMed Google Scholar, 13Studwell-Vaughan P.S. O'Donnell M. J. Biol. Chem. 1993; 268: 11785-11791Abstract Full Text PDF PubMed Google Scholar). This dimeric polymerase effectively couples synthesis of the leading and lagging strands (12McHenry C.S. J. Biol. Chem. 1982; 257: 2657-2663Abstract Full Text PDF PubMed Google Scholar, 14Kim S. Dallmann H.G. McHenry C.S. Marians K.J. J. Biol. Chem. 1996; 271: 21406-21412Abstract Full Text Full Text PDF PubMed Scopus (94) Google Scholar). The τ subunit binds tightly to α, but the shorter dnaX translation product (γ) does not. This observation suggested that the C-terminal portion unique to τ is critical for its interactions with the α subunit. Indeed, the α subunit and C-τ, an OmpT proteolytic fragment corresponding to the 215 C-terminal residues of τ, bind with a 1:1 stoichiometry (15Dallmann H.G. Kim S. Marians K.J. McHenry C.S. J. Biol. Chem. 2000; 275: 15512-15519Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar). Interactions between the τ subunit and DnaB helicase (DnaB) are critical for rapid movement at the replication fork (16Kim S. Dallmann H.G. McHenry C.S. Marians K.J. Cell. 1996; 84: 643-650Abstract Full Text Full Text PDF PubMed Scopus (303) Google Scholar, 17Yuzhakov A. Turner J. O'Donnell M. Cell. 1996; 86: 877-886Abstract Full Text Full Text PDF PubMed Scopus (157) Google Scholar). In systems using the reconstituted DNA polymerase III holoenzyme, τ subunit−DnaB interactions stimulate the rate of helicase unwinding more than 10-fold to levels approaching the rate of fork progression invivo. The C-terminal region found in τ but lacking in γ has been implicated in replication fork function. The C-τ fragment was shown to interact with DnaB and to effectively couple the leading strand polymerase with DnaB helicase at the replication fork (15Dallmann H.G. Kim S. Marians K.J. McHenry C.S. J. Biol. Chem. 2000; 275: 15512-15519Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar). The τ subunits bind γδδ′χψ to form the DnaX complex, τ2γ1(δδ′χψ) (18Glover B.P. McHenry C.S. J. Biol. Chem. 2000; 275: 3017-3020Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar, 19Pritchard A.E. Dallmann H.G. Glover B.P. McHenry C.S. EMBO J. 2000; 19: 6536-6545Crossref PubMed Google Scholar). The τ subunit also serves as a bridge between α and a χ-SSB interaction, strengthening the holoenzyme interactions with the single-stranded DNA-binding protein-coated lagging strand at the replication fork (20Kelman Z. Yuzhakov A. Andjelkovic J. O'Donnell M. EMBO J. 1998; 17: 2436-2449Crossref PubMed Scopus (154) Google Scholar,21Glover B.P. McHenry C.S. J. Biol. Chem. 1998; 273: 23476-23484Abstract Full Text Full Text PDF PubMed Scopus (129) Google Scholar). As part of the elongation complex, τ protects β2from removal by exogenous γ complex, increasing the processivity of the replicase to the megabase range (22Kim S. Dallmann H.G. McHenry C.S. Marians K.J. J. Biol. Chem. 1996; 271: 4315-4318Abstract Full Text Full Text PDF PubMed Scopus (37) Google Scholar). Clearly, τ mediates its functions through interactions with other subunits. To identify distinct structural domains that might mediate these multiple interactions, we performed limited proteolytic digestion of recombinant, biotin-tagged τ. Based on these findings, we constructed plasmids encoding truncated τ fusion proteins lacking one or more putative structural domains. The relative binding of each resultant purified fusion protein to the α subunit was determined by surface plasmon resonance. These studies enabled the identification of domain V (147 C-terminal amino acid residues) as the α subunit-binding domain of τ. E. coli DH5α and HB101 were used for initial molecular cloning procedures and plasmid propagation. E. coli BL21 and BL21(λ DE3) were employed for protein expression. All proteases were purchased from Roche Molecular Biochemicals or Sigma. d-Biotin was purchased from Sigma. SDS-polyacrylamide gel electrophoresis protein standards were obtained from Amersham Pharmacia Biotech, and prestained molecular mass markers were from Bio-Rad or Life Technologies, Inc. Ni2+-NTA1 resin, QIAquick Gel extraction kits, QIAquick PCR purification kits, and plasmid preparation kits were purchased from Qiagen (Valencia, CA). The Coomassie Plus Protein Assay Reagent and ImmunoPure Streptavidin are vended by Pierce. CM5 sensor chips (research grade), P-20 surfactant, NHS, EDC, and ethanolamine hydrochloride were obtained from BIAcore, Inc. (Piscataway, NJ). The N- and C-terminal fusion vectors pPA1-N0 and pPA1-C0 were constructed as previously described (23Kim D.R. McHenry C.S. J. Biol. Chem. 1996; 271: 20690-20698Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar). The fusion peptides contained a short 13-amino acid biotinylation sequence, a hexahistidine sequence, and a thrombin cleavage site. The induced fusion proteins are under the control of either the T7 promoter of pET-11C vector (Novagen, Madison, WI) or the PA1/04/03 promoter/operator (referred to as PA1). PA1 is a semi-synthetic E. coli RNA polymerase-dependent promoter containing twolac operators (23Kim D.R. McHenry C.S. J. Biol. Chem. 1996; 271: 20690-20698Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar, 24Lanzer M. Bujard H. Proc. Natl. Acad. Sci. U. S. A. 1988; 85: 8973-8977Crossref PubMed Scopus (288) Google Scholar). The dnaX gene was derived from the pRT610A plasmid in which the dnaX gene was modified at the frameshifting site. This modification results in the specific expression of the τ subunit; the alternative expression product, γ, is not encoded by this construct (25Dallmann H.G. Thimmig R.L. McHenry C.S. J. Biol. Chem. 1995; 270: 29555-29562Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar). PA1-N-Δ1τ plasmid (see Fig. 1 A) encodes the τ protein lacking only the first amino acid (methionine) with an N-terminal fusion peptide placed in frame. PA1-N-Δ1τ was generated by replacing the α-encoding gene dnaE from the vector used as the starting material (plasmid PA1-N0) with dnaX (see Fig. 1 A). Oligonucleotides 2782 and 2783 (Table I), which correspond to the codons for τ amino acids 2–10, were annealed and inserted into pBluescript (KS−) to generate pDG10. The remainder of thednaX gene sequence was from pDG50, which was derived from pRT610A via elimination of a Pst I restriction site. The 2187-base pair Nar I/Hin dIII restriction fragment from pDG50 was ligated to a similarly digested pDG10 vector to generate pDG100. The 2209-base pair Pst I/Sph I fragment from pDG100 replaced the corresponding fragment in the N-terminal fusion peptide-containing vector pPA1-N0 to yield pPA1-N-Δ1τ.Table IOligonucleotides used for construction of τ deletion fusion proteinsOligonucleotideUseSequence1-aThe underlined sequences are complementary to portions of the dnaX gene.2782N-Δ1τAGCTTGCTGTGGGCGCCATTTACGAGCCAGAACCTGGTAGCTCTGCA2783N-Δ1τGAGCTACCAGGTTCTGGCTCGTAAATGGCGCCCACAGCA3321C(0)τGGCGAAGCTAGCGGCAGAAGC3322C(0)τTACCAACTGCAGACTAGTAATGGGGCGGC-Δ147P1C-Δ147τTACAAVTTTVGCCTGCTGCACTTGC-Δ147P2C-Δ147τGGACTAGTCGCCTTCGGN-Δ413P1N-Δ413τAAAACTGCAGGCGGCGCGCCAGCAGTTGCN-Δ413P2N-Δ413τTGCAGACATACTGCGTTGTCGCTCTCCN-Δ496P1N-Δ496τAACTGCAGCTGAAAAAAGCGCTGGAACAN-Δ496P2N-Δ496τCTCGCATGGGGAGACCCCACAC1-a The underlined sequences are complementary to portions of the dnaX gene. Open table in a new tab Plasmid PA1-C(0)τ encodes the intact τ protein tagged with a C-terminal fusion peptide (see Fig. 1 B). TheNhe I/Pst I fragment at the C-terminal end of thednaX sequence within pRT610A was replaced with a PCR-generated fragment to produce pRT610AM, in which the stop codon was replaced with a Spe I cloning site. The 1963-base pairXba I-Spe I fragment from this plasmid was used to replace the corresponding fragment in the C-terminal fusion peptide-expressing vector pPA1-C0 to produce pPA1-C(0)τ. PCR was used to generate plasmid PA1-N-Δ413τ, which lacks the sequences encoding the N-terminal 413 amino acids of τ. Oligonucleotides N-Δ413P1 and N-Δ413P2 (Table I) were used with template pPA1-N-Δ1τ to PCR amplify a truncateddnaX fragment. N-Δ413P1 contained a Pst I site in the noncomplementary 5′ region followed by a complementary region extending from codons 414–419. N-Δ413P2 annealed to a region ofdnaX located 100 bases downstream of Nhe I site. To generate pPA1-N-Δ413τ, the amplified dnaX fragment was ligated into pPA1-N-Δ1τ following digestion of the plasmid with Pst I and Nhe I. Plasmid pET11-N-Δ496τ lacks the sequences encoding the N-terminal 496 amino acids of τ. Primers N-Δ496P1 and N-Δ496P2 were used with template pPA1-N-Δ1τ to generate a partialdna X fragment, which contained a Pst I site in the noncomplementary 5′ region and Kpn I restriction site more proximal to the 3′ end. The Kpn I restriction site was located downstream of the dnaX natural stop codon (see Fig.1 A). This PCR fragment replaced the dnaE gene inPst I/Kpn I-digested pET11-N0 (23Kim D.R. McHenry C.S. J. Biol. Chem. 1996; 271: 20690-20698Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar) to generate pET11-N-Δ496τ. Plasmid PA1-C-Δ147τ lacks the sequences encoding the C-terminal 147 amino acids of τ. PCR primer C-Δ147P1 annealed to adnaX sequence located 146 bases upstream of theRsr II cloning site. The other PCR primer C-Δ147P2 was complementary to the dnaX from codon 494–496, followed by a noncomplementary Spe I cloning site. The two primers were used with template pRT610A to generate a partial dnaX sequence. Following Rsr II and Spe I digestion, this fragment replaced the corresponding fragment in pPA1-C(0)τ (see Fig. 1 B) to generate pPA1-C-Δ147τ. For protein expression applications, E. coli strain BL21 was transformed with plasmids PA1-C(0)τ or PA1-N-Δ1τ. E. coli strain BL21(λ DE3) was used for each of the other expression plasmids. E. coli bearing plasmid PA1-N-Δ1τ was grown at 37 °C to an optical density of 0.8 in 6 liters of F medium (26Cull M.G. McHenry C.S. Methods Enzymol. 1995; 262: 22-35Crossref PubMed Scopus (41) Google Scholar) containing 100 μg/ml ampicillin. Bacteria transformed with each of the other plasmids were grown to the same density under the same conditions, except that the volume of F medium was 2 liters. The induction process was started by the addition of isopropyl-β-d-thio-galactoside (final concentration, 1 mm). Additional ampicillin (100 μg/ml) andd-biotin (10 μm) were added to the media at the same time. After 2 h of induction, cells were harvested by centrifugation at 5860 × g for 10 min at 4 °C and resuspended in 1 ml of Tris-sucrose buffer (50 mm Tris-HCl, pH 7.5, and 10% sucrose)/g of cells. Cells were quickly frozen in liquid N2 and stored at −80 °C. BL21 cells containing expression plasmids PA1-C(0)τ or PA1-N-Δ1τ were lysed in the presence of lysozyme (2 mg/g of cells), 2 mm EDTA, 5 mm benzamidine, and 1 mm PMSF for 1 h on ice followed by a 4-min incubation at 37 °C (26Cull M.G. McHenry C.S. Methods Enzymol. 1995; 262: 22-35Crossref PubMed Scopus (41) Google Scholar). For BL21(λ DE3) cells containing expression plasmids PA1-C-Δ147τ, PA1-N-Δ413τ, or pET11-N-Δ496τ, the lysis procedure was modified by increasing the concentrations of lysozyme (2.5 mg/g of cells) and EDTA (5 mm) and by extending the heat treatment step to 6 min at 37 °C. Lysates were centrifuged at 23,300 ×g at 4 °C for 1 h to remove debris. For purification of N-Δ1τ, 0.226 g of ammonium sulfate was added to each milliliter of the resulting supernatant and precipitant was collected by centrifugation at 23,300 × g at 4 °C for 1 h. Protein pellets were resuspended to ∼30 mg protein/ml in Buffer L (50 mm sodium phosphate, pH 7.6, 500 mm NaCl, 10% glycerol, 0.5 mm PMSF, 0.5 mm benzamidine, and 1 mm imidazole). Ni2+-NTA resin, previously equilibrated with Buffer L, was added to the suspensions for binding. Binding was conducted at 4 °C for 2 h with gentle shaking. Slurries of Ni2+-NTA resin/τ fusion protein complexes were then packed into columns. Columns were washed with 10 column volumes of Buffer L and then with roughly 30 column volumes of Buffer W (50 mm sodium phosphate, pH 7.6, 500 mm NaCl, 20% glycerol, 0.5 mm PMSF, and 0.5 mmbenzamidine) plus 23 mm imidazole. Bound N-Δ1τ protein was eluted with 10 column volumes of a 23–150 mm imidazole gradient in Buffer W. The peak fraction eluted at about 60 mm imidazole. Wash and elution steps were performed at 4 °C. The purification procedures for C(0)τ, C-Δ147τ, N-Δ413τ, and N-Δ496τ were the same as above, except for modifications of the precipitation, binding, and washing steps for N-Δ413τ and N-Δ496τ and a simplified elution step for each of these four proteins. For N-Δ413τ and N-Δ496τ, supernatant proteins were precipitated by adding 0.36 g of ammonium sulfate to each milliliter of cell lysates. For these two fusion proteins, the imidazole concentrations in the binding and washing steps were 2 and 15 mm, respectively. The N-Δ413τ, N-Δ496τ, C(0)τ, and C-Δ147τ proteins were each eluted in single steps with 150 mm imidazole in Buffer W. Proteins were separated by overnight electrophoresis at 65 V on a 10–17.5% SDS-polyacrylamide gradient gel (0.75 × 18 × 16 cm). Gels were stained with a 0.1% solution of Coomassie Brilliant Blue R-250 in 20% methanol and 10% acetic acid and then destained in a solution of 10% methanol and 10% acetic acid. After separation by SDS-polyacrylamide gel electrophoresis, proteins were transferred onto polyvinylidene difluoride membranes at 500 mA for 3 h in 25 mmTris-HCl, 192 mm glycine, pH 8.3, 20% methanol, and 0.01% SDS. Membranes were dipped in methanol and then air-dried for 20 min. Membranes were incubated with alkaline phosphatase-conjugated streptavidin (2 μg/ml) in TBS + 0.05% Tween 20 plus 0.5% nonfat milk for 1 h at room temperature and then washed three times in TBS +0.05% Tween20. Blots were developed in a substrate solution containing nitroblue tetrazolium chloride (0.33 mg/ml) and 5-bromo-4-chloro-3′-indolylphosphate p-toluidine salt (0.165 mg/ml) in 0.1 m Tris-HCl (pH 9.5), 0.1 m NaCl, and 50 mm MgCl2. Reactions were stopped by washing the membranes with distilled water. C(0)τ digestions were carried out in 50 mm HEPES-KOH (pH 7.4), 150 mm NaCl, 10% glycerol, and 0.1 mm EDTA. Proteolytic digestions of C-Δ213τ were in 50 mm Tris-HCl (pH 7.6), 100 mm NaCl, 5% glycerol, and 10 mmMg(CH3CO2)2. At different time points, 15-μl aliquots from reaction mixtures were removed, mixed with 8 μl of stop buffer (0.18 m Tris, pH 6.8, 30% sucrose, 6% SDS, 180 mm dithiothreitol), and then immediately boiled for 2 min. Each aliquot contained 3 μg of protein. Digestion products were separated by SDS-polyacrylamide gel electrophoresis and then stained with Coomassie Brilliant Blue or transferred onto a polyvinylidene difluoride membrane for biotin blots. After digestion, the selected biotinylated fragments were purified from others by using Ni2+-NTA chromatography with the same buffers used for N-Δ1τ except that urea was added to the binding and washing buffer at 8 m final concentration. These purified biotinylated fragments were resolved by SDS-polyacrylamide gel electrophoresis and transferred onto polyvinylidene difluoride membranes in 10 mm CAPS (pH 11.0) and 10% methanol at constant current (0.4 A) for 3 h. The membranes were washed with 20% aqueous methanol and then subjected to N-terminal sequence analysis using standard Edman chemistry (James McManaman, University of Colorado Cancer Center Protein Core Laboratory). Concentrations of all purified proteins were determined by UV spectroscopy using their extinction coefficients. Concentrations of τ fusion proteins were determined by the Pierce Coomassie Plus Protein Assay Reagent according to the manufacturer's instructions. Bovine serum albumin (fat-free; Sigma) was used as a standard. Activities of τ fusion proteins were measured by their requirement for reconstitution of holoenzyme activity and by measuring DNA synthesis from a primed M13Gori template (26Cull M.G. McHenry C.S. Methods Enzymol. 1995; 262: 22-35Crossref PubMed Scopus (41) Google Scholar). Assay mixtures (25 μl) contained 500 pmol of M13Gori (as nucleotide), 165 units (40 ng) of DnaG primase, 1.6 μg of E. coli single-stranded DNA binding protein, 250 fmol each of DNA polymerase III core (αεθ), β, δδ′, χψ, and the test τ fusion protein (20–50 fmol). Reactions were performed in a buffer containing 50 mm HEPES-KOH (pH 7.5), 10% (v/v) glycerol, 100 mm potassium glutamate, 10 mmdithiothreitol, 10 mmMg(CH3CO2)2, 200 μg/ml bovine serum albumin, 0.02% (v/v) Tween 20, 48 μm dATP, 48 μm dCTP, 48 μm dGTP, 18 μm[3H]TTP (specific activity, 520 cpm/pmol TTP), and 200 μm rNTP. Assay mixtures were incubated at 30 °C for 5 min, quenched by trichloroacetic acid precipitation, and then filtered through GF/C filters (26Cull M.G. McHenry C.S. Methods Enzymol. 1995; 262: 22-35Crossref PubMed Scopus (41) Google Scholar). One unit is defined as the amount of enzyme catalyzing the incorporation of 1 pmol of dNTPs/min at 30 °C. A BIAcoreTMinstrument was used for protein binding analyses. CM5 research grade sensor chips were used for all experiments. The carboxymethyl dextran matrix of the sensor chip was activated by the NHS/EDC coupling reaction as previously described (27Olson M.W. Dallmann H.G. McHenry C.S. J. Biol. Chem. 1995; 270: 29570-29577Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar). The matrix was activated using a 220-μl injection of a mixture of 0.2 m EDC and 0.05m NHS in water to maximize the conversion of the carboxyl groups of the sensor chip matrix to NHS esters. Streptavidin and bovine IgG were sequentially captured onto the matrix by injecting over the chip in 10 mm sodium acetate (pH 4.5) buffer at 0.2 and 0.1 mg/ml, respectively. IgG was used to partially block the negatively charged carboxyl groups on the sensor chip surface. Unreacted NHS ester groups were inactivated using 1 m ethanolamine-HCl (pH 8.5). Typically, 2000 response units (RU) of streptavidin were immobilized. The biotinylated τ proteins were then injected over the immobilized streptavidin in HBS buffer (10 mm HEPES, pH 7.4, 150 mm NaCl, 3.4 mm EDTA, and 0.005% P-20 surfactant). For kinetic analyses, less than 200 RU of τ fusion protein were immobilized. Binding studies of τ to the α subunit (12.5–50 nm) were performed at 20 °C in HKGM buffer (50 mm HEPES, pH 7.4, 100 mm potassium glutamate, 10 mm Mg(CH3CO2)2, and 0.005% P-20 surfactant). A flow rate of 25 μl/min was used for kinetic analyses. Kinetic parameters were determined using the BIAevaluation 2.1 software. We constructed plasmids PA1-C(0)τ (Fig.1 A) and PA1-N-Δ1τ (Fig. 1 B), which encode C(0)τ and N-Δ1τ, respectively. Our nomenclature system for truncated fusion proteins indicates the number of terminal residues deleted following the Δ; the preceding N or C indicates the terminus from which the amino acids were deleted. The fusion peptide is located at the truncated terminus. N-Δ1τ, for example, indicates that one amino acid was deleted from the N terminus of the τ sequence and that the fusion peptide was added to the new N terminus. The expressed C(0) τ and N-Δ1τ represented ∼5% of the total cell protein, as determined by densitometric scans of Coomassie-stained gels. C(0)τ and N-Δ1τ were purified via Ni2+-NTA metal chelating chromatography (Table II). The hexahistidine sequence within the fusion peptide specifically interacts with Ni2+ cheated to the column resin. Lysates (FrI) were prepared from 21 g of C(0)τ or 50 g of N-Δ1τ expression cells. Both C(0)τ and N-Δ1τ were recovered at >85% purity after Ni2+-NTA chromatography. The activity peaks of the eluted fractions of both C(0)τ and N-Δ1τ corresponded to each of their protein peaks (data not shown). Both C(0)τ and N-Δ1τ were fully active compared with wild-type τ protein in DNA polymerization assays. C(0)τ and N-Δ1τ were the only biotinylated proteins in the corresponding eluted fractions examined by the biotin blot analysis (data not shown).Table IIPurification of C(0)τ and N-Δ1τ from overproducing cellsProtein nameFractionTotal proteinActivitySpecific activitymgunits (106)units/mg (103)C(0)τILysate300150500IIAmmonium sulfate521102200IIINi2+-NTA (1 ml)8475700N-Δ1τILysate128040320IIAmmonium sulfate220291300IIINi2+-NTA (3 ml)35206000 Open table in a new tab Limited proteolyses were performed to identify protease-sensitive interdomain hinges of the τ protein. Eight different proteases that encompass a broad spectrum of substrate specificities were tested: chymotrypsin, endoprotease Glu-C (SV8), papain, subtilisin, trypsin, thermolysin, endoprotease Asp-N, and endoprotease Lys-C. We first investigated the effects of varying the protease: C(0)τ ratios and incubation times on the observed proteolytic products. Varying incubation times distinguished the initial cleavage products and also established the differences between stable and unstable fragments. Results from a typical experiment employing chymotrypsin proteolysis are shown in Fig.2. At short incubation times, 56-, 52-, 48-, and 24-kDa products were observed along with full-length τ (Fig.3, lanes 1–3 and8–10). At longer incubation times, the 56- and 52-kDa products were diminished, whereas the 48- and 24-kDa products and several small bands (<20 kDa) became more intense (Fig. 2, lanes 4–7 and 11–14).Figure 3Biotin blots of C(0)τ and C-Δ213τ proteolysis products. After digestion, samples were boiled immediately with the addition of SDS sample buffer, resolved on 10–17.5% SDS-polyacrylamide gel, and subjected to biotin blots as described under "Experimental Procedures." A, C(0)τ was subjected to limited proteolysis with six proteases. Abbreviations used for proteases and their dilution (w/w) and digestion temperature are: C, chymotrypsin, 1:100 at 20 °C; Th, thermolysin, 1:2000 at 37 °C; S, subtilisin, 1:1500 at 20 °C; T, trypsin, 1:2000 at 37 °C; P, papain, 1:500 at 20 °C; SV, endoprotease Glu-C, 1:2000 at 37 °C. Lane 13, C(0)τ, no protease. Arrows on the left indicate the bands (38 kDa, lane 9; 30 kDa, lane 1; 22 kDa, lane1) selected for sequencing. B, C-Δ213τ was subjected to limited proteolysis with five proteases for several different times. Abbreviations used for proteases and their dilution (w/w) and digestion temperature are: Asp-N, endoprotease Asp-N, 1:500 at 37 °C; Lys-C, endoprotease Lys-C, 1:50 at 37 °C;Glu-C, endoprotease Glu-C, 1:50 at 37 °C;papain, 1:500 at 20 °C; chymtry, chymotrypsin, 1:300 at 20 °C. Arrows on the left indicate the bands (45 kDa, lane 3; 27 kDa, lane 5; 8 kDa,lane 12) selected for sequencing.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Similar experiments were performed for six proteases, and optimal time points were selected for each of them. After separation on SDS-polyacrylamide gels, the digested products were transferred to membranes. Biotin blot analyses were used to identify terminal fragments (Fig. 3 A). Several cleavage products resulted from each protease digestion. Several bands with similar mobilities were generated via digestion with different proteases, suggesting that certain regions of C(0)τ were subject to cleavage by multiple proteases. For example, bands of roughly 38 kDa were obtained by digestion with thermolysin, papain, or subtilisin. Bands migrating at ∼30 kDa were obtained with either chymotrypsin or subtilisin, and products of about 22 kDa were obtained after digestion with chymotrypsin, SV8, or papain. These observations suggested that C(0)τ contains several protease-sensitive regions. To facilitate mapping of the cleavage sites closer to the N terminus of τ, C-Δ213τ, which is equivalent to the γ protein plus the fusion peptide at its C terminus, was subjected to limited proteolysis. The conditions for each protease were optimized as described above. Products with apparent molecular masses of 45 kDa were obtained after digestion with endoproteinase Asp-N, endoproteinase Lys-C, or chymotrypsin (Fig. 3 B). These and other cleavage products vanished after longer endoproteinase Ly
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