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J. Biol. Chem., Vol. 280, Issue 9, 7890-7900, March 4, 2005
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¶
From the
Replidyne, Inc., Louisville, Colorado 80027 and the Department of Biochemistry and Molecular Genetics, University of Colorado Health Sciences Center, Denver, Colorado 80262
Received for publication, October 29, 2004 , and in revised form, December 13, 2004.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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), single-stranded DNA-binding protein, a complex containing the polymerase (
) and exonuclease (
) subunits, and the essential components of the DnaX complex (
3
'). Efficient primer elongation requires the presence of , , and 3'. Pseudomonas aeruginosa can substitute completely for E. coli polymerase III in E. coli holoenzyme reconstitution assays. Pseudomonas and 3' exhibit a 10-fold lower activity relative to their E. coli counterparts in E. coli holoenzyme reconstitution assays. Although the Pseudomonas counterpart to the E. coli 
subunit was not apparent in sequence similarity searches, addition of purified E. coli 
and (components of the DnaX complex) increases the apparent specific activity of the Pseudomonas 3' complex 
10-fold and enables the reconstituted enzyme to function better under physiological salt conditions. |
INTRODUCTION |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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Extensive genetic and biochemical studies in E. coli have elucidated the functions of the critical replication proteins comprising pol III holoenzyme. pol III holoenzyme exhibits unique characteristics required for efficient replication, including high processivity, a rapid elongation rate, tolerance of physiological salt concentrations, and ability to utilize a long single-stranded template coated with SSB (7). Non-replicative polymerases cannot substitute effectively either in vitro or in vivo (8). Conditionally lethal mutations have been isolated for dnaE, dnaQ, dnaN, and dnaX genes that encode the , , , and subunits, respectively (9–13). Strains carrying a deletion of holD, the gene encoding the subunit, show chronic induction of the SOS response resulting in a temperature-sensitive phenotype (14). Knock-out mutants of holA and holB have shown that both and ' are essential for cell viability (15), validating them along with dnaE, dnaX, dnaQ, holD, and dnaN as targets for antibacterial development.
In Escherichia coli, pol III holoenzyme is composed of ten different subunits that function in concert to perform highly rapid and processive DNA chain elongation from a primed template. The subunit serves as the polymerization subunit; catalyzes a 3'-5' exonuclease activity that is necessary for proofreading. 
binds to the N-terminal region of (16, 17). Together, , , and associate tightly to form pol III (18). The subunit confers high processivity (19). It consists of a bracelet-shaped molecule that clamps around DNA, contacting the polymerase and preventing it from falling off of the template, thus ensuring high processivity (20, 21). The asymmetric DnaX complex is responsible for transferring the sliding clamp onto a primer terminus in an ATP-dependent reaction (22–27). The native holoenzyme appears to employ a DnaX complex containing two copies of the subunit and one copy of the shorter 
variant along with ancillary subunits (21') (as discussed in Ref. 7). The dnaX gene expresses two related proteins; is the full-length protein, and is a truncated version formed by frameshifting during translation of dnaX (28–30). binds the subunit of DNA polymerase III and causes it to dimerize, forming the scaffold upon which other auxiliary proteins can assemble to form a dimeric replicative complex (31, 32). and ' are required for processive elongation in addition to their role in initiation complex formation (15). forms a 1:1 heterodimeric complex with (33). binds tightly to domain III of (34), whereas alone does not bind to (35). The interaction of and is probably mediated through the conserved N-terminal region of (36). confers resistance to high salt on DNA synthesis catalyzed by holoenzyme, and this salt resistance requires the presence of SSB (37). interacts with the C terminus of SSB and enhances the binding of SSB to DNA, thereby preventing premature dissociation of SSB from the lagging strand and increasing holoenzyme processivity (38, 39).
DNA replication is central to the propagation of all bacteria. DNA replication components are highly conserved across bacterial genera and are essential to survival but are largely distinct from the human DNA replication components. To date, published work relating to DNA replication in PA has focused on either characterization of the origin of replication (40–42) or on the biophysical properties of the single-stranded binding protein (SSB) (43). No biochemical characterization of pol III holoenzyme from PA has been reported. Here we describe the reconstitution of the DNA replication elongation apparatus from PA. This system will enable us to screen for inhibitors of PA DNA replication that may possess novel antibacterial properties.
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EXPERIMENTAL PROCEDURES |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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– IN(rrn-D-rrnE)1 lexA3
(uvrD)::T rc(ompT)::K rm). The time course of expression of recombinant proteins at 37 °C following IPTG induction was compared for the strains (data not shown). The strain giving optimal protein expression was chosen for large scale growth. No deleterious effects on cell growth were observed with any of the clones following induction. Cells expressing PA recombinant proteins were grown in a 180-liter fermentor at 37 °C (or 30 °C for expression) in F broth (yeast extract, 14 g/liter; Tryptone, 8 g/liter; K2HPO4, 12 g/liter; KH2PO4, 1.2 g/liter; glucose 1%) plus 100 µg/ml ampicillin to an A600 of 0.75. The pH was maintained at 7.2 by the addition of ammonium hydroxide. Additional ampicillin was added (200 µg/ml), and expression was induced (except SSB) by addition of IPTG to 1 mM. Cells were harvested at 3 h post induction with simultaneous chilling to 14 °C in the harvest line. Cells were suspended in an equal volume of Tris-sucrose (50 mM Tris-HCl (pH 7.5), 10% sucrose) and frozen by liquid nitrogen. Cells constitutively expressing PA SSB were grown in the same conditions as described above, but without the IPTG induction step, and were harvested at stationary phase. Lysis was accomplished via creation of spheroplasts by treatment of cells with lysozyme in the presence of 10% sucrose (45). The presence of 18 mM spermidine kept the nucleoid condensed within partially disrupted cells and displaced DNA-binding proteins. Centrifugation (16,000 x g, 1 h, 4 °C) resulted in a DNA-free supernatant (Fraction I).
Activity Assays—Units are defined as picomoles of total nucleotide incorporated per minute at 30 °C. DNA synthesis activity was monitored during purification of , , and 3' using a reconstituted E. coli DNA pol III holoenzyme assay (33). M13Gori single-stranded phage DNA was used as template DNA and was purified as described previously (46). As purified PA subunits became available, these were substituted for their E. coli counterparts as indicated. Enzymes were diluted in EDB (50 mM HEPES (pH 7.5), 20% glycerol, 0.02% Nonidet P40, 0.2 mg/ml bovine serum albumin). Primase mix containing single-stranded DNA template, nucleotides, DnaG, and SSB was prepared first by incubating the following components for 20 min at 30 °C: 50 mM HEPES (pH 7.5), 20% glycerol, 0.02% Nonidet P40, 0.2 mg/ml bovine serum albumin, 13 mM Mg(OAc)2, 5 mM DTT, 0.27 mM each of rGTP, rUTP, rATP, and rCTP, 64 µM each of dATP, dCTP, dGTP, 24 µM dTTP, 60 cpm/pmol [3H]dTTP, 2.4 nM
M13Gori single-stranded DNA circles, 42 µg/ml E. coli SSB, and 1.0 µg/ml E. coli DnaG primase. Primase mix was then aliquoted and frozen for use in subsequent assays. Holoenzyme reactions were reconstituted by incubating 20 µl of primase mix with E. coli pol III (), 3', and in a 25-µl reaction volume for 5 min at 30 °C. Buffer conditions were comparable to the priming reaction except that each of the components was 25% more dilute. Incorporation of [3H]dTTP was measured by trichloroacetic acid precipitation on GF-C filters (18). For the component being assayed, the corresponding E. coli subunit was omitted, and other components were used at saturating concentrations. Enzyme titrations were performed to determine the linear range of the assay, and specific activities were calculated using points in the linear range. In some cases where it was not necessary to determine absolute incorporation rates, formation of double-stranded DNA was measured by fluorescent detection of double-stranded DNA using PicoGreen (Molecular Probes) (47). For these assays 100 RFU approximately equals 15 pmol of nucleotide incorporated. In this case, 25-µl reactions were performed in opaque 96-well plates and stopped with 25 µl of 100 mM EDTA. PicoGreen was diluted 1:150 in 10 mM Tris-HCl (pH 7.5); 150 µl was added to each well. Fluorescence emission at 538 nm was measured in a GeminiEM platereader (Molecular Dynamics) with excitation at 485 nm. DNA synthesis was measured as relative fluorescent units (RFUs).
Primer extension assays were performed on single-stranded DNA templates that had been primed with a synthetic DNA oligonucleotide, 5'-AGGCTGGCTGACCTTCATCAAGAGTAATCT-3'. The oligonucleotide primer was annealed to M13Gori single-stranded DNA circles by mixing a 1:1 molar ratio of primer to template in the presence of 0.1 M KCl and 50 mM HEPES (pH 7.5), heating to 95 °C for 4 min and slowly cooling to room temperature. Primer extension reactions were performed in a 25-µl reaction volume for 10 min in 50 mM HEPES (pH 7.5), 20% glycerol, 0.02% Nonidet P-40, 0.2 mg/ml bovine serum albumin, 10 mM Mg(OAc)2, 5 mM DTT, 0.2 mM rATP, 48 µM each of dATP, dCTP, and dGTP, 24 µM dTTP, 60 cpm/pmol [3H]dTTP, 2 nM primed template DNA, and enzyme components as indicated. Acid-precipitable 3H label was measured to determine the picomoles of DNA incorporated. Reaction temperatures are indicated in the figure legends.
Estimates of PA Protein Complex Concentrations—We assumed that the stoichiometry of PA protein assemblies was the same as their E. coli counterparts: , 2, 3', and SSB4. Protein concentrations were determined using the Coomassie Plus Protein Assay (Pierce) and a bovine serum albumin standard.
Gel Electrophoresis and Protein Analysis—SDS-PAGE analysis of , 3', and samples was performed using 4–12% acrylamide gradient pre-cast gels (Novex NuPAGE; Invitrogen) using MOPS running buffer (Invitrogen). Benchmark unstained protein molecular weight markers were used (Invitrogen). SDS-PAGE analysis of SSB samples was performed on 10–20% acrylamide Novex Tris-glycine pre-case gels (Invitrogen) using Tris-glycine running buffer (Invitrogen). Gels were stained with SimplyBlue SafeStain (Invitrogen) or with Coomassie Brilliant Blue. Comparable results were obtained with either staining method. Densitometry was performed using a Kodak Image Station 440CF. Peptide mass fingerprinting for protein identification was performed by Amprox, Inc. (Carlsbad, CA). N-terminal sequencing was performed at the Molecular Resource Center (National Jewish Medical Center) using Edman degradation.
Buffers Used in Purifications—Subscripts indicate millimolar concentration of NaCl in each buffer (i.e. A40 is A0 and 40 mM NaCl). BW is 20% glycerol, 50 mM Tris-HCl (pH 8.0), 1 mM EDTA, 1 mM EGTA, 0.1 M KOAc (pH 8.0), 0.19 g/ml ammonium sulfate (i.e. 34% ammonium sulfate saturation). A0 is 20 mM Tris-HCl (pH 8.0), 10% glycerol, 0.1 mM EDTA, 0.1 mM EGTA, and 0.5 mM DTT. B0 is 50 mM Tris-HCl (pH 7.5), 10% glycerol, 0.5 mM EDTA, and 1 mM DTT. C is 50 mM imidazole (pH 6.8), 10% glycerol, 50 mM NaCl, and 0.5 mM DTT. D is 25 mM HEPES (pH 7.5), 50 mM KCl, 10% glycerol, 0.1 mM EDTA, and 0.5 mM DTT. E0 is 25 mM Tris-HCl (pH 7.5), 10% glycerol, 0.1 mM EDTA, and 0.5 mM DTT. F is 50 mM Tris-HCl (pH 7.5), 10% glycerol, 5 mM DTT, and 0.5 mM EDTA. G100 is 25 mM Tris-HCl (pH 8.0), 10% glycerol, 0.2 mM EDTA, 1 mM DTT, and 100 mM NaCl.
Expression and Purification of —The vector pA1-CB-NdeI and the dnaQ PCR fragment were cut with NdeI and NheI, and the dnaQ PCR fragment was inserted, resulting in the plasmid pA1-PA-dnaQ. The vector pA1-CB-NcoI and the dnaE PCR fragment were cut with SpeI and PacI, and the dnaE PCR fragment was inserted, resulting in the plasmid pA1-PA-dnaE. An operon expressing dnaQ and dnaE was constructed by digesting pA1-PA-dnaE1 with HindIII and SpeI. The fragment containing the dnaE gene (3.5 kb) was inserted into pA1-PA-dnaQ that had been digested with the same two restriction enzymes, resulting in the plasmid pA1-PA-core1, containing the dnaQ gene upstream of the dnaE gene. Cells (2045 g) were grown, harvested, and lysed as described above. Ammonium sulfate (0.21 g to each initial milliliter of Fraction I; 37% saturation) was added slowly to Fraction I. The precipitate was collected by centrifugation (16,000 x g, 30 min, 4 °C). The pellet was resuspended using a Dounce homogenizer in 0.125 x Fraction I volume of BW buffer. The remaining precipitate was collected by centrifugation (16,000 x g, 45 min, 4 °C). The pellet was resuspended in buffer A0 and diluted to match the conductivity of buffer A40, forming Fraction II. Fraction II was applied to a 5-cm x 7-cm MacroPrep High S (Bio-Rad) column connected in series with a 5- x 35-cm DEAE-Sepharose Fast Flow (Amersham Biosciences) column equilibrated in A20. The columns were washed with 300 ml of buffer A20. Then the S column was disconnected, and the DEAE-column was washed with 3200 ml of buffer A40 followed by a 6.4-liter linear gradient from 40 to 400 mM NaCl. The PA complex eluted at 150 mM NaCl, and fractions with the highest specific activity were pooled to form Fraction III. Fraction III was precipitated with 50% ammonium sulfate. Fraction III pellet was resuspended in buffer A0 and was diluted to match the conductivity of buffer A30 and applied to a 2.5- x 41-cm heparin-Sepharose Fast Flow (Amersham Biosciences) column equilibrated in buffer A30. The column was washed with 17 column volumes of buffer A30, followed by a 2-liter linear gradient from 30 to 500 mM NaCl. eluted at 100 mM NaCl, and fractions with the highest specific activity were pooled to form Fraction IV.
Expression and Purification of —The vector pA1-CB-NdeI and the dnaN PCR fragment were cut with SpeI and PacI, and the dnaN PCR fragment was inserted, resulting in the plasmid pA1-PA-dnaN. Cells (40 g) were grown, harvested, and lysed as described above. Ammonium sulfate (0.197 g to each initial milliliter of Fraction I; 35% saturation) was added slowly to Fraction I, and the precipitate was removed by centrifugation (16,000 x g, 60 min, 4 °C). Ammonium sulfate was added to the resulting supernatant (0.153 g to each milliliter of 35% supernatant; 60% saturation), and the precipitate was collected by centrifugation (16,000 x g, 60 min, 4 °C). The pellet containing the 35–60% ammonium sulfate fraction was resuspended in buffer B0 diluted to match the conductivity of buffer B50, forming Fraction II. Fraction II was applied a 2.5- x 40-cm DEAE-Sepharose Fast Flow column (Amersham Biosciences) equilibrated in buffer B50. The column was washed with 400 ml of buffer B50, followed by a 2-liter linear gradient from 50 to 600 mM NaCl gradient. The subunit eluted at 75 mM NaCl, and fractions with the highest specific activity were pooled to form Fraction III. Fraction III was applied to a 5- x 20-cm hydroxyapatite column (Hypatite C, Clarkson Chemical Co.) equilibrated in buffer C. The column was washed with 800 ml of buffer C, followed by a 4-liter linear gradient from 0–200 mM potassium phosphate. The subunit eluted at 100 mM phosphate, and fractions with the highest specific activity were pooled to form Fraction IV. Fraction IV was dialyzed into buffer D for storage.
Expression and Purification of 3'—The vector pA1-CB-NcoI and the holB PCR fragment were cut with SpeI and NcoI, and the holB PCR fragment was inserted, resulting in the plasmid pA1-PA-holB. The vector pA1-CB-NcoI and the holA PCR fragment were cut with SpeI and PacI, and the holA PCR fragment was inserted, resulting in the plasmid pA1-PA-holA. The vector pA1-CB-NcoI and the dnaX PCR fragment were cut with SpeI and PacI, and the dnaX PCR fragment was inserted, resulting in the plasmid pA1-PA-dnaX. An operon expressing holB, and holA was constructed by digesting both pA1-PA-holA and pA1-PA-holB with NheI and SpeI. The fragment from pA1-PA-holA containing holA (1 kb) was then inserted into the digested pA1-PA-holB, resulting in the plasmid pA1-PA-holBA. A three-gene operon was then constructed by digesting both pA1-PA-dnaX and pA1-PA-holBA with HindIII and SpeI. The fragment of pA1-PA-dnaX containing the dnaX gene (2 kb) was then inserted into the digested pA1-PA-holBA plasmid, resulting in the plasmid pA1-PA-BAX, with the holB gene upstream, followed by holA and dnaX, respectively. Cells (400 g) were grown, harvested, and lysed as described above. Ammonium sulfate (0.197 g to each initial ml of Fraction I; 35% saturation) was added slowly to Fraction I, and the precipitate was collected by centrifugation (16,000 x g, 30 min, 4 °C). The ammonium sulfate pellet was resuspended in buffer E0 and diluted to match the conductivity of buffer E20, forming Fraction II. Fraction II was divided into three parts, and one-third was applied to 20-ml MacroPrep High S (Bio-Rad; 4- x 5-ml S Econo-Pac cartridges) connected in series to a 5- x 10.2-cm heparin-Sepharose 6 Fast Flow column (Amersham Biosciences) equilibrated in buffer E20. The columns were washed with 600 ml of buffer E20. The High S cartridges were removed, and the heparin column was washed with another 200 ml of buffer E20, followed by a 2-liter linear gradient from 20 to 1000 mM NaCl. 3' eluted at 150 mM NaCl, and fractions with the highest specific activity were pooled to form Fraction IIIA. The columns were then regenerated as per the manufacturer's recommendations, and additional Fraction II was purified, creating Fractions IIIB and IIIC. Ammonium sulfate (0.366 g/ml Fraction III; 60% saturation) was added slowly to each batch of Fraction III, and the precipitate was collected by centrifugation (16,000 x g, 30 min, 4 °C). Fraction III pellets were resuspended in buffer F0 and diluted to match the conductivity of buffer F20, and Fractions IIIA, IIIB, and IIIC were pooled and applied to a 2.5- x 20-cm DEAE-Fast Flow column (Amersham Biosciences) equilibrated in buffer F20. The column was washed with 300 ml of buffer F20, followed by a 1,000-ml linear gradient from 20 to 500 mM NaCl. 3' complex eluted at 250 mM NaCl, and fractions with the highest specific activity were pooled to form Fraction IV.
Expression and Purification of SSB—The vector pBluescript II KS (Stratagene) and the ssb PCR fragment were digested with SpeI and PstI, and the ssb PCR fragment was inserted, resulting in the plasmid pBlue-lac-Pa-ssb. Unlike the vectors used for expression of the genes described above, this vector gave constitutive expression of the recombinant gene. Cells (1000 g) were grown, harvested, and lysed as described above. Ammonium sulfate (0.136 g to each initial milliliter of Fraction I; 25% saturation) was added slowly to Fraction I, and the precipitate was collected by centrifugation (16,000 x g, 30 min, 4 °C). The ammonium sulfate pellets were resuspended in buffer G100 and diluted in buffer G0 to match the conductivity of buffer G100, forming Fraction II. Insoluble material was removed by centrifugation and Fraction II was applied to a 5- x 10-cm column packed with Q-Sepharose Fast Flow (Amersham Biosciences) and equilibrated in buffer G100. The column was washed with 1000 ml of buffer G100, followed by a 2-liter linear gradient from 100 to 1000 mM NaCl. PA SSB eluted at 320 mM NaCl, and fractions were pooled based on gel analysis of purity to form Fraction III.
Other Proteins—E. coli holoenzyme components were purified as described: pol III (44), 3' (48), (26), (49), ' (50), (46), and (33). 3' was reconstituted by mixing individual components at the specified molar ratio.
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RESULTS |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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subunit was apparent in the PA genome. Because is not essential (52), we assumed that the PA subunit would not be essential either. We found no apparent frameshifting site in the PA dnaX gene that would lead to formation of a subunit. This did not appear to be a concern, because the full-length E. coli protein can substitute for in vitro (26) and in vivo (53). In E. coli, is a binding partner to , and both associate with , , , and ' to form the DnaX complex. In PA, the holC gene, encoding , was apparent based on homology, but the holD gene, encoding , was not. The sequence of is not highly conserved across proteobacteria (36). The apparent lack of identifiable subunits in these bacteria and in PA may reflect sequence divergence rather than actual absence of the subunit. In E. coli, the reconstituted holoenzyme reaction is only moderately stimulated by the presence of (37). Thus, we expected that a functional holoenzyme could be reconstituted from PA in the absence of .
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Purification of PA —The (polymerase) and (proofreading exonuclease) subunits were co-expressed and purified as a complex. PA was purified from cells transfected with pA1-PA-core1 and induced with IPTG. In initial trial purifications, a gap-filling assay was used to monitor non-processive polymerase activity (18). In subsequent purifications, activity was monitored in a holoenzyme reconstitution assay (see "Experimental Procedures"). Both and expressed well with this system, comprising greater than 25% of the total cell protein. Unfortunately, when cells were grown and induced at 37 °C, was found largely in inclusion bodies. When growth and induction were performed at 30 °C, the yield of soluble increased 3-fold.2 Preliminary studies indicated that had exonuclease activity as expected (data not shown). The subunit from E. coli is insoluble when overproduced alone; upon co-expression with it forms a 1:1 complex that is soluble, but excess is in inclusion bodies (54). By contrast, co-expression of the - and -subunits from PA resulted in a large excess of soluble . Excess separated from the complex during purification (see below).
Initial purification attempts were complicated by the fact that exhibits a narrow pH tolerance, with optimal stability retained only at pH 7.5 or above. Exposure to pH 7.0 or below resulted in rapid loss of activity. In addition, we found that stability was markedly enhanced when EDTA or EGTA was included in the storage buffer (data not shown). Therefore the purification protocol described here was performed entirely at pH 8.0 in the presence of EDTA and EGTA.
As has been seen previously for E. coli pol III (55), PA was relatively insoluble in solutions containing ammonium sulfate. Fraction II was formed by precipitation with ammonium sulfate at 37% saturation, followed by backwashing with a 34% saturated ammonium sulfate solution. Fraction II was applied to an S-Sepharose column that was connected in series to a DEAE-Sepharose column. The S column served to remove the endogenous E. coli pol III and RNA polymerase as well as a number of other major contaminating proteins, while allowing the majority of the PA to pass through.3 The S column was then disconnected, and the DEAE-Sepharose column was developed with a linear salt gradient. The fractions showing the highest specific activity (Fig. 1, A and B, and Table III) were pooled to form Fraction III. Excess subunit eluted early in the gradient, whereas the complex eluted later. Fraction III was applied to a heparin-Sepharose column. The column was washed extensively and then developed with a linear salt gradient. The fractions showing the highest specific activity (Fig. 1, C and D, and Table III) were pooled to form Fraction IV. SDS-PAGE analysis of the purification steps showed that Fraction IV was at least 95% pure (Fig. 1E). The purified polymerase complex exhibited an : subunit stoichiometry of 1:1 based on densitometric scans of gels of Fraction IV. Purified eluted as a single peak when subjected to high resolution gel filtration with Superdex-200 resin (data not shown).
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—The subunit was purified from E. coli cells expressing the dnaN gene from PA. The crude lysate was adjusted to 35% ammonium sulfate to precipitate contaminating proteins including the E. coli pol III holoenzyme. The supernatant was then adjusted to 60% ammonium sulfate to precipitate , forming Fraction II. Fraction II was applied to a DEAE-Sepharose column; the column was washed and then developed with a linear salt gradient. The fractions showing the highest specific activity (Fig. 2, A and B, and Table III) were pooled to form Fraction III. Fraction III was applied to a hydroxyapatite column; the column was washed and then developed with a linear phosphate gradient. The fractions showing the highest specific activity (Fig. 2, C and D, and Table III) were pooled to form Fraction IV. SDS-PAGE analysis of the purification steps showed that was purified to near homogeneity (Fig. 2E).
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, , and '. Unlike DnaX complex from E. coli (56), this complex lacks the and subunits. The 3' complex4 was purified from cells transfected with pA1-PA-BAX and induced with IPTG. The crude lysate was adjusted to 35% ammonium sulfate to precipitate 3' and form Fraction II. Fraction II was applied to a MacroPrep S column connected in series to a heparin-Sepharose column. 3' passed through the S column and bound to the heparin column. The heparin column was washed and then developed with a linear salt gradient. The fractions showing the highest specific activity (Fig. 3, A and B, and Table III) were pooled to form Fraction III. Fraction III was applied to a DEAE-Sepharose column; the column was washed and then developed with a linear salt gradient. The fractions showing the highest specific activity (see Fig. 3, C and D, and Table III) were pooled to form Fraction IV. SDS-PAGE analysis of the purification steps is shown in Fig. 3E.5
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We were surprised to find that the subunit migrated with an apparent molecular mass that was almost 20 kDa higher than the expected 73 kDa. The identity of the protein was confirmed by N-terminal sequencing. Tryptic digest followed by mass spectrometry detected the expected C terminus of the protein and no additional peptides besides those encoded by the dnaX gene. Therefore, the anomalous migration of the subunit does not appear to be the result of translational readthrough or frame-shifting. The most likely explanation for the anomalous migration is that the protein has an unusually low isoelectric point (predicted pI 
4.6) resulting in reduced SDS binding under the pH conditions used in standard electrophoresis. The band migrated with a progressively lower apparent molecular mass as the pH of the gel running buffer was lowered (data not shown).
A protein of about 45 kDa co-purified with 3' activity. N-terminal sequencing showed that this protein was an N-terminal fragment of . Relative to the amount of full-length DnaX, the amount of the N-terminal fragment was between 10–30% in different preparations. In E. coli, the subunit is formed by translational frameshifting, creating an N-terminal fragment of (29). The PA dnaX gene lacks the consensus sequence thought to be responsible for the frameshift. The shorter protein is likely a proteolytic breakdown product of .6
Purification of SSB—Initial attempts to express PA SSB using an inducible pA1 promoter resulted in very low expression levels. An excellent overexpression of PA SSB using a constitutive expression vector that included substantial upstream and downstream genomic sequence flanking the ssb gene has been reported (43). Reasoning that the genomic sequence context may enhance transcription or translation and/or stabilize the mRNA transcript, we expressed PA SSB using a construct that included 178 nucleotides of upstream genomic sequence and 97 nucleotides of downstream genomic sequence. This system produced PA SSB constitutively at a level of about 10% of total cellular protein. Fraction II was prepared by precipitation of the crude lysate with 25% ammonium sulfate. The resulting Fraction II (>90% pure) was applied to a Q-Sepharose column. The column was developed with a linear salt gradient, and fractions containing SSB were pooled based on SDS-PAGE to form Fraction III (Fig. 4, A and B). The identity of the purified protein was verified by peptide mass fingerprinting.
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, , 3', and SSB were tested for their ability to reconstitute PA pol III holoenzyme activity (Fig. 5, A–D). The 8623-nucleotide single-stranded circular DNA template was primed by a single DNA oligonucleotide primer. As expected, nucleotide incorporation was completely dependent on ; under the conditions shown there was a 2- to 5-fold stimulation by each of the other components. We found that decreasing the concentration gave a concomitant increase in the dependence on both and 3' (data not shown). Using a sub-saturating amount of , the activity of each component was then measured alone and in combination with the other components (Fig. 5E). In the absence of SSB, only the combined presence of , , and 3 was sufficient to produce a signal above background. In the presence of SSB, modest levels of synthesis by subassemblies were observed ( plus or plus 3'), but a synergistic increase in synthesis was evident when all components were combined. Addition of both 3' and to gave a 70-fold increase in activity over alone, consistent with the reconstitution of a highly processive holoenzyme. SSB only stimulated the reconstituted holoenzyme elongation reaction by about 2-fold. Changing the order of addition of SSB did not change the observed activity (data not shown).
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, , , , and ' Form a Functional Complex That Co-purifies on Gel Filtration—We purified a subassembly of the PA pol III holoenzyme that contained , , , , and ' by co-lysing cells expressing with cells expressing 3'. Activity was assessed in the reconstitution assay using purified PA . The activity was purified by ammonium sulfate fractionation followed by Q-Sepharose chromatography and gel filtration (data not shown). The complex had an apparent molecular mass of 500–600 kDa based on gel filtration standards. The fact that these subunits formed a complex that was active in DNA synthesis and co-purified by gel filtration supports the functional relevance of the reconstituted system we have developed thus far. This complex was similar to the pol III* complex that has been reported from E. coli (57) except that it lacked and .
E. coli Stimulates DNA Synthesis by Minimal Reconstituted PA Replicase—In E. coli, the tightly associated and subunits of the DnaX complex play a role in supporting DNA synthesis, especially at high salt and in increasing the affinity of the DnaX complex subunits for one another (33, 37). In PA, was not apparent by sequence comparison, and therefore these studies were performed with a subassembly of only the essential , , and ' subunits. In reconstituted PA pol III holoenzyme elongation reactions in the presence of limiting 3' (10% of the amount required for saturation) E. coli was found to stimulate the reaction 10-fold (Fig. 6A). This suggested that E. coli , although quite divergent, binds to PA 3' and stimulates the replicative reaction. A large molar excess of E. coli was required to saturate the stimulatory response (ca. 100-fold), suggesting that the binding interaction was weak. Stimulation by E. coli was most evident at subsaturating concentrations of 3'. With addition of high concentrations of 3', the same levels of DNA synthesis could be obtained as in the -stimulated reactions (Fig. 6B). However, even when near saturating levels of 3' were used, the effect of E. coli could be readily observed at elevated salt concentrations where conferred an increase in salt tolerance (Fig. 6C).
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substituted on an equivalent molar basis for E. coli pol III (Fig. 7A). In contrast, when subunits were compared in the E. coli holoenzyme assay, 10-fold more PA was required to achieve activity levels comparable to that exhibited by E. coli , suggesting an impaired binding interaction between PA and other E. coli holoenzyme components (Fig. 7B). Identical results were obtained for the PA and titrations when was omitted from the reaction (data not shown). In the E. coli holoenzyme assay, 10-fold more PA 3' was required to achieve the same levels of synthesis relative to E. coli 3'. However, PA 3' could be rescued by addition of E. coli such that it functioned as well as the E. coli counterparts (Fig. 7C).
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. Equivalent levels of synthesis are obtained at nearly 10-fold lower concentrations of E. coli pol III (Fig. 8A). This difference is largely suppressed by addition of E. coli that results in stimulation of PA about 5-fold with negligible affect on E. coli pol III. Thus, as observed in the experiments reported in Figs. 6 and 7, the PA proteins are stimulated by E. coli more than the cognate system! This observation surprised us at first, because it appeared to contradict the results reported in Fig. 7A where PA and E. coli polymerases were interchangeable in the E. coli system. We explored this observation further using various combinations of PA and E. coli components and found that the critical variable was DnaX complex. Replacement of PA 3' with E. coli DnaX complex in the PA holoenzyme reconstitution assay yielded a system where E. coli and PA polymerases gave equivalent synthesis (Fig. 8A). This and the results shown in Fig. 7A argue that the difference observed is not due to contamination of PA with inactive enzyme.
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) in millimolar NaCl, the protein concentration (
) in µg/ml (x 10–1), and the polymerase activity (
) in RFU (100 RFU approximately equals 15 pmol of nucleotide incorporated). Polymerase activity was measured in the holoenzyme reconstitution assay in the presence of 3.8 µg/ml E. coli DnaX complex (
, titration of E. coli 
, titration of E. coli 
, titration of PA 