©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
DnaX Complex of Escherichia coli DNA Polymerase III Holoenzyme
THE bullet COMPLEX FUNCTIONS BY INCREASING THE AFFINITY OF AND FOR bullet` TO A PHYSIOLOGICALLY RELEVANT RANGE (*)

(Received for publication, June 2, 1995; and in revised form, August 23, 1995)

Matthew W. Olson (§) H. Garry Dallmann (¶) Charles S. McHenry (**)

From the Department of Biochemistry, Biophysics and Genetics and Graduate Program in Molecular Biology, University of Colorado Health Sciences Center, Denver, Colorado 80262

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
REFERENCES

ABSTRACT

An artificial operon that contains tandem holC-holD genes was used to overproduce a complex of the and subunits of the DNA polymerase III holoenzyme. Normally insoluble by itself, forms a tight soluble complex with . A purification procedure that yields pure, active bullet complex in 100-mg quantities suitable for biophysical studies is reported. Sedimentation equilibrium studies demonstrate that bullet is a 1:1 heterodimer. The presence of bullet dramatically lowers the level of bullet` required to reconstitute holoenzyme to levels expected in vivo. That bullet accomplishes this by binding to or and increasing their affinity for bullet` was demonstrated by surface plasmon resonance using a Pharmacia BIAcore instrument. In the absence of bullet`, bullet binds to either the or DnaX protein with K = 2 nM.


INTRODUCTION

The DNA polymerase III holoenzyme (^1)is the replicative polymerase responsible for synthesis of the Escherichia coli chromosome. Holoenzyme is composed of a DNA polymerase III core (alpha--) plus auxiliary subunits that confer the special properties expected of a replicative polymerase (for reviews, see McHenry(1991) and Kuriyan and O'Donnell(1993)). These include high processivity and the ability to communicate with primosomal proteins at the replication fork to permit coordinated replication (Wu et al., 1992a, 1992b ). The holoenzyme auxiliary subunits can be divided into two subassemblies: 1) beta forms a sliding clamp that apparently encircles DNA (Kong et al., 1992) and tethers the pol III core to the template by protein-protein interactions (LaDuca et al., 1986; Stukenberg et al., 1991); and 2) the DnaX complex sets the sliding clamp onto the template-primer (Wickner, 1976).

The DnaX clamp-setting apparatus contains either the or dnaX gene product complexed to bullet` and bullet (McHenry et al., 1986; Maki and Kornberg, 1988, Xiao et al., 1993b; Dallmann and McHenry, 1995). The and subunits are ATPases within the clamp-loading assembly (Lee and Walker, 1987; Hawker and McHenry, 1987; O'Donnell et al. 1993). Presumably, these subunits function to couple the energy achieved from ATP hydrolysis to the assembly of the beta sliding clamp. The subunit also functions to dimerize pol III by direct contact with the alpha subunit (McHenry, 1982; Studwell-Vaughan and O'Donnell, 1991). The ATPase activities of and are stimulated by the presence of bullet` or bullet (Onrust and O'Donnell, 1993; Xiao et al., 1993a), suggesting direct binding of one of the subunits. Gel filtration of mixed subunits show that and ` bind weakly to and to form a complex (Onrust and O'Donnell, 1993). In a minimal holoenzyme assembly, a strong requirement is observed for both the and ` subunits (Onrust and O'Donnell, 1993).

The and subunits have not been assigned a clear function. They were initially identified by their association with purified complex (McHenry et al., 1986; Maki and Kornberg, 1988), and it was not clear until they had been partially sequenced and their structural genes cloned that they were distinct proteins instead of proteolytic products of or ` (Xiao et al., 1993a; Carter et al., 1993a, 1993b). No requirement has been observed for bullet other than a modest stimulation of holoenzyme reconstituted with the DnaX protein in the presence of elevated levels of salt (Xiao et al., 1993b). It has been shown that and form a 1:1 complex (Xiao et al., 1993b). Gel filtration studies indicated that bullet forms a complex with or in solution and that bridges the interaction of with (Xiao et al., 1993b).

The insolubility of the subunit and its tendency to aggregate has limited its utility in physical and functional studies (Xiao et al., 1993a). required 6 M urea for all purification steps, and the resulting protein was inactive and aggregated when urea was removed. The resulting denatured purified was useful only if rapidly gel-filtered to remove urea immediately before conducting an experiment or if diluted to 0.5 M urea, clearly a complication for kinetic and biophysical experiments. Presumably as a result of the need to refold, assembly reactions proceeded slowly, typically requiring 30 min (Xiao et al., 1993a).

The discovery, cloning, overexpression, and purification of each subunit of the DnaX complexes has allowed us to study their contributions to the holoenzyme replicative reaction. We now report the purification and physical and functional characterization of the bullet complex. Exploiting an artificial operon that overproduces both and , we show that these subunits assemble in vivo to form a soluble 1:1 complex that is suitable for biophysical studies. We demonstrate that the most striking contribution of bullet to the holoenzyme reaction is its ability to bind or and increase their affinity for bullet` so that they can form a functional clamp-loading complex at physiological subunit concentrations.


EXPERIMENTAL PROCEDURES

E. coli Strains and Growth

The E. coli K-12 strain MC1061 (F`, hsd R2, mcrB1, ara D139 Dara-leu7696 (DEL), Dlac -174, gal U, gal K, rpsL, thi) containing the plasmid pMAF 310 (holC, hol D, amp (R), lacI (Q) (Carter, et. al. 1993a) was grown in a 250-liter fermentor (New Brunswick) in F-media + glucose and ampicillin at 37 °C. Cells from a growing 20-liter culture were diluted 1:10 in 180 liters of F-media in the 250-liter fermentor. F-media is composed of yeast extract (14 g/liter), tryptone (8 g/liter), K(2)HPO(4) (12 g/liter), KH(2)PO(4) (1.2 g/liter) (pH 7.2). Glucose and ampicillin are added to 1% and 50 µg/ml, respectively, at the beginning of the fermentation and at the point of induction with isopropyl-beta-D-thiogalactoside (1 mM final concentration at OD = 1.0). Three hours after induction (OD = 3.1), cells were chilled and harvested by passing the fermentation broth through cooling coils en route to a Sharples continuous flow centrifuge. The temperature of the effluent did not exceed 16 °C. Cells were resuspended with an equal volume (w/v) of cold (4 °C) Tris-sucrose (50 mM Tris-HCl (pH 7.5), 10% sucrose) and poured into liquid nitrogen as a stream. This procedure yielded 1280 g of cells.

Chromatographic Supports

Q-Sepharose, SP-Sepharose, and Sephacryl S100 were obtained from Pharmacia Biotech Inc.

Proteins

Purification of the beta subunit (Johanson et al., 1986) and of the and subunits (Dallmann et al., 1995) to homogeneity from overexpressing strains was carried out as previously described. SSB (1 mg/ml) and DnaG primase (4.2 times 10^6 units/mg) were purified from overproducing strains as described (Griep and McHenry 1989). DNA polymerase III core (alpha complex), , and ` were purified to homogeneity by methods developed in this laboratory. (^2)Bovine serum albumin (RNase-free) used in the buffer for activity assays was purchased from Intergen.

Nucleic Acids

Unlabeled and labeled nucleotides were obtained from Pharmacia and Amersham Corp., respectively. M13Gori single-stranded DNA (Kaguni and Ray, 1979) was prepared as described by Johanson et al.(1986).

DNA Polymerization Assay

One unit of bullet was defined as the amount needed to incorporate 1 pmol of (total) nucleotide/min during a 5-min incubation at 30 °C into acid-precipitable DNA under conditions where all other components were saturating. Assays contained 700 fmol of pol III core (alpha complex), 500 fmol of beta, 500 fmol of , or 500 fmol of , 600 fmol of , 600 fmol of `, and 500 fmol of bullet unless indicated otherwise. Protein concentrations were determined using their extinction coefficients. Reactions (25 µl) contained 50 mM HEPES (adjusted to pH 7.5 with KOH), 11.5% glycerol, 400 mM potassium glutamate (unless indicated otherwise), 10 mM DTT, 10 mM magnesium acetate, 542 pmol of single-stranded DNA (total nucleotide), 60 units of DnaG primase, 400 nM SSB tetramers, 48 µM dATP, dCTP, and dGTP, 18 µM [^3H]TTP (specific activity = 520 cpm/pmol TTP) and 200 µM rNTPs.

Protein Determinations

Protein was determined by the Pierce Coomassie plus protein assay reagent according to the manufacturer's instructions. Bovine serum albumin (fat-free) (Sigma) was used as the standard.

Buffers

These were buffer Q (20% glycerol, 50 mM Tris-HCl (pH 7.8), 1 mM EDTA, 5 mM DTT); buffer SP (20% glycerol, 50 mM Tris-HCl (pH 7.0), 1 mM EDTA, 5 mM DTT); buffer S (20% glycerol, 20 mM potassium phosphate (pH 7.5), 0.5 mM EDTA, 20 mM NaCl, 2.5 mM DTT); buffer HBS (10 mM HEPES-KOH (pH 7.4), 150 mM NaCl, 3.4 mM EDTA, 0.005% P-20 detergent (Pharmacia Biosensor)); and buffer HKGM (50 mM HEPES-KOH, pH 7.4, 100 mM potassium glutamate, 10 mM magnesium acetate, 0.005% P-20 detergent). Potassium glutamate (4 M) was adjusted to pH 7.5 with KOH.

SDS-Polyacrylamide Gel Electrophoresis

Proteins were separated by electrophoresis according to Laemmli(1970) on an SDS-15% polyacrylamide gel run in a Hoefer vertical gel electrophoresis apparatus for 18 h at 60 V. Protein was visualized by overnight staining with a 0.10% solution of Coomassie Brilliant Blue R-250 in 20% methanol and 10% acetic acid, and destaining in a solution of 10% methanol and 10% acetic acid.

Immobilization and Analysis of Proteins on BIAcore

Protein-protein interaction studies were performed using a Pharmacia Biosensor BIAcore instrument. CM5 research grade sensor chips (Pharmacia Biosensor) were used for all experiments. Proteins were immobilized in solutions of buffer HBS at a flow rate of 5 µl/min (20 °C). The carboxylmethyl dextran matrix of the sensor chip was activated using a 30-µl (6 min) injection of a mixture of 0.2 M 1-ethyl-3-[(3-dimethylamino)propyl]-carbodiimide and 0.05 MN-hydroxysuccinimide in water to convert the caboxyl groups of the sensor chip matrix to an N-hydroxysuccinimide ester. This ester is susceptible to nucleophilic attack by amino groups of proteins, resulting in an amide linkage of the protein to the sensor chip. The , , , and ` subunits were immobilized to activated sensor chips in buffers and at concentrations optimized for each subunit. Immobilization conditions were as follows: , 50 µg/ml, 5-µl injection, 20 mM sodium phosphate (pH 7.4); , 50 µg/ml; 10 µl-injection, 20 mM Mes (pH 6.0); , 300 µg/ml, 7-µl injection, 20 mM Mes (pH 6.0); and `, 100 µg/ml, 30-µl injection, 20 mM Mes (pH 6.0). Unreacted N-hydroxysuccinimide ester groups were quenched by a 30-µl injection of 1 M ethanolamine-HCl (pH 8.0). Under these conditions, typically 7000 response units (RU) of , 3000 RU of , and 1500 RU of and ` were immobilized. Protein-protein interaction studies were carried out in buffer HKGM at a flow rate of 5 µl/min at 20 °C.

Analytical Ultracentrifugation

Sedimentation velocity and sedimentation equilibrium experiments were performed using a Beckman model XLA analytical ultracentrifuge. All sedimentation experiments were performed at 4 °C using a Beckman four-hole An-60Ti rotor and were carried out in 1.2-cm path length double-sector cells with quartz windows. The protein absorbance was monitored at 230 nm and 280 nm. bullet complex (fraction V) was dialyzed overnight against 3 liters of 5% glycerol, 100 mM NaCl, 2 mM DTT, 20 mM Tris (pH 7.5), and 1 mM EDTA to a final concentration of 57 µM. The sample channel contained bullet in buffer (5% glycerol, 100 mM NaCl, 2 mM DTT, 20 mM Tris (pH 7.5) and 1 mM EDTA), and the blank channel contained buffer only. For sedimentation equilibrium studies, six-channel cells were used. Here, the bullet concentrations were 4 µM, 2 µM, and 1 µM, and radial absorbance scans were taken at 4-h intervals over 95 h (total) at 15,000, 25,000, and 35,000 rpm. The data were subjected to the single data set analysis model, ``IDEAL 1.'' For sedimentation velocity analysis, the bullet concentrations were 8, 4, and 2.5 µM, and scans were taken at 30-min intervals over 20 h at 40,000 rpm. Data were subjected to analysis using the second moment/boundary spreading method in the XLA-VELOC program.


RESULTS

In preliminary studies, we found that the subunit, when overproduced by itself, formed aggregates that require denaturing conditions for solubilization. O'Donnell and colleagues published a similar observation and developed a purification for that started with denatured material (Xiao et al., 1993a). They found that assays using required extensive incubations, presumably to permit proper folding and assembly of into complexes. We found that , when overproduced from an artificial operon with , forms a soluble, monodisperse complex with in vivo. We exploited the availability of this artificial operon (Carter et al., 1993a) to generate a bullet complex that could be purified intact without a requirement for denaturation and refolding.

Overproduction and Purification of bullet

Preparation of Cell Lysate and Ammonium Sulfate Precipitation

All operations used in the purification of bullet were performed at 0-4 °C. The lysate (fraction I) was prepared from 150 g of cells (300 g of cell paste) as described by Cull and McHenry(1995)) with the following exceptions: 0.258 g of ammonium sulfate for each milliliter (45% saturation) was added to the resulting supernatant, and precipitant (fraction II) was collected by centrifugation at 22,000 times g for 60 min. Densitometry of Coomassie-stained SDS-polyacrylamide gels indicated and constitute 9% and 6% of the total cellular protein (data not shown) and 7 and 4% of the total soluble protein in fraction I, respectively (Fig. 1, lane 1).


Figure 1: Purification of and as a complex. Fractions I-V were denatured and subjected to electrophoresis on a 15% SDS-polyacrylamide slab gel. Protein was detected by Coomassie Blue staining as described under ``Experimental Procedures.'' Lane 1, fraction I (cell lysate, 250 µg protein). Lane 2, fraction II (45% ammonium sulfate pellet, 200 µg protein). Lane 3, fraction III (Q-Sepharose peak, 40 µg protein). Lane 4, fraction IV (SP-Sepharose peak, 40 µg protein). Lane 5, fraction V (Sephacryl S-100 peak, 40 µg protein). The migration of marker proteins (phosphorylase B, bovine serum albumin, ovalbumin, carbonic anhydrase, soybean trypsin inhibitor and alpha-lactalbumin) is indicated on the left.



Q-Sepharose Chromatography

Fraction II (2310 mg) was dissolved in 60 ml of buffer Q and dialyzed against a total of 3 liters of buffer Q to an ionic equivalent of 20 mM NaCl (buffer Q + 20 mM NaCl). This material was loaded onto a Q-Sepharose column (750 ml; 5.8 cm times 30 cm) equilibrated with buffer Q at 0.3-column volume/h. The bullet activity eluted with an 8-column volume linear gradient from 20 to 200 mM NaCl in buffer Q at 0.5-column volume/h. Peak fractions (200 ml) were combined and precipitated with ammonium sulfate (65% saturation) for 3 h and collected by centrifugation at 28,000 times g for 60 min.

SP-Sepharose Chromatography

Fraction III (535 mg) was dissolved in 18 ml of buffer SP and dialyzed against 3 liters of buffer SP overnight to an ionic equivalent of 20 mM NaCl. This material was loaded onto an SP-Sepharose column (350 ml; 4 cm times 28 cm) previously equilibrated with 3.5 liters of buffer SP at 0.5-column volume/h. The bullet activity was eluted with an 8-column volume linear gradient from 20 to 200 mM NaCl in buffer SP at 0.5-column volume/h. Peak fractions (158 ml) were combined and precipitated with ammonium sulfate (65% saturation) for 3 h and collected by centrifugation at 28,000 times g for 60 min.

Sephacryl S-100 Gel Filtration Chromatography

Fraction IV (361 mg) was dissolved in 5 ml of buffer S and dialyzed against 2 liters of buffer S to an ionic equivalent of 180 mM NaCl. This material was loaded onto a Sephacryl S-100 column (570 ml; 3 cm times 82 cm) equilibrated with buffer S. The bullet activity was eluted with buffer S at 1/15-column volume/h. Active fractions containing at least 50% of the activity of the peak tube were combined to yield fraction V (40 ml, 248 mg). Homogeneous bullet was divided into aliquots, immediately frozen in liquid nitrogen, and stored at -80 °C. This protein is stable frozen for at least 1 year and for 10 days on ice. Table 1summarizes the overall purification. Purity of the major fractions was determined by SDS-polyacrylamide gel electrophoresis (Fig. 1).



Physical Characterization of bullet

Determination of the Molar Extinction Coefficient for bullet

The absorption spectrum was determined after a 40-fold dilution of bullet (fraction V) into 10 mM potassium phosphate (pH.7.5) with or without 6 M guanidine HCl. The instrument was blanked by adding a 1/40 volume of buffer S (fraction V buffer) to the same buffers used to dilute bullet. Absorbance, measured from 220-360 nm, reached a maximum at 280 nm. The molar extinction coefficient was calculated using the equation of Edelhoch(1967) ( = Ntrp(5690) + Ntyr(1280)). We used the amino acid composition available through the nucleic acid sequence of (Carter et al. 1993b) and (Carter et al. 1993a) to calculate the denatured molar extinction coefficient (53,200 liters mol cm) and the ratio of the absorbance of native bullet to bullet in 6 M guanidine (0.811) to calculate a molar extinction coefficient of 43,145 liters mol cm for the native protein.

Sedimentation Equilibrium Analysis

To determine the composition, native molecular weight, and equilibrium interactions within the bullet complex, we performed sedimentation equilibrium experiments at 1, 2, and 4 µM bullet. Four equilibrium boundary scans after 84, 88, 92, and 96 h at 35,000 rpm indicated that equilibrium had been achieved (Fig. 2A). From these data, the native molecular weight of the complex was determined using the Beckman IDEAL1 program which can be used to calculate an apparent weight average of single ideal species. If the system contains two or more species sedimenting independently at significant concentrations, then curve fits generated by the IDEAL1 model do not fit the data.


Figure 2: Sedimentation Equilibrium of bullet. The bullet complex was sedimented as described under ``Experimental Procedures.'' A, sedimentation equilibrium boundary scans from the XLA analytical ultracentrifugation runs at 84, 88, 92, and 96 h with bullet at a concentration of 4 µM. B, data and residual plot of bullet (4 µM) sedimented for 92 h at 35,000 rpm and fit to the IDEAL1 model. Residuals are expressed as A units. The curve fit to the data assumes a heterodimeric form of bullet. C, same as in B, except the curve fit to the data assumes and sediment independently as monomers. D, same as in B, except the curve fit to the data assumes bullet sediments as a trimer ((2)(1)or (1)(2)). E, same as in B, except the curve fit to the data assumes bullet sediments as a tetramer ((2)(2), (3)(1), or (1)(3)).



We modeled bullet as single species sedimenting independently, and as a dimer, trimer and a tetramer. Only the dimer (1:1 bullet complex) fit the data (Fig. 2B), yielding very low residuals (±0.02 A units) distributed around the theoretical curve. Sedimentation equilibrium data from all three concentrations and each angular velocity were in close agreement. They provided native molecular mass for bullet complex of 31,755 ± 178 daltons. The fit for the other models was unacceptable, giving nonrandom residuals that deviated as much as 0.2 A units from the theoretical curves (Fig. 2, C-E). Based on the amino acid composition predicted from the DNA sequence and the protein sequence of lacking its amino-terminal methionine, and are 16,599 and 15,043 Da, respectively (Xiao et al., 1993a; Carter et al., 1993a, 1993b). Thus, the species that best represents bullet is a heterodimer of 31,642 Da.

Sedimentation Velocity Analysis

bullet was subjected to sedimentation velocity analysis to further investigate its hydrodynamic properties. The sedimentation and diffusion coefficients were determined using the second moment/boundary spreading method (Muramatsu and Minton, 1988) at 2, 5, and 8 µM bullet. The calculated s(w) value for bullet was 2.6 ± 0.07, independent of protein concentration during sedimentation. The calculated diffusion coefficient (D(w)) was 7.6 times 10. The correlation coefficients for both determinations were >0.997. Applying the Svedberg equation, the calculated molecular mass of bullet was 31,400 ± 600 Da. The Stokes radius, 28 ± 0.1 Å, and frictional coefficient, 1.3 ± 0.01 were calculated from D(w) using Stokes' law and Fick's law, respectively.

Stimulation of Reconstituted Holoenzyme by bullet

Stimulation of the Reconstituted Holoenzyme Reaction in the Presence of 400 mM Potassium Glutamate

To develop a functional assay for use in monitoring the purification of bullet, we first exploited an earlier observation (Xiao et al., 1993a) that holoenzyme-like activity reconstituted with alpha-, , , and ` becomes more salt-resistant in the presence of bullet. We observed a modest but reproducible stimulation in the presence of bullet to a reaction using only the DnaX gene product (Fig. 3A). However, this reaction is much more sensitive than native holoenzyme to glutamate (Griep and McHenry 1989). Thus, we investigated the effect of the missing component .


Figure 3: bullet confers salt resistance to -reconstituted DNA polymerase III holoenzyme but not -reconstituted holoenzyme. DNA synthesis was measured as described under ``Experimental Procedures'' using holoenzyme reconstituted with or in the presence of the indicated potassium glutamate concentrations. A, each assay contained 600 fmol of pol III core (alpha complex), 500 fmol of beta, 500 fmol of , 600 fmol of , 600 fmol of `, and 500 fmol of bullet. B, same as in A, only holoenzyme was reconstituted with 500 fmol of instead of . Data represent DNA synthesis by reconstituted holoenzyme in the presence (bullet) or absence (circle) of bullet. Each data point represents the average of a duplicate determination.



Holoenzyme reconstituted with the product of the dnaX gene instead of , in the presence of bullet, is extremely resistant to increasing potassium glutamate concentrations up to 800 mM (Fig. 3B). The salt resistance is similar to that observed for native purified holoenzyme (^3)(Griep and McHenry, 1989). However, in the absence of bullet, the DNA polymerase activity of -reconstituted holoenzyme decreased dramatically as a function of increasing potassium glutamate concentration. At 400 mM potassium glutamate, in the presence or absence of bullet, the amount of dNTP incorporation was 190 and 21 pmol, respectively, a 9-fold difference. Thus the bulletbullet complex is the key component required for the salt resistance observed in native holoenzyme. This 9-fold dependence for bullet in the presence of 400 mM glutamate provided a convenient functional assay to monitor the purification of bullet reported in Table 1. The assay is linear over a broad range, from 10-50 fmol bullet per 25-µl assay (Fig. 4).


Figure 4: Dependence of DNA polymerase activity of -reconstituted holoenzyme on bullet in 400 mM potassium glutamate. DNA synthesis was measured with DNA polymerase III holoenzyme reconstituted as described under ``Experimental Procedures'' and in the Fig. 3legend using and the indicated amount of bullet. Each data point represents the average of a duplicate determination.



Under Standard Assay Conditions, bullet Decreases the Requirement for bullet` to Physiologically Relevant Levels

We next addressed the effect of bullet on the holoenzyme reaction under standard conditions (100 mM glutamate). Under conditions where components have been titrated to saturating levels, (^4)bullet reproducibly stimulates the reaction 30%, indicating a small, but real contribution of bullet to the intrinsic activity of the complex. However, we observed a requirement for high levels of bullet` in the absence of bullet (Fig. 5). Nearly 200 nM bullet` (^5)is required to saturate the assay in the absence of bullet. The [bullet`] (apparent K(d)) or bullet` dissociation from holoenzyme under these conditions, estimated from the amount required for half-maximal synthesis, is 40 nM in the absence of bullet. The actual value may be higher, since the high concentrations of some of the reaction components may shift the equilibrium. In any case, these estimates are useful for comparative purposes. In the presence of bullet, the [bullet`] and saturation level dropped to 2 and 20 nM, respectively. The cellular levels of most holoenzyme components including the alpha polymerase catalytic subunit and the DnaX proteins to which bullet binds are about 28 nM(^6)(Wu et al., 1984; Hawker, 1985). Thus, bullet is presumably required in vivo to permit activation of a significant portion of or by binding bullet`.


Figure 5: The presence of bullet reduces the level of and ` required to reconstitute holoenzyme. DNA synthesis was measured as described under ``Experimental Procedures'' except that 75 fmol of the dnaX gene products ( or ) were included, and the potassium glutamate concentration was 100 mM. Holoenzyme was reconstituted with (up triangle) and (circle) Closed and open symbols represent holoenyme reconstituted with or without bullet, respectively. Each data point represents the average of a duplicate determination.



The Major Function of bullet Is to Increase the Affinity of and for bullet`: BIAcore Analysis of bullet Interaction with and

To further understand the interactions of bullet with and and to study the effect of bullet on bullet` binding, we examined the interactions directly in the Pharmacia BIAcore. This instrument uses the optical phenomenon of surface plasmon resonance to monitor the interaction of an immobilized ligand to a protein in the flow solution that is passed over it (Malmqvist, 1993; Fagerstam et al., 1992) (see the companion study by Dallmann and McHenry(1995) for more details). Either or was immobilized to the sensor chip surface, dilute solutions of bullet (15-100 nM) were injected over each, and the binding signal was monitored (plotted as response units which are directly proportional to the mass bound to the chip) (Fig. 6). The buffer used in this analysis contained 100 mM potassium glutamate and the other ionic components of our standard holoenzyme assay. bullet rapidly bound to the - (Fig. 6) and -derivatized sensor chips (data not shown) with nearly equivalent rates (Table 2). Passing buffer over the chip permitted us to monitor the first-order dissociation of bullet from the immobilized DnaX protein (Fig. 6). bullet dissociated slowly from both and (t = 14-17 min). From the pseudo-first-order rate constant for association and the first-order rate constant of dissociation, we calculated nearly equivalent dissociation constants (1.8-2.5 nM) for both the - and -bullet interaction. The calculated stoichiometry of :bullet and :bullet was 4:1, using Equation 2 from Dallmann and McHenry(1995). This is the same stoichiometry reported within the native and complexes in solution (Dallmann and McHenry, 1995), indicating that immobilized and are properly folded and active.


Figure 6: BIAcore analysis of bullet interaction with immobilized . Sensorgram overlays of various concentrations bullet (15-100 nM) injected over immobilized are shown. The and subunits were immobilized to a CM5 sensor chip as described under ``Experimental Procedures.'' Solutions of bullet at the indicated concentrations in HKGM buffer were injected over immobilized . To completely dissociate bound protein, sensor chips were regenerated with two 1-min pulses of 1 M urea, 0.1 M KNO(3). These conditions allow for >95% retention of the original binding activity to the immobilized . Control injections of each bullet concentration over an underivatized flow cell were subtracted from the data to eliminate contributions due to minor refractive index changes Data were analyzed using the BIAEvaluation 1.0 and 2.0 software packages.





In preliminary BIAcore experiments, no high affinity interactions were observed between the following pairs of proteins: -, -`, -, -`, or bullet`. With or coupled to the BIAcore chip, injection of either or ` (up to 200 nM each) over the coupled DnaX subunit resulted in a signal essentially equal to a control injection over a blank chip. The same result was observed when either or ` was coupled to the BIAcore chip and or (up to 400 nM each) was injected. Injection of over immobilized `, or ` over immobilized also failed to show significant interaction at analyte concentrations up to 2 µM (data not shown). These observations did not rule out possible interactions between these subunit pairs at significantly higher concentrations, but might indicate that the establishment of these pairwise interactions is either kinetically slow or the resulting equilibrium is sufficiently unstable that these interactions do not represent central steps in the holoenzyme assembly pathway. Likewise, bullet (up to 400 nM) also did not appear to interact with either or ` when they were immobilized on BIAcore sensor chips (data not shown).

When equimolar mixtures (200 nM) of + or + were passed over a `-bound chip, no binding was observed (Fig. 7). The small signal observed was due to a refractive index change caused by residual glycerol from the protein storage buffer and is identical to the signal obtained from an injection over a blank sensor chip. When a mixture of and was injected over the ` chip, complex formation occurred (Fig. 7). Since neither or alone binds, the binding observed must represent a highly cooperative assembly of a bulletbullet` complex. When bullet was injected along with and , the rate and the extent of binding was greater, indicating that bullet stimulated the rate of binding of bullet` to . Identical binding curves were obtained when was used in place of or when was coupled to the BIAcore chip and ` was used in the mixture of analyte proteins (data not shown). Due to the complexity of this associating system, no kinetic and equilibrium constants could be obtained since the data could not be fit to the relatively simple binding models available in the BIAcore evaluation software. Nevertheless, the qualitative conclusion that bullet functions to stabilize the interaction between bullet` and DnaX is consistent with the interpretation of the functional experiments.


Figure 7: bullet increases the affinity of for bullet`. Sensorgram overlays of combinations of , bullet, and injected over immobilized ` are shown. ` was immobilized to a CM5 sensor chip as described under ``Experimental Procedures.'' All samples were injected at equimolar concentrations (200 nM) in HKGM buffer. The sensor chip was regenerated after each injection with a 15-s pulse of 10 mM NaOH. These conditions allow for >95% retention of the original binding activity to the immobilized `. Control injections over an underivatized sensor chip were done but were not subtracted from the data in order to illustrate the magnitude of the refractive index changes due to the injection.




DISCUSSION

and were purified to homogeneity as a tightly associated complex following overexpression of both subunits from a vector containing an artificial holC-holD operon. We pursued this strategy because of the insolubility of when overproduced individually. Having this material by itself permitted the important demonstration that binds to DnaX and bridges an interaction with (Xiao et al., 1993b), but more detailed biophysical experiments required defined folded material so that interactions could be studied without the complicating step of protein folding in assembly reactions.

bullet when overexpressed as a complex constitutes 15% of the total cellular protein and 11% of the total soluble protein. Together, ammonium sulfate fractionation and Q-Sepharose chromatography yielded nearly pure material. A trace 115-kDa contaminant and smaller molecular mass polypeptides were removed upon SP-Sepharose chromatography. Sephacryl S100 gel filtration chromatography provided only a marginal purification, but ensured that the final material was free of aggregates or unassembled subunits and permitted exchange into a defined buffer. Purified fraction V bullet complex, subjected to polyacrylamide gel electrophoresis, appeared as only two bands of 15,100 and 16,600 Da even when 40 µg of protein was loaded (Fig. 1, lane 5). Laser densitometry of a Coomassie-stained gel containing 1-10 µg of bullet demonstrated 99% purity.

The molar extinction coefficient of and purified independently were calculated based on the amino acid composition (Xiao et al., 1993a). The sum is the calculated extinction coefficient for the bullet complex 53,200 M. The actual native extinction coefficient is 43,145 M, a 20% difference from the calculated molar extinction coefficient. Use of this rigorously defined extinction coefficient will allow more precision in future experiments.

Sedimentation velocity analysis indicated an s(w) of 2.6, a Stokes radius of 28 Å, a native molecular mass of 31,400 daltons, and a frictional coefficient of 1.3, data that are in reasonable agreement with glycerol sedimentation of bullet (Xiao et al., 1993b) and the sedimentation equilibrium data presented here. The frictional coefficient of 1.3 suggests that bullet is an asymmetric molecule.

Sedimentation equilibrium experiments were conducted to examine the composition of bullet in solution. This technique is particularly powerful because at each position within the boundary established, all components are at sedimentation and chemical equilibrium. The shape of the curve at varying protein concentrations permits a particularly sensitive way to detect multiple molecular species in a mixture and to determine the equilibrium between them. bullet sediments as a single ideal species with native molecular mass of 31,755 Da, a difference of less than 1% from the calculated molecular mass for a 1:1 heterodimer. Although it is possible that bullet sediments independently as monomers, or as a trimer or tetramer, these curve fits do not correlate with the data generated by sedimentation equilibrium studies (Fig. 2).

Holoenzyme activity can be reconstituted without bullet under optimal levels of the other holoenzyme subunits. Thus, to develop a functional assay, we needed to define a set of conditions that provided a maximal stimulation by bullet. We reproduced the findings of O'Donnell and colleagues that a modest bullet requirement exists at elevated salt concentrations using reconstitution of complex and holoenzyme as an assay (Xiao et al., 1993a), but we found that the resulting -reconstituted holoenzyme did not exhibit the salt resistance observed for native holoenzyme (Griep and McHenry, 1989). In a search for conditions that permitted reconstitution of native holoenzyme at high salt levels (400 mM glutamate), we found that both and bullet are required. This result not only provided a convenient linear assay for bullet, but also suggested that a bulletbullet interaction occurs within native holoenzyme, providing additional evidence for our model that plays a central role as a clamp loader in holoenzyme (Dallmann et al., 1995; Dallmann and McHenry, 1995).

We examined the influence of bullet on the binding of bullet` to DnaX in the BIAcore. This instrument permits real-time direct monitoring of the binding of protein in the flow phase to a protein immobilized on a chip. By monitoring binding of bullet to and , we determined a K(d) of 2 nM. That the K(d) was roughly equivalent for and suggests that the site for bullet interaction is entirely within the amino-terminal domain of . The estimated K(d) is consistent with the requirement for 1 nM bullet in our functional assays.

Analyses using the BIAcore to monitor DnaX complex formation supported our model of bullet function. bullet` bound to DnaX to a greater extent and more rapidly in the presence of bullet than in its absence. Binding on the BIAcore is directly proportional to a change in the amount of mass bound to the chip. Because bullet constitutes 8% of the mass of the complex (Dallmann and McHenry, 1995), the 2-fold increase in binding is not due solely to the mass contribution by bullet.

The DnaX complex functions to load the beta sliding clamp onto DNA. A defined position with the initiation complex that results when pol III core assembles also suggests an elongation role and alpha-DnaX contacts (Reems et al., 1995). The function of , `, , and subunits within the complex remains poorly understood. From the results of this study, we propose that bullet, though not required for DNA synthesis per se, is an important component for proper holoenzyme function in vivo. High concentrations of bullet` can overcome most of the bullet requirement in vitro, but concentrations of 200 nM are required to saturate DnaX under our standard assay conditions (100 mM glutamate). To date, only the beta subunit is known to be present in excess. Other components of the complex, including alpha and the DnaX subunits with which bullet` interacts, are present at 20 copies per cell, which corresponds to 28 nM.^4 This level of bullet` would permit sequestration of DnaX in a complex in the presence of bullet. In the absence of bullet, only a fraction of the maximal amount of complex would be formed. The ability of bullet to alter the amount of functional clamp loader present in the cell could also enable bullet to serve a regulatory role or as a modulator of the holoenzyme assembly pathway.


FOOTNOTES

*
This work was supported, in part, by National Institutes of Health Research Grant GM35695 and facilities support from the Lucille P. Markey Charitable Trust. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
Supported by National Institutes of Health Postdoctoral Fellowship GM15685.

Supported by Postdoctoral Fellowship from the Natural Sciences and Engineering Research Council of Canada.

**
To whom correspondence should be addressed.

(^1)
The abbreviations used are: holoenzyme, E. coli DNA polymerase III holoenzyme; core pol III, E. coli DNA polymerase III core (alpha--); -complex, (4)--`--; complex, (4)--`--; DnaX complex, a complex containing either product of the dnaX gene, or with associated bullet` and bullet; Mes, 2-(N-morpholino)ethanesulfonic acid; SSB, E. coli single-stranded DNA binding protein; pol III, polymerase III; DTT, dithiothreitol.

(^2)
D.-R. Kim, J. Carter, M. Franden, and M. Olson, manuscripts in preparation.

(^3)
When both and are present in reconstitution mixes, the activity of the resulting holoenzyme is the same as the -reconstituted and native holoenzyme. However, does not readily enter the holoenzyme assembly under these conditions.

(^4)
Except or , which was fixed. High levels of or (400 nM) decrease the levels of bullet` required, presumably by mass action effects.

(^5)
Individual concentrations of and ` are given.

(^6)
The concentration was calculated based on a cell volume of 1 fl/cell during balanced growth (Ingraham et al., 1983). One molecule/cell would have a concentration of 1.4 nM.


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