©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
DnaX Complex of Escherichia coli DNA Polymerase III Holoenzyme
PHYSICAL CHARACTERIZATION OF THE DnaX SUBUNITS AND COMPLEXES (*)

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

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

A physical characterization of the and subunits of the Escherichia coli DNA polymerase III holoenzyme and their complexes with the , `, , and subunits is presented. The native molecular mass of the and subunits was determined to be 255,000 and 189,000 Da, respectively, by sedimentation equilibrium analytical ultracentrifugation. Both values indicate a tetrameric quaternary structure. The and complexes were reconstituted and purified using two different methods. Both complexes assembled readily and were reconstituted at subunit concentrations approaching physiological levels. The stoichiometries of the and complexes, as determined by quantitative densitometry of SDS-polyacrylamide gels, were found to be (4)(1)`(1)(1)(1) and (4)(1)`(1)(1)(1).

BIAcore analysis demonstrated that the formation of large multiprotein complexes of holoenzyme subunits depends on the presence of the subunit; could not substitute. We present a model for a -less form of DNA polymerase III holoenzyme that has asymmetrical structural features that may be responsible for the functional asymmetry observed in holoenzyme. The stoichiometry of the reconstituted DNA polymerase III* component of holoenzyme in this model is (alpha)(2)DnaX(4)(1)`(1)(1)(1).


INTRODUCTION

The DNA polymerase III holoenzyme, (^1)the replicative polymerase of Escherichia coli, contains three essential subassemblies: (i) a polymerase that is able to interact with other specialized proteins at the replication fork, (ii) a beta sliding clamp that tethers the polymerase to the template, enabling high processivity, and (iii) a DnaX complex that loads the sliding clamp onto the primer-template (see the companion study in this series, Dallmann et al.(1995), for a more thorough explanation and references). The holoenzyme is dimeric and functions asymmetrically, presumably because it contains distinct leading and lagging strand polymerases. We have demonstrated that the asymmetric function of the holoenzyme can be reconstituted using only the subunit, eliminating the possibility that an asymmetric placement of relative to is responsible for this asymmetry (Dallmann et al., 1995).

In addition to the finding that alone permits reconstitution of native holoenzyme, other evidence implicates as a key essential component and relegates to nonessential roles that can be replaced by . Olson et al.(1995) demonstrated that the salt resistance of native holoenzyme required a -- complex; -- would not substitute. This suggests that is found complexed with auxiliary clamp loading subunits within holoenzyme. Blinkova et al.(1994) showed that E. coli are viable when their dnaX gene is mutated so that they cannot frameshift and only the product is produced. DNA polymerase III* isolated from these strains starts out in the -less form, but during purification, is rapidly proteolyzed to . We (Dallmann et al., 1995) and others (Tsuchihashi and Kornberg, 1989) have demonstrated that overproduction of both and in vivo from the wild-type dnaX gene results only in assembly of and oligomers; no mixed - oligomers are detectable. We have also shown that only complexes function at low, physiologically relevant concentrations of pol III core and that addition of free , although it stimulates the reactions, does not efficiently recruit complexes into an efficient holoenzyme during the 5-min time course of the reaction, a time adequate for replication of 300 Okazaki fragments in vivo.

As a step toward resolving these issues, we analyzed formation of DnaX complexes under conditions that allow accurate determination of their stoichiometry and analysis of their interactions. We demonstrate that both and can form well defined complexes with -` and -, but that only the complex can efficiently enter holoenzyme. This work has led to a model for the DNA polymerase III* component of holoenzyme: (alpha--)(2)(beta(2))(2)(DnaX(4)--`--), where DnaX can be (4) with preservation of all of the known properties of native holoenzyme.


EXPERIMENTAL PROCEDURES

Sedimentation Equilibrium Analysis of the and Subunits

Sedimentation equilibrium analysis was carried out with a Beckman Optima XL-A analytical ultracentrifuge. The buffer used for all experiments was 20 mM HEPES-KOH (pH 7.5), 100 mM NaCl, 5% (v/v) glycerol. Experiments were done using six-sector cells with multiple protein concentrations for each subunit (2.5, 5 and 10 µM for ; 0.7, 1.4, 2.8, 7, 14, and 28 µM for ). Both and were sedimented over a 60-h period at 15,000 rpm (4 °C) in a Beckman An-Ti60 rotor. Radial absorbance scans (280 nm) were taken every 5 h. Only scans which indicated that equilibrium had been reached were used for analysis (typically >30 h). Data were analyzed using the IDEAL1 fitting program provided with the Beckman analysis software (version 2.0, 1993). The underlying model of this program assumes a single, ideal, sedimenting species. To calculate a native molecular weight the data were fit to :

On-line formulae not verified for accuracy

where A(x) is absorbance at radial position x; A(0) is absorbance at radial position x(0); H = (1-)^2/RT; = protein partial specific volume; = solvent density; = angular velocity; R = gas constant; T = temperature (K); M = native molecular weight, and E is the base-line offset.

Sedimentation Velocity Analysis of the and Subunits

Sedimentation velocity analysis of and was carried out in the same buffer as described above. Both and were sedimented at 30,000 rpm (4 °C) at concentrations of 20 µM and 33 µM, respectively. Radial absorbance scans (280 nm) were taken at 30-min intervals. Data were analyzed using the second moment/boundary spreading (Muramatsu and Minton, 1988) and g*(s) (Stafford, 1992) methods provided with the Beckman analysis software.

Reconstitution of DnaX Complexes

The and complexes were prepared by mixing the or subunits (10 nmol of monomer) with an excess of the and ` subunits and the - complex (20 nmol monomer, each) in a volume of 0.5 ml (2.5 µM DnaX complex equivalent) unless otherwise stated. Proteins were mixed by gentle vortexing and allowed to incubate at room temperature for 15 min before chromatography to remove unincorporated subunits from the complexes.

Superose 6 Gel Filtration

and complexes were isolated by gel filtration chromatography on a Superose 6 (Pharmacia Biotech Inc.) fast protein liquid chromatography column. The column was developed in buffer N (20 mM sodium phosphate (pH 7.4), 10% glycerol, 50 mM NaCl, and 5 mM MgCl(2)) at a flow rate of 0.2 ml/min, and 0.5-ml fractions were collected from the point of injection. Under these conditions complex eluted between fractions 25 and 28, and complex between fractions 27 and 30. Unincorporated , `, and eluted between fractions 33 and 39.

Mono Q Ion Exchange

The and complexes were also isolated by anion exchange chromatography on a Mono Q (Pharmacia) fast protein liquid chromatography column. The column was developed using a 15-ml linear gradient from 50 to 400 mM NaCl in buffer N at a flow rate of 0.5 ml/min. Fractions (0.5 ml) were collected from the point of injection. All subunits and complexes eluted at unique conductivites in the following order: (80 mM NaCl equivalent), ` (170 mM NaCl equivalent), (240 mM NaCl equivalent), ` complex (270 mM NaCl equivalent), and subunits (260 mM NaCl equivalent), and and complexes (280 mM NaCl equivalent).

Stoichiometry Determination of DnaX Complex Subunits

DnaX complexes prepared by the methods described above were analyzed by SDS-polyacrylamide gel electrophoresis (12% acrylamide gels, 0.75-mm thickness). Four samples (2.5, 5, 10, and 15 µg) of each purified complex plus eight standards were analyzed. The standards were made up of DnaX complex constituent subunits mixed according to the DnaX::`:: ratios of 2:1:1:1:1 and 4:1:1:1:1 (2.5, 5, 10, and 15 µg of each, as determined by extinction coefficient). The gels were stained for at least 12 h in 0.05% (w/v) Coomassie Brilliant Blue R-250 (Bio-Rad), 45% methanol, 10% acetic acid to ensure complete staining of the proteins. Gels were destained in three changes of 20% methanol, 5% acetic acid, and scanned using a Molecular Dynamics laser densitometer. The subunits in the purified DnaX complexes were quantitated by linear least squares fitting to standard curves derived from plots of the subunit standards.

DNA Polymerization Assays for and Complexes

Assays of the and complexes described above contained 4 pmol of core (unless otherwise stated), 400 fmol of beta, 540 pmol of primed (DnaG primase), and SSB-coated M13G (as nucleotide). Reactions were carried out in a volume of 25 µl in a buffer containing 50 mM HEPES-KOH, pH 7.5, 10% (v/v) glycerol, 0.1 M potassium glutamate, 10 mM dithiothreitol, 10 mM magnesium acetate, 200 µg/ml bovine serum albumin, 0.02% (v/v) Tween 20, 10 µM ATP, 48 µM dATP, dCTP and dGTP and 18 µM [^3H]TTP (specific activity = 520 cpm/pmol TTP). Assay mixtures were incubated at 30 °C for 5 min, and quenched by trichloroacetic acid precipitation. The unit definition for and complex activity is 1 pmol of total nucleotide incorporated into acid-insoluble DNA in a 5-min reaction at 30 °C.

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. Protein immobilizations to the sensor chip flow cells were done in buffer HBS (10 mM HEPES KOH, 150 mM NaCl, 3.4 mM EDTA, 0.005% P-20 detergent (Pharmacia Biosensor)) 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. This mixture reacts with caboxyl groups of the sensor chip matrix to yield an N-hydroxysuccinimide ester, which is susceptible to nucleophilic attack by amino groups of proteins and results 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 are 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. Protein-protein interaction studies were carried out in buffer HKGM (50 mM HEPES KOH (pH 7.4) 100 mM potassium glutamate, 10 mM magnesium acetate, 0.005% P-20 detergent (Pharmacia Biosensor)) at a flow rate of 5 µl/min at 20 °C unless otherwise stated. Since the binding signals obtained using the BIAcore are directly proportional to mass bound, stoichiometries could be estimated using :

On-line formulae not verified for accuracy

where RU is the measured response (units) obtained at binding saturation and M is the molecular weight.


RESULTS

Hydrodynamic Studies of and

The availability of large amounts of purified and allowed us to perform hydrodynamic studies using analytical ultracentrifugation. The stoichiometries of the and subunits relative to other subunits in holoenzyme subcomplexes have previously been estimated by densitometry of stained SDS-polyacrylamide gels (Maki and Kornberg, 1988; McHenry, 1982) and by gel filtration and glycerol gradient centrifugation (Tsuchihashi and Kornberg, 1989; Studwell-Vaughan and O'Donnell, 1991). These methods suffer inherent problems, such as differences in dye binding affinity between subunits when defined standards are unavailable and significant dependence on molecular shape, which often yield misleading results. By contrast, sedimentation equilibrium analysis in the analytical ultracentrifuge does not involve these problems and provides the most reliable estimate of native molecular weight (Hansen et al., 1994; Minton, 1990; Schachman, 1989). In combination with sedimentation velocity studies, a more accurate description of the hydrodynamic properties of a protein can be obtained. Sedimentation equilibrium and velocity experiments were performed on the and subunits. Native molecular weights were determined from equilibrium data by nonlinear curve fitting to in the IDEAL1 fitting routine. Sedimentation and diffusion coefficients (s(w) and D(w)) were calculated using the methods of Muramatsu and Minton(1988) and Stafford(1992) which were also provided with the data analysis software. The results are summarized in Table 1and representative data, curve fits, and residual plots are shown in Fig. 1, A and B. At the concentrations used in this analysis, both and sediment as single, ideally behaved particles with molecular weights equivalent to tetramers. The sedimentation coefficients, s(w), for the and subunits were found to be 8.1 and 7.3, respectively. The frictional ratios for both and were 1.6 and the Stokes radii were 66 and 47 Å, respectively (Table 1).




Figure 1: Sedimentation equilibrium of and . Sedimentation equilibrium studies of and were done using the Beckman Optima XL-A analytical ultracentrifuge as described under ``Experimental Procedures.'' Representative data, curve fit, and residual plots are shown for (14 µM) sedimented at 15,000 rpm for 40 h (A) and for (10 µM) sedimented at 15,000 rpm for 40 h (B). Data were fit to the IDEAL1 model (). Residuals are expressed in absorbance units (280 nm).



Preparation and Purification of DnaX Complexes

and complexes (DnaX complexes) were prepared by mixing and incubation of their constituent subunits and purified by either gel filtration or Mono Q anion exchange chromatography. In all cases a 2-fold molar excess of , `, and over the DnaX subunit was used to ensure that all of the DnaX protein entered a complex. Both purification methods allowed base-line separation of the resulting DnaX complex from unincorporated , `, and . However, gel filtration chromatography could not separate the DnaX complexes from unincorporated DnaX subunits. Anion exchange chromatography was used to separate DnaX subunits from their resulting complexes and to assess the degree of incorporation of and into complexes under the conditions used. The complete inclusion of both DnaX proteins into their complexes when prepared by Mono Q anion exchange demonstrated that significant amounts of free DnaX protein did not contaminate DnaX complexes prepared by gel filtration (data not shown). Both preparation methods yielded complexes with comparable specific activities (Table 2). Furthermore, assay of these complexes for DNA synthesis with additional amounts of , `, and revealed no stimulation of their activities (Fig. 2), indicating that the complexes were replete in these subunits.




Figure 2: Activity of reconstituted and complexes are not stimulated by additional , `, and . The () and (bullet) complexes were assayed as described under ``Experimental Procedures.'' Both DnaX complexes were present at 100 fmol/assay (4 nM), and core and beta were present at 480 fmol/assay.



Preliminary studies on the reconstitution of DnaX complexes indicated no preferential order of addition of subunits and no requirement for prolonged stepwise preincubations or for ATP or Mg in obtaining maximal activity yields. The DnaX complexes could be quantitatively assembled at various concentrations in buffer N at room temperature for 15 min before purification (Fig. 3).


Figure 3: Assembly of complexes at low protein concentrations. The (2.5 nmol of monomer), (5 nmol) and ` (5 nmol) subunits, and the complex (5 nmol) were diluted into three different volumes of buffer N (0.5, 3.5, and 12.5 ml) at three different concentrations (1.25 µM, 180 nM, and 50 nM complex equivalent) in order to reconstitute the complex. After mixing, samples were incubated at room temperature for 15 min, then chromatographed over a Mono Q ion-exchange column as described under ``Experimental Procedures.'' Samples of complexes prepared at each concentration were denatured in SDS sample buffer and subjected to electrophoresis on a 12% SDS-polyacrylamide gel. Following staining in Coomassie Brilliant Blue G-250, subunits were quantitated by laser densitometry to determine subunit stoichiometry. This was compared to the subunit stoichiometry of complex prepared by gel filtration which was run on the same gel. Lane 1, complex prepared at 500 nM by Mono Q (8 µg); lane 2, complex prepared at 180 nM by Mono Q (8 µg); lane 3, complex prepared at 50 nM by Mono Q (8 µg); lane 4, complex prepared at 2.5 µM by Superose 6 gel filtration (8 µg).



DnaX Complex Stoichiometry

Stoichiometries of the and complexes prepared by both gel filtration and ion exchange chromatography were determined by laser densitometry of the complexes and known amounts of standard proteins (determined using their extinction coefficients) subjected to electrophoresis on SDS-polyacrylamide gels. To ensure complete staining of all subunits, thin gels (0.75 mm) were run and subsequently stained with Coomassie Blue for at least 12 h to allow complete diffusion of the dye into the gels. Gel destaining was monitored, and gels were scanned immediately after destaining was complete. Stoichiometries of the and complexes were identical regardless of the purification method and were (4)(1)`(1)(1)(1) and (4)(1)`(1)(1)(1) (Table 2).

The stoichiometries of the DnaX complexes were consistent with the subunit stoichiometries determined for the DnaX subunits by analytical ultracentrifugation, but inconsistent with previously reported stoichiometries, which assumed two copies of the DnaX component in the DnaX complexes (Maki and Kornberg, 1988; Onrust and O'Donnell, 1993). The reconstitutions of the and complexes described above were carried out at fairly high protein concentrations (2.5 µM DnaX tetramer equivalent), raising the possibility that the observed stoichiometry reflected the use of nonphysiological protein concentrations far exceeding the DnaX dimer-tetramer equilibrium dissociation constant. To rule out this possibility, complexes were reconstituted and prepared by ion-exchange chromatography at concentrations approaching the estimated intracellular holoenzyme subunit concentration (30 nM assuming 20 copies of holoenzyme/cell) (^2)(McHenry and Kornberg, 1981) to determine whether the stoichiometry of decreased in these complexes. Visual inspection of an SDS-polyacrylamide gel of samples of complex prepared at 1.25 µM, 180 nM, and 50 nM complex equivalents, along with a standard of complex prepared by gel filtration (Fig. 3), indicated no obvious changes in the stoichiometry of the complexes. Laser densitometry of each of these samples and comparison to defined standards indicated that stoichiometry of all complexes was the same as the values reported in Table 2, with four to five copies of for each of `, , and .

BIAcore Analysis of Multiprotein Complexes Dependent on

A Pharmacia Biotech BIAcore instrument (Malmqvist and Granzo, 1994; Malmqvist, 1993; Fagerstam et al., 1992) was used to investigate whether the inclusion of in various complexes precluded the binding of other subunits to . The BIAcore instrument uses the optical phenomenon of surface plasmon resonance to monitor biospecific interactions in real time. A ligand protein is chemically linked to a sensor chip made up of a sandwich of gold film between glass and a carboxylmethyl dextran matrix to which the ligand is immobilized. The sensor chip is mounted on a fluidics cartridge which forms flow cells through which analyte proteins can be injected. Ligand-analyte interactions on the sensor chip are detected as changes in the angle of a beam of polarized light reflected from the chip surface. The binding of any mass to the chip alters surface plasmon resonance in the gold layer. This is essentially a change in the electrical field in the gold layer which interacts with the reflected light beam and alters the angle of reflection proportional to the amount of mass bound. The reflected light is detected on a diode array and translated to a binding signal expressed as response units (RU). Because the response is directly proportional to the mass bound, it is possible to measure kinetic and equilibrium constants, and binding stoichiometries for protein-protein interactions.

Binds and Core Simultaneously

If the role of in holoenzyme includes loading beta clamps (in concert with the , `, and subunits) and dimerizing core, a single assembly must be able to bind both the core polymerase and DnaX complex subunits. The complex activity assays described in Dallmann et al.(1995) strongly implied that core and bind the same assembly, and we have shown that the K(d) for the - interaction is 2 nM (Olson et al. 1995) and the -core interaction is <1 nM. (^3)We used BIAcore analysis to directly determine whether immobilized simultaneously binds both core and the complex. was immobilized and core and were injected sequentially over the immobilized (Fig. 4). In the first phase of the injection in sensorgram 1 ( injection), it was clear that the immobilized was completely saturated with (as shown by the plateau in the injection phase indicating no further binding), yet in the next phase (core injection), it was also clear that a large amount of core bound to the - complex. Sensorgram 2 shows the sequential injection of core followed by . Again was saturated with core as indicated by the plateau in the binding signal. In this sensorgram, the binding signal was partially obscured due to its relatively small magnitude and the simultaneous dissociation of core. Subtraction of sensorgram 1 from sensorgram 2, aligned such that the background of core dissociation could be eliminated, was done to obtain a representation of the binding signal. This yielded a binding curve (indicated by the thick line) identical to that seen in sensorgram 1. In both sensorgrams, the : binding ratio was estimated from the plateau of the binding phase using and was found to be approximately 4:1, consistent with a tetramer binding a heterodimer. The :core binding ratio was similarly estimated to be 2:1, consistent with a tetramer dimerizing two copies of core. These results indicate that binds core and simultaneously at nearly the expected stoichiometries determined above and in Olson et al.(1995).


Figure 4: Interaction of with does not block interaction of with core. Sensorgram overlays are shown for (200 nM) and core (200 nM) sequentially injected over immobilized (3800 RU immobilized). The subunit was immobilized to a CM5 sensor chip as described under ``Experimental Procedures.'' Proteins were diluted in HKGM buffer. To completely dissociate bound protein, the sensor chip was regenerated with a 15-s pulse of 10 mM NaOH, which allowed for >95% retention of the original binding activity of the immobilized . Control injections over an underivatized flow cell were performed but not subtracted from the data. Background contributions due to these injections were equal to 50 RU for the injection and 400 RU for the core injection. Sensorgram 1, sequential injection of (200 nM, 20 µl) and core (200 nM, 40 µl). Sensorgram 2, sequential injection of core (200 nM, 40 µl) and (200 nM, 20 µl). The sensorgram at the bottom of the figure shows the subtraction of sensorgram 1 from sensorgram 2 to extract a representation of binding to without the background of core simultaneously dissociating from . The thick line indicates binding of to in the presence of core bound to .



Complex Binds Core Tightly but Complex Does Not

and complexes were reconstituted on the BIAcore via or ` immobilized to the sensor chips (Fig. 5) (Olson et al., 1995). Both complexes had dissociation half-lives of 1.4 h and were stable over the time course of all experiments (typically 30 min). The calculated relative binding stoichiometries for both complexes were inconsistent with either the DnaX(4)(1)`(1)(1)(1) or DnaX(2)(1)`(1)(1)(1) stoichiometries, suggesting that a significant proportion of the immobilized or ` did not participate in or complex formation on the BIAcore chip. This was likely due to a preferential orientation of a subpopulation of these proteins on the sensor chip during coupling that did not favor complex formation. However, the amount of DnaX complex that was ultimately formed appeared to be capable of binding stoichiometric quantities of other components as described below. The DnaX complexes were tested for their ability to bind core, beta, and the subunit (Fig. 5). The complex bound core rapidly to form a stable complex with a dissociation half-life of 1.5 h and an apparent binding stoichiometry consistent with 2 core moieties bound to a (4)(1)`(1)(1)(1) complex. The complex did not bind core (Fig. 5). The signal observed for the injection of core over the complex was equal to the signal seen when the core sample was injected over a blank sensor chip, indicating no specific binding. Additionally, core did not bind to immobilized or `. Clearly, the participation of in a complex with the , `, , and subunits did not prevent the binding and dimerization of core by .


Figure 5: Participation of in a DnaX complex does not block the binding of core. Sensorgram overlays are shown for mixtures of , , `, or , , ` (200 nM each subunit) sequentially injected before core (200 nM), beta (200 nM), or (200 nM monomer) over immobilized (660 RU immobilized), The subunit was immobilized to a CM5 sensor chip as described under ``Experimental Procedures.'' All proteins were diluted in HKGM buffer. To completely dissociate bound protein, the sensor chip was regenerated with a 15 s pulse of 10 mM NaOH, allowing for >95% retention of the original binding activity of the immobilized . Control injections over an underivatized flow cell were performed but not subtracted from the data. Background contributions due to these injections were equal to 200 RU for the (), , and ` injections, 400 RU for the core injection, 10 RU for the beta injection, and 100 RU for the injection. Similar sensorgrams were obtained when ` was immobilized to the sensor chip and was injected with the other subunits (not shown).



The DnaX complexes act to load the beta subunit onto primed DNA. To determine whether the and complexes bind beta, the beta subunit was injected over the and complexes assembled on the BIAcore. As expected, beta rapidly bound to both complexes (400 RU) and dissociated fairly rapidly (as indicated by a more pronounced exponential decay of the signal seen after the end of the beta injection) (Fig. 5). The apparent binding stoichiometry was calculated to be 1 beta monomer per DnaX(4)(1)`(1)(1)(1) complex, although this represents a minimal estimate since the beta concentration was not varied to ensure that it was saturating. To test the postulate that the subunit functions to recruit the complex into holoenzyme (O'Donnell, 1994; Stukenberg et al. 1994), we analyzed whether the subunit could bind to either or complexes assembled on the BIAcore (Fig. 5). Under the conditions used, no binding of either or complex to immobilized subunit was detected. Additional experiments showed no binding of to immobilized on the BIAcore, or to immobilized on the BIAcore (data not shown).

Reconstitution of a -dependent Holoenzyme-DNA Complex by BIAcore

Because complex could bind all components of a -reconstituted holoenzyme, we attempted to reconstitute a -dependent ``initiation complex'' on a BIAcore sensor chip via immobilized . As shown in Fig. 6, the beta subunit and primed, SSB-coated, M13G bound strongly to the complex-core assembly (dissociation half-life, 1.5 h). We do not know whether binding of the primed M13 template requires the presence of the RNA primer. This experiment showed that all the components of an initiation complex can participate in a large multiprotein-DNA assembly which is dependent on the presence of the subunit.


Figure 6: Reconstitution of a -dependent initiation complex on BIAcore. A sensorgram is shown for sequential injections of , , and ` (200 nM each subunit), core (200 nM), beta (200 nM), and primed, SSB-coated M13G (30 fmol, 10 nM as circles) over immobilized (660 RU immobilized). All proteins were diluted in HKGM buffer + 200 µM ATP. Immobilization and regeneration conditions and control injections were done as in Fig. 5, and under ``Experimental Procedures.'' Background contribution due to the injection of primed, SSB-coated M13G was 1300 RU (1000 RU were bound).




DISCUSSION

We have used sedimentation equilibrium analytical ultracentrifugation to determine the native molecular weights of and (Fig. 1, Table 1). Both proteins sedimented as ideally behaved particles at molecular weights consistent with a tetrameric quaternary structure. Numerous previous studies implied stoichiometries from dimer to trimer for DnaX (Tsuchihashi and Kornberg, 1989; Studwell-Vaughan and O'Donnell, 1991; O'Donnell, 1994). These studies were conducted using gel filtration and glycerol gradient centrifugation which are affected by molecular properties other than molecular weight (such as particle shape and frictional ratio), whereas sedimentation equilibrium is dependent only on the weight of the sedimenting particle and not on molecular shape factors (van Holde, 1971). Thus, our determination of the native molecular weights of and must be considered the most reliable estimate to date.

Sedimentation velocity analytical ultracentrifugation of and was also performed (Table 1). For , the sedimentation coefficient, Stokes radius and frictional ratio determined here were comparable to those determined by Studwell-Vaughan and O'Donnell(1991). The frictional ratios for both and indicate that they are asymmetric.

The purified and subunits were used to reconstitute DnaX complexes along with the and ` subunits and the complex. These complexes were readily assembled with no requirement for stepwise preincubation of subunits or requirement for ATP or Mg before purification. Previous reports (Blinkova et al., 1993; Stukenberg et al., 1994) have indicated that a stepwise preincubation of subunits is required for the reconstitution of complex and holoenzyme before purification. We attribute this requirement to the use of that had been purified in 6 M urea from inclusion bodies (Xiao et al., 1993). Presumably this procedure reflects the time required for the refolding of and its recruitment into a reconstituted holoenzyme. The used in our study was co-expressed with the subunit as a soluble complex which has been shown to readily interact with the DnaX proteins and is required for cooperative assembly of and ` into DnaX complexes (Olson et al., 1995).

The and complexes were purified by either gel filtration or anion exchange chromatography. Both methods yielded complexes that were indistinguishable by activity and subunit stoichiometry (Table 2). Stoichiometries of the and complexes were found to be (4)(1)`(1)(1)(1) and (4)(1)`(1)(1)(1), respectively, inconsistent with a previous report of complex stoichiometry ((2)(1)`(1)(1)(1)) (Maki and Kornberg, 1988), which was estimated by densitometry of stained SDS-polyacrylamide gels using relative staining intensity for quantitation. We believe that the stoichiometries determined here are reliable and can be used to infer the stoichiometry of DnaX complexes in vivo for a number of reasons. (i) Defined amounts of each subunit (determined from subunit extinction coefficients) were used as standards in the quantitation, not relative staining intensity. (ii) Reconstitutions of complex at low protein concentrations (down to 50 nM complex) approaching the estimated intracellular holoenzyme subunit concentration (30 nM^2 assuming 20 copies of holoenzyme/cell) (McHenry and Kornberg, 1981) produced complexes with stoichiometries identical to complexes prepared at high concentrations (Fig. 3). (iii) Addition of , `, and to and complex assays (Fig. 2) caused no stimulation, indicating that the complexes were replete in these subunits. This last point implies that the dissociation constant for the DnaX dimer-tetramer equilibrium is no higher than 4 nM, the concentration of DnaX complex used in a typical assay and below the estimated intracellular concentration. If the (4)(1)`(1)(1)(1) or (4)(1)`(1)(1)(1) stoichiometry were an artifact due to high protein concentration favoring DnaX tetramer formation, the observed stoichiometry might have been caused by the use of protein concentrations far exceeding the dimer-tetramer dissociation constant. In that case, addition of , `, and to and complex assays (Fig. 2) should have caused a stimulation of DnaX complex activity at the high dilutions used in the assay as the DnaX tetramer reequilibrated to the dimer form. This was not observed.

The participation of the and subunits in higher order multiprotein complexes with other holoenzyme subunits and subcomplexes was qualitatively examined using the Pharmacia Biosensor BIAcore instrument (Malmqvist and Granzow, 1994). Optimally, the BIAcore can be used to determine kinetic and equilibrium constants for simple A + B AB associating systems (Fagerstam et al., 1992). For the interactions studied in this report, where binding cooperativity (Olson et al., 1995) and the assembly and dissociation of multisubunit complexes are involved, kinetic and equilibrium constants could not be determined without more extensive experimentation and mathematical modeling. The qualitative results presented here can be considered analogous to protein-protein cross-linking.

We have shown that the subunit, when immobilized to the BIAcore, can simultaneously interact with core and the complex (Fig. 4), as expected based on the ability of complex to reconstitute holoenzyme with stoichiometric amounts of core and beta (Dallmann et al., 1995). We are confident that the final complexes assembled in this experiment represent a core-- complex and not a mixed population of - and -core complexes since the intermediate - and -core complexes were formed to completion, leaving no free subunit present during the next injection phase. The core-- complex also appeared to assemble at the expected 2:4:1 core:: ratio.

The and complexes were reconstituted on BIAcore via or ` coupled to the sensor chip (Olson et al., 1995). Not surprisingly, both and complexes bound beta (Fig. 5). The complex readily bound a stoichiometric amount of core to form a complex with a dissociation half-life of 1.5 h; the complex did not (Fig. 5), consistent with previous results (Dallmann et al., 1995) showing that the complex requires a large excess of core for activity. These results can be interpreted as a mass action effect. The activity of complex increases with increasing core since the interaction of core with beta (required for high processivity) (Fay et al., 1982) on the primed DNA will be diffusion-limited in the absence of any core- complex interaction. With complex, core-beta interaction can be established rapidly since the polymerase, beta-clamp loader and beta are all associated in one large complex ( Fig. 5and Fig. 6), effectively increasing the local concentration of core relative to beta.

We do not detect any interaction between the complex and the subunit on the BIAcore (Fig. 5). The small effect of the subunit on complex activity assays (Dallmann et al., 1995) also supports our present observation. All the components of a -less initiation complex can be assembled on the BIAcore via the subunit as a large, stable multiprotein-DNA complex dependent on the presence of the subunit (Fig. 6). Furthermore, the apparent binding stoichiometry of core was consistent with the presence of tetrameric DnaX complexes, as expected based on the DnaX subunit stoichiometry determined by analytical ultracentrifugation and DnaX complex stoichiometry determined by quantitative gel scanning.

The results presented in this report and elsewhere (Olson et al., 1995; Dallmann et al., 1995; Blinkova et al. 1993) indicate is a dispensible subunit, both in vitro and in vivo. Asymmetry in the function of holoenzyme can be conferred by the subunit alone (Dallmann et al., 1995) without apparent requirement for the subunit/complex. In Fig. 7, we present a model for a -less DNA polymerase III holoenzyme that possesses all of the properties of native holoenzyme. When is present in this complex, it must replace two s at a position away from the two s that dimerize pol III core. This model is also consistent with the results of Blinkova et al.(1993), who reconstituted and purified a functional -less holoenzyme from the alpha, , , , , `, , and subunits, and additionally found that expression of , but not , was essential for cell viability. We have also observed that holoenzyme reconstituted from all 10 subunits fractionates upon gel filtration into a 9-subunit holoenzyme, lacking (data not shown), indicating that the mechanism of entry is complex.


Figure 7: Model of a -less DNA polymerase III holoenzyme. A model for DNA polymerase III holoenzyme lacking the subunit is shown. The model is based on observations in this report and others (Blinkova et al., 1993; Olson et al., 1995; Dallmann et al., 1995). In -containing holoenzyme, must replace 2 s distal to those that dimerize core pol III, since is not a participant in this interaction.



The model shown in Fig. 7remains an asymmetric complex, considering that the interaction of the single copy , `, , and subunits with the tetrameric subunit is an inherently asymmetric arrangement. An analogous situation can be observed in the recently determined crystal structure of bovine heart mitochondrial F(1)-ATPase (Abrahams et al. 1994) where the interaction of the single copy , , and subunits impose asymmetry in the tertiary structures in each copy of the 3 alpha and 3 beta subunits. The asymmetric placement of , `, , and on the tetrameric subunit could then confer the asymmetric properties of the leading and lagging strand polymerase halves of holoenzyme.

While this manuscript was under review, a paper by O'Donnell and colleagues (Onrust et al., 1995a) concluded that and exist principally as dimers and that the stoichiometry of the and complexes are (2)(1)`(1)(1)(1) and (2)(1)`(1)(1)(1), respectively. Our data consistently indicate a tetrameric assembly for both and , based on the molecular weight determinations for and obtained by the more rigorous method of sedimentation equilibrium. Note that no discrepancy exists when the stoichiometry assigned by O'Donnell and colleagues for the complex (their Table 2) is divided by the yield obtained in the HPLC experiment used to separate them (HPLC section of their ``Experimental Procedures'').

In the same series of reports (Onrust et al., 1995b), O'Donnell and co-workers described a method for reconstituting into holoenzyme that depends on the presence of limiting amounts of and a molar excess of in the reconstitution mix. Furthermore, the presence of -` during the early stages of reconstitution blocked the entry of into holoenzyme. Those results are also consistent with a tetrameric DnaX assembly within holoenzyme. Our observations that DnaX assemblies are tetrameric and that -` bind to (and presumably stabilize) this tetrameric complex provide additional insight into holoenzyme assembly. With present in limiting concentrations and present in excess, the formation of a mixed - complex (^4)is the only way to accommodate the maximal quantity of energetically favorable alpha- contacts (K(D)<1 nM^3) while preserving a tetrameric DnaX arrangement. If -` binding to stabilizes a tetrameric assembly, it may not be able to dissociate to (2), a requisite for assembly with alpha(2)(2). It seems unlikely to us that a lengthy stepwise incubation in the absence of -` represents a biologically relevant pathway for holoenzyme assembly. Given our finding that (4) is the favored assembly state and that pol III can bind and form pol III` containing a dimer (McHenry, 1982; Studwell-Vaughan and O'Donnell, 1991), formation of this complex may provide an assembly intermediate for the entry of into holoenzyme. In any case, appears to be dispensable, since all of the known functional properties of DNA polymerase III holoenzyme, including functional and structural asymmetry can be reconstituted in -less holoenzyme.


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 a 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, E. coli DNA polymerase III core (alpha--); complex, (--`--); complex, (--`--); DnaX complex, a complex containing either product of the dnaX gene ( or ) with associated , `, and ; SSB, E. coli single-stranded DNA binding protein; Mes, 2-(N-morpholino)ethanesulfonic acid; HPLC, high performance liquid chromatography; pol III, polymerase III.

(^2)
Concentration 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.

(^3)
D.-R. Kim and C. McHenry, manuscript in preparation.

(^4)
Even if energetically unfavorable, such an arrangement would occur if the -alpha interaction is stronger than the energy lost in converting a (2)(2) interaction into an (2)(2) interaction.


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