©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Assembly of a Chromosomal Replication Machine: Two DNA Polymerases, a Clamp Loader, and Sliding Clamps in One Holoenzyme Particle
I. ORGANIZATION OF THE CLAMP LOADER (*)

Rene Onrust (1)(§), Jeff Finkelstein (2), Vytautas Naktinis (1)(¶), Jennifer Turner (1), Linhua Fang (1), Mike O'Donnell (1) (2)(**)

From the (1) Microbiology Department and the Hearst Research Foundation and the (2) Howard Hughes Medical Institute, Cornell University Medical College, New York, New York 10021

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
REFERENCES

ABSTRACT

The complex of DNA polymerase III holoenzyme, the replicase of Escherichia coli, couples ATP hydrolysis to the loading of sliding clamps onto primed DNA. The sliding clamp tethers the holoenzyme replicase to DNA for rapid and processive synthesis. In this report, the complex has been constituted from its five different subunits. Size measurements and subunit stoichiometry studies show a composition of `. Strong intersubunit contacts have been identified by gel filtration, and weaker contacts were identified by surface plasmon resonance measurements. An analogous complex has also been constituted and characterized; it is nearly as active as the complex in clamp loading activity, but as shown in the fourth report of this series, it is at a disadvantage in binding the , `, , and subunits when core is present (Xiao, H., Naktinis, V., and O'Donnell, M. (1995) J. Biol. Chem. 270, 13378-13383). The single copy sub-units within the complex provide the basis for the structural asymmetry inherent within DNA polymerase III holoenzyme.


INTRODUCTION

The replicative polymerase of Escherichia coli, DNA polymerase III holoenzyme (holoenzyme), contains 10 nonidentical subunits (reviewed in Refs. 1 and 2). The subunit is the DNA polymerase; the subunit is the proofreading 3`-5`-exonuclease; and the subunit is a dimer in the shape of a ring with a central cavity for encircling duplex DNA. The function of the ring is to slide freely along duplex DNA while tethering the polymerase machinery to the template for highly processive synthesis (3, 4) .

Several subassemblies of the holoenzyme can be purified from cell lysates. The smallest of these is the core polymerase, a heterotrimer of (5) . The polymerase (Pol)() III` subassembly is composed of four different subunits: a dimer and two core polymerases (core-) (6) . The complex, composed of five different subunits (`), is a molecular matchmaker that couples ATP hydrolysis to load clamps onto primed DNA. The largest subassembly of the holoenzyme is Pol III*, which contains nine different subunits: two cores, , and the complex (9) . Two DNA polymerases in one molecular particle fits nicely with the hypothesis that replicative polymerases act in pairs for coordinated synthesis of both leading and lagging strands of a chromosome (7, 8) .

The core polymerase is neither highly processive nor rapid in DNA synthesis; it polymerizes nucleotides at a rate of 10/s and dissociates from DNA after incorporating 11 nucleotides (10, 11) . The Pol III` and Pol III* assemblies are not very processive either, but are severalfold more efficient than core (10, 12) . The subunit sliding clamp is needed for rapid and highly processive DNA synthesis. Once the ring is assembled onto DNA, it confers efficient synthesis on all these polymerase subassemblies (3, 13, 14) .

The subunit does not assemble onto DNA by itself, but requires the ATP-dependent clamp loading activity of the complex (3, 13, 15, 16) . The individual functions of complex subunits are largely unknown. Past studies have shown that binds ATP (17) and can function with to place onto DNA (18, 19) , although the ` subunit stimulates this reaction considerably (19, 20) , and and are needed at physiological ionic strength (18) . Elucidating the exact function of each subunit and the role(s) of ATP is an important goal.

For several years, the genes encoding five of the holoenzyme subunits have been known. The subunit is encoded by dnaE, by dnaQ (also mutD), by dnaN, and and are both encoded by dnaX. The subunit is approximately the amino-terminal two-thirds of and is generated by a -1 translational frameshift that results in only one unique amino acid in , the C-terminal Glu residue (21, 22, 23) . Two years ago, the remaining five genes of the holoenzyme were identified (19, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32) and used to produce large amounts of pure subunits (19, 24, 25, 26, 27) . With all 10 subunits in hand, detailed questions of structure and function can be addressed. This series of reports presents five related topics in which the pure subunits are used to assemble the holoenzyme. Studies of the assembly process have outlined the subunit contacts and the overall structure of this replicative machine.

This first report of the series focuses on the complex. The complex has been constituted in quantity from its five separate subunits, revealing several subunit contacts and providing a framework for its overall organization. Compositional studies show that four of the five subunits are present in single copy, which defines the complex as a structure lacking a 2-fold axis of symmetry (i.e. it is asymmetric). In the second report, the point of contact between the clamp and the complex is identified as residing nearly completely, if not solely, in the subunit (41) . Surprisingly, ATP is needed for the complex to ``present'' the subunit for interaction with , providing insight into the role for ATP and the mechanism of clamp loading. The third report defines the assembly path of the nine-subunit Pol III* assembly (38) . Study of Pol III* and its subunit composition reveals that contact between and is the central touch point between the clamp loader ( complex) and the twin polymerase (Pol III`) within the holoenzyme. Compositional study of Pol III* shows that only one copy of some of the subunits is present, and therefore, the holoenzyme must be structurally asymmetric. The first report shows that the single copy subunits can assemble with either or to form a clamp loader. The fourth report utilizes ATP-binding site mutants of and to show that in Pol III*, the single copy subunits of the clamp loader reside on (39). The preference of over appears to be caused by association of core with (but not ), which puts the association of with the single copy subunits at a kinetic disadvantage. The last report shows that four different forms of Pol III can be assembled in the presence of primed DNA (42) . These distinct polymerase forms may perform different tasks in DNA metabolism.


EXPERIMENTAL PROCEDURES

Materials

Radioactive nucleotides were obtained from DuPont NEN, and unlabeled nucleotides were from Pharmacia Biotech Inc. Proteins were purified as described: , , , and (33) ; (4); and ` (24) ; and (25) ; and (27) . Pol III* was purified from cell lysates (9) ; the complex was purified from cell lysates (16) ; and the complex was constituted and purified as described (33) . Protein concentrations were determined from their extinction coefficients at 280 nm, except for Pol III* and the complex purified without overproduction, which were quantitated using the protein assay from Bio-Rad and bovine serum albumin as a standard. M13mp18 ssDNA was phenol-extracted from phage that was purified by two consecutive bandings (first down and then up) in cesium chloride gradients as described (43) . M13mp18 ssDNA was primed with a DNA 30-mer (map positions 6817-6846) as described (33) . DNA oligonucleotides were purchased from Oligos Etc. Buffer A contained 20 mM Tris-HCl (pH 7.5), 0.1 mM EDTA (pH 7.5), 20% glycerol, and 5 mM DTT. Replication buffer contained 20 mM Tris-HCl (pH 7.5), 4% glycerol, 0.1 mM EDTA, 40 µg/ml bovine serum albumin, 5 mM DTT, 8 mM MgCl, 0.5 mM ATP, 60 µM dCTP, 60 µM dGTP, 60 µM dATP, and 20 µM [-P]TTP (specific activity of 2000-4000 cpm/pmol). Column buffer contained 20 mM Tris-HCl (pH 7.5), 10% glycerol, 2 mM DTT, 0.1 mM EDTA, and 100 mM NaCl. SPR buffer contained 10 mM Hepes-NaOH (pH 7.4), 150 mM NaCl, 3.4 mM EDTA, and 0.005% Tween 20. Sedimentation buffer contained 20 mM Tris-HCl (pH 7.5), 2 mM DTT, 0.1 mM EDTA, and 100 mM NaCl.

Replication Assays

Replication assays contained 63 ng (32 fmol) of singly primed M13mp18 ssDNA, 0.82 µg of SSB, 8.8 ng (56 fmol) of complex, and 20 ng (246 fmol as dimer) of in a final volume of 25 µl of replication buffer (after the addition of remaining proteins). All proteins were added to the assay on ice and then shifted to 37 °C for 5 min. DNA synthesis was quenched and quantitated using DE81 paper as described (34) . When needed, proteins were diluted in 20 mM Tris-HCl (pH 7.5), 2 mM DTT, 0.5 mM EDTA, 20% glycerol, and 50 µg/ml bovine serum albumin.

Gel Filtration

Gel filtration of the reconstituted and complexes was performed at 4 °C (unless indicated otherwise) using a Superose 6 HR 10/30 column (Pharmacia Biotech Inc.) equilibrated with column buffer containing 5 mM MgCl and 0.2 mM ATP. To constitute these complexes, (78 µg, 0.88 nmol as dimer) or (118 µg, 0.83 nmol as dimer) was incubated with (39 µg, 2.4 nmol as monomer) and (30 µg, 2 nmol as monomer) for 30 min at 15 °C. To this protein mixture was added (77 µg, 2 nmol as monomer) and ` (74 µg, 2 nmol as monomer) for 30 min at 15 °C. The protein mixture was then concentrated to 100 µl by spin dialysis using a Centricon 30 apparatus (Amicon, Inc.) and gel-filtered. After the first 7 ml, fractions of 200 µl were collected and analyzed on 15% SDS-polyacrylamide gels (100 µl/lane) stained with Coomassie Blue R-250. Densitometry of stained gels was performed using a Pharmacia Ultrascan XL laser densitometer. Replication assays of column fractions were performed by first diluting a 2-µl aliquot 20-fold and then adding 2 µl to the assay.

Gel filtration of mixtures containing less than all five subunits of the complex (i.e.Fig. 5 ) were performed using a Superose 12 column (in one case, Superdex 75 was used as indicated in the legend to Fig. 5). Subunit mixtures were incubated for 30 min at 15 °C in 200 µl of column buffer and included (when present) the following: , 84 µg (0.9 nmol as dimer); , 57 µg (1.5 nmol); `, 56 µg (1.56 nmol); , 41.8 µg (2.52 nmol); and , 25 µg (1.67 nmol). Subunit mixtures were gel-filtered as described above, except that after collecting the first 6.0 ml, fractions of 170 µl were collected. Additions of in the absence of required 0.5 M urea as described (26) . To remove urea, mixtures containing both and were first mixed using a 1.5-fold excess of to in a final concentration of 0.5 M urea in buffer A and then dialyzed against sedimentation buffer.


Figure 5: Search for subassemblies of the complex. Gel filtration analysis of mixtures of complex subunits was performed as described under ``Experimental Procedures.'' The SDS-polyacrylamide gels of the column fractions were stained with Coomassie Blue. Fraction numbers are shown at the top; positions of molecular weight markers are noted on the left; and complex subunits are identified on the right. A-G were analyses on Superose 12, and H was an analysis on Superdex 75.



Protein standards (Bio-Rad and Sigma) were a mixture of 50 µg each in 100 µl of column buffer. The K value was calculated using the following equation: K = (V- V)/(V- V), where V is the observed elution volume, V is the included volume, and V is the exclusion volume. The V value for both Superose 6 and 12 columns was 24 ml (manufacturer's specifications), and the V value, determined using M13mp18 ssDNA saturated with SSB (total molecular mass of 25 MDa), was 6.0 ml for Superose 12 and 7.0 ml for Superose 6.

Glycerol Gradient Sedimentation

The ` and ` complexes were constituted as described above for gel filtration analysis and then layered onto separate 11.6-ml 10-30% glycerol gradients in sedimentation buffer. Protein standards (50 µg each in 100 µl of sedimentation buffer) were layered onto a parallel gradient, and the gradients were centrifuged at 270,000 g for 30 h at 4 °C. Fractions of 170 µl were collected from the bottom of the tube and analyzed on a 13% SDS-polyacrylamide gel (100 µl/lane) stained with Coomassie Blue and analyzed in replication assays as described for the gel filtration column fractions. Stability of the [H] Complex-The and subunits were tritiated by reductive methylation as described (3, 44) to specific activities of 7.5 10 and 1.4 10 cpm/pmol, respectively. The [H] complex was constituted upon mixing 4 mg of with 0.125 mg of [H], 2.7 mg of unlabeled , 0.25 mg of [H], and 1.7 mg of unlabeled in a final volume of 3.0 ml of buffer A (0.5 M urea final concentration due to the urea in ). After 1 h at 15 °C, the mixture was applied to a 1-ml Mono Q column and eluted with a 19-ml linear gradient of 0-0.4 M NaCl in buffer A. The [H] complex was the last to elute (0.22 M NaCl). Fractions containing [H] were pooled (3.1 mg; specific activity of 1.8 10 cpm/pmol), dialyzed against 4 liters of buffer A, and then aliquoted and stored at -70 °C. Dissociation of the [H] complex was assayed by gel filtration on Superose 12, which resolves [H] from dissociated [H]. A total of 8.9 µg (71 pmol) of [H] complex was incubated for 5 min at the indicated temperature in either the absence or presence of 5.5 µg (141 pmol) of and 5.25 µg (140 pmol) of ` in 100 µl of column buffer containing 0.3 M NaCl, 0.5 mM ATP, and 8 mM MgCl. After incubation, reactions were analyzed by gel filtration on Superose 12 essentially as described above, except that the column buffer contained 300 mM NaCl, 8 mM MgCl, and 0.5 mM ATP. The amount of [H] in each column fraction was determined by liquid scintillation counting. Different temperatures of gel filtration were achieved by immersing the column and the injection loop in a water bath containing ice or held at 28 or 37 °C.

Preparative Constitution of the Complex

A mixture of 11.1 mg of (118 nmol as dimer), 13.7 mg of (355 nmol as monomer), 8.7 mg of ` (235 nmol as monomer), 5.9 mg of (354 nmol as monomer), and 3.6 mg of (237 nmol as monomer in 4 M urea) was incubated in a final volume of 63.75 ml of buffer A for 30 min at 15 °C (incubation was started with and proteins combined together in a final volume sufficient to bring the urea concentration to 0.5 M, and then the other subunits were added). This mixture was loaded onto an 8-ml Mono Q HR 10/10 column (Pharmacia Biotech Inc.) and eluted with a 180-ml linear gradient of 0-0.4 M NaCl in buffer A at 0.3 ml/min. Fractions of 2.5 ml each were collected. Fractions 46-52, which contained `, were pooled (18.5 ml, 18 mg), dialyzed against 4 liters of buffer A, and then aliquoted and stored at -70 °C.

HPLC

Reversed-phase HPLC analysis was performed on a Waters system. The reconstituted complex (30 µg) in 100 µl of buffer A was adjusted to 1% trifluoroacetic acid and injected onto a Dynamax C (butyl) reversed-phase HPLC column (250 4.6 mm) with a pore size of 300 Å equilibrated with 40% HPLC-grade acetonitrile (J. T. Baker Inc.) in 0.125% trifluoroacetic acid. Subunits were eluted with a 48-55% acetonitrile gradient in 0.125% trifluoroacetic acid over 3 min followed by a 55-100% acetonitrile gradient in 0.125% trifluoroacetic acid over 12 min at a flow rate of 1 ml/min. Elution of subunits was monitored at 280 nm and recorded on chart paper. The areas under the peaks were measured manually by cutting the peaks out and weighing them. The molar extinction coefficients of the subunits calculated from their amino acid sequences are as follows: , 20,340 M cm; , 46,130 M cm; `, 60,136 M cm; , 29,160 M cm; and , 24,040 M cm).

The recoveries of complex subunits from the HPLC column were determined by passing a mixture of the subunits of known concentration (measured by their absorbance at 280 nm and known extinction coefficient) over the same HPLC column. The total absorbance recovered for each subunit was as follows: , 75.6%; , 87.3%; `, 100.4%; , 90.6%; and , 79.1%. Hence, all the subunits were recovered from the HPLC column in a reasonably high yield. These recoveries were not used to correct the observed molar ratio of subunits in the complex as the recovery measurements have an error of their own.

SPR

Immobilization of subunits was performed on the carboxymethyldextran matrix-coated sensor chip CM5 by carbodiimide covalent linkage following the manufacturers' instructions (Pharmacia Biosensor AB) using 30-µl solutions of subunits in SPR buffer in 10 mM sodium acetate at the following subunit concentrations and pH values: , 1.24 µM and pH 5.5; `, 0.35 µM and pH 5.5; and , 1.13 µM and pH 4.5. To immobilize the complex, the subunit was immobilized, and then a 35-µl solution of (0.5 µM in SPR buffer containing 0.5 M urea) was passed over the chip for 3 min, followed by washing with SPR buffer (28% of immobilized became complexed with ). The final change in response units for each immobilization was as follows: , 3542 response units; `, 3562 response units; , 2963 response units; and , 3683 response units (2936 response units of plus 747 response units after adding ). SPR analysis was performed by injecting 30 µl of a 1 µM solution of the indicated protein (as monomer, except for and , which are expressed as dimers) in SPR buffer for 3 min at 25 °C. All proteins were dialyzed against SPR buffer to reduce buffer-related artifacts. To obtain a value for the k, injections were followed for 3 min by SPR buffer lacking protein. After completing each analysis, the surface of the chip was regenerated by injecting 10 µl of 0.1 M glycine (pH 9.5), which released remaining bound protein without decreasing the capacity of the immobilized protein to bind in future injections. When the complex was immobilized, regeneration was performed by consecutive injection of 30 µl of 6 M urea and 30 µl of 2 M urea in SPR buffer, followed by de novo binding of to as described above. Data are presented as the observed change in response units divided by the molecular mass of the subunit in the mobile phase (, `, and as monomer; and as dimer; core as ; as ; and ` as `). All apparent k and k values were determined through nonlinear curve fitting using the Pharmacia Biosensor kinetics software (BIAevaluation 2.0) assuming the simplest case: A + B AB.


RESULTS

Constitution of the Complex

To determine if the complex (`) can be reconstituted from purified overproduced subunits, was mixed with a 2-fold molar excess each of , `, , and and then gel-filtered (Fig. 1). A complex of ` formed as indicated by comigration of all five subunits (Fig. 1A, panel 1, fractions 21-25); excess proteins eluted later as the ` complex (fractions 40-49) and the complex (fractions 46-49). Column fractions containing the ` complex were active in assembly of the clamp onto primed DNA, leading to processive DNA synthesis by the polymerase (Fig. 1A, panel3).


Figure 1: Native mass of constituted ` and ` complexes. A, either (panel 1) or (panel 2) was incubated with , `, , and , followed by gel filtration on a Superose 6 column as described under ``Experimental Procedures.'' Column fractions (Frx) are identified above and below the Coomassie Blue-stained SDS-polyacrylamide gels. The first lane of each gel contains protein standards, and their molecular weights (MW) are indicated to the left. The , , , `, , and subunits are identified to the right of each gel. Panel3 shows the activity of the column fractions of ` () and ` (). Panel4 shows the elution of ` and ` relative to protein standards of known Stokes radii calculated from their diffusion coefficients (as described in Ref. 36). B, shown is the glycerol gradient sedimentation analysis of ` (panel1) and ` (panel2). Either or was incubated with , `, , and , followed by sedimentation in a 10-30% glycerol gradient as described under ``Experimental Procedures.'' Panel3 shows the activity assays of glycerol gradient fractions of ` () and ` (). Panel4 compares the migration of protein standards of known s values with the migration of ` and `. Tgb, thyroglobulin (670 kDa, 85.0 Å); Apf, horse apoferritin (440 kDa, 59.5 Å); Amy, -amylase (200 kDa, 8.9 S); IgG (158 kDa, 52.3 Å, 7.4 S); BSA, bovine serum albumin (67 kDa, 34.9 Å, 4.41 S); Ova, chicken ovalbumin (43.5 kDa, 27.5 Å, 3.6 S); Myo, horse myoglobin (17.5 kDa, 19.0 Å, 2.0 S).



Comparison with protein standards showed that the complex has a Stokes radius of 67 Å (Fig. 1A, panel4), close to the 61-Å radius determined for the complex purified from E. coli lysates (25) . The constituted complex sedimented in a glycerol gradient with an s value of 8.7 S (Fig. 1B, panels1, 3, and 4), similar to the value of 8.2 S for the complex purified from E. coli lysates (16) . Trailing of the complex in the sedimentation analysis indicates that some dissociation occurs during the 30-h procedure. Hence, the s value should be taken as a minimum estimate.

Combining the Stokes radius and the s value in the mass equation of Siegel and Monty (35) yields a minimum estimated mass of 256 kDa for the reconstituted complex (), similar to the value of 210 kDa for the complex purified from E. coli(16) .

For structural studies to be described below, the complex was prepared in quantity and purified away from excess subunits that were not bound in the complex. Preparation of the complex is shown in Fig. 2, in which the subunits were mixed using limiting , followed by chromatography on a fast protein liquid chromatography Mono Q column eluted with an NaCl gradient. The complex was tightly retained on the column and was the last to elute. This method is highly efficient as the 11 mg of present initially was recovered as 18 mg of complex for an overall yield of 79%.


Figure 2: Preparative constitution and purification of the complex. A, the subunits of the complex were incubated, and the complex was separated from free components on a fast protein liquid chromatography Mono Q column as described under ``Experimental Procedures.'' B, 7-µl aliquots of the indicated column fractions were analyzed by 13% SDS-polyacrylamide gel electrophoresis as described under ``Experimental Procedures.''



Molar Ratio of Subunits in the Complex

Two techniques were applied to determine the molar ratio of subunits within the complex. The simplest was to scan the Coomassie Blue-stained SDS-polyacrylamide gels of the complex that was either constituted from pure subunits or purified as a complex from E. coli lysates. The molar ratio of complex subunits from this analysis is presented in after correcting the areas under the peaks for the relative molecular mass of each subunit and normalizing to the ` subunit. The results suggest a molar ratio of `, similar to the conclusions of an earlier study using the complex purified from cell lysates (16) . Different proteins may take up different amounts of Coomassie Blue dye, and therefore, we used as standards individual subunits of known concentration determined by absorbance from their extinction coefficients at 280 nm.

The molar ratio of complex subunits was also determined by an HPLC approach. The pure constituted complex was treated with 1% trifluoroacetic acid to disperse the subunits and then applied to an HPLC column with a four-carbon chain as a ligand. Elution of the column was monitored at 280 nm (Fig. 3), and the peaks were assigned by collecting fractions and analyzing them on an SDS-polyacrylamide gel. The 280 nm absorbance is due mainly to the Trp and Tyr residues in each protein. From the subunit gene sequences, the number of Trp and Tyr residues in each subunit allowed us to calculate their relative molar ratio from the area under their respective 280 nm absorbance peaks. The results () indicate a molar ratio of `. An HPLC analysis of the complex purified intact from E. coli cell lysates was also performed. Due to insufficient material, only one analysis was performed at 280 nm, but the observed molar ratio of subunits (`) was consistent with the subunit molar ratio measurements of the complex constituted from pure subunits. The composition of the complex most consistent with the measured molecular mass (256 kDa) and the observed subunit molar ratios is as follows: one each of , `, , and and two or three of (`) (). A simple explanation of the value of two to three subunits in the complex is that the complex preparation is a mixture of two complexes: ` and `. As discussed in the third report of this series, and migrate on gel filtration columns as homotetramers and a heterotetramer of (38) . Only one copy each of , `, , and can associate with the tetramer (e.g. to form `, `, or `) (38). The and subunits can also form homodimers as they appear as such in glycerol gradient analysis (17, 36) and are present as two copies each in Pol III* (38) . If the , `, , and subunits first associate with a dimer (of or ), then the formation of the tetramer is prevented (i.e. yielding a complex of either ` or `). Hence, the value of two to three subunits in the complex may reflect the proportions of and that the single copy subunits become associated with. However, a trimer as the basis for the observed stoichiometry of in the complex cannot be rigorously ruled out.


Figure 3: Molar ratio of subunits in the complex. Separation of the subunits of the pure complex on a C reversed-phase HPLC column was performed as described under ``Experimental Procedures.'' Elution of subunits was monitored at 280 nm. The assignment of a peak with its respective subunit, shown above each peak, was determined upon collecting the peak, evaporating the sample to dryness, and analysis on a 15% SDS-polyacrylamide gel.



The Complex

The subunit (72 kDa) is encoded by the same gene as (47 kDa) and therefore contains the amino acid sequence of within it (except for the C-terminal amino acid of ) and an additional extension of 213 amino acids at the C terminus (21-23). Hence, can probably bind , `, , and to form a `` complex'' (`). Indeed, our previous studies showed that the subunit was capable of forming and ` complexes (19, 26) . Upon mixing with , `, , and , a complex was formed as anticipated. Fig. 1(panel2 in A and B) shows the gel filtration and sedimentation analyses of the complex. The replication assays in panel3 show that the activity corresponds to the position of the complex. Combining the Stokes radius (75 Å) and the s value (8.3 S) of the complex in the equation of Siegel and Monty yields an observed mass of 271 kDa ().

The molar ratio of subunits in the complex, determined by densitometry of the Coomassie Blue-stained SDS-polyacrylamide gel, yielded a subunit stoichiometry similar to that of the complex (). A stoichiometry of two protomers and one monomer each of , `, , and predicts a mass of 250 kDa for the complex, consistent with the observed mass of 271 kDa (). The value of two to three protomers in the complex is possibly due to a mixture of two complexes (` and `) due to the variable aggregation state of as discussed above for in the complex.

Replicative Activity of Constituted Complexes

Are these constituted clamp loader complexes as active as the naturally purified complex in loading onto primed DNA for processive synthesis by the core polymerase? In Fig. 4, the complexes were titrated into a replication assay containing the subunit and core polymerase in saturating amounts. The DNA template is a long (7.2 kilobases) M13mp18 circular ssDNA primed with a single synthetic DNA oligonucleotide and coated with SSB. In this assay, there is no detectable DNA synthesis unless the clamp has been assembled onto DNA for use by the core polymerase. The results show that the activity of the constituted complex is comparable to (in fact, slightly higher than) the complex purified intact from E. coli cell lysates. In contrast, the complex appears to be slightly less active and does not reach the same plateau level as the complex.


Figure 4: Activity of constituted and complexes. Replication assays were performed as described under ``Experimental Procedures.'' Shown is the stimulation of DNA synthesis upon the addition of increasing amounts of the following: the complex purified from E. coli lysates (), the constituted complex (), or the constituted complex ().



Strong Intersubunit Contacts within the Complex

Gel filtration is a nonequilibrium technique, and thus, only strong subunit contacts survive. To determine which subunits of the complex are in strong contact with one another, different combinations of subunits were incubated together and analyzed by gel filtration. Previously, we analyzed all combinations of , , and ` with the finding that a ` complex was stabile to gel filtration, as was a ` complex, but the + and + ` mixtures did not result in gel-filterable complexes (19) . Also, all combinations of , , and have been analyzed by gel filtration with the result that , , and complexes were observed, but the + mixture did not result in a stabile complex (26) .

In Fig. 5 , the remaining combinations of subunits were analyzed by gel filtration, specifically those containing and either one or both of and ` plus one or both of and . In Fig. 5A, the mixture of all five subunits (i.e. the entire complex) was analyzed as a basis of comparison for the other panels. The analysis shows a similar pattern as in Fig. 1A in which the complex (fractions 16-22) eluted ahead of the ` complex (fractions 34-44) and the complex (fractions 40-48). In Fig. 5B, a mixture of the , , , and subunits did not result in a four-subunit complex, but assorted as a complex (fractions 18-28) and a free subunit (fractions 40-46).

The mixture of , `, , and in Fig. 5C resulted in a stabile four-subunit ` complex in fractions 18-28, eluting earlier than the excess ` subunit (fractions 40-46) and the complex (fractions 40-48). This result was somewhat surprising as ` does not coelute with either or during gel filtration. A mixture of , `, and in Fig. 5D resulted in a stabile heterotrimer of ` (fractions 20-28), but a mixture of , `, and (Fig. 5E) showed no complex. Hence, is not needed for ` to form a stabile complex with .

Does the ` complex bind ? In Fig. 5F, a mixture of , , `, and was analyzed, but was not assimilated into the ` complex (i.e. migrated alone in fractions 50-58 and not with ` in fractions 20-26). Since both ` and are stabile to gel filtration (19, 26) , a ` complex should also be stabile. Fig. 5G shows the anticipated formation of a gel-filterable ` complex (fractions 18-26).

Perhaps a ` complex can form in the absence of . This four-subunit mixture was tested in Fig. 5H using a Superdex 75 column (hence its own set of fraction numbers). The pattern on the SDS-polyacrylamide gel showed formation of the ` and complexes, but not a ` complex.

The analysis of Fig. 5shows that strengthens the binding of ` to . In Fig. 6 , we examined this cooperative binding further. We used a complex that was reconstituted using [H] and [H] to easily follow (and that dissociated from it) in the column fractions. The [H] complex was gel-filtered at three different temperatures (Fig. 6, A-C). At 0 °C, the [H] complex remained nearly completely intact, but as the temperature was increased to 37 °C, most of the [H] dissociated from . The addition of ` provided stabile association of [H] with at 37 °C (Fig. 6D), consistent with cooperative binding of and ` to . Analysis of column fractions by 13% SDS-polyacrylamide gel electrophoresis followed by fluorography to visualize H-labeled subunits showed that the dissociated peak was an equal mixture of [H] and [H], and therefore, both dissociated from simultaneously (data not shown).


Figure 6: Cooperativity between ` and in binding to . The [H] complex was gel-filtered at different temperatures as described under ``Experimental Procedures.'' The earliest peak to elute was the [H] complex, and the second peak was the [H] complex. A, analysis of [H] at 0 °C; B, analysis of [H] at 28 °C; C, analysis of [H] at 37 °C; D, analysis of [H]` at 37 °C.



Surface Plasmon Resonance Analysis

Next, the weaker intersubunit interactions were analyzed using the SPR technique in a Biosensor instrument. In SPR, one protein is immobilized on a sensor chip, and a second protein is injected over the surface in a mobile phase. If the two proteins interact, the resulting increase in mass on the chip is detected and plotted as an increase in response units over time, allowing calculation of the apparent k value. After the injection of protein in the mobile phase is complete, buffer is passed over the chip, and the dissociation of the proteins is observed as a loss in mass over time from which the apparent k value can be calculated. From these data, the apparent dissociation constant (K ) is obtained (K= k/k). It is important to note that these constants apply to the specific conditions used in these experiments and could vary under other conditions. In this report, similar levels of immobilized protein and 1 µM concentrations of protein in the mobile phase were used. Hence, the values should be approximately comparable for the different experiments.

In each of the panels in Fig. 7, a different subunit of the complex was attached to the sensor chip, and then 1 µM solutions of the other subunits were serially passed over the chip. The subunit was also included in the analysis, as was the core polymerase. In Fig. 7, openarrowheads mark the start of protein injection, and closed arrowheads mark the end. Since is insoluble by itself, the complex was analyzed, and therefore, subunits that bind specifically to must be inferred upon comparison to results using alone. The immobilization of appeared to inactivate it, and therefore, SPR analysis could not be performed.() The data are plotted as response units divided by the molecular mass of the protein in the mobile phase to normalize the results for the different masses of subunits in the different injections. In general, the stoichiometry of protein in the mobile phase bound to immobilized protein was 15-30% of the known stoichiometries. This level of ligand binding to immobilized protein often occurs when using the SPR technique. Control experiments showed no detectable binding of these subunits to a sensor chip that was pretreated with the immobilization reagents.


Figure 7: Identification of weak interactions within the complex by SPR. The scheme for protein injection and buffer (buf.) injection over an immobilized protein is shown in the panel at the top. In each panel, the immobilized complex subunit on the chip is indicated at the top left corner, and the subunits in the fluid phase are indicated in the panel. The closed arrowheads (above the data line) mark the start of protein injection, and the openarrowheads (below the data line) mark the start of buffer injection. A, immobilized ; B, immobilized `; C, immobilized ; D, immobilized complex. RU, response units.



All the gel-filterable complexes were also observed in the SPR analysis, but we hoped that the sensitive SPR technique would detect new interactions that are too weak to detect by gel filtration. For example, a mixture of and will assemble onto DNA, although the reaction is feeble, indicating that and may weakly interact. Also, a mixture of and ` results in DNA-dependent ATPase activity, suggesting that they interact. Another possible weak interaction is one between and ` as an explanation of how these subunits cooperate in binding to . In summary, SPR analysis detected the predicted weak `- interaction, but no interaction was observed between and or between ` and . Hence, these latter two subunit combinations probably cooperate by means other than direct contact (discussed below). Furthermore, a putative - interaction was observed for which there is no other supporting evidence. The detailed experiments were as follows.

In Fig. 7A, was attached to the chip, and each subunit of the complex was passed over it. Included in the analysis were the subunit and the core polymerase. SPR analysis detected the previously identified strong interaction of with `, but no other significant interaction was observed, not even with the subunit.

In experiments using ` immobilized on the chip (Fig. 7B), an interaction with was observed as anticipated from the ATPase assays. A particularly strong interaction was observed for , consistent with the previously identified ` complex.

Analysis of immobilized , in Fig. 7C, showed a slight, but reproducible, interaction with , but not with ` or . To determine if ` would bind tighter, the ` complex was passed over the chip, but it showed only the same interaction with as alone. These results indicate that may bind to , but it is important to note that experiments with immobilized failed to detect significant interaction with or the complex (see Fig. 7A). Perhaps the surface on that interacts with may be preferentially cross-linked to the sensor chip, preventing interaction with . The subunit binds tightly to , but this is not shown in Fig. 7since is insoluble. However, the interaction of with immobilized was observed by injecting in 0.5 M urea over the chip (data not shown). This resulted in a huge increase in response units due to the change in refractive index caused by urea in the preparation. Nonetheless, upon finishing the injection and replacing urea with SPR buffer, the final response units indicated that had bound to (28% of immobilized bound ).

Since is inactive and insoluble by itself, but is soluble when bound to , we immobilized (Fig. 7D) by first coupling to the chip and then adding to it as described above. In the SPR analysis of immobilized (Fig. 7D), we expected to observe a weak interaction between and ` or the ` complex as an explanation of their cooperativity in binding , but no interaction between and ` was observed. A slight interaction of with and ` was observed, presumably due to weak contact between and . Although this putative contact may lend cooperativity between ` and in binding , it does not explain the cooperative nature of ` and in forming a ` complex.

The apparent k and k values for the interactions observed in Fig. 7 were determined from the kinetic traces. It is important to note that since one ligand is immobilized and thus not free to diffuse in three dimensions, the apparent rates may differ from those measured by other means. Since the experiments in Fig. 7were performed using a uniform concentration of protein in the mobile phase and similar amounts of immobilized protein, the apparent rates should be comparable within the set of experiments. The association and dissociation rates of with immobilized ` and of ` with immobilized are similar (within 2-fold), and the apparent K value of 68-124 nM is consistent with the ability to detect the ` complex in the gel filtration approach. The kinetic parameters for binding of ` to and are similar (within 3-fold), and the apparent K value for ` and ` complexes is in the 100-300 nM range, apparently too weak to detect by gel filtration. The putative k value for the interaction of with is 1-2 µM. Likewise, the k for the interaction of with ` and of with and ` is also 1-2 µM. Hence, the putative interaction between and ` appears to reside in the -to- contact. The and subunits bind tightly to (38-150 nM), consistent with the ability to gel filter and complexes. These experiments were performed in the absence of MgCl and ATP. We have performed a similar set of experiments in the presence of 0.5 mM ATP and 10 mM MgCl, but no significant differences were observed (data not shown).

Lack of Interaction between the Complex and Core

In a previous study, we showed that bound core tightly, but did not (36) . Now that we have reconstituted the complex in quantity, we re-examined whether the complex binds to core by gel filtration analysis, but still did not detect an interaction (data not shown). In the event that core binds a subunit of the complex weakly, we have used SPR to look for an interaction of core with , `, , or , but no interaction was detected (Fig. 7).


DISCUSSION

Structure of the Complex

Individual overproduced subunits have been used to constitute the complex in quantity for structural studies. The constituted complex is as active as the complex purified from E. coli lysates. The hydrodynamic measurements and subunit molar ratios indicate a stoichiometry of `, in agreement with estimates of a preliminary study (16) . As the aggregation state appears to consist of tetramers (i.e. in gel filtration analysis) and dimers (i.e. observed in glycerol gradients) (17, 36) , the value of two to three subunits in the complex likely has its basis in a mixture of two species, ` and `.

The schematic in Fig. 8depicts a subunit organization of the complex consistent with the stoichiometry of subunits in the complex and the observed interactions among them. The subunit is an oligomer, probably a tetramer, by itself (17, 37) , but stoichiometry and native mass measurements indicate that is a dimer in the complex. Only two subunits of the complex appear to have appreciable affinity for the dimer. The subunit has strong affinity for and was detected in an earlier study by gel filtration (26). Interaction of ` with was inferred by DNA-dependent ATPase activity that depends upon both ` and (19, 20) . Interaction of ` with was not detected by gel filtration, but was detected by the SPR technique. Once is bound to , association of ` becomes strong enough to survive gel filtration. One explanation for this is direct contact between and `. However, no interaction between and ` or between and ` was observed in this study. Hence, the subunit may induce a conformational change in such that binds ` tighter. The and ` subunits are shown as lacking direct contact in Fig. 8 to reflect this possibility. The and subunits are placed in contact with one another in Fig. 8to reflect the weak interaction detected by SPR analysis.


Figure 8: Putative subunit arrangement of the complex. A dimer of binds one monomer each of and `. A monomer forms a firm contact with . A monomer of associates with ` and .



Because is a dimer, it should have two binding sites for ` and two sites for . There are at least four obvious explanations of how the dimer may bind a single monomer of ` and . 1) The two sites may overlap. 2) Binding may be negatively cooperative, whereupon binding one ` to a protomer induces a conformational change in the other protomer, lowering its affinity for a second ` (likewise for ). 3) The binding sites of and ` overlap such that once ` is bound to , it occludes one site, and conversely, once binds , it occludes one ` site. 4) The dimer may not be isologous, and thus, one site may be formed by two protomers, and this site would not be repeated elsewhere in the dimer. The third possibility predicts that in the absence of `, two subunits would bind a dimer. Our earlier study observed a ratio of subunits in the complex of , indicating that the third explanation is not the basis for the binding of only one to a dimer (26) .

A direct interaction between and was expected on the basis of the clamp loading activity that depends upon both and (18, 19), but no interaction between these subunits was detected in this study. Hence, and act separately to load onto DNA, or their interaction is very transient, or the subunit and/or DNA present in clamp loading assays is needed for direct interaction of with . However, the -` contact is a strong interaction observed in our earlier study (19) . Hence, in Fig. 7C, is shown to associate with indirectly through association with the ` subunit. The subunit is known to bind strongly (26) , and the present report suggests that may also bind .

The Complex

The subunit contains the amino acid sequence of (and an extra 213 residues at the C terminus) and therefore may be expected to bind the , `, , and subunits. Indeed, a complex (`) can be constituted and is within 2-fold of the activity of the complex. The native aggregation state of , like , appears to consist of dimers and tetramers (17, 36) , and thus, we propose that the complex (like the complex) is a mixture consisting predominantly of ` with some `. It is important to note that a complex has yet to be purified from E. coli cells. Furthermore, the , `, , and subunits are not present in Pol III` (core-) (6) . The third report of this series demonstrates that only one each of the , `, , and subunits assembles into Pol III*, and therefore, these subunits must reside on either or , but not both (38) . The location of these single copy subunits in Pol III* is the subject of the fourth report of this series (39) .

Implication of Complex Subunit Stoichiometry to the Asymmetric Structure of Pol III Holoenzyme

Single copy subunits bound to a dimer would eliminate the 2-fold axis of symmetry relating the two protomers of the dimer, thereby imposing a structural asymmetry to the complex. In other words, since monomeric proteins are asymmetric structures, the complex must also be asymmetric. This has important implications for the putative asymmetric holoenzyme as discussed more fully in the third report on the constitution of Pol III* (38) . The Pol III* assembly process indicates that at the center of the structure, a dimer forms a heterotetramer with a dimer. The dimer also binds two molecules of the core polymerase, presumably one core for each protomer. Hence, in this arrangement, the two core polymerases are likely to be related by a 2-fold rotational axis of symmetry to the two protomers, and since and would be 2-fold symmetric, the cores would have a 2-fold symmetry relative to the two protomers also. Only one copy each of , `, , and is assimilated into Pol III*, and therefore, , `, , and must each be disposed asymmetrically relative to the two core polymerases. Thus, it is these single copy subunits of the complex that confer the structural asymmetry on the entire holoenzyme structure. Whether they all confer special properties on the lagging strand core or whether one or more function with the leading strand core is a topic for future studies.

  
Table: Native mass of ` and `

The Stokes radii and s values of the ` and ` complexes were determined from the gel filtration and glycerol gradient analysis in Fig. 1. Molecular masses and frictional coefficients were calculated from the Stokes radii and s values as described (35). These calculations require the partial specific volumes of ` and `, which were calculated by summation of the partial specific volumes of the individual amino acids for ` and ` (assuming and as dimers) (40). Molecular masses of the ` and ` complexes were calculated from the gene sequences of , , `, , and .


  
Table: Molar ratio of subunits in the and complexes

Molar ratios of subunits in the constituted and complexes purified by gel filtration as in Fig. 1 were determined by densitometry of Coomassie Blue-stained 15% SDS-polyacrylamide gels. The values are the average of three determinations and were normalized to `. In the HPLC analysis, the molar ratio of subunits of the constituted complex and of the complex purified from E. coli lysates was determined by monitoring their elution from the HPLC column at 280 nm. The areas under the peaks of 280 nm absorbance (Fig. 3) were divided by their respective values (see ``Experimental Procedures'') and normalized relative to the value for `. Each value is an average of three independent analyses.



FOOTNOTES

*
This work was supported by National Institutes of Health Grant GM38839 and National Science Foundation Grant MCB-9303921. 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.

§
Present address: Dept. of Pharmacology, University of California, San Francisco, CA 94122.

On leave from the Institute of Biotechnology Fermentas, Vilnius 2028, Lithuania.

**
To whom correspondence should be addressed: Howard Hughes Medical Inst., Cornell University Medical College, 1300 York Ave., New York, NY 10021. Tel.: 212-746-6512; Fax: 212-746-8587.

The abbreviations used are: Pol, polymerase; DTT, dithiothreitol; ssDNA, single-stranded DNA; SPR, surface plasmon resonance; HPLC, high pressure liquid chromatography; SSB, single-stranded DNA-binding protein of E. coli.

The immobilized subunit still bound the complex, but showed no detectable interaction with `, yet ` is a tight gel-filterable complex. In addition, immobilized did not show interaction with `. Hence, appears to be largely inactive during the immobilization procedure (probably due to the low pH needed for the carbodiimide chemistry).


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