©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
III. INTERFACE BETWEEN TWO POLYMERASES AND THE CLAMP LOADER (*)

Rene Onrust (1)(§), Jeff Finkelstein (2), Jennifer Turner (1), Vytautas Naktinis (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 nine-subunit DNA polymerase (Pol) III* coupled to its sliding clamp is a rapid and highly processive replicating machine. The multiple subunits are needed for the complicated task of duplicating the Escherichia coli chromosome. In this report, Pol III* was constituted from individual pure proteins, and its structure was studied. Constitution of the Pol III* particle requires an ordered addition of the subunits, and the final structure contains 14 polypeptides in the ratio `. The structure can be summarized as being composed of two core polymerases () held together by a dimer of and one complex clamp loader (`) for loading onto DNA. At the center of the structure, the related and subunits form a heterotetramer upon which the two core polymerases and clamp loader proteins assemble. The single copy nature of the , `, , and subunits confers a structural asymmetry with respect to the two polymerases, presumably for the different functions of replicating the leading and lagging strands.


INTRODUCTION

The Escherichia coli replicase, DNA polymerase III holoenzyme (holoenzyme), has traditionally been difficult to obtain in large quantity due to its low cellular concentration (10-20 molecules/cell) (1, 2) . All 10 subunits of the holoenzyme are now available in abundance by molecular cloning of their genes and high level expression techniques (3, 4, 5, 6, 7, 8, 9, 10, 11, 12) . In this report, we have used the pure subunits to determine how to assemble the nine-subunit polymerase (Pol)() III* and to obtain it in large quantity.

Previously, we described the constitution of the four-subunit Pol III` subassembly (), which consists of a dimer that serves, among other things, as a scaffold to hold together two core polymerases (13, 14) . In the first report of this series, we described the constitution and subunit composition of the five-subunit complex clamp loader (`), the molecular matchmaker that assembles clamps onto DNA (15) . Since the complex and Pol III` together account for the full complement of subunits of the nine-subunit Pol III* assembly, we presumed that Pol III* would be constituted upon mixing these two subassemblies. Hence, we were surprised to find that Pol III` and the complex did not associate with each other even when mixed at high concentrations (25 µM each). The fact that Pol III` and the complex do not easily associate with one another suggests that once they are formed in the cell, they stay separated. Perhaps their inability to associate serves the purpose of isolating their separate functions for specialized tasks with other proteins such as in repair or recombination.

In this study, methods by which Pol III* can be assembled in vitro are described. Assembly depends upon staging the addition of subunits in a defined order. The resulting Pol III* remains associated even when diluted to 30 nM. Thus, once pol III* is assembled, it is tightly associated and does not easily fall apart. The ability to assemble Pol III* from overproduced subunits provided the material to analyze the stoichiometry of the subunits in the particle. The assembly process, the stoichiometry of subunits, and the implications of the results to the asymmetric structure and function of Pol III holoenzyme are the subject of this report.


EXPERIMENTAL PROCEDURES

Materials and Methods

All methods, materials, and sources not described here were described in the first report of this series (15) . Gel filtration was performed as described in the first report, except when Superose 6 was used, 4.8 ml was collected before the start of collecting fractions. Buffer B contained 20 mM Hepes-NaOH (pH 7.5), 2 mM dithiothreitol, 0.5 mM EDTA, and 10% glycerol.

Replication Assays

Assays were performed as described for the complex in the first report (15) , except that in Pol III* assays, the complex was omitted (only was added to the assay). Assays of column fractions from the analysis of 30 nM Pol III* and complex required the addition of 10 µl of undiluted column fraction to each assay. Assays comparing the specific activities of Pol III* forms were only allowed to proceed for 20 s, sufficient time to finish one primed template.

Preparation of Reconstituted Complexes

Proteins were incubated in buffer A for 30 min at 15 °C unless stated otherwise. The concentrations of all subunits except and are expressed as monomer. The concentrations of and are expressed as dimer since this is their final aggregation state when assembled with the other subunits.

The Core Polymerase

A mixture of 10 mg (78 nmol) of , 6.4 mg (229 nmol) of , and 6 mg (698 nmol) of was incubated in a volume of 7.4 ml and then chromatographed on a Mono Q HR 5/5 column eluted with a 32-ml linear gradient of 0-0.4 M NaCl in buffer A. Fractions of 0.5 ml were collected and analyzed on a Coomassie Blue-stained SDS-polyacrylamide gel. The core polymerase eluted at 0.25 M NaCl (11 mg final concentration).

The ` Complex

The ` complex was made in two steps. First, the complex was constituted and purified as described (6, 19) and then ` was added, and the resulting ` complex was purified (however, the ` complex can also be made by mixing all four subunits, followed by purification on Mono Q). The complex (1.5 mg, 52.5 nmol) was incubated with ` (3.89 mg, 105 nmol) in 3.06 ml, and then the mixture was chromatographed on a Mono Q HR 5/5 column equilibrated with buffer A. The ` complex was eluted from the column with a 32-ml linear gradient of 0-0.4 M NaCl in buffer A. The ` complex eluted last at 0.28 M NaCl. Column fractions containing ` were pooled (4.2 mg in 3 ml) and stored at -70 °C.

The Complex

The complex was constituted and purified by chromatography on a Mono Q column as described in the first report of this series (15) .

Pol III`

Pol III` was constituted by mixing , , , and at a molar ratio of 3:4.5:6.75:1 (molarity of as dimer, the rest as monomer) in buffer A and incubating for 30 min at 15 °C (see figure legends for total protein concentrations). Pol III` was concentrated to 100 µl by spin dialysis using a Centricon 30 apparatus (Amicon, Inc.) and isolated by gel filtration on a Superose 6 column to remove excess core, complex, and subunit.

-less Pol III*

Pol III* lacking was constituted upon incubating (239 µg, 0.84 nmol), (40.5 µg, 2.44 nmol), and (30 µg, 1.97 nmol, in 6 µl of 4 M urea) in a final volume of 98.1 µl, after which (92 µg, 2.38 nmol) and ` (73.7 µg, 1.99 nmol) were added. This mixture was further incubated for 30 min at 15 °C, at which time (239 µg, 1.99 nmol), (67.6 µg, 2.46 nmol), and (24.13 µg, 2.8 nmol) were added to a final volume of 260 µl containing 5 mM MgCl and 0.2 mM ATP. This mixture was further incubated for 30 min at 15 °C and then concentrated to 100 µl at 4 °C with a Centricon 30 apparatus, followed by purification through gel filtration on a Superose 6 column.

Pol III*

Two general methods were used to constitute Pol III*. In Method 1, first the complex was constituted and dialyzed to remove urea in the preparation by mixing 1.414 mg (85 nmol) of with 0.864 mg (57 nmol) of in a final volume of 4.2 ml (urea was present at 0.7 M) for 1 h and then dialyzing against buffer A. The subunit (504 µg, 3.5 nmol) was incubated for 60 min with the complex (224 µg, 7 nmol) in 488 µl. The subunit (1.0 mg, 10.6 nmol) was incubated with the complex (676 µg, 22 nmol) for 60 min in 1.953 ml. The mixtures were then combined and incubated for 1 h, after which a 665-µl mixture of 787 µg of ` and 823 µg of (21.3 nmol each) was added, followed by a further incubation for 30 min. During this time, the core polymerase was constituted in a separate tube containing (2.77 mg, 21.3 nmol), (878 µg, 31.9 nmol), and (412 µg, 47.9 nmol) in 5.4 ml for 1 h. Then core was combined with the ` mixture and incubated for 1 h. The mixture was then loaded onto a 4-ml heparin-Sepharose column (Pharmacia Biotech Inc.) equilibrated with buffer A and eluted with a 40-ml linear gradient of 0-325 mM NaCl in buffer A. Eighty fractions of 0.5 ml each were collected. Column fractions were analyzed on a 15% SDS-polyacrylamide gel, and those containing Pol III* were pooled (fractions 32-50), concentrated with the Centricon 30 apparatus to 230 µl, and loaded onto a 24-ml Superose 6 column equilibrated with buffer A containing 0.1 M NaCl. Fractions of 200 µl were collected and analyzed on a 15% SDS-polyacrylamide gel. Fractions containing Pol III* were pooled (total protein, 760 µg in 2 ml) and stored at -70 °C. Assuming a mass of 670 kDa for Pol III*, if all the had been incorporated, there would have been 2.39 mg of Pol III*. Hence, the recovery by this procedure is 32%. Two other preparations of Pol III* were assembled by this same general method with the following exceptions. The complex was initially placed exclusively on by starting with the pure complex (and was not incubated with ). In the other variation, was initially placed only on by using pure (prepared similarly to ), and was not incubated with .

In Method 2, 6.6 mg (52.5 nmol) of complex (prepared as described (6) ) was mixed with 3.89 mg (105 nmol) of ` in a volume of 3 ml, followed by incubation for 30 min. The mixture was chromatographed on a Mono Q HR 5/5 column equilibrated with buffer A and eluted with a 32-ml linear gradient of 0-0.4 M NaCl in buffer A (see Fig. 3 ). Fractions 40-43 were pooled to yield 4.2 mg of ` complex. The subunit (0.19 mg, 1.34 nmol) was mixed with 0.8 mg of ` complex in 1.6 ml for 1 h followed by the addition of 0.36 mg (9.33 nmol) of in a final volume of 2.75 ml and incubated for an additional 30 min. During this time, core was constituted upon incubating (0.516 mg, 4 nmol), (162 µg, 6 nmol), and (77 µg, 8.95 nmol) in 183 µl for 30 min. Core was mixed with the ` mixture and incubated for 60 min and then was loaded onto a 2-ml heparin-Affi-Gel column (Bio-Rad) equilibrated with buffer B and eluted with a 30-ml linear gradient of 0-0.325 M NaCl in buffer B. Fractions of 0.5 ml were collected and analyzed on a 13% SDS-polyacrylamide gel for Pol III* and the complex. Fractions 38-54 were pooled and loaded onto a Mono Q HR 5/5 column and then eluted as described for the ` complex. Fractions were analyzed for Pol III* on a 15% SDS-polyacrylamide gel, and fractions 45-50 were pooled, concentrated to 200 µl using a Centricon 30 apparatus, and gel-filtered on a 24-ml Superose 6 column in buffer A containing 100 mM NaCl. Fractions of 190 µl were collected and analyzed by SDS-polyacrylamide gel electrophoresis, and fractions 25-30 were pooled (41 µg of Pol III*) and stored at -70 °C. Recovery by this procedure is 5%. Another preparation of Pol III* was assembled by this method using a ` complex instead of ` and adding later.


Figure 3: Scheme of the two general methods used to constitute Pol III* for the experiments of this report. Method 1 requires no purification of subassemblies until after all the subunits are added. Method 2 requires the complex containing ` to be purified from excess ` before the further addition of subunits. See ``Discussion'' for the numerous variations of these assembly schemes.



Chemical Cross-linking

Solutions of 32 fmol of (as dimer), 32 fmol of (as dimer), or a mixture of 16 fmol each of and were incubated at 15 °C for 30 min in 80 µl of 50 mM Hepes-NaOH (pH 8.1), 1 mM EDTA, 10% glycerol, and 0.1, 0.3, 0.5, 1, or 2 M NaCl. The solutions were shifted to 25 °C, and 8 µl of dimethyl suberimidate (DMS) at 11 mg/ml in the same buffer (but at pH 10) was added. After 30 min at 25 °C, the reactions were quenched upon adding 8 µl of 1 M glycine and analyzed by electrophoresis on an 8% SDS-polyacrylamide gel.


RESULTS

Pol III` and the Complex Do Not Assemble into Pol III*

To constitute Pol III*, we initially mixed Pol III` () and the complex (`) together under the assumption they would associate into Pol III* (`). In replication assays, the mixture of Pol III` and the complex is essentially as effective in providing processive DNA synthesis with as the Pol III* assembly is. Hence, to assay formation of Pol III*, we could not use simple replication assays, but needed to follow the assembly by physical methods. In Fig. 1, we assayed Pol III* assembly by gel filtration and analyzed the column fractions by Coomassie Blue staining of an SDS-polyacrylamide gel. In Fig. 1(A and B), the complex and Pol III` assembly were analyzed separately. The complex elutes in fractions 26-34 (Fig. 1A), and Pol III` peaks in fractions 14-22 (Fig. 1B). If Pol III` and the complex associate into Pol III*, then all nine subunits should coelute in a position earlier than either complex alone. Analysis of the mixture of Pol III` and the complex in Fig. 1C shows that they do not associate to a significant extent; the subunits neither comigrate nor elute substantially earlier. However, the slightly earlier elution of the complex in the presence of Pol III` may indicate a weak interaction with Pol III` that is not stabile to gel filtration.


Figure 1: Pol III` does not associate with the complex. Pol III` and the complex were analyzed by Superose 12 gel filtration either alone or after incubating them together as described under ``Experimental Procedures.'' A, the constituted and purified complex (40.6 µg, 0.20 nmol) in 200 µl; B, Pol III` constituted upon incubating (42.3 µg, 0.33 nmol) with (13.4 µg, 0.50 nmol), (6.4 µg, 0.75 nmol), and (28.9 µg, 0.2 nmol) in 200 µl; C, mixture of Pol III` and the complex at the same concentrations as in A and B in a 200-µl final volume and further incubated for 60 min at 15 °C before gel filtration. The firstlane contains molecular mass standards, and their masses are shown to the left. The , , , , `, , , , and subunits are identified to the right. Frxn, fraction number.



One explanation for this result is that the association of Pol III` with the complex is too weak to isolate the Pol III* complex under the nonequilibrium conditions of gel filtration. An earlier report showed that Pol III* isolated naturally from E. coli was isolable as a complex on the same gel filtration matrix; however, it appeared to partially dissociate as its concentration was decreased (16). The experiment of Fig. 1was performed at a concentration of 1 µM each Pol III` and complex and thus may be less concentrated than needed. However, we have performed gel filtration of these complexes at a concentration of 28 µM with the same result as in Fig. 1(17) . In the experiments below, we show that Pol III* can indeed be assembled by the appropriate order of subunit addition and that once formed, Pol III* is stabile to gel filtration even at a concentration of 30 nM.

Constitution of Pol III*

After numerous studies using different orders of subunit addition, methods to constitute Pol III* were discovered. In overview, formation of Pol III* requires that the and subunits associate with each other, and this - association does not occur when using intact Pol III` and the complex. The underlying reason that (in Pol III`) could not bind (in the complex) rested with the subunit, which prevented their interaction; needed to be added after and were preincubated.() In fact, the - contact could be made upon incubating Pol III` with a complex of ` ( complex lacking ), and subsequent addition of resulted in formation of Pol III*. In the course of these studies, we identified an additional reaction that inhibited interaction of with , thus preventing Pol III* formation. The ` subunit can bind both and as shown previously ( must also be present on () for ` to stably associate with () (15) ), but if ` is allowed to bind both and , it prevents the essential association of with . The results of adding and ` at different points in the assembly scheme are summarized in Fig. 2. Study of these necessary orders of addition is presented below (see Fig. 10 and Fig. 11). Mixtures of complexes that do not result in productive assembly are shown in Fig. 2with an X. Although the addition of and is shown near the beginning of the assembly scheme and core () is added near the end, , , and core can be added at any stage without preventing the formation of a Pol III* complex.


Figure 2: Assembly schemes showing possible paths leading to Pol III* and paths leading to dead-end complexes. Openwhitecircles indicate pure subunits. Shadedellipses indicate mixtures of subunits that result in the indicated complex. These mixtures contain excess free subunits and/or subcomplexes. The twohatchedellipses indicate the ` and ` complexes, which must first be purified to remove excess unbound ` subunit before continuing the assembly process. The productive paths are shown as solidlines that end in another complex. The unproductive paths converge to an X. Core () and the and subunits can be added at any point in the assembly scheme. See ``Results'' for details.




Figure 10: Chemical cross-linking analysis of the - interaction. Solutions of , , and a mixture of and were treated with DMS and then analyzed on an 8% SDS-polyacrylamide gel stained with Coomassie Blue. The firstlane contains molecular mass markers; the nextfivelanes show treatment of with DMS at increasing concentrations of NaCl (0.1, 0.3, 0.5, 1, and 2 M). The middlefivelanes show treatment of with DMS at 0.1, 0.3, 0.5, 1, and 2 M NaCl. The lastfivelanes show treatment of the and mixture with DMS at 0.1, 0.3, 0.5, 1, and 2 M NaCl. The positions of the and monomers and dimers are shown to the right, as is the cross-linked form ( heterodimer).




Figure 11: The subunit inhibits interaction of with . A, in one tube, Pol III` was constituted by incubating core (54.8 µg, 0.33 nmol) and (28.4 µg, 0.20 nmol) for 30 min at 15 °C in 77 µl and then adding the ` complex (32.6 µg, 163 nmol) in a 98-µl final volume, followed by gel filtration on a Superose 12 column. Column fractions were analyzed on a Coomassie Blue-stained 15% SDS-polyacrylamide gel. B, the ` complex (32.6 µg, 163 nmol) was gel-filtered as described for A. C, Pol III` was constituted and gel-filtered as described for A. D, Pol III`, constituted as described for A, was incubated 30 min at 15 °C with the complex (40.3 µg, 0.2 nmol) in 117 µl of buffer A and then gel-filtered. E, Pol III* was constituted upon mixing Pol III` (constituted as described for A) with the ` complex (32.6 µg, 163 nmol) in 97.8 µl of buffer A for 30 min at 15 °C followed by the addition of 19 µl of (11.6 µg, 0.3 nmol) and incubated for an additional 30 min at 15 °C before gel filtration. Column fractions are shown at the top; positions of molecular mass markers are indicated to the left; and subunits are identified to the right. Frxn, fraction number.



The schemes in Fig. 3outline the two methods by which Pol III* was assembled for the studies in this report. In Method 1, the key feature is that and are premixed before the addition of and `. The determining feature of Method 2 is that ` is first assembled onto (or ); then excess ` is removed by ion-exchange chromatography before adding (or ) so that the - interaction is productive, and then is added subsequently. Provided the requirements described above are satisfied in the assembly scheme, the rest of the subunits can be added at any step (or together), and a nine-subunit Pol III* will assemble. For example, in Method 1, the subunits and the core polymerase can be added to and/or before or after mixing with . In Method 2, (or ) must be added initially to stabilize the association of ` with (or ), but and core can be added at any point in the scheme. Staging the addition of subunits in various ways may, in principle, result in different subunit orientations within Pol III* (see ``Discussion''). We have compared five preparations of Pol III* assembled using three variations of Method 1 and two variations of Method 2 (see ``Discussion''). The Pol III* used in most of these studies was assembled by Method 1 (unless stated otherwise).

In any assembly scheme, the ``outer'' subunits such as and must be added in excess over subunits more ``central'' to the structure such that all complexes are driven to completion. The subunit is the most central since it binds both the complex and core, and therefore, must be limiting in the assembly scheme. The - interaction appears to be an equilibrium. We typically push the - association by adding a 3-fold excess of over ; however, the excess complex that forms is difficult to separate from Pol III* (explained below). If insufficient is used to push this equilibrium, then an eight-subunit form of Pol III assembles that has all the subunits except (referred to below as ``-less Pol III*''). It is desirable to reduce the amount of -less Pol III*, even at the expense of forming excess complex, since it is most difficult to separate -less Pol III* from Pol III*.

After all the subunit additions, Pol III* can be purified from excess subunits and smaller subassemblies as shown in Fig. 4 . Fig. 4A shows the profile of elution from a heparin column, which partially resolves excess complex (fractions 23-38) from Pol III* (fractions 32-54). Fractions 38-54 were pooled, resulting in some loss of Pol III* at the expense of removing the bulk of excess complex. Excess and ` coelute with Pol III* on the heparin column, but are cleanly separated from Pol III* by chromatography on a Mono Q column (Fig. 4B). Fractions 45-50 were pooled, concentrated, and passed over a Superose 6 gel filtration column to remove the remaining complex (Fig. 4C). The persistent complex contaminant (fractions 32-38) now resolves from Pol III* (fractions 22-28), and the -less Pol III* contaminant becomes apparent (fractions 16-20). -less Pol III* has a higher molecular mass and elutes earlier than Pol III*.() To remove -less Pol III* and the complex from the preparation, we pooled a narrow range of fractions (fractions 25-30).


Figure 4: Purification of constituted Pol III*. Pol III* was constituted by Method 2 as described under ``Experimental Procedures.'' Purification of the resulting Pol III* from contaminating single subunits and subassemblies was achieved by chromatography on a heparin-Affi-Gel column (A) and on a Mono Q column (B) and by gel filtration on a Superose 6 column (C) as described under ``Experimental Procedures.'' Column elution was monitored at 280 nm (leftpanels) and by Coomassie Blue-stained 13% SDS-polyacrylamide gel electrophoresis (rightpanels).



Characterization of Constituted Pol III*

The size of constituted Pol III* was estimated by gel filtration analysis and comparison to size standards in Fig. 5. Pol III* peaks at fractions 23-27, followed by remaining excess complex in fractions 29-35 (Fig. 5A). Pol III* activity assays (Fig. 5B) show that the active fractions correspond to Pol III* with a Stokes radius of 85 Å for a mass of 670-680 kDa (see ). The mass of Pol III* purified from E. coli cell lysates (without protein overproduction) was also analyzed by gel filtration (Fig. 5C). It eluted in the same position as constituted Pol III*, reasonably consistent with a previous study estimating the mass of naturally purified Pol III* at 800 kDa (16) .


Figure 5: Size analysis of Pol III*. Pol III* (600 µg, 0.9 nmol), constituted by Method 1, was gel-filtered on Superose 6 as described under ``Experimental Procedures.'' A, analysis of column fractions by Coomassie Blue-stained 13% SDS-polyacrylamide gel. The firstlane contains molecular mass standards. The positions of the , , , , `, , , , and subunits are identified to the right. The positions and masses of gel filtration standards analyzed separately are shown above the gel. B, activity assays of the column fractions. C, elution of constituted Pol III* and Pol III* purified from E. coli relative to protein standards of known Stokes radii. The Stokes radius was calculated from the diffusion coefficient using the following equation: Stokes radius = kT/6D, where k is the Boltzmann's constant, T is absolute temperature, is viscosity, and D is the diffusion coefficient. Tgb, thyroglobulin (670 kDa, 85 Å); Apf, horse apoferritin (440 kDa, 59.5 Å); IgG (158 kDa, 52.3 Å); BSA, bovine serum albumin (67 kDa, 34.9 Å); Ova, chicken ovalbumin (43.5 kDa, 27.5 Å); Myo, horse myoglobin (17.5 kDa, 19.0 Å).



We tried to determine the molar ratio of subunits in Pol III* by the HPLC technique (14) , but could not identify a gradient that resolved all the subunits.() Hence, we estimated the molar ratio of subunits in constituted Pol III* and naturally purified Pol III* by laser densitometry of a Coomassie Blue-stained SDS-polyacrylamide gel in which the differences in staining for each subunit were corrected by comparison to individual subunits of known concentration analyzed on the same gel (Fig. 6). The scan of constituted Pol III* (Fig. 6A) is similar to that of naturally purified Pol III* (Fig. 6B). The concentrations of the individual Pol III* subunits used as standards were determined from their extinction coefficients at 280 nm. The quantitation of the analysis is shown in . All values are normalized to the subunit.


Figure 6: Subunit molar ratio in Pol III*. Pure Pol III* was gel-filtered on a Superose 6 column, and the fractions were analyzed on a Coomassie Blue-stained SDS-polyacrylamide gel, followed by densitometric scanning. Densitograms are presented. A, 600 µg of Pol III* constituted and purified by Method 1; B, 330 µg of Pol III* purified from E. coli.



The ratio of the , , and subunits was approximately equimolar for both constituted Pol III* and Pol III* purified from cell lysates. The intensity of the subunit was too weak to be determined accurately in this study; but previous studies showed that the stoichiometry of the complex is 1:1, and therefore, it is assumed here that is equimolar to in Pol III* (7) . The subunit in both reconstituted Pol III* and Pol III* purified from E. coli lysates is also approximately equimolar to , , and . However, even though the , `, , and subunits were added in excess during the assembly of Pol III*, these subunits are present at approximately half the amount of the , , , and subunits in both constituted Pol III* and E. coli purified Pol III*.

The results for both constituted and naturally purified Pol III* are most consistent with a dimer of and , two each of the core subunits (), and only one each of the , `, , and subunits. The single copy nature of and ` can be seen by simple inspection of the scans in Fig. 6by comparing the greater peak height of the double copy subunit (27.5 kDa) relative to the heights of the larger molecular mass but single copy (38.7 kDa) and ` (37.0 kDa) subunits. This result is also consistent with previous studies on the composition of Pol III` () that showed it to be composed of two of each subunit (13, 14) . Although an earlier study of Pol III* purified from cell lysates suggested there were two of each subunit, analysis of the holoenzyme in that same study gave the following composition (after setting to two): ` (16), quite consistent with the subunit ratios obtained in this report. The subunit stoichiometry predicts the molecular mass of Pol III* to be 673 kDa as summarized in , consistent with the size estimated by gel filtration.

The activity of constituted Pol III* in the -dependent replication of singly primed M13mp18 single-stranded DNA coated with SSB was compared with that of Pol III* purified from cell lysates. The results (Fig. 7) show that constituted Pol III* is slightly more active than Pol III* purified from cell lysates; their specific activities were 3.4 10 and 2.3 10 pmol of nucleotide incorporated per mg/min, respectively. Constituted Pol III* is similar to Pol III* purified from lysates with respect to size, subunit stoichiometry, and replication activity, thus indicating that constituted Pol III* is authentic.


Figure 7: Activity of constituted Pol III* and of Pol III* purified from cell lysates. Shown are -dependent replication activity assays of Pol III* on singly primed and SSB-coated M13mp18 single-stranded DNA of Pol III* purified from E. coli lysates () and Pol III* constituted by Method 1 ().



How tightly associated is Pol III*? In the cell, there are 10-20 molecules of Pol III* for a concentration of 17-34 nM(1) . In Fig. 8, we tested the ability of the Pol III* particle to remain intact at a concentration similar to that in the cell. At 30 nM Pol III*, the subunits are far below the limit of detection in column fractions by Coomassie Blue staining (or silver staining) of an SDS-polyacrylamide gel. Hence, Pol III* was identified in column fractions by assaying for -dependent replication of singly primed M13mp18 SSB-coated single-stranded DNA. If Pol III* falls apart, the complex, upon dissociating from within Pol III*, should elute much later. Hence, the column fractions were also assayed for the complex. The results show that Pol III* and the intrinsic complex activities comigrate in fractions 16-28, consistent with the size of Pol III* and with the particle remaining intact during gel filtration at these dilute conditions. As a control, a second gel filtration analysis using 30 nM complex showed the expected elution position of complex activity had Pol III* dissociated (Fig. 8, dashedline).


Figure 8: Pol III* remains intact at cellular concentrations. Pol III* was constituted and purified by Method 1 and then diluted to a concentration of 30 nM (assuming a mass of 670 kDa) in a final volume of 200 µl of column buffer and gel-filtered on Superose 6. The replication activities of Pol III* () and the complex () were followed in the column fractions. Also shown is a separate experiment in which a solution of 30 nM constituted complex was gel-filtered and followed in the column fractions by activity assays ().



Structural Insights from the Assembly Process

The and Subunits Interact

The initial observation that Pol III` and the complex did not assemble into Pol III* implied that extra subunits might be needed. For example, perhaps the complex was meant to assemble with -less Pol III* to form a dimeric polymerase with a complex and a complex. However, these assemblies did not interact either. After that, we pursued a line of investigation based on the assumption that one of the subunits of either Pol III` or the complex was preventing assembly of Pol III*, an assumption that turned out to be correct.

The approach was to mix combinations of two subunits, one from Pol III` (, , , or ) and one from the complex (, , `, , or ), and to assay for an interaction by gel filtration (except for the previously established interactions of with ). Using this approach, a contact of with was identified (Fig. 9). The complex is somewhat difficult to observe by gel filtration because each subunit migrates in gel filtration columns as a higher order oligomer, consistent with a tetrameric state (14, 17), but upon mixing them, they migrate as a heterotetramer. The heterotetramer elutes earlier than alone, but later than alone. A study of the - interaction is presented in Fig. 9. Panel A shows the subunit alone, and panelD shows alone. PanelB shows the result of mixing with a 4-fold excess of . Comparison of the position in panelB with alone in panel A shows that causes to elute earlier (bigger) than alone (compare fractions 27-33 in panelB with fractions 33-37 in panel A). When is in a 4-fold excess over (panel C), it causes to elute later (smaller) than alone (fractions 29-33 in panel C relative to fractions 27-31 in panel D). The mixed heterotetramer does not resolve from the homotetramers, and therefore, the stoichiometry of and in the heterooligomer cannot be judged from the polyacrylamide gels.


Figure 9: The and subunits interact. The and subunits were incubated for 60 min at 15 °C and then gel-filtered on a Superose 6 column. A, the subunit (94 µg, 1.0 nmol as dimer); B, mixture of the subunit (32.9 µg, 0.35 nmol as dimer) and (198.8 µg, 1.4 nmol as dimer); C, mixture of (132 µg, 1.4 nmol as dimer) and (50 µg, 0.35 nmol as dimer); D, the subunit (142 µg, 1.0 nmol as dimer); E, mixture of (94 µg, 1.0 nmol as dimer) and (142 µg, 1.0 nmol as dimer). Column fractions are indicated at the top. In the firstlane are molecular mass standards, and their masses are indicated to the left. The and subunits are identified to the right. Frxn, fraction number.



That and form a mixed oligomer is not surprising as and are related structures; contains the sequence plus an additional 24 kDa of polypeptide at the C terminus. Since they both form homooligomers, it is reasonable to expect them to form a heterooligomer. The complex does not appear to be disproportionately favored relative to homotetramer formation as an equimolar mixture of and does not yield a heterotetramer as the sole product (Fig. 9E). The positions of and are both affected, but they do not fully comigrate, indicating the presence of all three tetramers: , , and .

In Fig. 10 , the interaction between and is shown by chemical cross-linking. The second through sixthlanes show cross-linking of at increasing concentrations of NaCl. The monomer and dimer are clearly visible; the trimeric and tetrameric forms are present, but on the gel of Fig. 10, they are unresolved at the top. The seventh through eleventhlanes show cross-linking of , and again at all salt concentrations, the monomer and dimer are evident, while the higher states are unresolved at the top. The lastfivelanes show cross-linking of an equimolar mixture of and . One band labeled heterodimer is unique to the mixture, and it migrates between the and positions. The presence of both and in this cross-linked band has been confirmed using either [H] or [H] (H label is present in this position regardless of which subunit is labeled (data not shown)). Attempts to resolve the higher molecular mass cross-linked products in lower percentage gels resulted in smeared bands and did not appreciably resolve the higher order cross-linked products. Presumably, these proteins are too large and the various inter- and intramolecular cross-links are too heterogeneous to give sharp and clearly resolved higher molecular mass products.

The Subunit Inhibits the Interaction of with

The assumption that a subunit of the complex or Pol III` prevents their association into Pol III* led to the identification of the - interaction (described above). Armed with the knowledge that and interact, we turned the argument around and asked which subunit, when added to or , prevents their interaction? The binding of , the complex, or the entire core () to before (or after) the addition of did not inhibit formation of the complex (Ref. 17 and data not shown). Likewise, prior formation of , , and ` did not prevent interaction with or Pol III` (summarized in Fig. 2 ).() Hence, by exclusion, it would appear that is the culprit. The subunit does not stabily interact with in the absence of `, and thus, the smallest complex that can be used to test the prediction is the ` complex. As expected, the ` complex did not bind to Pol III`, implying that indeed prevents interaction between Pol III` and the complex. Fig. 11illustrates this finding. Pol III` and ` ( complex) do not interact, but if Pol III` and ` are premixed and is added last, Pol III* is formed. The effect of on Pol III* assembly is also summarized in Fig. 2.

Panel A in Fig. 11 is the mixture of Pol III` with a 3-fold molar excess of the ` complex; some of the ` complex binds to and comigrates with Pol III` (in fractions 16-20).() The ` complex alone migrates much later (fractions 26-32; panel B) and is well resolved from Pol III` (fractions 16-20; panel C).

In panel D in Fig. 11 , the complex is mixed with Pol III`, and the filtration analysis shows that they do not associate to a significant extent, as described more fully in Fig. 1. In panel E, the addition of has been delayed until ` and Pol III` have been given time to associate. Then is added, and the filtration analysis shows that Pol III* has formed. Thus, the binding of to ` prevents the formation of Pol III* presumably by interfering with the - contact.

The ` Subunit Bound to Both and Prevents the - Interaction

Both and can bind the , `, , and subunits, and both the and complexes are active as clamp loaders in replication assays (15) . Do both and complex clamp loaders exist within Pol III*? To study this, we tried to construct a Pol III* assembly containing both a complex and a complex. Obviously, the subunit must be excluded from the reaction until after the - contact has been formed. Hence, we assembled a Pol III`` complex and a ` complex and analyzed the mixture by gel filtration (Fig. 12). The results show that they did not interact (Fig. 12A), as is most simply observed by examining the different peak positions of (fractions 16-20) and (fractions 26-32). However, a Pol III` complex was able to bind the ` complex (Fig. 12B), as evidenced by some comigration of with in fractions 16-22. Hence, even though both and can bind `, if ` is on both and , they cannot establish the - contact as though there is only enough room in the heterotetramer for one molecule of `. We have also confirmed the ` inhibition of the - contact using the minimal assemblies possible: ` does not bind `, but binds `, and binds `.() The effect of ` on the assembly of Pol III* is also summarized in Fig. 2.


Figure 12: The ` subunit bound to both and inhibits the interaction of with . A, the Pol III`` complex was formed upon incubating constituted Pol III` (formed as described in the legend to Fig. 11), and then ` (11.1 µg, 0.3 nmol) and (9.5 µg, 0.3 nmol) were added (final volume of 90 µl) and incubated for 30 min. Then the ` complex (32.6 µg, 163 fmol) was added, and the mixture was incubated for another 30 min at 15 °C (111 µl) and gel-filtered as described in the legend to Fig. 11. B, the Pol III` complex was formed as described for A, except that ` was omitted from the incubation (final volume of 108 µl) followed by the addition of the ` complex as described for A, and then gel-filtered. Column fractions are shown at the top; positions of molecular mass markers are indicated to the left; and subunits are identified to the right of the gels.



This study of Pol III* assembly showing ` can be on or , but not both, is consistent with the observed stoichiometry of one ` (and , , and ) in Pol III*. Furthermore, the single copy of in Pol III* follows from the fact that the stoichiometry of the ` complex is 1:1, and the point of attachment of to the complex is through interaction with ` (3). The fact that there is only one ` in Pol III*, coupled with the fact that ` stabilizes the complex (15) , provides a ready explanation for why Pol III* retains only one copy of and even though their presence on both and does not prevent Pol III* assembly. Hence, only the subunit ( or ) that receives ` will retain the complex due to the stabilization incurred by `.


DISCUSSION

The Pol III* Structure

The nine-subunit Pol III* assembly has been constituted from individual pure proteins; it is similar in size, subunit composition, and replication activity to Pol III* purified from E. coli cell lysates. The particle contains 14 polypeptides in an arrangement consisting of two core polymerases and a complex clamp loader all connected to each other through as illustrated in Fig. 13. The two core polymerases bound to the dimer are most likely arranged with one core on each protomer of . Assuming the subunits of the dimer are related by a 2-fold axis of rotation, then each polymerase is also related to the other polymerase by a 2-fold rotational axis. The dimer forms a heterotetramer with , and assuming the heterotetramer is also symmetric, each (core- protomer- protomer) unit will be related to the other unit by a 2-fold rotational axis. However, the single copy each of , `, , and imposes a structural asymmetry onto the structure of Pol III* such that there can be no overall 2-fold rotational axis. Presumably, the structural asymmetry created by these single copy subunits imposes a functional asymmetry onto the two polymerases for the different actions needed on the leading and lagging strands. Whether these subunit are all on or whether some or all are on is discussed below. The subunit associates with the subunit of the core polymerase (14) and with the subunit of the complex (19) , and therefore, in principle, a total of three dimers may associate with Pol III*. A previous study indicated that two dimers were present in the holoenzyme, and in Fig. 13 , they are placed on the two core polymerases.


Figure 13: Subunit arrangement of Pol III holoenzyme. The and dimers are each shown in an isologous arrangement, and the heterotetramer is also shown as isologous. The two core polymerases are attached to the dimer, presumably one on each protomer. Depending on which face of the dimer they bind, they could point in the same direction (as shown) or in opposite directions. If and form a heterologous closed tetramer, the two cores could be oriented 90° with respect to each other. The single copy subunits, , `, , and , are drawn on , although they may be shared physically or in time with (see ``Discussion'' for details). ` is positioned between the and interface to explain the observation that only one ` is accommodated in the heterotetramer, yet one ` is present in the complex (contains a dimer) and in the complex (contains a dimer) (15). Two dimers are shown bound to the two cores.



It is quite interesting to see how the subunit stoichiometry and native aggregation state of the various Pol III subunits have been utilized to form this machine. All the subunits of Pol III*, except and , are monomers in isolation. The oligomeric structure of is needed to bring two copies of the core polymerase into the Pol III* structure. Furthermore, the holoenzyme architecture has been sculpted such that it contains only one each of the , `, , and subunits. Since either or can bind these single copy subunits, it seems plausible that the holoenzyme would obtain at least two copies of each of them: a set on and a set on . However, the holoenzyme architecture precludes this by preventing the complex from binding more than one ` monomer, perhaps by allotting only enough space inside the junction of the heterotetramer to fit one monomer of ` (as suggested in Fig. 13). The restriction of assembling only one ` monomer into Pol III* is consistent with the observed stoichiometry of one ` in Pol III*. It is also in keeping with the single copy of in Pol III* as it has been shown previously that ` is a 1:1 complex (3) .

A set of and subunits can be bound to both and without inhibiting the - interaction, and thus, it seems possible that Pol III* could, in principle, have two copies of and . Nevertheless, the stoichiometry studies suggest that only one of each is present in Pol III*. The first report in this series showed that the complex dissociated easily from (and ) at 37 °C, but that ` greatly stabilized the - interaction (15) . Hence, it may be presumed that whichever dimer, or , that the ` complex is associated with in Pol III* will also be the dimer that the complex stabily associates with.

The subunit arrangement of Pol III* based on an earlier study (16) differs from that proposed here. In the earlier study, the core polymerase () was proposed to be dimerized by the subunit of core. It is now known that the subunit binds tightly and dimerizes the core polymerase (14) . Furthermore, is a monomer, and core does not dimerize even at a concentration of 100 µM(7) . Also in the earlier study, the and subunits were proposed as the basis for asymmetry within Pol III*, in which one core polymerase was on and the other core was on . This asymmetric arrangement of subunits was proposed to endow each polymerase with different properties, one suited for continuous synthesis of the leading stand and the other for discontinuous synthesis of the lagging strand. The arrangement in Fig. 13 differs from the earlier study in that a dimer bridges both core polymerases and binds the polymerase indirectly, through the dimer. In support of this, we detected no interaction of core with (14) or with any other subunit of the complex (15) or with the entire complex (data not shown). Both models propose that the , `, , and subunits are associated with the dimer, and this report shows that the asymmetric structure of Pol III* is based in the single copy nature of these subunits. The earlier study of Pol III* structure proposed two copies of each subunit in Pol III*. However, this stoichiometry does not match the observed 2:1:1:1:1 stoichiometry of the complex (`) (15, 20) . Perhaps some complex was present in the Pol III* prepared from cell lysates, which would have increased the apparent stoichiometry of the , `, , and subunits. The stoichiometry of subunits in the holoenzyme determined in the earlier study is consistent with the results of this report, in which the holoenzyme consists of one subunit each of , `, , and and two subunits each of , , , , and (16).

Location of the Single Copy Subunits

Since the , `, , and subunits can be added after mixing with , there is an ambiguity in the exact position of these single copy subunits (i.e. see Fig. 2). Are they on or , split between them, or shared? The ` subunit can be placed on either or , but not on both at the same time or else assembly of Pol III* is halted (i.e. as in Fig. 2). Furthermore, the subunit must be added after and are mixed, and thus, it too may assemble with either or (presumably the one that has `). In the fourth report of this series, we found that the complex forms in preference to the complex in Pol III* (21) . Apparently, the presence of core on decreases the efficiency of ` and association with (21) . Consistent with as the preferred target of association of these single copy subunits, Pol III` purified from E. coli lysates does not contain the , , , or ` subunit, nor has a complex been purified from E. coli lysates. Despite these arguments, it is still conceivable that and share these subunits either in time (e.g. by dissociation and reassociation of ` between and ) or physically, in which the , `, , and subunits bridge the / boundary with some present on and some on .

Although the complex has not been purified from wild-type E. coli, a -less form of Pol III* can be reconstituted from individual subunits, and these assemblies are active in replication assays (15, 23) . Whether such complexes exist in vivo is unknown; however, it has recently been reported that E. coli cells are viable, even when the signal for the -1 translational frameshift in dnaX that produces is removed (23) . These cells contain only the subunit, and -less Pol III* appears to be present in them, implying that -less Pol III* can function at a replication fork (23) . -less Pol III* appears to contain two dimers, and we propose that one dimer replaces both functionally and structurally. Why both and are present in the holoenzyme when alone can do the job of both is unknown; speculation on this issue is included in the fourth report of this series (21) .

Forms of Pol III* Resulting from Different Methods of Assembly

By staging the assembly of Pol III* in vitro, it should be possible to selectively place the , `, , and subunits on in a preincubation with in the absence of (see Fig. 2as an aid in understanding the assembly paths discussed below). For example, we have constituted Pol III* starting from the ` complex, followed by adding and then and core. Provided the subunits do not exchange from to during the assembly time (a real possibility), the resulting Pol III* should at least have ` on rather than on .

We have constituted Pol III* using five variations of the two methods described here. The three variations of Method 1 were as follows: 1a) placing on both and before mixing them (as in Fig. 2 ), 1b) mixing the pure complex with (this may direct the assembly of ` to ), and 1c) mixing the pure complex with (this may direct the assembly of ` to ). The two variations of Method 2 that we have performed include the following: 2a) starting with ` (as in Fig. 3; this may direct to ) and 2b) starting with ` (this may direct to ). Methods 1b and 2a may be expected to direct , `, , and onto , and Methods 1c and 2b should direct , `, , and onto . Method 1a is the least assuming of the five constitution reactions (even less assuming is to premix and and then add a mixture of all the rest of the subunits).

These Pol III* preparations have been compared for differences in their DNA synthetic activity with on singly primed single-stranded DNA. However, all five forms were within 2-fold of the activity of Pol III* purified from cell lysates, and they all had the same subunit composition as determined by scanning of Coomassie Blue-stained SDS-polyacrylamide gels (data not shown). Perhaps these forms would show different behavior in more complete assays including initiation at an origin and function with the helicase and primase during propagation of the replication fork. It is also possible that all these forms reverted to a single species due to subunit rearrangement over the time scale used to assemble them.

Comparison of the Pol III* Structure with Replicases of T4 Bacteriophage and Eukaryotes

The T4 replicase has a two-subunit complex (gene 44/62 protein complex) that is thought to act as a clamp loader of the gene 45 protein clamp onto primed DNA, but unlike the holoenzyme, it does not appear to have a homolog to bring the polymerases and clamp loader into one macromolecular assembly in the absence of DNA (24, 25, 26) . The structure of eukaryotic DNA Pol is even more similar to the E. coli holoenzyme (25, 26, 27) . Pol is composed of two subunits containing the polymerase and 3`-5`-exonuclease activity and may be compared with the E. coli core. Pol becomes highly processive upon assembly with the PCNA clamp encircling DNA. The PCNA clamp is assembled onto DNA by activator-1 (or RF-C), a five-subunit clamp loader that couples ATP to load PCNA clamps onto DNA. Hence, the human replicase machinery is quite similar to the E. coli holoenzyme. At the current state of knowledge, Pol is not organized into a twin polymerase, and the clamp loader is not physically connected to Pol in solution. Hence, the human system lacks the equivalent of the E. coli subunit for organizing its polymerases and clamp loader into one particle.

  
Table: Subunit stoichiometry of constituted Pol III* and Pol III* purified from E. coli cell lysates

Coomassie Blue-stained polyacrylamide gels were scanned as described under ``Experimental Procedures.'' The differences in subunit staining by Coomassie Blue were corrected by comparison with subunits of known concentration (determined by absorbance at ) on a Coomassie Blue-stained gel. Values for constituted Pol III* are the average of four different preparations, two by Method 1 and two by Method 2. Values for Pol III* purified from cell lysates are from three adjacent lanes of one preparation. All values are normalized to a value of 2 for . The error represents one standard deviation.


  
Table: Average subunit stoichiometry and calculated mass of Pol III*

Molecular masses of the subunits were obtained from the gene sequences.



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; DMS, dimethyl suberimidate; HPLC, high pressure liquid chromatography; SSB, single-stranded DNA-binding of protein E. coli.

In fact, any assembly containing (i.e. `, `, `) will not bind . The converse is also true: a assembly containing (i.e. `, `, `) will not bind (17) (R. Onrust and M. O'Donnell, unpublished data).

-less Pol III* has a Stokes radius of 97 Å for a mass of 1 MDa and a specific activity of 4.9 pmol/min/mg. We propose that -less Pol III* is composed of a tetramer of in which a dimer replaces the dimer in associating with , `, , and and the other dimer has two core polymerases.

The and ` subunits did not resolve on the HPLC column by any gradient or elution program that we tried. For those subunits that did resolve, the molar ratio was (where ND is not determined).

The reverse is also true: Pol III`, Pol III`, or Pol III`` did not prevent interaction of with (in Pol III`).

The reason that Pol III` does not elute much earlier upon binding ` is that Superose 12 was used in these experiments. Pol III` already elutes in the excluded volume of this column.

R. Onrust and M. O'Donnell, unpublished data.


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