©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
V. FOUR DIFFERENT POLYMERASE-CLAMP COMPLEXES ON DNA (*)

P. Todd Stukenberg (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
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Several different subassemblies of DNA polymerase III holoenzyme can be purified from Escherichia coli. Toward the goal of understanding the functional significance of these subassemblies, we have used the complex clamp loader and the ring to assemble each different polymerase onto DNA. Through use of radioactive labeled proteins, the subunit structure of each resulting processive polymerase has been determined. Use of DNA polymerase III core, the complex, and results in a core- complex on DNA; the complex is not incorporated into the structure. The addition of to the assembly reaction to form either core- or core- results in a more efficient polymerase and more stabile association of core- on DNA, although the complex still does not remain on DNA. The complex clamp loader was retained on DNA with the other subunits only if it was first assembled into the polymerase (Pol) III* structure. The clamp loader within Pol III* appeared to be capable of loading two clamps onto DNA for both core polymerases within Pol III*, consistent with the hypothesis that one replicase can simultaneously replicate both strands of a duplex chromosome. These findings extend those of an earlier study showing that distinctive polymerases can be assembled depending on the presence or absence of (Maki, S., and Kornberg, A.(1988) J. Biol. Chem. 263, 6561-6569). The significance of these distinct polymerases in separate paths of DNA metabolism is discussed.


INTRODUCTION

DNA polymerase III holoenzyme (holoenzyme) is the replicative polymerase of the Escherichia coli chromosome (reviewed in Refs. 1 and 2). Besides the holoenzyme, three different polymerase subassemblies can be purified from cells. 1) The three-subunit core polymerase is a 1:1:1 complex of (DNA polymerase), (proofreading 3`-5`-exonuclease), and . 2) The four-subunit polymerase (Pol)() III` contains two molecules of core polymerase attached to a dimer of the subunit (core-). 3) The Pol III* assembly consists of nine different subunits (`) and lacks only the sliding clamp. These polymerase subassemblies do not have the rapid and processive character of the holoenzyme. Generally, they incorporate residues at a speed of 10-20 nucleotides/s, and their processivity ranges from 10 to 50 residues/binding event (3, 4) . However, when combined with the sliding clamp, each of these polymerases becomes rapid (750 nucleotides/s) and processive (1, 2) . Assembly of the ring-shaped clamp (5, 6) onto DNA requires the complex (`), which couples ATP hydrolysis to load clamps onto DNA (1, 2) . The sliding clamp confers a high degree of processivity onto the holoenzyme by tethering it to the primed template for continuous DNA synthesis (5) .

Besides its role as a replicase, Pol III also functions in other areas of DNA metabolism (1) . Perhaps the different polymerase subassemblies serve distinct functions in DNA metabolism. Consistent with this is the curious observation that Pol III* appears to be composed of one Pol III` and one complex (7) , yet Pol III` and the complex do not associate to form Pol III* unless a specific order of addition is followed (8) . If these subassemblies are formed separately within the cell, then this order of addition requirement may serve the purpose of providing the cell with a stabile quantity of each of these assemblies for action in other areas of DNA metabolism such as repair, recombination, and mutagenesis. Alternatively, Pol III` and the complex may associate with each other in the presence of primed DNA and . For example, proximity at a primed site may lead to their association. Yet another possibility is that the association of Pol III` with the complex may be facilitated in vivo by a chaperonin.

Toward the goal of understanding the functional significance of these subassemblies to replication and chromosome maintenance, these subassemblies have been used to assemble processive polymerases onto DNA using the complex clamp loader and clamp, and each resulting polymerase on DNA has been analyzed for its structural composition and activity. These subassemblies were studied earlier using small amounts of isolated core, the complex, and overproduced and subunits with the finding that distinctive polymerases (+) could be assembled onto a primed template (9) . Now that each subunit is available in quantity and the subassemblies can be constituted from them, we have tagged each subassembly with a subunit of known specific radioactivity, providing accurate stoichiometry measurements of each subassembly on DNA. Depending on the relative amounts of core and present in the cell, one may expect either one core (limiting core) or two cores (excess core) to associate with the dimer (10) . Hence, we have constituted and studied both of these polymerase forms (core- and core-).

The results show that use of different polymerase subassemblies results in different processive polymerase structures attached to a clamp on DNA. Furthermore, comparison of the polymerase formed using Pol III* plus to that formed using Pol III` plus the complex and showed polymerases of different compositions on DNA even though all 10 holoenzyme subunits were present in both reactions. If these several different forms of DNA polymerase III also assemble onto primed DNA sites in the cell, then it seems likely that they could be used in different processes of DNA metabolism.


EXPERIMENTAL PROCEDURES

Materials and Methods

Materials, methods, and sources not described here were described in the first report of this series (11) . A gapped circular DNA template was prepared by nicking M13mp18 plasmid DNA (376 µg) at a specific site using 35 units of M13 gene protein II (a gift of Dr. Peter Model (The Rockefeller University) and purified as described (12) ) in a 20-min incubation at 37 °C in 3 ml of 40% sorbitol, 25 mM Tris-HCl (pH 8.1), 6.7 mM MgCl, 6.7 mM dithiothreitol, and 5 µg/ml BSA (13) . This treatment results in a single nick at the M13 origin in 60% of the DNA templates. A gap was produced at the nick upon treatment with 1000 units of exonuclease III for 1 min at 37 °C followed by phenol/chloroform extraction and ethanol precipitation. The average size of the gap was estimated to be 500 nucleotides from the amount of radioactive nucleotide incorporated by the holoenzyme using [-P]dTTP. Reaction buffer contained 5% glycerol, 20 mM Tris-HCl (pH 7.5), 8 mM MgCl, 40 µg/ml BSA, 0.5 mM ATP, 50 µM dCTP, 50 µM dGTP, 0.1 mM EDTA, and 5 mM dithiothreitol (ATP was omitted where indicated).

Radioactive Labeled Subunits

The , , , `, and subunits were H-labeled by reductive methylation as described (5, 15, 27) . Briefly, 1 ml of protein at a concentration of 1-3 mg/ml was incubated with 10 mM formaldehyde and 2 mM NaBH (74 Ci/mmol) on ice for 15 min. The H-labeled protein was separated from unspent reagents by gel filtration in the fume hood on a 10-ml column of Sephadex G-25. The excluded fractions were counted, pooled, and stored at -70 °C. The specific activities were as follows: [H], 15 cpm/fmol; [H], 29 cpm/fmol as dimer; [H], 29 cpm/fmol as dimer; [H]`, 20 cpm/fmol as monomer; and [H], 67 cpm/fmol as dimer. The replication activity of labeled protein relative to unlabeled protein was assessed by comparative titrations in replication assays as follows (14) . [H], assayed for replication of singly primed SSB-coated M13mp18 ssDNA with Pol III*, was indistinguishable in activity from unlabeled . The activity of [H] was determined by reconstituting core- using either unlabeled or [H] and then comparing their replication activity with and the complex on singly primed SSB-coated ssDNA. Core- constituted using [H] was within 2% of the activity of that constituted using unlabeled . The replication activity of [H] was determined by constituting Pol III* using either [H] or unlabeled and then testing their activity with on singly primed SSB-coated circular ssDNA. Pol III* constituted using [H] was within 80% of the activity of that constituted using unlabeled . The activity of [H]` was within 10% of that of unlabeled ` as determined by constituting the complex using either labeled or unlabeled ` and then comparing their replication activity with and core- on singly primed SSB-coated ssDNA. We initially tried labeling core using [H], but [H] was only 40% as active as core reconstituted using unlabeled . Therefore, core was H-labeled by reconstituting it using the [H] subunit. The presence or absence of has no effect on holoenzyme activity; however, [H] was as effective as unlabeled in forming a tight nondissociable complex with as determined by comigration of [H] subunits during gel filtration analysis.

A derivative of containing an additional six amino acids at the C terminus (NH-RRASVP-COOH) serves as a substrate for phosphorylation by cAMP-dependent protein kinase (15, 27) . This derivative was labeled using [-P]ATP and protein kinase as described in the second report of this series (16) . Although this derivative could be labeled to very high specific activity, the typical specific activity of [P] used in this report was low (20-65 cpm/fmol) such that both P and H could be assayed in the same sample by liquid scintillation.

Assembly of Multiprotein Complexes Using H-Labeled Subunits

Subassemblies containing H-labeled subunits were constituted by incubating purified unlabeled subunits with one labeled subunit in buffer A at 15 °C for 1 h, and then the resulting complex was purified from free subunits by chromatography. Fractions from the chromatography columns were analyzed on an SDS-polyacrylamide gel stained with Coomassie Blue. Fractions containing the desired complex of proteins were pooled, dialyzed against 1 liter of buffer A, aliquoted, and stored frozen at -70 °C. All complexes used here retain full activity for over 1 year when stored in this manner. Specific activities of complexes were determined by scintillation counting, and protein concentration was measured using the protein assay from Bio-Rad with BSA as a standard.

[H] was assembled upon incubating 180 µg (2.5 nmol) of [H], 960 µg (7.5 nmol) of , 510 µg (18 nmol) of , and 294 µg (33 nmol) of in 1.44 ml and then purified from free subunits by chromatography on a 1-ml Mono Q column eluted with a 19-ml gradient of 0-0.5 M NaCl in buffer A. The fractions containing core-[H] were pooled, concentrated to 200 µl using a Centricon 30 apparatus, and then gel-filtered on a 24-ml Superose 6 column (Pharmacia Biotech Inc.) developed in buffer A containing 300 mM NaCl to remove any remaining excess core (43.2 µg of final complex recovered). Core-[H] had a specific activity of 30 cpm/fmol (474.2 kDa), consistent with the specific activity of [H].

[H]Core- was constituted by the same method as core-[H] (except [H] and unlabeled were used), and it was purified on a Superose 6 column only. Its specific activity was 24 cpm/fmol, approximately half the specific activity of , since is only a monomer and two subunits are present in the [H]core- complex.

[H]Core- was assembled by incubating 522 µg (3.75 nmol) of , 132 µg (5 nmol) of , 58.8 µg (7.5 nmol) of [H], and 801 µg (11.25 nmol) of in 1.6 ml and then was concentrated using a Centricon 30 apparatus to 7.6 mg/ml and purified by Superose 6 chromatography as described above (final amount of 67 µg). [H]Core- had a specific activity of 18 cpm/fmol (304 kDa), consistent with the presence of only one molecule of /core- complex.

The [H] complex ([H]`) was assembled by mixing 710 µg (9 nmol) of complex (reconstituted as described (11, 17) ), 392 µg (11 nmol) of , and 340 µg (9 nmol) of ` in 2.045 ml and then was purified on a 1-ml Mono Q column using a 19-ml gradient of 0-0.5 M NaCl in buffer A. The fractions containing ` were pooled (302 µg), dialyzed against buffer A, and stored at -70 °C. The specific radioactivity of the [H] complex was 20 cpm/fmol, consistent with the presence of only one ` subunit within the complex.

Three preparations of Pol III* were constituted differing only in which subunit was labeled. Pol III* was assembled in stages essentially as described in the third report of this series (8) . The subunits were added in the following molar ratios before purification (relative to ): , 3.0; , 4.5; , 7.0; , 1.0; , 3.0; , 7.0; `, 6.0; , 7.0; and , 6.0. Detailed procedures were as follows.

[H]Pol III* labeled with [H] was assembled upon incubating 352 µg (3.7 nmol) of [H] with 178 µg (1.25 nmol) of , 145 µg (8.73 nmol) of , and 114 µg (7.5 nmol) of in 789 µl. After 30 min, 328 µg (8.48 nmol) of and 278 µg (7.53 nmol) of ` were added, bringing the volume to 1394 µl, and then incubated for an additional 30 min. During this time, a separate tube containing 200 µg (1.55 nmol) of , 154 µg (5.6 nmol) of , and 76 µg (8.84 nmol) of was incubated for 30 min in 283 µl. Then the two tubes were mixed and incubated for an additional 30 min. Pol III* was separated from free subunits and subassemblies by chromatography on a 1-ml heparin-agarose column eluted with a 15-ml gradient of 0-325 mM NaCl in buffer A. Fractions containing Pol III* were pooled, dialyzed against buffer A, and then chromatographed on a 1-ml Mono Q column eluted with a 30-ml gradient of 0-0.4 M NaCl in buffer A. Fractions containing Pol III* were pooled, dialyzed against buffer A, and stored at -70 °C. The specific activity of [H]Pol III* ([H]) was 29 cpm/fmol, consistent with one dimer of within Pol III*.

[H]Pol III* labeled with [H] was assembled as described above for Pol III* labeled with [H], and its specific activity was 18 cpm/fmol, consistent with two molecules of in the Pol III* structure (final yield of 142 µg).

[H]Pol III* labeled with [H] was assembled as described for Pol III* labeled with [H], except that a pre-purified complex was incubated with [H]. A further difference was the use of Superose 6 gel filtration in place of the Mono Q column, which removes the small amount of core-([H]) side product of the assembly. Use of Superose 6 required concentration of the sample after heparin-agarose chromatography to 70 µl using a Centricon 30 apparatus (final yield of 23.8 µg).

Assembly of Subunits on DNA and Analysis by Gel Filtration

In general, subunits and subassemblies were added to DNA templates in 75 µl of reaction buffer on ice and then incubated at 37 °C for 4 min to allow time for assembly onto primed DNA. Reactions were then gel-filtered on 5-ml columns developed in column buffer, and fractions of 200 µl were collected over a period of 30 min at 4 °C. Tritium and P were quantitated in the same fraction by analyzing aliquots of 150 µl in 5 ml of Aquasol (DuPont NEN) by liquid scintillation counting (H window was 0-350; P window was 400-1000). The specific activities of [P] and of the H-labeled subunits were comparable in all the experiments, and therefore, bleed-over of one isotope into the window of another was <1%. Reaction buffer contained 50 µM each dCTP and dGTP in all experiments to prevent the 3`-5`-exonuclease activity of the subunit from digesting primed templates during assembly reactions and on gel filtration columns. The individual volumes of reactions and incubation times varied and are detailed in the figure legends. The recovery of [P] was typically 75-90% using either Sephacryl S-400 or Bio-Gel A-15m. The recovery of the [H] complex and complexes containing [H] was only 50-70% on Bio-Gel A-15m, but was 56-80% on Sephacryl. Therefore, after the experiment of Fig. 2, subsequent gel filtrations were performed with Sephacryl columns.


Figure 2: stimulates the binding of core to a sliding clamp. [P] was assembled onto gapped M13mp18 DNA using the complex and ATP as described under ``Experimental Procedures'' (see scheme at top) with the exception that 2.7 pmol of DNA was present in the 382-µl reaction (instead of 1.8 pmol) before dividing it into 87-µl aliquots. Then 1.77 pmol of [H]core (A), [H]core- (B), or [H]core- (C) was added and analyzed by gel filtration on agarose A-15m as described under ``Experimental Procedures.'' Panels to the right are controls that were performed just as those to the left except ATP was omitted. , [P]; , the [H]polymerase complex. In D, aliquots of reactions (containing ATP) were replicated and analyzed on a 0.6% native agarose gel as described under ``Experimental Procedures.''



In experiments using [P] assembled onto gapped DNA by the complex, the complex (0.76 µg, 3.78 pmol) was preincubated with [P] (0.76 µg, 3.78 pmol) and gapped DNA (4.1 µg, 1.8 pmol) saturated with SSB (28.8 µg) in 382 µl of reaction buffer for 4 min at 37 °C. Then the reaction was split into 85-µl aliquots in 1.5-ml Eppendorf tubes that contained 15 µl of core (290 ng, 1.8 pmol), core- (540 ng, 1.8 pmol), or core- (840 ng, 1.8 pmol) in buffer A with 40 µg/ml BSA and incubated for an additional 2 min at 37 °C prior to analysis of 75 µl by gel filtration.

Replication Reactions

Replication reactions contained 130 ng (30 fmol) of gapped M13mp18 DNA, 0.32 of µg SSB, 22 ng (272 fmol) of , 6 ng (30 fmol) of complex (omitted when Pol III* was used), and one of the following: core, core-, core-, or Pol III* (amounts are indicated in the legend to Fig. 1) in 23.5 µl of reaction buffer. Reactions were shifted to 37 °C for 5 min to allow time for Polymerase assembly onto DNA, and then a 15-s pulse of DNA synthesis was initiated upon the addition of 1.5 µl of dATP and [-P]dTTP (final concentrations of 60 and 20 µM, respectively). Synthesis was quenched after 15 s upon spotting onto DE81 filter paper and quantitated as described (11) .


Figure 1: Replication activity of four different polymerases. Polymerase subassemblies were titrated into replication reactions containing a clamp on gapped M13mp18 DNA as described under ``Experimental Procedures.'' , Pol III*; , core-; , core-; , core.



In gel filtration experiments, polymerase-DNA complexes were quantitated for DNA synthesis as follows. An 11-µl aliquot of the reaction was removed, and a 1.5-µl aliquot of dATP and [-P]dTTP (final concentrations of 60 and 20 µM, respectively) was added to initiate DNA synthesis. The reaction was shifted to 37 °C, and then after 15 s, the reaction was quenched with 12.5 µl of 1% SDS and 40 mM EDTA. The extent of replication in the reaction was analyzed by two methods. First, a 10-µl aliquot was spotted onto DE81 paper, and the amount of incorporated nucleotides was quantitated as described (11) . Second, a 10-µl aliquot was analyzed on a 0.6% native agarose gel, which separates unreplicated template from replicative form II product. The DNA was visualized by UV-induced fluorescence of ethidium bromide.


RESULTS

Experimental Strategy

Throughout this report, the physical presence of protein on DNA was identified and quantitated through use of radioactive subunits of known specific radioactivity. H-Labeled protein and primed DNA were mixed, and then the protein-DNA complexes were gel-filtered on a large pore molecular sieving column. Protein bound to DNA comigrates with the large DNA template in the excluded volume and resolves from unbound proteins in the included volume. The molar amount of H-labeled protein bound to DNA was calculated from the known specific radioactivity. There are 10 different proteins in the holoenzyme, and we have not labeled each of them. Instead, we have labeled the clamp, the subunit, the subunit of core, the ` subunit of the complex, either the or subunit of Pol III`, and the , , or subunit of Pol III*. Thus, some conclusions drawn in this study rest on the assumption that these subassemblies remain tightly associated through gel filtration. This assumption is supported by the tight association between the subunits of these H-labeled complexes through liquid chromatography resins and gel filtration columns (15, 18) and during their preparation (see ``Experimental Procedures'').

The dimer binds two core polymerases provided core is supplied in excess. If core is limiting, then only one core is present on the dimer (10) . Therefore, we have prepared both forms for these studies (core- and core-). To follow the clamp and H-labeled proteins in the same experiment, was P-labeled by engineering a six-residue kinase recognition motif onto the C terminus, followed by P radiolabeling with a protein kinase. This derivative of is as active as unmodified (15, 27) .

Pol III* and the complex interact nonspecifically with SSB-coated ssDNA (i.e. the interaction is independent of ATP, , and a primed site) (5, 19) . The basis for this interaction is weak binding between the subunit and SSB that depends on ssDNA.() This particular interaction must be reduced for quantitative studies of other specific protein-DNA complexes. Thus, the template used in these studies is an M13mp18 plasmid with a ssDNA gap of 500 nucleotides. This template minimizes the amount of SSB-coated ssDNA in the assay and reduces to a low background level this ``nonspecific'' interaction of the complex and Pol III* with SSB-coated ssDNA (15, 18) .

Activities of Four Different Polymerases

In Fig. 1 , the replication activities of core, core-, core-, and Pol III* were compared on the SSB-coated gapped DNA template containing a clamp (assembled onto DNA by the complex prior to initiating replication). The results show that Pol III* is the most active of these polymerases, core is least active, and the core- polymerases are intermediate in activity. Similar results are observed if the polymerase subassemblies are included during the assembly of the clamp onto DNA and onto primed ssDNA (data not shown). This hierarchy is consistent with earlier studies showing that stimulates the activity of core with a clamp (9) and is also consistent with studies in the absence of , in which polymerase subassemblies of increasing subunit complexity are also increasingly active in DNA synthesis (3, 4) . The differences may lie in different processivity or speed of chain elongation or in differential efficiencies of polymerase binding to a clamp on primed DNA. Previous work has already shown that the complex is similar to holoenzyme in its rapid and processive chain elongation (5) . Therefore, we have examined the relative binding stability of different polymerase subassemblies to a clamp on DNA.

The Subunit Provides Stoichiometric Interaction of Core with a Clamp

In Fig. 2, a [P] clamp was assembled onto circular gapped DNA using the complex and then incubated for 2 min with [H]core, [H]core-, or [H]core- prior to analysis by gel filtration (see scheme at the top of Fig. 2). Because only two of the four dNTPs were included, the polymerase was unable to replicate the template and was stalled in an elongation mode. Proteins bound to the large DNA template elute early (fractions 9-14) and resolve from free proteins that elute later (fractions 15-30). The number of clamps assembled onto DNA is approximately equimolar to the input DNA in all three experiments (Fig. 2, A-C, left panels). The amount of core- and core- bound to DNA in the excluded fractions was nearly equimolar to the clamp (Fig. 2, B and C, respectively). However, in the absence of , only half as much core was retained with on DNA (Fig. 2A, left panel). Hence, provides stoichiometric interaction between core and on DNA. In the absence of ATP, the clamp is not assembled onto the DNA, and none of the polymerases comigrate with the DNA in appreciable amounts (Fig. 2, A-C, rightpanels). This result implies that these polymerases require a clamp on DNA for productive coupling to the template, consistent with their need for a clamp to become highly processive in DNA synthesis.

Prior to applying the reactions of Fig. 2 onto the gel filtration column, a sample of each reaction was analyzed for ability of these polymerases to fill the ssDNA gap upon initiating DNA synthesis. The agarose gel analysis (Fig. 2D) shows conversion of the gapped DNA to the slower migrating replicative form II. Core- and core- fill the gap in all the circular DNA molecules, whereas core fills the gap in some DNA molecules, but leaves others untouched (Fig. 2D, compare second through fourth lanes). This action is consistent with core being processive with a clamp (5) , but unable to bind to all the DNA molecules as indicated by gel filtration (Fig. 2A), and is consistent with the lower replication activity of core with in Fig. 1.

In the gel filtration experiments of Fig. 2, the polymerase is in molar excess over DNA, and therefore, even with the core- dimer, which may be capable of binding two clamps/molecule, the limited availability of clamps provides only one clamp/molecule of core-. We address the question of whether a dimeric polymerase can bind two clamps in experiments below using Pol III*.

The Subunit Is Present on DNA with Core and

How does increase the amount of core bound to ? Is action stoichiometric, or does it act as a molecular matchmaker by transferring core to a clamp and then dissociating from the DNA to transfer other cores to clamps? To distinguish between these possibilities, we assembled a core-[H] complex onto DNA containing the [P] clamp, followed by gel filtration analysis. The results show approximately equimolar association of core-[H] with [P] clamps (Fig. 3A). The interaction of core-[H] with DNA requires a clamp on the DNA as there is little binding in a reaction where ATP is omitted (Fig. 3B). Hence, acts in a stoichiometric fashion and is present with core and on DNA rather than dissociating from DNA and acting as a catalytic matchmaker.


Figure 3: acts in stoichiometric fashion to increase the efficiency of core binding to on DNA. A, [P] was assembled onto gapped DNA by the complex in the presence of core-[H] as described under ``Experimental Procedures,'' followed by gel filtration. B, the analysis of A was repeated, but ATP was omitted from the reaction. , core-[H]; , [P].



A simple mechanism by which may increase the amount of core bound to on DNA is that it may act as a brace between core and and/or the DNA. To test whether can bind to on DNA without core, we mixed [H] with a [P] clamp on primed DNA and analyzed the mixture by gel filtration (Fig. 4). The results show that very little [H] binds to the clamp. This low level of [H] does not require the clamp (data not shown) and therefore can be ascribed to the previously documented weak association of with DNA (18) . Presumably, the weak binding of to DNA is sufficient to provide the extra stabilization to the core- contact needed to observe stoichiometric comigration of the core- complex to clamps on DNA. Alternatively, may change the conformation of core such that it binds tighter.


Figure 4: does not bind a clamp in the absence of core. Shown is the analysis of [H] interaction with [P] sliding clamps in the absence of core. [P] was placed onto gapped DNA by the complex as described under ``Experimental Procedures'' and then treated with 1.77 pmol of [H] before gel filtration analysis. , [P]; , [H].



Does the Complex Assemble with the Core Polymerase and on DNA?

Previous experiments have shown that after the complex assembles onto DNA, the complex can be removed from the reaction by gel filtration (5) . After gel filtration, core can be added to the clamp on DNA, resulting in highly efficient synthesis similar to that of the entire holoenzyme (5) . But would the complex have stably associated with the clamp on DNA if the core polymerase were present before gel filtration? In Fig. 5, we used the [H] complex and [P] to determine whether the complex remains on DNA with when core is added to the reaction prior to gel filtration. The gel filtration analysis showed that very little complex remained on DNA with even in the presence of these polymerase subassemblies (Fig. 5A).


Figure 5: The complex does not remain associated with the core- or core- complex on DNA. [P] was assembled onto gapped DNA using the complex as described under ``Experimental Procedures'' with the exception that the [H] complex (labeled in `) was used in place of unlabeled complex. A-C are gel filtration analyses of reactions containing unlabeled core, core-, and core-, respectively. , [P]; , the [H] complex.



The subunit is known to interact directly with (8) . The complex interaction is essential for the complex to be assimilated into the Pol III* structure (8) . However, the complex does not associate with (or Pol III`) unless a particular order of subunit addition is followed, such as adding the and ` subunits after the - contact has been established (8) . However, it remains possible that Pol III` and the complex will associate in the presence of a clamp and a primed template. This experiment is shown in Fig. 5C. The results show that the [H] complex does not remain with the [P] clamp on DNA through the gel filtration column and thus did not assemble into Pol III*. In Fig. 5B, core- was incubated with the complex, , and DNA. Perhaps the unoccupied protomer of will associate with the complex and hold it to DNA with the polymerase and clamp. However, the results show that the [H] complex does not assemble with the other proteins on the primed DNA.

Does the Complex within Pol III* Remain on DNA with ?

A previous study indicated the physical presence of all the subunits of the holoenzyme on DNA through gel filtration (7) . However, the large amounts of SSB-coated ssDNA present in that study may have led to the nonspecific binding of Pol III* to DNA (e.g. through the -SSB contact). This early study also required use of silver-stained polyacrylamide gels to identify subunits and thus was qualitative. Here we have re-examined the composition of Pol III* on DNA with using radioactive labeled proteins and the gapped plasmid to minimize nonspecific binding of Pol III* to DNA (Fig. 6). To perform these experiments, Pol III* was reconstituted using [H] (to follow the complex), [H] (to follow core), or [H]. In Fig. 6(A-C), a 3-fold molar excess of [H]Pol III* over gapped template was incubated with . Approximately equimolar amounts of [H]core (there are two cores in one Pol III*), [H] complex, and [H] were bound to [P] on gapped DNA in the excluded fractions. Therefore, when the complex clamp loader is already part of the polymerase structure, it remains associated with the polymerase and on DNA.


Figure 6: The complex within Pol III* is retained with core, , and the clamp on DNA. Pol III*, labeled in different subunits, was incubated with [P] and gapped DNA, followed by gel filtration. Proteins were incubated for 3 min at 37 °C in 100 µl of reaction buffer with 30 mM NaCl. Experiments in A-C contained 1.2 µg (260 fmol) of gapped M13mp18 DNA, 3.2 µg of SSB, 81 ng (1 pmol) of [P], and 1 µg (1.4 pmol) of [H]Pol III* constituted with the indicated tritiated subunit. Experiments in D-F contained 0.57 µg (400 fmol) of gapped DNA, 3.2 µg of SSB, 67 ng (830 fmol) of [P], and 277 ng (400 fmol) of [H]Pol III* reconstituted with the indicated tritiated subunit. A and D, Pol III* and [H]core; B and E, Pol III* and [H]; C and F, Pol III* and [H]. , [H]Pol III*; , [P].



Up until now, the polymerase has been used in molar excess over clamps on gapped DNA, and therefore, the assemblies containing two core polymerases (i.e. core- and Pol III*) could bind only one of the clamps, even though these polymerases may be capable of binding two clamps/polymerase molecule. In the experiments presented in Fig. 6(D-F), less Pol III* was added, and the concentration of the gapped plasmid was increased, resulting in an excess of clamps over dimeric polymerases. The results show that each molecule of [H]Pol III* (labeled in core, , or ) binds approximately two [P] clamps.

In these experiments, Pol III* contains only one molecule of complex, yet two clamps are formed per Pol III*. Hence, these results further indicate that the single complex within Pol III* can assemble two clamps on DNA, one for each core within Pol III*.

Multiple clamps may be assembled onto one primed template (5) , and if this occurs, in the experiments of Fig. 6, the amount of each polymerase binds would be an overestimate. The clamp freely slides off linear DNA unless it is held to DNA through stoichiometric association with the polymerase (5, 18) . Fig. 7shows an experiment in which an excess of DNA was used to assemble [H]Pol III* with [P] clamps. The circular DNA was then linearized in a 1-min incubation with BamHI prior to gel filtration in order to remove clamps not associated with a polymerase. The results show a stoichiometry of one [H]Pol III* bound to 1.7 [P] clamps, consistent with Pol III* cross-linking two clamps on separate DNA templates. These experiments are technically difficult, and thus, these intriguing conclusions remain to be rigorously tested in future studies. The possibility that each core within Pol III* binds a clamp on DNA would be consistent with the hypothesis that both strands of the chromosome are replicated concurrently by the twin polymerase within the holoenzyme (20, 21) .


Figure 7: Pol III* binds two sliding clamps. Initiation complexes were assembled onto 600 fmol of gapped M13mp18 template using 200 fmol (135 ng) of [H]Pol III* (labeled in ) and 250 fmol (20 ng) of [P] and incubated for 3 min at 37 °C in 100 µl of reaction buffer with 30 mM NaCl. After a 1-min treatment with 100 units of BamHI to linearize the DNA, reactions were gel-filtered, and the amount of [H]Pol III* and [P] in the column fractions was quantitated. , [H]Pol III*; , [P].




DISCUSSION

Prior to this work, it had been shown that the complex is a clamp loader that assembles a clamp on DNA, but then dissociates and is not needed for to tether core to DNA for processive elongation of a 7-kilobase pair template (1, 2) . This may have led to the conception that the complex is not present in the holoenzyme structure on DNA and that processive synthesis is performed only by a complex of the clamp with the core polymerase (Fig. 8A). This study has shown that the complex is retained with the other subunits (core, , and the clamp) on primed DNA (Fig. 8D) provided that Pol III* is used instead of separate subassemblies of the complex and Pol III`. Hence, even though the complex is not essential to the polymerase- contact, the clamp loader is present with the other proteins on DNA when Pol III* is used (via contact with ). Presumably, Pol III* is the form that replicates the chromosome since the function of the complex in assembling onto DNA is needed repeatedly during discontinuous synthesis of the lagging strand (15, 22, 23) .


Figure 8: Models of the four different initiation complexes that DNA polymerase III can assemble onto DNA templates. A, core- clamp; B, core- clamp; C, core- clamp; D, Pol III*- clamp. In C and D, both cores are presumed to bind a clamp, but for clarity, only one clamp is shown. See ``Results'' for details. 3`, the 3` termini of the DNA being extended. The model of core is based on the crystal structure of DNA polymerase I and duplex DNA (26).



An interesting aspect of this study is that the subunit structure of the holoenzyme on DNA can be very different depending on how the subunits are added, even though all the same subunits are present. Hence, the addition of core- (Pol III`) to the complex and results in a structure containing the two polymerases and bound to the clamp, but without the complex (Fig. 8C). However, if these subunits are first assembled into Pol III* before mixing with and primed DNA, the complex is retained on DNA with the other proteins (Fig. 8D). Consistent with this result, we showed in the third report of this series that the constitution of Pol III* from pure subunits requires a strict order of addition and that if the four-subunit Pol III` is added to the five-subunit complex, the nine-subunit Pol III* does not form (8) . In this study, we show that these assemblies still do not associate with each other even in the presence of primed DNA and the clamp. Hence, , either on or off DNA, does not circumvent the order of addition needed to incorporate the complex into the holoenzyme structure.

The minimal processive polymerase, shown in Fig. 8A, contains one core polymerase attached to a clamp. Whether the complex is removed from solution by gel filtration or is present in free solution, this minimal polymerase is highly processive and is nearly as efficient in DNA synthesis as the holoenzyme (5) . The subunit lends extra stability to the interaction of core with a clamp on primed DNA, consistent with a previous observation that in the presence of , duplex structures in the path of replication are more easily traversed by the polymerase (9) . Use of the subunit in molar excess over the core polymerase results in a core- complex instead of core- (Pol III`) (10) . Study of the core- complex showed that it too binds a clamp, but like the core- complex, it fails to retain the complex, leading to the structures shown in Fig. 8(B and C, respectively). Hence, the availability of one protomer lacking core does not lead to assimilation of the complex into the holoenzyme structure. For simplicity, the dimeric polymerases in Fig. 8are shown with only one clamp on DNA, even though both polymerases can bind clamps. Whether both polymerases within Pol III* are equally active in DNA synthesis is under investigation.

Our observations of core- and core- complexes lacking the complex appear to contrast with results of an earlier study that showed the complex to be bound along with core and on singly primed ssDNA coated with SSB (7) . However, in that study, the authors also formed a processive polymerase that was presumed to consist of only and core, as in Fig. 8A (the complex appeared catalytic in their reactions). When a large excess of complex was added, two molecules of complex were observed bound to DNA along with one core and a dimer, suggesting that the complex may bind core (7) . However, the complex binds nonspecifically to SSB-coated ssDNA (5, 19) , and therefore, the complex may have been bound nonspecifically to SSB-coated DNA, while core- was bound specifically at the primer terminus. For this reason, we have used in this report a duplex template with a gap of only 500 nucleotides, which essentially eliminates the nonspecific background of the complex bound to SSB-coated DNA. An apparent competition between and the complex has been observed in which appeared to compete the complex off the DNA (7) . Although we find that , like the complex, binds nonspecifically to SSB-coated ssDNA (18) , it is not clear why would compete the complex off DNA since one might expect a large number of nonspecific binding sites on these SSB-coated ssDNA templates for both and the complex to bind at the same time. Perhaps there are sites of preferential association, such as at areas of secondary structure, for which and the complex compete.

An earlier study showed that at least two distinct polymerases, either lacking or containing , could be assembled onto DNA (9) . In the presence of , the polymerase that formed was probably core- (e.g.Fig. 8B) since was present in 10-fold molar excess over core. The function of the two polymerases (+ or - ) was hypothesized to be in replicating the leading (+) or the lagging (-) strand of a chromosome. The presence of made the polymerase more efficient at traversing obstacles (i.e. duplexes in the path of replication) and thus was an attractive candidate for action with the leading strand polymerase. Now, in light of the dimeric structure of core-, it seems more likely that one protomer is on each core polymerase to increase the efficiency of their interaction with their respective clamps on both the leading and lagging strands. Another important function of the dimer is to hold the complex to the holoenzyme structure (8) .

There are 40 molecules of core polymerase in E. coli, only half of which are incorporated into the holoenzyme (24) . Likewise, there is an excess of complex in cell lysates relative to that within the holoenzyme (25) . Hence, it seems possible that the different processive polymerase structures outlined in Fig. 8 could be present in the cell and may be useful in other areas of DNA metabolism such as repair, recombination, and mutagenesis. For example, the core- polymerase in Fig. 8A may be useful in short gap repair, where its lower stability on DNA would allow it to more easily dissociate from DNA upon finishing a gap. The core- assemblies that lack the complex (Fig. 8, B and C) may be useful in repair of longer gaps such as in mismatch repair. Furthermore, the ability of to bind ssDNA and double-stranded DNA (18) and its dimeric structure (i.e. may cross-link two DNA molecules) make it an attractive candidate as a player in replicative recombination.

During discontinuous synthesis on the lagging strand, the holoenzyme must be capable of rapidly cycling from the end of a completed Okazaki fragment to a new primed site to extend the next fragment. We have shown previously that Pol III* remains tightly associated with its clamp on DNA during chain extension, but upon completing a template to the last nucleotide (i.e. upon completing a lagging strand fragment), it rapidly dissociates from DNA and cycles to a new primed site (15, 22) . However, this rapid transfer of polymerase to another primed site requires that site to be endowed with a clamp. Hence, the structure of Pol III* is ideally suited for chromosome replication (Fig. 8D). The complex matchmaker is physically associated with the polymerases by mutual interaction with , and therefore, its clamp loading activity is ever present and available for repeated action in clamp assembly onto the multiple RNA primers of the lagging strand.


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 Cell Biology, Harvard Medical School, Boston, MA 02115.

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; BSA, bovine serum albumin; SSB, single-stranded DNA-binding protein of E. coli; ssDNA, single-stranded DNA.

The -SSB interaction requires no other proteins, and the ssDNA is not absolutely required, but strengthens the -SSB interaction considerably (Z. Kelman and M. O'Donnell, manuscript in preparation).


ACKNOWLEDGEMENTS

We are grateful to Dr. Vytautas Naktinis for construction and purification of the subunit containing the kinase recognition sequence and the conditions for labeling it.


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