©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
II. INTERMEDIATE COMPLEX BETWEEN THE CLAMP LOADER AND ITS CLAMP (*)

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

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

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

The Escherichia coli replicase, DNA polymerase III holoenzyme, derives its processivity from the subunit sliding clamp that encircles DNA and tethers the replicase to the template. The dimer is assembled around DNA by the complex clamp loader in an ATP-dependent reaction. In this report, the essential contact between the clamp loader and is identified as mediated through the subunit of the complex. The subunit appears to contact the face of the dimer ring that contains the two C termini. Surprisingly, ATP is required for the complex to bind , but not for to bind . This indicates that is buried in the complex and suggests a role for ATP in exposing for interaction with . A protease protection assay has been developed to specifically probe the subunit within the complex. The results of the assay are consistent with an ATP-induced conformational change in the complex that alters the state of the subunit within it. The implication of these key features to the clamp loading mechanism of the complex is discussed.


INTRODUCTION

The cellular replicases of prokaryotes and eukaryotes attain high processivity by use of circular proteins that behave as DNA sliding clamps (1, 2) . There is an increasing number of proteins that encircle DNA. For example, Escherichia coli topoisomerase I ( protein) has a cavity through which DNA strands are passed (3) , and the RuvB recombination protein appears to encircle two DNA molecules during Holiday junction movement (4) . The DNA polymerase and recombination clamps require accessory protein clamp loaders (also called molecular matchmakers), which couple ATP hydrolysis to the assembly of the clamp onto DNA.

Little is known about the mechanism by which circular proteins are assembled onto DNA. As a model system, we have studied the E. coli complex (`), the clamp loader of DNA polymerase III holoenzyme that assembles clamps onto DNA. Studies of individual subunits of the complex have shown that no one subunit can assemble onto DNA. Minimally, both and are required (5) , although ` is needed for an efficient reaction at low ionic strength (6) , and the and subunits are also needed at physiological ionic strength (5) . The multiple protein requirement suggests that the assembly of a protein ring around DNA is a multistep process. Consistent with the concept of a multistep reaction, the clamp loaders of the T4 bacteriophage (gene 44/62 protein) and eukaryotic (RF-C, activator-1) systems are also composed of multiple proteins and require ATP for efficient assembly of their respective clamps onto DNA (gene 45 protein and PCNA, respectively) (7, 8, 9, 10) . The T4 gene 44/62 protein complex contains five subunits, four protomers of gene 44 protein and one copy of gene 62 protein. The eukaryotic RF-C (activator-1) clamp loader consists of five different subunits, like the E. coli complex. The amino acid homology between some of the subunits of these clamp loaders from prokaryotic, eukaryotic, and viral systems suggests that their basic mechanism of action may be the same (11) .

The goal of this study was initially to identify which subunit(s) of the complex bind to to further the understanding of the overall structure of DNA polymerase III holoenzyme. This goal was quickly reached: the subunit forms the major (if not sole) contact between the complex and . But as the study progressed, we found that the complex required ATP to bind , whereas , at a comparable concentration to the complex, did not require ATP. This observation indicates that is masked from binding by other subunits of the complex. In the presence of ATP, the masking is relieved, and is exposed for interaction with . This complex- interaction is likely an early intermediate on the mechanistic path of assembly of the protein ring around DNA.


EXPERIMENTAL PROCEDURES

Materials and Methods

All sources and procedures not described here are described in the first report of this series (12) . Streptomyces griseus Pronase and the serine protease inhibitor Pefabloc SC were from Boehringer Mannheim; the autoradiography enhancer ENHANCE was from DuPont NEN. The protein kinase (EC 2.7.1.37, ATP:protein phosphotransferase) catalytic subunit of murine cAMP-dependent protein kinase (recombinant expressed in E. coli(13) ) was kindly provided by Dr. Susan S. Taylor (Department of Chemistry, University of California at San Diego, La Jolla, CA).

Preparative Constitution of the Complex

The subunit (5.64 mg, 340 nmol as monomer) was incubated with (3.9 mg, 257 nmol as monomer) in 3 ml of buffer A (urea was present at 0.5 M due to the need for urea in the purification of as described (14, 15) ) for 60 min at 15 °C, at which time (8.0 mg, 85 nmol as dimer) was added. This mixture was further incubated for 60 min at 15 °C in a final volume of 7 ml of buffer A containing 100 mM NaCl. This mixture was loaded onto a 8-ml Mono Q HR 10/10 column (Pharmacia Biotech Inc.) and eluted with a 180-ml linear gradient of 0-0.4 M NaCl in buffer A. Eighty fractions of 2.5 ml each were collected and analyzed on an SDS-polyacrylamide gel stained with Coomassie Blue. Fractions 44-48 containing were pooled (7.7 mg, 11.5 ml) and dialyzed against buffer A to a conductivity equal to 29 mM NaCl.

Preparative Constitution of the ` Complex

The ` complex was constituted from pure , , and ` as described previously (14) .

Preparative Constitution of the ` Complex

The ` complex was assembled in two steps. First, the complex (1.5 mg, 52.5 nmol) was constituted as described above and then was incubated with ` (3.89 mg, 105 nmol) in 3.06 ml of buffer A, followed by chromatography 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. Preparative Constitution of the [H]` Complex-The subunit was tritiated by reductive methylation as described (17) to a specific activity of 12.5 10 cpm/µg (24) . The complex was constituted from pure subunits using [H] in place of and was purified from free subunits as described in the first report of this series (12) . The specific activity of the [H]` complex was 3.3 10 cpm/µg, and its activity was >90% of the activity of the unlabeled complex in replication assays with and the core polymerase.

Kinase Protection Assays

The six-residue protein kinase recognition sequence was engineered onto the C terminus of as described (17, 24) . Then 30 pmol (as dimer) of the derivative was incubated either with 1) 240 pmol (9.3 µg) of and 18.7 µg of bovine serum albumin or with 2) 30.5 µg of bovine serum albumin in 13 µl of buffer A at 15 °C for 30 min. Each mixture was then adjusted to a final volume of 30 µl in 20 mM Tris-HCl (pH 7.5), 2 mM dithiothreitol, 100 mM NaCl, 12 mM MgCl, 10 mM NaF, 60 µM ATP, and 5 µCi of [-P]ATP. The reaction was incubated at 37 °C, and phosphotransfer was initiated upon the addition of 0.0015 units of protein kinase. Aliquots of 3 µl were withdrawn and brought to a final concentration of 5 mM ATP and 50 mM EDTA in 10 µl, followed by analysis on a 12% SDS-polyacrylamide gel. The gel was dried and exposed to x-ray film, and the autoradiogram was quantitated using a PhosphorImager (Molecular Dynamics, Inc.).

Protease Protection Assays

Limited proteolytic digestion of 0.4 µg of [H] or 1.5 µg of complex constituted using the [H] subunit was performed in 25 µl of buffer A containing 100 mM NaCl and, when present, 1 mM ATP and 10 mM MgCl. To equalize the total amount of protein in each assay, 1.1 µg of bovine serum albumin was added to the reaction containing only [H]. Proteolytic digestion was initiated upon adding Pronase to a concentration of 5.6 µg/ml on ice, and then the reaction was shifted to 25 °C for 10 min. Digestion was quenched upon adding 2 µl of buffer A containing 10 mg/ml protease inhibitor (Pefabloc SC) and 2% SDS, followed immediately by boiling for 5 min. Samples were analyzed by electrophoresis on a 15% SDS-polyacrylamide gel. The gel was equilibrated with ENHANCE according to the manufacturer's protocol and fluorographed on Biomax MR x-ray film at -70 °C for 60 h.

Surface Plasmon Resonance

Methods of immobilization of and SPR() experiments were as described in the first report of this series (12) . For analysis of the - binding kinetics, the chip contained 1185 response units of immobilized , and the mobile phase (SPR buffer) contained at the following concentrations: 5, 10, 20, 40, 80, and 160 nM (as dimer). When 0.5 mM ATP and 10 mM MgCl were present in the mobile phase, the concentrations of were 25, 100, and 250 nM. After the initial immobilization and between injections, noncovalently bound proteins were removed with 10 µl of 0.1 M glycine (pH 9.5). For analysis of the complex- binding kinetics, the chip contained 810 response units of immobilized , and then the complex was reconstituted into immobilized by injecting 35 µl of 4 µM ` complex in SPR buffer followed by 35 µl of 1 µM ` and finally 35 µl of 20 nM `. The use of 20 nM ` in the mobile phase resulted in a stabile signal of 660 response units (over the initial 810 response units) and was included in the mobile phase in experiments using 1280 and 5120 nM (use of 5, 20, 80, and 320 nM produced insignificant signals). For analysis in the presence of 0.5 mM ATP and 10 mM MgCl, the mobile phase also included 20 nM ` and the following concentrations of : 20, 40, 80, and 160 nM. Treatment with 0.1 M glycine was not required between injections in experiments using the immobilized complex as the base line returned to the initial level during the dissociation phase. The k and k values were determined from the result of each injection using the nonlinear curve fitting BIAevaluation 2.0 software (based on Ref. 18).


RESULTS

The Subunit Is the Major Contact Point in the Complex for

Initially, we used gel filtration to study the interaction of with the complex.During gel filtration, components are not at equilibrium, and therefore, only strong complexes with relatively slow dissociation rates are observed. In Fig. 1A, the complex was mixed with and then gel-filtered. Analysis of the column fractions on an SDS-polyacrylamide gel shows that comigrates with the five subunits of the complex in fractions 21-27, much earlier than the position at which elutes alone (Fig. 1D, fractions 35-39). Hence, binds to the complex with high enough affinity to survive gel filtration. To determine which subunit(s) of the complex interact with , we studied two subassemblies of the complex. Analysis of a mixture of with the ` complex (Fig. 1B) shows that comigrates with `, indicating that and are not essential for interaction with . This is substantiated in Fig. 1C, where analysis of a mixture of with the complex showed no comigration of with .


Figure 1: The subunit interacts with the complex. The subunit was incubated with ` (A), ` (B), or (C) or alone (D) and then gel-filtered on Superose 12. A, mixture of (36 µg, 0.45 nmol as dimer) and constituted ` (360 µg, 1.8 nmol); B, mixture of (36 µg, 0.45 nmol as dimer) and 3.2 nmol of ` complex constituted by mixing 300 µg of (3.2 nmol as dimer), 70 µg of (1.8 nmol as monomer), and 66.7 µg of ` (1.8 nmol as monomer); C, mixture of (36 µg, 0.45 nmol as dimer) and 1.8 nmol of complex constituted by mixing 169 µg of (1.8 nmol as dimer), 37.4 µg of (2.25 nmol as monomer), and 30 µg of (2.0 nmol as monomer); D, the subunit alone (36 µg, 0.45 nmol as dimer). 50-µl aliquots of column fractions were analyzed. Fraction (Frx) numbers are indicated above each gel. The first lane of each gel contains molecular weight standards (MW), and their weights are indicated to the left. The , , `, , , and subunits are identified to the right.



Which subunit(s) of the ` complex bind ? In Fig. 2, the , , and ` subunits were analyzed individually for interaction with . In Fig. 2A, a mixture of and showed no interaction ( and did not comigrate), but instead migrated in the same position as when analyzed alone. Likewise, in Fig. 2B, a mixture of ` and showed no interaction. However, in Fig. 2C, an interaction was observed between and ; the two proteins comigrated in fractions 29-35, significantly earlier than alone (fractions 35-41), indicating that they form a complex. The dimer is 81 kDa, and is a monomer of 38.7 kDa. Thus, these two proteins should have resolved from each other if they did not interact. Consistent with complex formation, alone elutes much later (Fig. 2D). Scans of the Coomassie Blue-stained SDS-polyacrylamide gel of Fig. 2C were performed to estimate the stoichiometry of the complex. However, the ratio of to increased continuously from fractions 29 to 35, indicating that the complex dissociates during gel filtration, thus precluding an accurate determination of their molar ratio by this technique.


Figure 2: The subunit interacts with the subunit of the complex. The subunit was incubated with , `, or and then gel-filtered (Superose 12) as described under ``Experimental Procedures.'' A, mixture of (36 µg, 0.45 nmol as dimer) and (169 µg, 1.8 nmol as dimer); B, mixture of (144 µg, 1.8 nmol as dimer) and ` (16.6 µg, 0.45 nmol as monomer); C, mixture of (91 µg, 1.1 nmol as dimer) and (293.5 µg, 7.6 nmol as monomer); D, the subunit alone (293.5 µg, 7.6 nmol as monomer). 50-µl aliquots of column fractions were analyzed. Column fractions are indicated above each gel. Positions of molecular weight standards (MW) and their weights are indicated to the left. The , , `, and subunits are identified to the right.



The subunit is the only complex subunit to form a gel-filterable complex with , indicating that is the major contact point between the complex and . Consistent with this, a ` complex showed no interaction with in a gel filtration analysis (data not shown). This result rules out the existence of multiple weak interactions that add up to a gel-filterable interaction between and two or more subunits of the complex (other than ).

We have also studied individual subunits of the complex for weak interaction with using the SPR technique. In these experiments, the dimer was immobilized on the sensor chip, and 1 µM solutions of individual subunits of the complex were serially passed over the top. No interaction was observed between and the , `, , or complex (data not shown). The - interaction was observed as shown below in Fig. 6and was also utilized as a control in our previous report on interaction between PCNA and the p21 cyclin kinase inhibitor (19) .


Figure 6: SPR analysis of the effect of ATP on interaction of with and the complex. The influence of ATP on the kinetics of interaction between either or the complex with was examined by SPR analysis as described under ``Experimental Procedures,'' except that in these experiments, was at 80 nM tested in all four panels. Openarrowheads denote the start of injection, and closedarrowheads mark the start of the buffer wash. In A and B, the subunit was immobilized on the sensor chip. In C and D, the complex was reconstituted on the sensor chip, and the mobile phase was supplemented with 20 nM `. In B and D, the buffer contained both ATP and MgCl; in A and C, the buffer contained neither ATP nor MgCl. RU, response units.



The minimum molecular mass of the complex was estimated to be 125 kDa by gel filtration upon comparing its volume of elution relative to that of size standards (Fig. 3). This mass is consistent with a complex (81 + 38.7 kDa = 119.7 kDa), although in light of the apparent dissociation of the complex during gel filtration, a complex cannot be excluded. Several attempts have been made to assess the exact stoichiometry of the complex, but the complex lacks sufficient stability for an accurate determination. However, the complex binds to the dimer and contains one monomer of . Thus, it appears that the association of one subunit with a dimer is sufficient for function during clamp loading by the complex.


Figure 3: Size estimate of the complex. The arrow shows the elution position of the complex, relative to protein standards of known molecular mass, from a Superose 12 gel filtration column. Also shown are the elution positions of and alone. Thy, bovine thyroglobulin (670 kDa); -G, bovine -globulin (158 kDa); Ova, chicken ovalbumin (44 kDa); Myo, horse myoglobulin (17 kDa); B-12, vitamin B-12 (1.35 kDa).



The Kinase Protection Assay Reveals That Interacts with the C-terminal Face of the Ring

A six-amino acid recognition sequence for cAMP-dependent protein kinase was engineered onto the C terminus of for the purpose of radiolabeling with P (17, 24) . Both C termini of the dimer protrude from one face of the ring as shown in Fig. 4A. In the experiment of Fig. 4B, this derivative was incubated with and then treated with protein kinase and [-P]ATP. At the times indicated, the reaction was quenched, and the extent of phosphorylation was determined by autoradiography of an SDS-polyacrylamide gel (Fig. 4B). In the absence of , the derivative is phosphorylated within 1 min, but in the presence of , phosphorylation is nearly completely blocked.() These results suggest that interacts with the C-terminal face of the dimer.


Figure 4: Kinase protection assay of interacting with the clamp. The dimer structure is shown in A. The view on the left shows the central cavity through which the DNA fits. On the right, the dimer is turned 90° to show the two C termini, which extrude out from the same side of the ring. The boxes placed on the C termini denote the location of the protein kinase recognition sequence engineered into (denoted as ). In B, was treated with protein kinase and [-P]ATP either in the presence or absence of (see scheme), and aliquots were withdrawn at the indicated times, followed by analysis on an SDS-polyacrylamide gel and autoradiography. The autoradiogram at the top shows the time course of phosphorylation in the absence of , and the bottom autoradiogram is in the presence of . Quantitation of the autoradiograms is shown below. , absence of ; , presence of .



ATP Is Required for Interaction of the Complex with , but Not for with

In the course of these studies, we noticed that ATP and magnesium were required in the gel filtration buffer to observe the interaction between the complex and (ATP and MgCl were present in the experiments of Fig. 1 and Fig. 2). However, in experiments with , ATP and MgCl were not needed to observe the - interaction. Furthermore, study of the effect of ATP and MgCl on the interaction of the complex (and ) with showed that without ATP and MgCl, the complex- interaction is much weaker than the - interaction, while with ATP and MgCl, the complex- interaction is comparable in affinity to the - interaction.

We demonstrated the effect of ATP in two ways, gel filtration and SPR. In Fig. 5 , was incubated with a substoichiometric amount of either the complex or and then gel-filtered in either the presence or absence of ATP and MgCl in the column buffer. PanelsB and E are analyses of and the complex, respectively, with performed in the presence of ATP and MgCl. PanelsA and F are analyses of and the complex, respectively, with performed in the absence of ATP and MgCl. It is apparent that in the presence of ATP and MgCl, comigrates with both the complex (fractions 22-30; panel E) and (fractions 36-38; panel B) (see panels C and G for elution position of alone and panelD for alone). In the absence of ATP, interacts only with (fractions 34-38; panel A), but essentially no interaction is observed with the complex (panel F). An experiment described below (see Fig. 8) shows that both ATP and MgCl are required to observe the interaction between the complex and .


Figure 5: ATP is required for the complex- interaction, but not for the - interaction. The dimer (4.8 nmol) was incubated with 1.2 nmol of either or complex in 200 µl of column buffer (with or without 1 mM ATP and 10 mM MgCl) at 15 °C for 30 min. Mixtures were gel-filtered on Superose 12 as described in the first report of this series (12) (ATP and MgCl, when present in the incubation mixture, were included in the column buffer as well). Aliquots (50 µl) of column fractions were analyzed on 12% SDS-polyacrylamide gels as described (12). A, and without ATP and MgCl; B, and with ATP and MgCl; C and G, alone; D, alone; E, the complex and with ATP and MgCl; F, the complex and without ATP and MgCl. Fraction numbers are at the top of the gels; molecular mass standards are in the first lane with their respective masses (in kilodaltons) shown to the left of each gel; and subunits of the complex and are indicated to the right of each gel.




Figure 8: Metal and nucleotide requirements for the complex- interaction. The [H] subunit (52 pmol as dimer) was incubated with 104 pmol of complex in 100 µl of column buffer for 5 min at 37 °C before injection into a Superose 6 column equilibrated with column buffer and developed at 37 °C. After the first 2.8 ml, fractions of 200 µl were collected and analyzed for [H] by scintillation counting. Present in the incubation and gel filtration buffer during the analysis were the following: A, 0.2 mM ATP and 8 mM MgCl; B, no addition; C, 8 mM MgCl; D, 0.2 mM ATP and 8 mM MgCl; E, 0.2 mM dATP and 8 MgCl; F, 0.2 mM ATPS and 8 mM MgCl; G, 0.2 mM AMP-PNP and 8 mM MgCl; H, 0.2 mM ADP and 8 mM MgCl; I, 0.2 mM dTTP and 8 mM MgCl.



In Fig. 6, the binding kinetics of with and of with the complex were compared by the surface plasmon resonance technique. In panels A and B, the subunit was immobilized on the sensor chip, and then was passed over immobilized either in the absence (panelA) or in the presence (panelB) of ATP and MgCl. After 3 min of observation of the association phase, buffer either lacking or containing ATP and MgCl was passed over the chip to observe the dissociation rate (panelsA and B, respectively). The results show that binds whether ATP and MgCl are present or not. These experiments were repeated using different concentrations of the subunit in the buffer, and the apparent kinetic parameters (k and k) and the apparent dissociation constant (K ) were calculated from these data. These values are listed in . The apparent K value for interaction of with was 7-10 nM with or without ATP.

Next, the complex was immobilized on a sensor chip, and the experiments were repeated. To immobilize the complex, the ` complex was passed over immobilized to reconstitute the complex, and then was passed over the immobilized complex. In the absence of ATP and MgCl, very little interaction of with the complex was observed (Fig. 6C), but with ATP and MgCl present in the buffer, a clear signal was obtained (Fig. 6D). Similar experiments were repeated using different concentrations of in the buffer, and the kinetic and equilibrium constants for the complex- interactions were calculated as described above for the complex (). The results show that associates very slowly with the complex in the absence of ATP and MgCl (250-fold slower than with ), but in the presence of ATP and MgCl, associates with the complex as fast as with . In contrast, the off rates of from the complex were affected only 3.5-fold by the presence of ATP and MgCl. The calculated K value for the complex- interaction in the presence of ATP and MgCl was similar to that of the complex, but in the absence of ATP and MgCl, the K value was increased 1000-fold.

A Protease Protection Assay Reveals a Conformational Change in the Complex Induced by ATP

The ATP requirement for the complex to bind , but not for to bind , could be interpreted as being buried within the complex and ATP inducing the complex to expose for interaction with . If ATP induces the complex to expose , then should become more susceptible to proteolysis. To test this, was H-labeled and reconstituted into the complex. The [H]` complex was treated with Pronase in the presence or absence of ATP (and MgCl), and the partial digestion pattern of [H] was analyzed on an SDS-polyacrylamide gel (Fig. 7).


Figure 7: ATP influences the protease digestion pattern of [H] within the complex. The [H] subunit (lanes2 and 3) and the complex constituted using [H] (lanes5 and 6) were treated with Pronase in the presence or absence of 1 mM ATP and 10 mM MgCl as specified at the top. After treatment, reactions were analyzed on an SDS-polyacrylamide gel, followed by fluorography as described under ``Experimental Procedures.'' Untreated samples of [H] and the complex containing [H] were analyzed in lanes1 and 4, respectively. For the complex, arrows indicate new or enhanced cleavages in the presence of ATP (lane6) relative to in the absence of ATP (lane5). Circles indicate cleavages present in the absence of ATP (lane5) relative to in the presence of ATP (lane6).



Fig. 7 shows the [H] subunit alone, either untreated (lane1) or treated with Pronase without ATP (lane2) or with ATP (lane3). As expected, ATP had no detectable influence on the digestion pattern of the isolated [H] subunit. Study of [H] assembled into the complex is shown in lanes 4-6. Lane4 is the complex untreated with Pronase. Lanes5 and 6 are the complex treated with Pronase in the absence or presence of ATP, respectively. Several ATP-dependent changes in the digestion pattern of [H] within the complex are observed, and they assort into two categories, enhanced cleavages (arrows) and decreased cleavage (circles). Hence, ATP induces several changes in the digestion pattern of [H] within the complex, consistent with ATP inducing a conformational change. These Pronase digestion experiments were performed in the absence of , and therefore, is not required for the ATP-induced conformational change in the complex.

Metal and Nucleotide Requirements for the Complex- Interaction

A study of the nucleotide requirements for induction of the complex- interaction is presented in Fig. 8 . In these experiments, [H] was used to follow its position of elution during gel filtration. Nucleotide and MgCl, when present, were in the reaction and the column buffer. PanelA shows the elution of [H] bound to the complex, and panelB shows the elution of [H] alone (no complex added). Both MgCl and ATP are needed for the complex- interaction as [H] is not bound to the complex if either ATP or MgCl is omitted (panelsC and D). PanelE shows that dATP induces the complex- interaction, consistent with the ability of dATP to support the complex-catalyzed assembly of onto DNA. The nonhydrolyzable analog AMP-PNP is not an effector of the complex- interaction (panelG), nor are ADP and TTP (panelsH and I, respectively). With ATPS, only one-half of the [H] was bound to the complex (panelF). This result did not change with longer incubation times. Furthermore, the ATPS was present at a saturating level as lower concentrations of ATPS did not result in a decrease in the extent of complex formation between [H] and the complex (data not shown).


DISCUSSION

Mechanism of the Complex in Assembly of Clamps onto DNA

This study has identified the subunit of the complex as the main (if not sole) contact point with . Using individual and subunits, the observed K value for the - interaction is 7-10 nM. However, when is assembled into the complex, the K value for interaction with is increased 350-fold (K 3 µM) and requires ATP and MgCl to achieve the high affinity interaction with (K 3 nM). A simple interpretation of these data is that is sequestered within the complex, and ATP induces a conformational change that presents for interaction with (as in Fig. 9). Consistent with this hypothesis, the Pronase digestion pattern of in the complex is changed by the addition of ATP. We propose this ATP-activated interaction of the complex with is an early step in the assembly of the dimer onto a primed template. Downstream events include recognition of the primed template and assembly of around the DNA.


Figure 9: Activation of the complex by ATP. The diagram of the complex is consistent with the stoichiometry of subunits and the intermolecular contacts between them (12). In the first diagram, the interface of the subunit that interacts with is shown as being partially buried within the complex to explain its inability to bind in the absence of ATP. In the presence of ATP, a conformational change exposes the subunit (second diagram) for binding the dimer (third diagram), followed by transfer of onto primed DNA (fourth diagram).



These studies in the E. coli system can be compared with studies in the phage T4 replication system. In the T4 system, the clamp loader is a complex of two proteins, the gene 44/62 protein complex. Laser cross-linking studies on the gene 44/62 protein complex and the gene 45 protein clamp suggest that the clamp loader undergoes a large conformational change during the process of loading the clamp onto primed DNA (20) .

When Does the Ring Open?

Although the dimer is very stabile, it is conceivable that the ring is opened in this ATP-activated complex (as hypothesized in Fig. 9 ).() It has been reported that the holoenzyme can act processively in the absence of ATP, provided is present at a high concentration (21, 22) . Perhaps a high concentration of circumvents the need for ATP by simply driving the unfavorable interaction of with the complex in the absence of ATP. Processive synthesis in the absence of ATP implies that ATP is not essential to open the ring; perhaps the binding energy between and opens the ring. However, it is important to note that the templates were linear in the earlier studies on holoenzyme activity in the absence of ATP, and therefore, the ring may have loaded onto DNA independent of the complex by threading over DNA ends (i.e. without opening at the dimer interface).

The Activated Complex

Whether the activated state of the complex (ATP-induced conformational change) requires ATP binding or hydrolysis is still uncertain. Further studies using ATP-binding site mutants of constituted into the complex are in progress to distinguish the roles of ATP binding and hydrolysis. In this report, the use of the nonhydrolyzable ATP analog AMP-PNP in place of ATP did not activate the complex for interaction with , indicating that hydrolysis may be necessary to achieve the activated state. However, the complex is partially activated for interaction with by ATPS, suggesting that ATP hydrolysis is not necessary as ATPS is generally nonhydrolyzable. However, the holoenzyme has been shown to be capable of hydrolyzing ATPS (23) . The extent of complex activation by ATPS is approximately half that observed using ATP. Previous studies have shown that ATPS results in one-half the amount of holoenzyme clamped to DNA relative to the use of ATP, and it was suggested that there were two populations of the holoenzyme, one that can hydrolyze ATPS and another that cannot (23) . The results of this report suggest that the observed value of one-half may be rooted entirely in the complex. Perhaps there are two populations of the complex, one that can be activated by ATPS and another that cannot. The results of this report suggest yet another explanation. Perhaps the complex hydrolyzes ATPS slower than ATP, thus giving time between binding and hydrolysis of ATP during which one-half of the activated complex re-laxes back to the form in which is unavailable for interaction with .

Activation of the complex by ATP may explain how the complex achieves its catalytic capability in assembling multiple clamps onto DNA. In the ATP-activated state, is exposed for interaction with , and upon relaxing from the activated state, the subunit is resequestered into the complex, thus severing the - interaction. Hence, after loading the clamp onto DNA, the relaxation of the complex would sever the - contact, resulting in a loss of affinity of the complex for the ring on DNA. This loss of affinity of the complex for would promote complex dissociation from DNA, thus freeing it to load other clamps onto DNA.

Dissociation of the complex from after assembling it onto DNA is also essential for yet another function. The core polymerase must interact with the ring on DNA to achieve high processivity. We observe that point mutants in the C terminus of bind neither (or the complex) nor core.() Hence, core and the complex have overlapping binding sites on , and they both interact with the C terminus of . Both core and the complex recognize a primed template single-stranded/double-stranded DNA junction for their action. Hence, it seems likely that the ring is positioned on the duplex portion of a primed template such that its C-terminal face points toward the 3` terminus, where the complex acts to assemble it. Loss of the activated state of the complex, such that its tie to is broken, would clear from the C termini of for the next interaction, namely that of tethering core to DNA for highly processive synthesis.

  
Table: Apparent kinetic constants for binding of to and of to the complex

Observed on and off rates were determined by surface plasmon resonance as described under ``Experimental Procedures.''



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.

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

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

**
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: SPR, surface plasmon resonance; AMP-PNP, adenyl-5`-yl imidodiphosphate; ATPS, adenosine 5`-O-(thiotriphosphate).

The inhibition is not due to an inhibitor of the kinase in the preparation since the same kinase recognition sequence has been engineered onto the C terminus of the EBNA1 protein, and does not inhibit its phosphorylation, nor is phosphorylation of the EBNA1 derivative inhibited in the presence of and the derivative.

Another possibility, besides the dimer ring opening at one or both interfaces, is that the complex acts by cutting DNA and threading it through the ring. This possibility would be similar to the action of topoisomerases in cutting DNA followed by strand passage through the cut.

Substitution of alanine for the five C-terminal residues of inactivates in replication assays and prevents interaction of with and core (V. Naktinis and M. O'Donnell, manuscript in preparation).


ACKNOWLEDGEMENTS

We are grateful to Dr. Susan S. Taylor for the catalytic subunit of cAMP-dependent protein kinase.


REFERENCES
  1. Kelman, Z., and O'Donnell, M.(1994) Curr. Opin. Genet. Dev. 4, 185-195 [Medline] [Order article via Infotrieve]
  2. Kuriyan, J., and O'Donnell, M.(1993) J. Mol. Biol. 234, 915-925 [CrossRef][Medline] [Order article via Infotrieve]
  3. Lima, C. D., Wang, J. C., and Mondragon, A.(1994) Nature 367, 138-146 [CrossRef][Medline] [Order article via Infotrieve]
  4. Stasiak, A., Tsaneva, I. R., West, S., Benson, C. J. B., Yu, X., and Egelman, E. H.(1994) Proc. Natl. Acad. Sci. U. S. A. 91, 7618-7622 [Abstract]
  5. O'Donnell, M., and Studwell, P. S.(1990) J. Biol. Chem. 265, 1179-1187 [Abstract/Free Full Text]
  6. Onrust, R., Stukenberg, P. T., and O'Donnell, M.(1991) J. Biol. Chem. 266, 21681-21686 [Abstract/Free Full Text]
  7. Young, M. C., Reddy, M. K., and von Hippel, P. H.(1992) Biochemistry 31, 8675-8690 [Medline] [Order article via Infotrieve]
  8. Jarvis, T. C., Paul, L. S., and von Hippel, P. H.(1989) J. Biol. Chem. 264, 12709-12716 [Abstract/Free Full Text]
  9. Tsurimoto, T., and Stillman, B.(1989) Mol. Cell. Biol. 9, 609-619 [Medline] [Order article via Infotrieve]
  10. Lee, S.-H., Kwong, A. D., Pan, Z.-Q., and Hurwitz, J.(1991) J. Biol. Chem. 266, 594-602 [Abstract/Free Full Text]
  11. O'Donnell, M., Onrust, R., Dean, F. B., Chen, M., and Hurwitz, J. (1993) Nucleic Acids Res. 21, 1-3 [Medline] [Order article via Infotrieve]
  12. Onrust, R., Finkelstein, J., Naktinis, V., Turner, J., Fang, L., and O'Donnell, M. J.(1995) Biol. Chem. 270, 13348-13357 [Abstract/Free Full Text]
  13. Slice, L. W., and Taylor, S. S.(1989) J. Biol. Chem. 264, 20940-20946 [Abstract/Free Full Text]
  14. Xiao, H., Crombie, R., Dong, Z., Onrust, R., and O'Donnell, M.(1993) J. Biol. Chem. 268, 11773-11778 [Abstract/Free Full Text]
  15. Xiao, H., Dong, Z., and O'Donnell, M.(1993) J. Biol. Chem. 268, 11779-11784 [Abstract/Free Full Text]
  16. Stukenberg, P. T., Studwell-Vaughan, P. S., and O'Donnell, M.(1991) J. Biol. Chem. 266, 11328-11334 [Abstract/Free Full Text]
  17. Stukenberg, P. T., Turner, J., and O'Donnell, M.(1994) Cell 78, 877-887 [Medline] [Order article via Infotrieve]
  18. O'Shannessy, D. J., Brigham-Burke, M., Soneson, K. K., Hensley, P., and Brooks, I.(1993) Anal. Biochem. 212, 457-468 [CrossRef][Medline] [Order article via Infotrieve]
  19. Flores-Rozas, H., Kelman, Z., Dean, F. B., Pan, Z.-Q., Harper, J. W., Elledge, S. J., O'Donnell, M., and Hurwitz, J.(1994) Proc. Natl. Acad. Sci. U. S. A. 91, 8655-8659 [Abstract]
  20. Hockensmith, J. W., Kubasek, W. L., Evertsz, E. M., Mesner, L. D., and von Hippel, P. H.(1993) J. Biol. Chem. 268, 15721-15730 [Abstract/Free Full Text]
  21. Crute, J. J., LaDuca, R. J., Johanson, K. O., McHenry, C. S., and Bambara, R. A.(1983) J. Biol. Chem. 258, 11344-11349 [Abstract/Free Full Text]
  22. Kwon-Shin, O., Bodner, J. B., McHenry, C. S., and Bambara, R. A. (1987) J. Biol. Chem. 262, 2121-2130 [Abstract/Free Full Text]
  23. Johanson, K. O., and McHenry, C. S.(1984) J. Biol. Chem. 259, 4589-4595 [Abstract/Free Full Text]
  24. Kelman, Z., Naktinis, V., and O'Donnell, M.(1995) Methods Enzymol. 262, in press

©1995 by The American Society for Biochemistry and Molecular Biology, Inc.