Mechanism of beta  Clamp Opening by the delta  Subunit of Escherichia coli DNA Polymerase III Holoenzyme*

Jelena StewartDagger , Manju M. HingoraniDagger §, Zvi Kelman||, and Mike O'DonnellDagger **DaggerDagger

From Dagger  The Rockefeller University and ** Howard Hughes Medical Institute, Laboratory of DNA Replication, New York, New York 10021 and  Department of Microbiology, Cornell University Medical College, New York, New York 10021

Received for publication, January 22, 2001


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

The beta  sliding clamp encircles the primer-template and tethers DNA polymerase III holoenzyme to DNA for processive replication of the Escherichia coli genome. The clamp is formed via hydrophobic and ionic interactions between two semicircular beta  monomers. This report demonstrates that the beta  dimer is a stable closed ring and is not monomerized when the gamma  complex clamp loader (gamma 3delta 1delta 1chi 1psi 1) assembles the beta  ring around DNA. delta  is the subunit of the gamma  complex that binds beta  and opens the ring; it also does not appear to monomerize beta . Point mutations were introduced at the beta  dimer interface to test its structural integrity and gain insight into its interaction with delta . Mutation of two residues at the dimer interface of beta , I272A/L273A, yields a stable beta  monomer. We find that delta  binds the beta  monomer mutant at least 50-fold tighter than the beta  dimer. These findings suggest that when delta  interacts with the beta  clamp, it binds one beta  subunit with high affinity and utilizes some of that binding energy to perform work on the dimeric clamp, probably cracking one dimer interface open.


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

The DNA polymerase III (Pol III)1 holoenzyme is primarily responsible for replicating the 4.4-megabase Escherichia coli genome (1, 2). Pol III holoenzyme performs this task with high speed and accuracy with the help of ten component subunits. These are alpha  (the DNA polymerase (3)), epsilon  (the proofreading 3'-5' exonuclease (4)), and theta  (unknown function) that form the DNA polymerase III core (5); beta  (the sliding clamp (6, 7)); and the multisubunit DnaX complex (gamma tau delta delta 'chi psi ) that functions as the clamp loader (8-10) and contains at least two subunits of the tau  "organizer" that binds two core polymerases (11-13) and connects to the DnaB helicase at the replication fork (14, 15).

Rapid and processive DNA synthesis by Pol III holoenzyme is dependent on the interaction between the alpha  subunit of the core polymerase and the beta  clamp (6). beta  is a ring-shaped protein that encircles double-stranded DNA and can slide freely along its length (6, 7). By itself, core polymerase can extend a primer by only a few nucleotides before dissociating from DNA (16). When beta  is bound to the polymerase and topologicaly linked to the primer-template, it serves as a mobile tether to keep the enzyme associated with DNA, facilitating replication of several thousand nucleotides at a time. Similar mechanisms for processive DNA synthesis by replicative polymerases have been discovered in a variety of other organisms (reviewed in Refs. 2, 17, 18, and 19), including eukaryotic DNA polymerase delta  (tethered to DNA by the PCNA sliding clamp (20, 21)) and bacteriophage T4 DNA polymerase, gp43 (tethered by the gp45 sliding clamp (22)).

The crystal structure of beta  shows it to be a ring-shaped dimer formed by the head-to-tail interaction of two semicircle-shaped monomers (7). A continuous beta -sheet forms a scaffold around the outer surface of the ring that supports 12 alpha -helices lining the inside of the ring. The central cavity is about 35 Å in diameter, which is large enough to encircle double-stranded DNA as well as one or two layers of water molecules. Moreover, although the inside of the beta  ring is positively charged, it lacks specific contact with DNA, allowing beta  to form a stable topological link with the DNA and yet slide freely along the duplex. At the two identical dimer interfaces, a continuous beta -sheet formed by hydrogen bonding between beta  strands from each monomer stabilizes the ring structure in addition to a small hydrophobic core formed by packing of Ile272 and Leu273 of one monomer with Phe106 and Leu108 on the other monomer. Charged amino acids at the interface are also in position to form six ion pairs (these interactions are detailed in Fig. 4). These numerous and potentially strong interactions between the two beta  subunits presumably underlie the highly stable dimeric structure of beta  and its ability to remain bound to DNA with a half-life of over 100 min (23, 24). Yet the closed circular clamp must be opened frequently during DNA replication for assembly on DNA to initiate processive replication as well as for disassembly of the beta  ring from DNA when replication is complete.

The gamma  complex clamp loader (gamma delta delta 'chi psi ) assembles beta  clamps on primer-template DNA (where they can be used by the polymerase) and can also remove clamps from DNA when necessary (23-26). The process of clamp assembly requires that the gamma  complex open the beta  clamp, guide DNA into the central cavity, and facilitate closure of the clamp around DNA. Crystal structure analysis,2 and a recent biochemical study (27) reveals that the gamma  complex contains three copies of gamma ; the other subunits (delta , delta ', chi , psi ) are each present in a single copy (10, 13). The delta  subunit of gamma  complex binds to beta  and destabilizes or opens the dimer interface (28, 29). The gamma  subunits are the only ones that hydrolyze ATP (30-32). The delta ' subunit is homologous to gamma  and appears to play a role in modulating the access of delta  to beta  (10, 33, 34). In the absence of ATP, the affinity between the gamma  complex and beta  is low compared with the affinity between the delta  subunit and beta  (28). Clamp assembly initiates when ATP binds the gamma  subunits and induces a change in conformation of the gamma  complex that results in ability of delta  to bind beta  (28, 29, 32). The delta ' subunit appears at least partially responsible for modulating the access of delta  to beta , since a previous study indicated that delta ' and beta  compete for interaction with delta  (29). The ATP-induced conformational change of gamma  complex may entail removing a surface of delta ' from delta , allowing delta  to bind and open the beta  clamp. In the presence of a nonhydrolyzable ATP analogue, the clamp loader-beta complex binds primer-template DNA with high affinity (32, 36). Interaction of gamma  complex with DNA, especially primed template, triggers ATP hydrolysis and is stimulated by the presence of beta  (29, 32, 36, 37). ATP hydrolysis is coupled to closure of the clamp around DNA and gamma  complex turnover. The chi  subunit of gamma  complex binds to SSB and helps coordinate the switch between the primase, clamp loader, and polymerase proteins at the primer template (38, 39), and psi  enhances the stability of the gamma  complex; however, these two proteins are not absolutely essential for clamp assembly (40-43).

Although all three subunits, gamma , delta , and delta ', are required for loading beta  onto DNA, the single delta  subunit appears to be the predominant contact between beta  and the gamma  complex (28). It remains possible that weaker interactions between beta  and the other gamma  complex subunits exist.3 However, our previous studies demonstrated that delta  alone can open and remove beta  clamps from circular DNA molecules with nearly the same efficiency as gamma  complex (kunloading gamma -complex = 0.015 s-1; kdelta unloading = 0.011 s-1) (24). We were therefore curious as to how the delta  subunit generates the leverage required to part the apparently tightly closed beta  dimer interfaces. Previous studies indicate that beta  opening at just one interface is sufficient to allow passage of DNA into (or out of) the central cavity (29). Experiments herein measure the exchange of labeled beta  subunits as they are utilized by the gamma  complex, and the results support the conclusion that the dimeric clamp is not split apart into monomers but rather stays intact during clamp assembly, presumably opening at only one interface for entry of DNA. In the simplest possible mechanism, the clamp loader could prompt clamp opening merely by perturbing one of the dimer interfaces and transiently reducing its stability.

Study of the delta -beta interaction in this report provides insight into how the delta  and gamma  complex might open the beta  ring. We demonstrate here that the beta  ring retains its dimeric structure when bound by one delta  subunit. Furthermore, we have mutated two hydrophobic residues in the beta  dimer interface to produce a stable monomeric version of beta . Only one delta  subunit binds the beta  monomer, which is surprising, given the one delta /two beta  stoichiometry of the wild type delta -beta complex. This suggests that the binding site of delta  on the beta  ring is located primarily on one of the two beta  subunits. The affinity of delta  for the beta  monomer mutant is about 50-fold greater than for the beta  dimer, implying that the binding energy of delta  to a single beta  subunit of the dimer is harnessed to perform work, namely to force open one of the dimer interfaces. The delta  subunit binds beta  at the carboxyl terminus, which lies in the vicinity of the dimer interface (44). Therefore, it is conceivable that delta  binding to one beta  protomer disrupts the contacts in a nearby dimer interface that hold the ring closed.

    EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Nucleotides, DNAs, and Buffers-- Radioactive nucleotides were purchased from PerkinElmer Life Sciences. Unlabeled nucleotides were purchased from Amersham Pharmacia Biotech. M13mp18 ssDNA was prepared by phenol extraction of purified M13mp18 phage that had been banded twice in CsCl gradients (45) and primed with a 30-nucleotide primer (Life Technologies, Inc.) as described (46). Buffer A contained 20 mM Tris-HCl (pH 7.5), 0.5 mM EDTA (pH 8.0), 100 mM NaCl, and 10% glycerol. DNA replication buffer contained 20 mM Tris-HCl (pH 7.5), 0.1 mM EDTA, 40 µg/ml bovine serum albumin, 5 mM dithiothreitol, 8 mM MgCl2, 4% glycerol, 0.5 mM ATP, 60 µM dGTP, and 60 µM dCTP. Surface plasmon resonance (SPR) buffer contained 10 mM Hepes-NaOH (pH 7.4), 150 mM NaCl, 3.4 mM EDTA, and 0.005% Tween 20.

Proteins-- Proteins were purified as described: alpha , epsilon , gamma  (46), delta , delta ', chi , psi  (33), theta  (47), and SSB (46). gamma  complex and Pol III* (a subcomplex of Pol III holoenzyme lacking the beta  subunit) were reconstituted from individual subunits and purified as described in Refs. 9 and 13, respectively. Mutant beta  proteins were constructed using DNA oligonucleotide site-directed mutagenesis. Various N-terminal tagged versions of beta  (described below) were purified according to the previously described protocol for wild type beta  (7). Radiolabeling of tagged beta  with 32P was performed using [alpha -32P]ATP and cAMP-dependent protein kinase to a specific activity of ~100 cpm/fmol as described (48). The catalytic subunit of cAMP-dependent protein kinase produced in E. coli was a gift from Dr. Susan Taylor (University of California, San Diego). 3H-beta was labeled by reductive methylation as described (48).

Gel Filtration Analysis of beta , delta , L273A-beta , and I272A/L273A-beta -- The beta , L273A-beta , and I272A/L273A-beta proteins (3 µM as dimer) were sized by gel filtration (at 4 °C) on an FPLC HR 10/30 Superose 12 column (Amersham Pharmacia Biotech) equilibrated with Buffer A. The proteins were incubated in a final volume of 200 µl of Buffer A for 15 min at 15 °C and then applied to the column. After collecting 6-ml, 170-µl fractions were collected, and 25-µl aliquots of the indicated fractions were analyzed by SDS-polyacrylamide gel electrophoresis (15% gels); proteins were visualized by Coomassie Blue staining. For size standards, alpha  (130 kDa), bovine serum albumin (66 kDa), and delta  (39 kDa) were analyzed similarly.

Interaction between delta  and beta  was analyzed by incubating 9 µM delta  with 12.5 µM wild type beta  (as dimer) or 25 µM I272A/L273A-beta , (as monomer) for 15 min at 15 °C in a final volume of 200 µl of Buffer A, followed by gel filtration chromatography and SDS-polyacrylamide gel electrophoresis analysis as described above.

DNA Replication Assays-- Singly primed M13mp18 ssDNA (20 fmol), 0.8 µg of SSB, 75 fmol of Pol III*, and 750 fmol of beta  (wild type and mutant concentrations are calculated as monomer) were incubated at 37 °C for 2 min in 25 µl (final volume) of DNA replication buffer (this buffer contains ATP, dCTP, and dGTP). DNA synthesis was initiated upon the addition of the remaining two deoxyribonucleoside triphosphates (60 µM dATP, 20 µM dTTP (final concentrations), and 1 µCi of [alpha -32P]dTTP). After 20 s, reactions were quenched with 25 µl of 40 mM EDTA and 1% SDS. Aliquots (20 µl) of the quenched reactions were analyzed by electrophoresis on a 1% TBE-agarose gel, and the radiolabeled DNA was visualized on a PhosphorImager (Molecular Dynamics, Inc., Sunnyvale, CA). Synthesis was quantitated by spotting 20-µl aliquots of the reaction on DE81 filters, followed by liquid scintillation counting as described (49).

SPR Analysis of beta -delta Interaction-- The delta  subunit (10 µl of 0.6 µM) was immobilized on a carboxymethylated dextran matrix-coated sensor chip (CM5; Biacore) by carbodiimide coupling in 10 mM sodium acetate (pH 5.5). SPR analysis was performed at 23 °C by injecting 15 µl of beta  or I272A/L273A-beta (0.25 and 1.23 µM; concentrations for both are given as monomer) in SPR buffer, at a flow rate of 5 µl/s. After each analysis was complete, the chip surface was regenerated by injecting 10 µl of 0.1 M glycine (pH 9.5) over the chip, which releases bound beta  with no significant effect on the binding capacity of the immobilized delta  protein.

The kinetic constants for interaction between delta  and beta  were determined by nonlinear curve fitting, using the BIAevaluation 2.1 software. The rate of dissociation (koff) was calculated by fitting the curves to a single exponential decay described by Equation 1,
R=R<SUB>0</SUB>e<SUP><UP>−</UP>k<SUB><UP>off</UP></SUB>(t−t<SUB>0</SUB>)</SUP> (Eq. 1)
where R0 represents the response and t0 represents the time at the start of the dissociation phase. The association rate (kon) was calculated using the binding model A + B= AB and Equation 2,
R=R<SUB><UP>eq</UP></SUB>(1−e<SUP><UP>−</UP>k<SUB><UP>on</UP></SUB>C+k<SUB><UP>off</UP></SUB></SUP>)(t−t<SUB>0</SUB>) (Eq. 2)
where Req is the response at steady state, C is the concentration of beta , and t0 is the time at the start of the association phase. The dissociation constant (Kd) for interaction between beta  and delta  was calculated as koff/kon.

Protomer Exchange Assay-- The two beta  mutants for this assay were constructed by placing the beta  gene into either the pHKEp vector or the pHKEpmut vector (50). Both of these vectors place a 34-amino acid tag onto the N terminus of the protein. The tags contain a protein kinase site (to label the protein with 32P) and either a functional (pHKEp) or a nonfunctional (pHKEpmut) hemagglutinin (HA) epitope. The nonfunctional epitope was formed by replacing two amino acids; YPYDVPDYA was changed to YPYDVPAAA. After expression and purification, one beta  contains a functional HA epitope (habeta 2) and the other beta  contains a nonfunctional HA epitope, which we use in this report in the phosphorylated form and refer to as 32P-beta 2. The beta  with the mutated HA-epitope was labeled with 32P (32P-beta ) as described (48). Titrations of these beta  variants showed that they were as active as wild type beta  in replication assays with Pol III* on SSB-coated M13mp18 ssDNA primed with a single oligonucleotide. Monoclonal antibody to the HA epitope was purchased from BabCo, and Protein A-Sepharose 4B was from Zymed Laboratories Inc. The HA antibody was conjugated to Protein A beads by incubation for 15 min at 25 °C in 400 µl of 20 mM Hepes (pH 7.4), 150 mM NaCl, 0.1% Triton X-100, 10% glycerol.

Spontaneous protomer exchange was measured (i.e. no other proteins besides beta ) in 50-µl reactions containing 2 pmol of 32P-beta 2 and 2 pmol of habeta 2 in 20 mM Hepes (pH 7.5), 150 mM NaCl, and 10% glycerol. Reactions were incubated at 37 °C for 0, 1, 2, 4, 6, or 8 h before the addition of 50 µl of HA antibody-conjugated beads and placed at 4 °C for a further 30 min. Beads were pelleted, washed three times with 1 ml of 50 mM Tris-HCl (pH 8.0), 500 mM NaCl, 5 mM EDTA, 0.1% SDS, and 0.1% Triton X-100; resuspended in Eco-Lume (ICN); and counted in a scintillation counter. Control experiments were performed similarly except that either no antibody was conjugated to the beads or the habeta 2 was not added to the reaction.

To measure the effect of gamma  complex on beta  protomer exchange during clamp assembly onto DNA, 250 fmol each of habeta 2 and 32P-beta 2 were incubated for 5 min at 37 °C with 500 fmol of gamma  complex and 1.8 pmol of nicked pBS DNA in 70 µl of 20 nM Tris-HCl (pH. 7.5), 0.1 mM EDTA, 4% glycerol, and 8 mM MgCl2. The reaction was then applied to a 5-ml A15 M gel filtration column equilibrated with the same buffer plus 0.15 M NaCl. Fractions of six drops each were collected, and those containing beta  on DNA were identified by scintillation counting and pooled (420 µl), and then the DNA was linearized upon treatment with 700 units of BamHI for 3 min at 37 °C to release beta . To confirm that linearization was complete within this time, an aliquot (20 µl) was removed, quenched with 20 µl of 1% SDS, 40 mM EDTA, and then analyzed in a native agarose gel. Then 50 µl of HA antibody beads were added to the reaction, and incubation was continued for a further 30 min at 4 °C. The beads were pelleted; washed three times with 1 ml of 50 mM Tris-HCl (pH 8.0), 500 mM NaCl, 5 mM EDTA, 0.1% SDS, and 0.1% Triton X-100; resuspended in Eco-Lume (ICN); and counted in a scintillation counter. In a control experiment, the above procedure was repeated except that heterodimeric beta  was used in the assembly reaction by first preincubating 250 fmol of each beta  in one tube for 5 h at 37 °C before adding to the reaction containing gamma  complex and DNA.

Nickel Column Affinity Assay for delta -beta 2 Complex-- Reactions contained 67.5 pmol of 3H-beta 2 (wild type beta  labeled by reductive methylation), 1.7 nmol of hisbeta 2, which contained a six-residue histidine tag on a 23-residue N-terminal leader (beta  was cloned into the pHK vector in Ref. 50), and 6.6 nmol of delta  (where present) in 200 µl of 20 mM Tris-HCl (pH 7.5), 10% glycerol, 8 mM MgCl2, and 100 mM NaCl. A control reaction utilized 1.7 nmol of unlabeled wild type beta 2 in placed of the hisbeta 2 derivative. Reactions were assembled on ice and then shifted to 37 °C, and aliquots of 20 µl were removed at 2 and 24 h of incubation. Upon removal of an aliquot, NaCl was added to a final concentration of 0.5 M, and the reaction was applied to a 1-ml nickel chelate column (HiTrap; Amersham Pharmacia Biotech) equilibrated in 20 mM Tris-HCl (pH 7.9), 5 mM imidazole, 8 mM MgCl2, and 10% glycerol. The column was washed with 5 ml of the same buffer and then eluted with 3 ml of 20 mM Tris-HCl (pH 7.9), 1 M imidazole, 8 mM MgCl2, and 10% glycerol. Fractions of 1 ml were collected. The flow-through (wash) and bound (elution) fractions were analyzed by liquid scintillation counting and analyzed in a 10% SDS-polyacrylamide gel to confirm the presence of delta  with beta  in the bound fractions. The typical yield of 3H-beta 2 off the column was greater than 85%.

    RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
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beta Is Not Monomerized during Assembly onto DNA-- We have shown previously that the beta  clamp is a tight dimer and remains a dimer even when diluted to a concentration of 50 nM (23). Nonetheless, it is possible that gamma  complex dissociates the beta  dimer into monomers using the energy of ATP hydrolysis and then reassembles the beta  dimer onto DNA in a second step. To test this possibility, we constructed two chemically distinct beta  mutants; one was phosphorylated and contained a protein kinase tag (32P-beta pk), and the other had a hemagglutinin epitope tag (habeta ). If the gamma  complex monomerizes beta  dimers and reassembles them onto DNA, then it should act upon a mixture of 32P-beta 2 and habeta 2 to form 32P-beta -habeta heterodimers on DNA.

As a prerequisite for an experiment of this type, it is important that the 32P-beta 2 and habeta 2 mixture does not undergo spontaneous protomer exchange to form heterodimers during the time of the experiment. The time course for spontaneous heterodimer formation was measured in the experiment of Fig. 1 by mixing equal amounts of 32P-beta 2 and habeta 2, followed by removal of aliquots at time intervals and immunoprecipitation of the mixture using Protein A beads to which an antibody to hemagglutinin is attached. Initially, 32P-beta will not be precipitated, since it lacks the epitope. But as protomer exchange occurs, the 32P-beta -habeta heterodimer will be formed, which should result in the appearance of radioactivity in the pellet. The result, shown in Fig. 1, demonstrates that the time scale of spontaneous subunit exchange is on the order of hours (t1/2 ~ 2 h). As the clamp-loading reaction only requires 5 min, spontaneous protomer exchange during the reaction should be nearly negligible. Control reactions not shown here have been performed that demonstrate requirements for both the antibody and the presence of the habeta to detect radioactivity attached to the beads in the pellet. In both controls, the pellets lacked radioactivity above background levels (0.5 fmol of 32P-beta 2).


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Fig. 1.   Time course of beta  protomer exchange. Two chemically distinct species of beta , one containing a hemagglutinin epitope and one labeled with 32P, were mixed together to initiate formation of heterodimers as indicated. After various times of incubation, aliquots were withdrawn, and beads to which antibody to the hemagglutinin tag were attached were added. Heterodimeric beta  consists of a 32P-beta protomer attached to a habeta protomer that should be trapped by the hemagglutinin beads. Radioactivity in the pellet, representing heterodimeric beta , is plotted with respect to time. I.P., immunoprecipitation.

Next, we examined how the gamma  complex loads a mixture of these two beta  variants onto DNA to determine whether it catalyzes protomer exchange during the clamp assembly process (i.e. whether gamma  complex breaks beta  dimers apart and reassembles them onto DNA as illustrated in the scheme of Fig. 2). To test this possible action, 32P-beta 2 and habeta 2 were mixed together, and gamma  complex was added immediately along with ATP and circular plasmid DNA containing a single nick to initiate clamp assembly. After 5 min, the reaction was applied to a gel filtration column to separate clamps that had been assembled on DNA from those remaining in solution. Following this, the isolated beta -DNA complex was treated with BamHI to rapidly linearize the DNA, allowing the clamps to slide off DNA into solution. Then the reaction was analyzed for heterodimer formation by immunoprecipitation using the hemagglutinin antibody beads (see the scheme in Fig. 2).


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Fig. 2.   gamma complex does not monomerize beta  during clamp assembly onto DNA. The gamma  complex and ATP were used to assemble a mixture of 32P-beta 2 and habeta 2 onto nicked circular plasmid DNA. As indicted, the beta -DNA complex was isolated from free beta  and then linearized to release beta  into solution. Hemagglutinin antibody bound to beads was used to quantitate heterodimers that were assembled onto DNA by the gamma  complex. Lane 1, 32P-beta 2 and habeta 2 were mixed immediately prior to their transfer to DNA by gamma  complex; lane 2, 32P-beta 2 and habeta 2 were premixed and incubated 5 h to form heterodimers before being transferred to DNA by gamma  complex.

The results of this experiment (Fig. 2, lane 1) demonstrate that very little heterodimeric beta  is formed in the reaction, indicating that gamma  complex does not catalyze beta  monomerization during clamp loading. In control reactions not shown here, we confirmed that gamma  complex loads approximately equal amounts of 32P-beta 2 and 32P-habeta 2 on DNA, and both variants of beta  were as active as wild type beta  in replication assays with PolIII*. In another control experiment 32P-beta 2 and habeta 2 were premixed for 6 h to form the 32P-beta -habeta heterodimer prior to use by gamma  complex in assembly onto DNA. The result, shown in Fig. 2, lane 2, demonstrates that the experimental strategy is functional in detecting heterodimers that are assembled on DNA. Thus, it would appear that gamma  complex does not monomerize beta  but probably only opens one interface of the ring during the clamp opening process. This conclusion is consistent with a previous finding that showed that gamma  complex was capable of assembling a beta  dimer onto DNA that was cross-linked at one interface by a disulfide bond (i.e. indicating that gamma  complex does not need to open both interfaces to assemble beta  onto DNA (29)).

In the study of Fig. 3 we designed another experiment to examine the oligomeric state of beta  during clamp assembly, this time while it is in complex with delta , the clamp-opening subunit of the gamma  complex. Previous studies indicated that one delta  monomer binds to the beta  dimer, consistent with the single copy of delta  in gamma  complex (28). The delta  subunit is capable of removing beta  rings from circular DNA (24, 29) and thus must either destabilize one interface or perhaps transiently dissociate beta  into monomers. In either case, one may expect delta  to accelerate the rate of protomer exchange. We examined these possibilities in a variation of the protomer exchange assay. The assay utilized a hexahistidine-tagged beta 2 (hisbeta 2) and tritiated wild type beta 2 (3H-beta 2). The 3H-beta 2 was mixed with a 25-fold molar excess of hisbeta 2 in the presence or absence of a 4-fold molar excess of delta  (over total beta ), and then the mixture was analyzed at either 2 or 24 h for heterodimer formation by nickel chelate chromatography. Homodimeric 3H-beta 2 should not bind to the column (flow-through fraction), and heterodimeric 3H-beta -hisbeta should be retained (bound fraction) and detected by elution from the nickel chelate column, followed by scintillation counting.


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Fig. 3.   delta does not monomerize beta 2. A mixture of 3H-beta 2 and a histidine-tagged beta  (hisbeta ) were mixed in the presence or absence of a 4-fold excess of delta  subunit (over total beta ). At either 2 h (lanes 2 and 3) or 24 h (lanes 5 and 6), aliquots were removed and loaded onto nickel-chelate columns. After washing the columns, bound protein was eluted with buffer containing 1 M imidazole. Since 3H-beta lacks a His tag, 3H-beta in the bound fraction represents 3H-beta -hisbeta heterodimers. Controls in which wild type beta 2 was substituted for hisbeta are shown in lanes 1 and 4.

The results of this experiment, shown in Fig. 3, illustrate that similar amounts of heterodimer are formed within 2 h in the presence or absence of delta , indicating that delta  does not appreciably speed up protomer interchange. Also, the fact that 3H-beta is retained on the column in the presence of delta  supports the delta 1-beta 2 stoichiometry, since if delta  monomerized beta 2, heterodimer would not be present for retention on the column. As a control, wild type beta 2 was substituted for hisbeta 2, which should form a 3H-beta -beta wt heterodimer, but should not bind the nickel chelate column. The result of this control showed that 3H-beta was not retained on the column, as expected (not shown).

How Does delta  Open One Interface of the beta  Ring?-- The experiments described above demonstrate that the gamma  complex does not catalyze the exchange of beta  protomers during the clamp loading operation. The results also demonstrate that delta  does not monomerize the beta  dimer. These results support and extend earlier studies that indicate that only one interface of the beta  dimer ring is cracked open during assembly onto DNA. The delta  subunit is the clamp-opening subunit of gamma  complex. How does delta  open an interface of the beta  ring? To gain insight into how delta  performs its ring opening task, we mutated beta  to form a stable beta  monomer. Initially, we set out to determine whether delta  mainly binds only one protomer of the beta  dimer, in which case delta  should still bind a beta  monomer about as well as a beta  dimer. Alternatively, delta  may need to associate with elements on both protomers of beta 2 in order to establish a firm grip on the beta  ring. The results of this line of investigation were unexpected and provided significant insight into the clamp opening function of delta .

To form a stable monomer of beta , we utilized the crystal structure to design site-specific mutations that would destabilize the dimer interface. The crystal structure of the dimeric beta  clamp revealed a small interface between the two beta  subunits that, despite its size, has an abundance of potentially strong interactions (see the diagrams in Fig. 4, A and B) (7). These interactions facilitate the formation of a highly stable circular clamp that maintains its dimeric structure even at low nanomolar concentrations. In particular, a small hydrophobic core of four amino acid residues (Phe106, Leu108, Ile272, and Leu273) at the dimer interface appears to play an important role in the stability of the clamp structure. Initially, we constructed three single residue mutants in which Ala was substituted in place of either Phe106, Leu108, or Leu273 (we could not obtain the I272A mutant). Each of these point mutants migrated as a dimer in gel filtration analysis and retained 70-100% activity with PolIII* (not shown). However, a double mutant, I272A/I273A, behaved as a monomer and lacked replication activity (explained below). The experiments to follow focus on the double mutant and compare it with wild type beta .


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Fig. 4.   Mutation of the beta  interface to form a stable beta  monomer. A, the crystal structure of the beta  dimer shows that it is a ring-shaped structure, with a 35-Å central hole, wide enough to encircle double-stranded DNA (7). The beta  dimer interface, in particular the amino acid residues forming the hydrophobic core, is highlighted on the right. B, the dimer interface consists of two beta  sheets, beta 8 and beta '4 (one from each subunit), that form an antiparallel sheet across the dimer interface and neighboring amino acid contacts that stabilize the clamp structure. The lines connect amino acid residues predicted to form six ion pairs across the interface. The hydrophobic core residues, Phe106, Leu108, Ile272, and Leu273, are indicated, and gray circles highlight Ile272 and Leu273, which were mutated to Ala in this study. C, analysis of wild type beta  and mutants of beta  on a sizing column followed by SDS-polyacrylamide gel electrophoresis. The results show that wild type beta  (top panel) and L273A-beta (middle panel) elute as dimers (81.2 kDa). In contrast, the I272A/L273A-beta (bottom panel) elutes as a smaller, monomeric protein (40.6 kDa), indicating that the double mutation severely disrupts the beta  dimer interface. D, quantitation of DNA synthesis by Pol III* on SSB-coated singly primed M13mp18 ssDNA in the presence of either no beta , I272A/L273A-beta , or wild type beta .

In Fig. 4C, wild type beta  and the beta  mutants, L273A-beta and I272A/L273A-beta , were examined by gel filtration to determine their oligomeric state. Fig. 4C shows the SDS-polyacrylamide gel electrophoresis analysis of column fractions from the gel filtration analysis of wild type beta , L273A-beta , and I272A/L273A-beta , in the top, middle, and bottom panel, respectively. Wild type beta  elutes as a dimer in peak fraction 21, as does the L273A-beta mutant (calculated mass = 81.2 kDa). In contrast, the double mutant I272A/L273A-beta migrates more slowly through the column, indicative of a smaller size, and elutes as a monomer (calculated mass = 40.6 kDa). The gel filtration experiments were performed with 3 µM beta  (as dimer). Therefore, even at high protein concentration, I272A/L273A-beta is unable to form a stable dimer.

It has long been presumed that the circular structure of the beta  dimer is required for its action as a DNA polymerase processivity factor. There are, however, single subunit processivity factors that do not appear to encircle DNA, particularly the herpes simplex virus UL42 protein, which in fact is structurally similar to the eukaryotic PCNA clamp but does not oligomerize into a ring (51). To determine if a monomeric form of beta  can serve as a processivity factor, the monomeric I272A/L273A-beta mutant was tested for DNA replication activity with PolIII* using primed M13mp18 ssDNA as substrate. The result, in Fig. 4D, demonstrates that the monomeric beta  mutant is inactive with PolIII*. The dimeric single mutants (L273A, L108A, and F106A) retained 70-100% the activity of wild type beta  (not shown). Thus, a beta  monomer that does not form a circular clamp is not capable of tethering Pol III* to DNA for processive DNA replication.

delta Binds the beta  Monomer with Higher Affinity than the beta  Dimer-- Only one copy of the delta  subunit is present in the gamma  complex, consistent with the stoichiometry of one delta  to two beta  in the delta -beta complex. The stoichiometry of only one delta  subunit per beta  dimer invokes the question of whether delta  interacts with both beta  protomers or can stably attach to one beta  protomer, perhaps somehow preventing a second delta  from binding the other beta  protomer (e.g. by steric occlusion). Interaction of delta  with the beta  monomer was tested in Fig. 5 by mixing delta  with an excess of either wild type beta 2 or the momeric beta 1 mutant, followed by gel filtration analysis on a sizing column. The elution profiles of the proteins were analyzed by SDS-polyacrylamide gel electrophoresis. As expected from previous studies, Fig. 5A shows that delta  and wild type beta 2 form a stable complex with an apparent molecular mass of 111 kDa, consistent with the delta 1beta 2 complex observed in our previous study (38.7-kDa delta  + 2 × 40.6-kDa beta  = 119.9 kDa) (28). Fig. 5B shows that delta  and the I272A/L273A-beta mutant also interact, forming a smaller delta 1beta 1 complex that migrates at an intermediate position between the delta 1beta 2 complex (Figs. 5A) and the free I272A/L273A-beta monomer (Fig. 5E). This result reveals that the binding site for delta  on the beta  clamp resides within one monomer and demonstrates that delta  need not bind both subunits of the dimer to form a stable contact with the clamp.


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Fig. 5.   delta binds the beta  monomer mutant. delta -beta interaction was analyzed by gel filtration on a sizing column, and the proteins were visualized in column fractions by SDS-polyacrylamide gel electrophoresis and Coomassie staining. The complex of the wild type beta  dimer with delta  (A) elutes faster than free delta  (C) and free beta  (D), indicative of its large size (81.2 + 38.7-kDa delta 1beta 2 complex). B, the monomeric I272A/L273A-beta protein also interacts stably with delta , forming a delta 1beta 1 complex that elutes in an intermediate position between the delta 1beta 2 complex (A) and either delta  (C) or free beta  monomer (E). The elution positions of molecular weight standards are shown at the bottom of the gel.

The gel filtration analysis revealed that delta  can bind a single beta  protomer, but the possibility remained that the affinity of delta  for beta  may be affected by disruption of the dimeric structure. In particular, we noticed that during gel filtration delta  trails as free protein from a complex with wild type beta  (fractions 24-31 in Fig. 5A), whereas in Fig. 5B most of the delta  appears in complex with the beta  monomer, suggesting that delta  may bind beta 1 tighter than beta 2. Next, we used the SPR technique to examine more closely the relative affinity between delta  and the beta  dimer versus the I272A/L273A-beta monomer mutant (Fig. 6). The delta  subunit was immobilized on a sensor chip, and a solution of beta  in buffer (at different concentrations) was passed over it. The increase in mass (response units) resulting from interaction between delta  and beta  was measured over time; this is the association phase from which the association rate (kon) can be calculated. Next, buffer lacking beta  was passed over the delta -beta complex on the chip, and the resulting decrease in mass over time provides information from which the dissociation rate (koff) can be calculated. Fig. 6, A and B, shows sensorgrams of the interaction between delta  and two different concentrations of beta  and I272A/L273A-beta , respectively.


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Fig. 6.   delta binds the beta  monomer tighter than the beta  dimer. The sensorgrams of wild type beta  (A), and I272A/L273A-beta (B) were obtained by measuring the increase in response units when a 0.25 or 1.23 µM solution of either beta  (as dimer for wild type beta , as monomer for mutant beta ) was passed over delta  immobilized on a sensor chip. The sensorgrams were analyzed for kinetic and equilibrium parameters of the delta -beta interaction as described under "Experimental Procedures" (summarized in Table I). The Kd values indicate that delta  binds I272A/L273A-beta with ~80-fold higher affinity than the wild type beta  dimer.

Kinetic analysis of the SPR data yielded the association (kon) and dissociation (koff) rate constants, from which the equilibrium dissociation constant for the delta -beta interaction could be calculated (Kd = koff/kon). The parameters, summarized in Table I, reveal that delta  binds the beta  monomer mutant with substantially higher affinity than the wild type beta  protein. The average Kd value for interaction between delta  and beta  is about 0.46 µM (average of values determined at 0.25 and 1.23 µM beta  concentrations), which is ~57-fold higher than the Kd for the interaction between delta  and I272A/L273A-beta (average Kd = 0.0075 µM). The tighter interaction between delta  and the beta  monomer is particularly striking because the beta  monomer has only one potential delta  binding site, in contrast to the beta  dimer.

                              
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Table I
Kinetic and equilibrium constants describing the interaction between beta  and delta


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

The beta  Subunit Remains Dimeric during Clamp Loading-- This study has examined whether gamma  complex monomerizes beta  during the clamp loading operation but could detect no evidence for splitting of beta  dimers during their assembly onto DNA. Consistent with retention of the beta  dimeric state, the delta  subunit does not appear to monomerize beta 2 or to significantly increase the rate of protomer exchange among beta  dimers. Hence, it seems likely that the delta  subunit opens only one interface of the beta  dimer during clamp loading, consistent with the ability of gamma  complex to load a beta  dimer onto DNA that has been cross-linked via a disulfide bond across one of the two interfaces (29).

The delta  subunit can open beta  and remove it from DNA but cannot load beta  onto DNA. The gamma  and delta ' subunits of the gamma  complex, along with delta , may orient DNA inside the open ring. The delta ' subunit, possibly also assisted by gamma , must also sever the delta -to-beta contact, allowing the ring to close around DNA. Release of the gamma  complex and closure of the beta  clamp around the DNA are tied to ATP hydrolysis and are probably coordinated with sensing the appropriate structure of DNA.

The Critical Role of Hydrophobic Interface Contacts in the beta  Clamp Structure-- Two beta  monomers contact each other in a head-to-tail fashion at two small, identical interfaces to form the ring-shaped, dimeric beta  clamp (Ref. 7; see also Fig. 4). A central feature of the beta  clamp structure is the continuous layer of sheet around the entire molecule, including the dimer interfaces. Further, particular hydrophobic amino acid side chains, contributed by each monomer, pack to form a small hydrophobic core within each interface. There are also six potential ion pairs formed at the interface, which may further strengthen the dimer. Earlier studies demonstrated that the beta  clamp retains its dimeric structure even when it is highly dilute (23). It is possible that the beta  clamp may "breathe" by alternate opening and closing of one or the other interface. However, the observed long lifetime of beta  on circular DNA when topologically linked to it (23, 24) indicates that if there is breathing at the interfaces, opening a wide distance (i.e. to slip off DNA) is a rare occurrence.

This study confirms an important role of the hydrophobic residues at the interface in maintaining a stable beta  clamp structure. Although beta  retained its dimeric status upon mutation of only one hydrophobic residue at the interface,4 mutation of two of the amino acids at the hydrophobic core, Ile272 and Leu273, to Ala destabilizes the two interfaces to such an extent that the beta  dimer exists as a stable monomer in solution.

The delta -to-beta Binding Energy Opens the beta  Clamp-- In this study, we show that delta  binds the beta  monomer about 50-fold tighter than the beta  dimer. Moreover, the tight delta -beta monomer complex has a 1:1 stoichiometry, indicating that delta  has a binding site for only one beta  protomer. Thus, in the delta 1-beta 2 complex, delta  probably binds only one of the beta  subunits. The apparent higher affinity of delta  for the beta  monomer mutant compared with wild type beta  also indicates that some of the binding energy of delta  to a beta  protomer is put into performing work on the beta  dimer, thus lowering the observed affinity. Given that upon binding of delta  to beta , the dimer opens, the work of the "lost" binding energy is probably utilized to part one of the beta  dimer interfaces.

The amount of work to open one interface can be calculated to be ~2.4 kcal, assuming a difference of 57-fold in the equilibrium binding constants for delta  binding to either beta 1 or to beta 2 (since beta  does not monomerize, there is no entropy component, and the free energy represents work). It is interesting to note that beta  remains a dimer well below 50 nM (23), and thus the free energy for dissociation to monomers is in excess of 10 kcal. These calculated free energies imply that the amount of work required to open one interface (i.e. ~2.4 kcal) is far less than the free energy to open the second interface (i.e. the full 10 kcal needed for beta  to monomerize). These results imply that the beta  dimer is constructed in such a way as to ensure preservation of a dimeric structure, even after one interface has been pried open.

The gamma  Complex Mechanism-- The high stability of the wild type beta  dimeric clamp explains the need for a clamp loader/unloader protein during DNA replication. The gamma  complex serves this function by binding and opening the beta  clamp when it must be loaded onto primer-template DNA or unloaded from a newly replicated duplex (25, 45). The gamma  complex utilizes energy from ATP binding and hydrolysis to perform its function. However, to our surprise, we found in earlier studies that gamma  complex opens the clamp simply on binding ATP and that energy from ATP hydrolysis is not necessary to crack the beta  dimer interface open (29, 32). The ATP binding energy is not utilized for opening the clamp but rather to expose the delta  subunit in the gamma  complex (28, 29), which then opens one interface of the beta  dimer, prior to ATP hydrolysis. In fact, free delta  protein appears to open the clamp almost as well as the ATP-using gamma  complex, as evidenced by the fairly similar rates at which they catalyze unloading of beta  from DNA (kgamma -complex unloading = 0.015 s-1; kdelta -unloading = 0.0115 s-1) (24). These results are consistent with the above conclusions, that the binding energy of the interaction between delta  and beta  is sufficient to open the clamp.

The scheme in Fig. 7 illustrates our current view of the gamma  complex mechanism. Only the gamma , delta , and delta ' subunits are shown, since previous studies have demonstrated that the chi  and psi  subunits of the gamma  complex are not essential for clamp loader action (40). The stoichiometry of gamma  in the gamma  complex has recently been demonstrated by crystal structure analysis of gamma delta delta ' to be three per complex,2 which is also consistent with the conclusions of a recent biochemical study (27). delta  and delta ' are each present in single copy (10, 13). The delta ' subunit is composed of three domains organized in a C-shape (52). The crystal structure shows that the gamma  subunits are three domain proteins like delta ', consistent with their known homology to delta ' (10, 33, 34). Although the delta  subunit shares no recognizable homology to gamma  and delta ', the crystal structure of delta  has recently been solved, and it has a domain structure similar to that of delta '.5 The affinity of the gamma  complex for beta  is quite reduced in comparison with the affinity of delta  for beta , indicating that delta  is sequestered when it is in the gamma  complex. But in the presence of ATP, the affinity of gamma  complex for beta  is enhanced, suggesting that delta  becomes more available to bind beta . This is illustrated in Fig. 7, going from diagram A to B, as a conformational change that increases the exposure of delta  for beta . The illustration is consistent with previous studies that indicate that ATP induces a conformation change in the gamma  complex (28, 32) and that delta ' competes with beta  for delta  (29). These earlier observations indicate that delta ' binding to delta  may partially occlude delta  in the gamma  complex and that, upon binding ATP, gamma  may relieve this occlusion via a conformational change. Hence, diagram B shows a separation between delta ' and delta , due to an ATP-induced conformation change in gamma .


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Fig. 7.   Scheme of gamma  complex action. The five subunits of gamma  complex needed for clamp loading, gamma 3delta 1delta '1, are shown as related C-shaped proteins. A, The area on delta  that binds beta  is in contact with delta ' to indicate that it is blocked for beta  binding. B, ATP binding to gamma  subunits induces a conformational change pulling delta ' from delta , so that delta  can bind to beta . C, the delta  subunit contacts one protomer of beta , and the binding energy of this interaction is placed into wedging open one interface of the beta  ring. D, gamma  complex/beta locate a proper DNA structure for loading beta , which triggers ATP hydrolysis, leading to dissociation of the gamma  complex and leaving beta  to close around DNA.

Upon interaction of delta  with beta  (Fig. 7, diagram C), the ring opens. This report demonstrates that delta  binds only one protomer of the beta  dimer in performing this ring-opening action. Further, as described above, delta  binds the beta  monomer tighter than the beta  dimer, indicating that the binding energy between delta  and one beta  protomer is used to perform work on the dimer, to open or destabilize one interface. The remaining single interface of an open beta  dimer is stronger when beta  is in the open conformation, thereby preventing decay to monomers.

Events in proceeding from diagram C to D, where the beta  ring is closed around DNA, are relatively unknown. Presumably, primed template is recognized and positioned within the open ring, at which time the ATP is hydrolyzed. Hydrolysis is stimulated by beta  and primed template and is associated with dissociation of gamma  complex from DNA, leaving the beta  ring closed around the duplex (29, 32, 37). At this time, we propose that the energy of ATP hydrolysis is utilized to pull delta  off of beta , allowing beta  to close. Particular roles of gamma  complex subunits in DNA recognition, orientation of DNA inside beta , and ring closure await further study.

Comparison with the Eukaryotic PCNA Clamp and RFC Clamp Loader-- The eukaryotic clamp, the PCNA ring, has essentially the same shape and structure as beta , except that each monomer is composed of only two domains, and therefore PCNA trimerizes to form a six-domain ring (20, 21). PCNA, like beta , is highly stable on DNA, exhibiting a half-life of ~24 min for spontaneous dissociation from circular DNA at 37 °C (23). The eukaryotic clamp loader, RFC, is composed of five different subunits, but each are homologous to gamma /delta ' and thus are probably shaped and arranged like the five subunits of the E. coli gamma 3delta delta ' clamp loader (34, 53). Given these striking similarities, it seems likely that the internal workings of RFC and the mechanism by which it opens PCNA will be quite similar to the E. coli gamma  complex and beta . Thus, one subunit of RFC may contact one protomer of PCNA and through the energy of this protein-protein interaction may force the ring open. Multiple RFC subunits appear to bind PCNA, making it seem different from the gamma  complex. However, we have recently determined that the gamma  and chi  subunits of gamma  complex bind beta , albeit much weaker than delta .3 Perhaps these other beta  and PCNA interactive subunits function in positioning beta  on DNA, aid delta  in ring opening, or function in the ring closure step. These and many other possible functions for additional beta  interactive subunits must await future studies.

    ACKNOWLEDGEMENTS

We are grateful to Drs. David Jeruzalmi and John Kuriyan for information on crystal structures in advance of publication and for the diagram of the beta  interface in Fig. 4A.

    FOOTNOTES

* This work was supported by National Institutes of Health Grant GM38839.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§ Present address: Molecular Biology and Biochemistry Dept., Wesleyan University, Middletown, CT 06459.

|| Present address: Center for Advanced Research in Biotechnology, 9600 Gedelesky Dr., Rockville, MD 20850.

Dagger Dagger To whom correspondence should be addressed: The Rockefeller University and Howard Hughes Medical Inst., Laboratory of DNA Replication, 1230 York Ave., New York, NY 10021.

Published, JBC Papers in Press, March 6, 2001, DOI 10.1074/jbc.M100592200

2 The crystal structure of gamma delta delta ' complex has been solved (D. Jeruzalmi and J. Kuriyan, personal communication). The stoichiometry is gamma 3delta 1delta '1, and the five subunits form a pentameric ring.

3 Weak interaction between gamma  and beta  and between chi  and beta  can be detected by surface plasmon resonance (A. Yuzhakov and M. O'Donnell, unpublished observations).

4 J. Stewart and M. O'Donnell, unpublished data.

5 The crystal structure of delta  in complex with a monomer of beta  has been solved (D. Jeruzalmi and J. Kuriyan, personal communication). delta  has the folding pattern of delta '.

    ABBREVIATIONS

The abbreviations used are: Pol III, DNA polymerase III; PCNA, proliferating cell nuclear antigen; ssDNA, single-stranded DNA; SPR, surface plasmon resonance; HA, hemagglutinin; 3H- and 32P-beta , 3H- and 32P-labeled beta  subunit, respectively; habeta and beta pk, subunit beta  with HA epitope tag and protein kinase tag, respectively; RFC, replication factor C.

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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
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Copyright © 2001 by The American Society for Biochemistry and Molecular Biology, Inc.