Clamp loading, unloading and intrinsic stability of the...

Clamp loading, unloading and intrinsic stability of thePCNA, ¯ and gp45 sliding clamps of human, E. coli andT4 replicases

Nina Yao1, Jennifer Turner1, Zvi Kelman1,a, P. Todd Stukenberg2,b Frank Dean1,David Shechter1, Zhen-Qiang Pan2, Jerard Hurwitz2 and Mike O’Donnell1, *1Microbiology Department and Hearst Research Foundation, *Howard Hughes Medical Institute, Cornell University Medical College,1300 York Avenue, New York, HY 10021, USA, and 2Graduate Program in Molecular Biology, Memorial Sloan-KetteringCancer Centre, 1275 York Avenue, New York, NY 10021, USA

Communicated by: Martin Gellert

AbstractBackground: The high speed and processivity ofreplicative DNA polymerases reside in a processivityfactor which has been shown to be a ring-shapedprotein. This protein (‘sliding clamp’) encirclesDNA and tethers the catalytic unit to the template.Although in eukaryotic, prokaryotic and bacter-iophage-T4 systems, the processivity factors arering-shaped, they assume different oligomericstates. The Escherichia coli clamp (the ¯ subunit) isactive as a dimer while the eukaryotic and T4 phageclamps (PCNA and gp45, respectively) are active astrimers. The clamp can not assemble itself on DNA.Instead, a protein complex known as a clamp loaderutilizes ATP to assemble the ring around theprimer-template. This study compares propertiesof the human PCNA clamp with those of E. coli andT4 phage.

Results: The PCNA ring is a stable trimer down to aconcentration below 100 nM (Kd� 21 nM). On DNA,the PCNA clamp slides freely and dissociates fromDNA slowly (t1/2� 24 min). ¯ is more stable insolution (Kd < 60 pM) and on DNA (t1/2� 1 h) thanPCNA which may be explained by its simpler

oligomeric state. The T4 gp45 clamp is a muchless stable trimer than PCNA (Kd� 250 nM) andrequires association with the polymerase to stabilizeit on DNA as observed previously. The consequenceof this cooperation between clamp and polymeraseis that upon finishing a template and dissociation ofthe polymerase from DNA, the gp45 clamp sponta-neously dissociates from DNA without assistance.However, the greater stability of the PCNA and ¯clamps on DNA necessitates an active process fortheir removal. The clamp loaders (RF-C and °complex) were also capable of unloading theirrespective clamps from DNA in the presence of ATP.

Conclusions: The stability of the different clamps insolution correlates with their stability on DNA.Thus, the low stability of the T4 clamp explains theinability to isolate gp45 on DNA. The stability of thePCNA and ¯ clamps predicts they will require anunloading factor to recycle them on and off DNAduring replication. The clamp loaders of PCNA and¯ double as clamp unloaders presumably for thepurpose of clamp recycling.

IntroductionThe sliding clamps of chromosomal replicases are ring-shaped proteins that encircle DNA and tether thereplicase to the template for highly processive chainelongation (reviewed in Kuriyan & O’Donnell 1993).The clamp loader recognizes a primed templatejunction and couples ATP hydrolysis to assemble the

# Blackwell Science Limited Genes to Cells (1996) 1, 101–113 101

* Corresponding author: Fax: +1 212 746 8587.a Present address: Department of Molecular Biology and Genetics,John Hopkins University, School of Medicine, 725 N. WolfeStreet, Baltimore, MD 21205-2195, USA.b Present address: Department of Cell Biology, Harvard MedicalSchool, Boston, MA 02115, USA

clamp around DNA. The eukaryotic clamp is theproliferating cell nuclear antigen (PCNA), the pro-karyotic clamp is the � subunit and the T4 phage clampis the product of gene 45 (gp45) (reviewed in Kuriyan& O’Donnell 1993). The E. coli � sliding clamp is adimer (Stukenberg et al. 1991; Kong et al. 1992) whilethe eukaryotic and T4 phage sliding clamps are formedby trimers (Krishna et al. 1994; Jarvis et al. 1989). In eachcase, the clamps have been shown to slide onDNA freely (Stukenberg et al. 1991; Gogol et al.1992; Burgers & Yoder 1993; Tinker et al. 1994). Theclamp loaders in each of these systems are composedof multiple subunits. The E. coli clamp loader is thefive-subunit complex, the eukaryotic clamp loader isthe five protein RF-C (also called activator-1) and theT4 clamp loader is the gene protein 44/62 complex(also a five subunit structure, reviewed in Kelman &O’Donnell 1994). A major difference among thesesystems is that the � and PCNA clamps can beisolated on DNA by gel filtration whereas the T4gp45 clamp requires association with its DNApolymerase, the product of gene 43 (gp43), to beisolated on DNA.

In E. coli, the complex acts catalytically to assemblethe � clamp on DNA and can be removed from theclamp-DNA complex (Wickner 1976; Maki & Korn-berg 1988; Stukenberg et al. 1991). The DNApolymerase III (Pol III core) then associates with the �clamp on DNA for processivity. However, an additional‘connector’ protein called � binds two molecules of PolIII core and one complex to produce a tightlyassociated particle called Pol III* (Onrust et al. 1995).The two Pol III core polymerases are thought toreplicate the leading and lagging strands concurrently(Sinha 1980; Kornberg & Baker 1992).

In eukaryotes and T4 phage, no such particlecontaining both polymerase and clamp loader hasbeen identified. Although it is presumed that theclamp loader and polymerase assemble together withthe clamp on the DNA in these systems, it remainspossible that the clamp loader acts catalytically resultingin only the clamp-polymerase complex, as observed inthe E. coli system. Indeed, the catalytic action of the T4gp44/62 complex has been documented (Kaboord& Benkovic 1995). In eukaryotes, both DNA poly-merase � (pol �) and DNA polymerase � (pol �) interactwith the PCNA clamp (Burgers 1991; Lee et al. 1991a).

In this report the inherent strength of the oligomericstructure of the clamps in solution, and their stability onDNA have been examined. Despite the apparentsimilarities of PCNA and T4 gp45 (both are trimersand less stable oligomers than the � dimer), PCNA is

similar to � in its stabilityon DNA and requirement for aclamp unloader for its removal.

ResultsTo directly follow the properties of PCNA, � and gp45in these studies we have radiolabelled them. All threewere tritiated by reductive methylation, a modificationthat results in a 3H-methyl group on one or two lysineresidues of each molecule (the charge is retained atphysiological pH) (Kelman et al. 1995a). Additionally,some studies were performed using 32P-labelled clampsusing � and PCNA containing a kinase recognitionmotif on the C- or N-terminus, respectively.

In solution, the trimer clamps are less stableoligomers than the dimer clamp

Previous studies have shown that upon transfer of slidingclamps to DNA by their respective clamp loaders, thePCNA and � clamps can be isolated on DNA by gelfiltration, but the T4 gp45 clamp can not (Wickner1976; O’Donnell 1987; Maki & Kornberg 1988; Lee &Hurwitz 1990; Burgers 1991; Capson et al. 1991;Richardson et al. 1991). One possible explanation forthe difference (in ability to form stable clamps on DNA)amongst the three systems could be the inherent stabilityof the oligomeric structure of the clamps themselves. InFig. 1 the oligomeric structure of these clamps wereexamined by gel filtering a mixture of all three insolution. They were analysed at a high concentration(2.5�M, Fig. 1A) and then at a 50-fold lower concen-tration (50 nM, Fig. 1B). To follow these proteins weadded a small amount of 3H-protein and analysed theirelution patterns by fluorography after SDS-polyacryl-amide gel electrophoresis. At 2.5�M, each clamp elutedat a position consistent with its native oligomeric state:PCNA as a trimer (86.7 kDa, fractions 28–31), b as adimer (81.2 kDa, fraction 31–34) and gp45 as a trimer(74.1 kDa, fractions 31–34). However, at 50 nM, theoligomeric states of the trimeric clamps begin to change,but � remained a stable dimer. At 50 nM, PCNA showedtwo distinct peaks; one correlated with its trimeric form,and the other was a smaller species that eluted infractions 37-40. At this low concentration, gp45 elutedentirely as a smaller species in fractions 37–43. 32P-� ofhigh specific activity was used to examine the behaviourof � at even more dilute conditions. 32P-� remained astable dimer even upon dilution to 67 pM and incubationat 37 8C with 1 M NaCl in buffer D for 6 h (J.Andjelkovic & M. O’Donnell, unpublished data). Thepossibility that the � monomer elutes at the same

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position as a dimer due to the unique shape of these ringproteins was examined. For this purpose, a mutation inthe dimer interface was introduced (Leu108Pro) and themutant was purified. This mutant �, which exists only asa monomer, was soluble but inactive in replicationassays. The Leu108Pro mutant of � was gel filtered at aconcentration of 2.5�M. The Coomassie Blue stainedSDS-polyacrylamide gel (Fig. 1C) showed that themonomer � eluted in fractions 38–42. Hence, themonomer form of � elutes in a different position than

the � dimer, as expected, supporting the conclusion thatthe wild type � dimer remains a stabile oligomer at thelowest concentrations tested.

Ring shaped trimer oligomers are expected to be lessstabile oligomers that ring shaped dimers (Kelman et al.1995b). In a protein ring each protomer has twocontacts, one with each adjacent protomer. In the caseof the trimer, upon dissociation of one protomer, theremaining two protomers now have only one contact,which should lead to cooperative disassembly of theentire ring. Thus, it seems reasonable to expect thatthe smaller species observed upon dilution of PCNAis the monomer. Further dilution did not result in athird, smaller species, as would be expected ifPCNA disassembled in stages from trimer-to-dimer-to-monomer. A recent study has shown that the PCNAtrimer dissociates into a putative dimer form upondilution (Zhang et al. 1995). In light of the abovereasoning, a dimeric form of PCNA that is more stabilethan the trimer would require a conformational changeleading to a tighter contact between the monomers ofthe dimer than exists in the trimer (e.g. perhaps a dimerring in which the two protomers form a smaller centralcavity). 100 nM radioactive PCNA was included in thegel filtration analysis of the monomeric � mutant ofFig. 1C. A PCNA monomer should elute later than a �monomer, and a PCNA dimer should elute earlier thana � monomer. However, the autoradiogram of the gelfiltration fractions showed that the disaggregated form ofPCNA comigrated with the monomer � in this column(not shown). This result is consistent with the smallerspecies of PCNA as a dimeric form, but a monomercan not be ruled out. A linear form of the PCNAtrimer (i.e. an open ring) can be excluded on the basisthat this would be an intramolecular reaction whichshould be concentration independent and not affectedby dilution.

The ability to observe the PCNA trimer and itsdisaggregated form in one sample provided the means toapproximate the dissociation constant of the PCNAring. Figure 2A shows that as PCNA is diluted from250 nM to 10 nM, the proportion of the smaller form ofPCNA increases at the expense of the trimer. Figure 2Bshows a double reciprocal plot of the fraction of PCNAin trimeric and disaggregated form as a function ofPCNA concentration. The x-intercept corresponds to aKd value of 21 nM.

The gp45 trimers started to dissociate at concentra-tions greater than 300 nM. However, unlike PCNA, thegel filtration profile of gp45 showed a single peak whichshifted in a continuous fashion to the lower molecularweight species as its concentration was decreased

Comparison of DNA sliding clamps


Figure 1 Trimer clamps (PCNA and gp45) dissociate at lowconcentration while the b ring remains a dimer. A mixture of thethree clamps (PCNA, b and gp45) was gel filtered on Superose 12at 4 8C at two different concentrations: (A) 2.5�M and (B) 50 nM.In both experiments, 5 pmol of 3H-clamp was present to followprotein by autoradiography. (C) A mixture of 2.5�M b monomermutant (b Leu108Pro) and 100 nM radiolabelled PCNA was gelfiltered and a SDS polyacrylamide gel of the column fractions wasstained with Coomassie Blue.

(data not shown). This suggests a rapid equilibriumbetween dissociation and reassociation of the gp45trimer. For this reason, the dissociation constant forgp45 could not be accurately determined, but can beestimated to be � 200–400 nM from the shift in thepeak.

In summary, these experiments demonstrate that insolution, the � dimer is more stable than the PCNAtrimer, which in turn is more stable than gp45.

E. coli � and human PCNA form stablepreinitiation complexes on DNA, but T4 gp45does not

Next, PCNA, gp45 and � were compared for theirability to be placed on singly primed M13mp18 ssDNAduring gel filtration. This is a non-equilibriumtechnique and thus only stable protein–DNA com-plexes survive this procedure. In these experiments, eachclamp was assembled onto the primed template by itsrespective clamp loader and ATP. The ssDNA wascoated with E. coli SSB except in the case of T4 proteinsin which the ssDNA was coated with T4 gp32.

Figure 3 shows a comparison of the gel filtrationprofiles resulting from the clamp loading reactions in thethree systems. The clamps were radiolabelled, thereforeenabling us to track their distribution during gelfiltration over a Bio-Gel A15 m column which resolveslarge DNA molecules, and 3H-proteins bound to them(fractions 9–13), from proteins not bound to DNA(fractions 20–30). The results show that � 1.5–2 �clamps were assembled onto each DNA molecule.Slightly less PCNA, 1–1.5 clamps per DNA molecule,was observed in the human system. Association of theT4 gp45 with DNA was too weak to be detected by gelfiltration [Fig.3C (circles)]. However, when the poly-merase, gp43, was included in the loading reaction someof the gp45 remained with the DNA during gelfiltration. The amount of gp43 in this experiment wasstoichiometric to the DNA template and the gp45 wassupplied in a 15-fold molar excess over the DNA.Under these conditions of excess gp45 and limitinggp43, � 0.25–0.5 gp45 clamps were retained per DNAmolecule. Due to the inability to isolate gp45 on DNAin the absence of the T4 DNA polymerase, we couldnot analyse the dynamic behaviour of gp45 in theexperiments of Figs 4–6 described below.

Human PCNA has similar sliding dynamicson DNA as the E. coli � clamp

Previously we developed DNA sliding assays for clamp

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Figure 2 Kd measurement for disaggregation of the PCNA ring.(A) The indicated concentrations of 3H-PCNA as trimers wereincubated at 37 8C for 1.5 h before gel filtration on Superose-12at 4 8C as described in Experimental proceedures. (B) Doublereciprocal plot of PCNA trimer and ‘monomer’ as a function ofprotein concentration. 1/r represents the ratio of total fmol of‘monomer’ PCNA to fmol of trimer PCNA.

proteins (Stukenberg et al. 1991), and these assays havebeen applied to human PCNA in Fig. 4. Resultsobtained with b in side-by-side experiments areshown for comparison. In panel A, 3H-PCNA (or

3H-�) was assembled onto a plasmid containing a singlenick using RF-C (or complex) and ATP, then thereaction was gel filtered to remove clamps not bound toDNA (not shown). The 3H-PCNA (or 3H-�) on DNAwas then divided into separate tubes and one half wastreated with BamHI to linearize the plasmid. The tworeactions were then gel filtered a second time. Theresults, in panel A, show that 3H-PCNA remainedbound to the circular DNA, but dissociated from linearDNA. These results are comparable to those observedwith the � clamp [bottom plot in panel A and inprevious work on � (Stukenberg et al. 1991)].

To further test that 3H-PCNA dissociated from linearDNA by sliding over the ends, a protein blockexperiment was performed (panel B). 3H-PCNA wasassembled onto a nicked plasmid containing oriP, thelatent origin of replication of the Epstein-Barr virus.The oriP sequence contains two elements, each ofwhich bind several molecules of the viral encodedEBNA-1 protein (Frappier & O’Donnell 1991).Sufficient EBNA-1 was added to saturate both sites.Then the reaction was gel filtered and the 3H-PCNAbound to DNA was collected from the excludedfractions of a gel filtration column. The pool ofDNA-bound clamps was divided into two tubes andone was treated with EcoRV to linearize the DNAbetween the EBNA-1 sites, then the two reactions weregel filtered side-by-side. The result shows that just asmuch 3H-PCNA was retained on the linear DNA byprotein bound near the ends, as was retained on thecircular DNA. This result supports the conclusion thatPCNA slides off the ends of DNA, and is comparable tothe result of similar experiments using 3H-� (panel B,lower plot) and observed in our previous study(Stukenberg et al. 1991).

This sliding behaviour of PCNA is consistent withearlier reports. Briefly, yeast PCNAwas shown to conferprocessivity to pol � on a linear template, in the absenceof clamp loader and ATP, by slipping onto the ends ofthe DNA and sliding to the primer-template junction(Burgers & Yoder 1993). Further, a protein-DNAcrosslinking study showed that PCNA could be cross-linked to DNA as it tracked along the DNA (Tinker etal. 1994). Additionally, it was found that PCNA couldbe stably isolated on circular but not linear DNA (Podustet al. 1995).

Human PCNA and E. coli � are slow todissociate from DNA

The time required for the PCNA and � rings todissociate from DNA was measured in the experiment



Figure 3 PCNA and b form stable clamps on circular DNA butgp45 requires DNA polymerase for stability. Clamps were loadedonto 800 fmol (8 nM) singly primed M13mp18 ssDNA using12 pmol (120 nM) of either 3H-b, 3H-PCNA, or 3H-gp45; and1 pmol (10 nM) complex, RF-C or gp44/62 complex,respectively. The reactions were incubated at 37 8C for 10 min.before applying to Bio-Gel A15 m columns equilibrated in bufferE containing 100 mM NaCl. The 3H-gp45 reaction was repeatedin the presence of 864 fmol gp43, and 60�M each of dGTP anddCTP.

shown in Fig. 5. Either radiolabelled PCNA, or �, wereassembled onto a plasmid containing a single nick usingtheir respective clamp loader and ATP. Radioactiveclamps bound to DNAwere isolated by gel filtration anddivided into several tubes. Each tube was then incubatedat 37 8C for a different length of time ranging from 0 to60 min before analysis through a second gel filtrationcolumn. The results show a 72 min half-life fordissociation of � from DNA, and a 24 min half-lifefor PCNA dissociation from DNA. It is important tonote that the E. coli complex is extracted from thereaction by the first gel filtration column (Stukenberg etal. 1991), but the human RF-C may not have beenremoved. This is vital because these clamp loaders canalso catalyse the removal of their respective clamps from

DNA (as will be shown later). Hence, if RF-C ispresent, it may speed the dissociation of PCNA fromDNA. In fact, PCNA dissociation from DNA appears tolevel off with time which may indicate an approach to anew equilibrium of PCNA on and off DNA as catalysedby residual RF-C. An alternative explanation for thebiphasic dissociation of PCNA from DNA is that thereare two different populations of clamps on the DNAwhich differ in stability. For example, some PCNAclamps may still be associated with RF-C. The observedhalf-life of PCNA dissociation from DNA is similar tothat of another study (t1/2 = 22 min.) (Podust et al.1995). In this latter study, ATP--S was used to halt RF-C catalysed loading of PCNA, but the effect of ATP--S on RF-C clamp unloading is uncharacterized and

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Figure 4 Dynamics of the PCNA ring on DNA. (A) ‘Sliding assay’: 3H-PCNA (1�g, 11.5 pmol as trimer) was incubated with nickedplasmid (3.4�g, 914 fmol as circles) and 200 ng (675 fmol) RF-C in a 100�L reaction for 30 min. at 37 8C and then gel filtered on a Bio-Gel A15 m column at 4 8C equilibrated with buffer E containing 50 mM NaCl. The excluded fractions (nos 11 and 12, 280�L)containing 3H-PCNA on DNA were pooled and divided into two tubes. One tube was treated with 6�L (120 U) BamHI, and the othertube was treated with 6�L of BamHI storage buffer. The reactions were incubated for 1 min. at 37 8C followed by gel filtration asdescribed above. In the bottom panel, the experiment was repeated using 3H-b (1�g, 12 pmol as dimer) and complex (336 ng, 1680fmol) in place of 3H-PCNA and RF-C. (B) ‘Protein-block assay’: Either 3H-PCNA or 3H-b was assembled onto 2�g (615 fmol) ofnicked pGEMoriP plasmid containing the latent origin (oriP) of the Epstein-Barr virus as described for the ‘sliding assay’. Then 4.3�g(179 pmol) of EBNA-1 (DNA binding domain) was added to saturate the two elements of oriP and the reaction was gel filtered to removefree protein. The excluded fractions containing 3H-PCNA (or 3H-b) on DNA were pooled and divided into two tubes. One tube wastreated with 10�L (200 U) of EcoRV and the other tube was treated with 10�L of EcoRV storage buffer [10 mM Tris-HCl (pH 7.4),0.1 mM EDTA, 1 mM DTT, 50 mM NaCl, 50% glycerol, 200�g/mL BSA]. After 1 min. at 37 8C the reactions were gel filtered andfractions were analysed for 3H-protein as described in Experimental proceedures.

may not halt RF-C catalysed release of PCNA fromDNA.

RF-C and complex are also clamp unloaders

We have observed that the complex, besides loading �on DNA, also unloads � clamps from DNA (Stukenberg1993; Stukenberg et al. 1994). We have examined RF-Cfor PCNA clamp unloading activity in Fig. 6. First, 32P-PCNA was loaded onto a nicked plasmid by RF-Cand ATP, and then gel filtered to remove unbound32P-PCNA (data not shown). The column fractions

containing 32P-PCNA on DNA were divided intotwo tubes and one was treated with RF-C and ATP.Then both reactions were gel filtered a second time.The result showed that RF-C removed most of the32P-PCNA clamps from DNA. Panel B shows a similarexperiment in the E. coli system using 3H-� and complex.

In both systems, this clamp unloading process isdependent on the presence of ATP (data not shown).



Figure 5 Rate of decay of PCNA and b rings from DNA at37 8C. (A) 32P-PCNA was loaded onto a nicked plasmid in a200�L reaction containing 24 pmol (120 nM) 32P-PCNA, 1.8units of RF-C and 1.66 pmol (8 nM) singly nicked plasmid DNA.The mixture was incubated at 37 8C for 15 min. before applyingto a Bio-Gel A15 m column at 4 8C equilibrated with buffer Econtaining 100 mM NaCl. The excluded peak fractions (nos 10–13) were pooled and then divided into five tubes of 100�L eachand incubated at 37 8C for the indicated amounts of time beforeanalysis on the second Bio-Gel A15 m column. (B) Theexperiment was repeated using 24 pmol (120 nM) 3H-b and1 pmol (5 nM) complex in place of PCNA and RF-C.

Figure 6 Clamp loaders of E. coli and human replicases are alsoclamp unloaders. (A) Radiolabelled PCNA was loaded ontosingly nicked plasmid in a 100�L reaction containing 12 pmol(120 nM) 32P-PCNA, 0.9 units of RF-C, and 1.2 pmol (12 nM)singly nicked plasmid. After 10 min. at 37 8C, the reaction was gelfiltered on a Bio-Gel A15 m column at 4 8C equilibrated withbuffer E containing 100 mM NaCl. Excluded peak fractions (10-13) were combined and then divided into two tubes (160�Leach). The two reactions were incubated 6 min at 37 8C either inthe presence or absence of 0.45 units of RF-C and then analysedfor clamp removal on a second Bio-Gel A15 m column at 4 8Cequilibrated in buffer E containing 100 mM NaCl. (B) Theexperiment was repeated using 384 nM complex to load 32P-bclamps on DNA, gel filtered, divided into two tubes, followed byincubation for 10 min. at 37 8C either in the presence or absenceof 192 nM complex prior to a second gel filtration analysis at4 8C.

A possible explanation of how the seemingly opposingactions of clamp loading and unloading can beperformed by one protein complex, and the physio-logical relevance of this reaction are presented in theDiscussion section.

T4 gp45 spontaneously cycles off DNA afterreplication is complete

As was shown in Fig. 3, T4 DNA polymerase is requiredto stabilize the gp45 clamp on DNA through analysisby gel filtration. In the experiment described in Fig. 7A,the 3H-gp45 clamp was placed on a plasmid with a 1 kbgap of ssDNA coated with gp32 in the presence ofthe gp44/62 complex and the DNA polymerase.A complete set of dNTPs was added to to one halfof this reaction to allow the gap to be filled and thenboth reactions were gel filtered side-by-side. The resultsshow that the 3H-gp45 clamp was efficiently releasedupon completion of the gap.

These findings are in contrast to our previousdemonstration that the � clamp is left on DNA afterthe polymerase finishes a template, although thepolymerase itself dissociates into solution (Stukenberget al. 1994). For comparison with the T4 system, anexperiment using 32P-� and Pol III* on gapped DNAbefore and after replication is shown in Fig. 7B.

Discussion

Stability of clamps in solution correlates withtheir stability on DNA

This study shows that �, a ring shaped protein composedof two identical subunits, is a highly stable oligomer insolution and remains associated at concentrations aslow as 67 pM. The PCNA and gp45 clamps, composedof three identical subunits, dissociate as their con-centration is reduced, but the PCNA trimer is an orderof magnitude more stable than the gp45 trimer (Kdvalues, 21 nM and 200–400 nM, respectively). Thisdifference in stability probably underlies the abilityto isolate b and PCNA, but not gp45, on DNA bythe non-equilibrium technique of gel filtration. Thisresult is consistent with previously described resultsof yeast PCNA (Burgers & Yoder 1993) and humanPCNA (Lee & Hurwitz 1990; Podust et al. 1995).

The half-life of the � clamp on DNA is� 72 min andthat of the PCNA ring is � 24 min. Spontaneousdissociation of these clamps from DNA requires at leastone subunit–subunit interface to open, although the

process may involve complete protomer dissociationfrom the rings.

Unlike PCNA and �, the T4 gp45 ring is unstableboth in solution and on DNA and requires the DNApolymerase to stabilize it on DNA through a gelfiltration column as observed previously (Richardson etal. 1991). The relative instability of gp45 relative to �and PCNA suggests differences among these systems as

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Figure 7 Fate of the T4 clamp after completing replication of atemplate. (A) The T4 holoenzyme was assembled onto gappedplasmid DNA using 3H-gp45 in a 100�L reaction containing12 pmol (120 nM) 3H-gp45, 3.6 pmol gp44/62 complex, 570fmol gapped plasmid, 149 pmol gp32, 864 fmol (8.6 nM) gp43 and60�M each of dGTP and dCTP. After 5 min at 37 8C, thereaction was filtered. The excluded peak fractions (10–12) werecombined and 200�L withdrawn into each of two tubes, one foranalysis of gp45 stability during idling, and the other for analysisafter replication. Idling reaction: The T4 holoenzyme on DNA wasincubated for 1 min at 37 8C before analysis on a second gel fil-tration column. Termination reaction: The T4 holoenzyme onDNA was supplemented with 60�M each of dATP and dTTP,incubated for 1 min at 37 8C, and then analysed at 4 8C on a secondBio-Gel A15 m column equilibrated in buffer E containing all 4dNTPs. (B) The experiment was repeated using 3.6 pmol 3H-b,3.2 pmol 3H-Pol III*, 12.6�g E. coli SSB in place of the T4proteins, and 1.34 pmol gapped plasmid DNA in a 200�L reaction.

illustrated in Fig. 8. The � and PCNA clamps are stableoligomers: � remains stably bound to DNA in theabsence of complex (Stukenberg et al. 1991) andPCNA likely remains stable without RF-C as indicatedby its ability to slide off DNA upon linearizing the DNA(if RF-C were bound to PCNA and the 3’ terminus,PCNA would not be released for sliding over the end ofDNA). This is reflected in step I of Fig. 8A. Associationof DNA polymerase could occur even several minuteslater (step II) as observed in these systems (O’Donnell1986; Burgers & Yoder 1993). The gp45 clamp is not asstable on DNA although it can exist for short periods oftime without the clamp loader as indicated by DNAcrosslinking studies (Tinker et al. 1994) and a cryoelec-tron microscopy study which observed a structure onDNA the size of � (presumably gp45) that disappearedseconds after removal of ATP (Gogol et al. 1992).Hence, gp45 can exist alone on DNA, but must bequickly captured by the polymerase as reflected inFig. 8B, step II. The instability of the gp45 trimer insolution suggests that it may be assembled fromprotomers to encircle the DNA as illustrated inFig. 8B, step I.

Are dimers inherently more stable than trimers?

The one feature that most distinguishes the stable � ringfrom the less stable PCNA and gp45 rings is its nativeoligomeric state; � forms a ring composed of twoprotomers while PCNA and gp45 form rings composed

of three protomers. A trimer oligomer is entropically lessfavoured than a dimer. Hence if the subunit-subunitinterfaces of dimer and trimer rings were of equivalentstrength, a trimer would tend to dissociate at higherconcentration than a dimer. However, the crystalstructure shows the interface of � is significantlydifferent from that of yeast PCNA. The � interfacesare composed of an antiparallel sheet four residues inlength whereas the sheet at the interface of yeast PCNAis twice as long, presumably to counteract the effects ofentropy (Krishna et al. 1994). It may be predicted thatthe forces that bind the gp45 trimer together will be lessextensive than those in PCNA.

Why eukaryotes and T4 utilize trimer clamps ratherthan a dimer like prokaryotes remains to be elucidated.In both T4 and eukaryotes the clamp is involved inprocesses other than replication. In T4 phage, the gp45clamp activates late gene transcription (Herendeen et al.1989) and in mammals the p21 protein interacts withPCNA as a means of stopping long chain synthesis(Flores-Rozas et al. 1994; Waga et al. 1994). PCNA isalso found with p21 in complex with cdk and cyclin D,implying a role in cell cycle control (Xiong et al. 1992).PCNA has recently been shown to interact with DNaseIV (also called FEN-1) in higher eukaryotes and RTH1in S. cerevisiae and stimulates its nucleolytic activity (Li etal. 1995). DNase IV has been shown to play a criticalrole in maturation of Okazaki fragments in laggingstrand DNA synthesis (Robbin et al. 1994). Theseobservations of trimer clamps utilized by other proteins



Figure8 Models of clamp assembly and release in different systems. (A) In the E. coli and human system, the clamp loader assembles theclamp onto DNA (Step I). The clamp tethers the polymerase to DNA for processive synthesis (Step II). After replication is complete, thepolymerase dissociates from the clamp (Step III) and the clamp loader removes the clamp from DNA (Step IV). It is not yet clear whetherRF-C remains associated with the PCNA clamp in Step I, or whether pol � dissociates from the clamp in Step III. (B) The gp44/62complex assembles the gp45 trimer onto DNA (Step I), but the gp45 clamp requires association with the gp43 polymerase for enhancedstability on DNA (Step II). Upon termination of replication the polymerase dissociates from DNA and gp45 looses its gel filterablestability on DNA (Step III).

may indicate a particular use of trimers in other cellularprocesses such as transcription, repair and cell cyclecontrol. For example, perhaps a metastabile trimer-monomer equilibrium is more amenable to controlprocesses than a clamp that exists as a stable dimer.

Two different molecular strategies of clamprecycling

In replication of the leading strand, a DNA polymeraseheld tightly to DNA for continuous synthesis isadvantageous. However, the lagging strand is synthe-sized as a series of numerous fragments, and uponcompleting each fragment the polymerase must rapidlycycle off the DNA and back onto the next primed site.This rapid cycling off and on the DNA is a process thatwould conceptually be hindered by too tight a grip onthe DNA such as incurred by a protein ring. Themechanism for this rapid polymerase cycling has beenelucidated in the E. coli and T4 phage systems. TheDNA polymerase is tightly held to the sliding clampduring chain extension, but upon completing a templatethe polymerase rapidly dissociates from its clamp(Hacker & Alberts 1994a,b; Stukenberg et al. 1994).

In the E. coli system, the � clamp remains on thecompleted DNA after the polymerase departs asillustrated in step III of Fig. 8A. Although the stoichio-metric use of � clamps for lagging strand fragments isconsistent with the intracellular abundance of � over PolIII, there are still 10 times more Okazaki fragments than� clamps. Hence these clamps must be recycled. Thestability of � on DNA implies clamp unloading will bean active process. Indeed, the complex removes �clamps from DNA as illustrated in step IV of Fig. 8A.The observed stability of PCNA clamps on DNAsuggests that clamp recycling may be an active process ineukaryotes as well. A simple calculation supports thisidea. The amount of PCNA in the cell nucleus (HeLa)has been estimated by immunoblot to be in the 3–6� 105 range (as monomer) [1–2� 105 trimers](Morris & Mathews 1989). The total number ofOkazaki fragments in replication of four billion basepairs, assuming a 200 nucleotide length, is 2� 107

fragments. Hence PCNA must be reused about 100times per cell cycle (2� 107/2� 105). Assuming an Sphase of 6 h, the time of a PCNA cycling event is� 3.6 min, much faster than the observed stability ofPCNA on DNA (t1/2 � 24 min). This report shows thatRF-C, like complex, efficiently removes PCNAclamps from DNA.

It seems a dilemma that the seemingly opposedactions of clamp loading and unloading are performed

by the same protein complex. We presume that thisapparent contradiction is explained by a dynamicequilibrium of clamps on and off the DNA catalysedby these clamp loaders. If one starts with all the clampsoff the DNA, the forward reaction of clamp loading isobserved. If on the other hand, one starts with all theclamps on the DNA (i.e. by placing clamps on DNA andthen removing those left in solution by gel filtration)then clamp unloading is observed. In both cases the endresult is an equilibrium of clamps on and off the DNA,but the direction in which the equilibrium isapproached determines whether clamp loading orunloading is observed.

In contrast to the E. coli and human system, the T4phage system may use a different strategy of clamprecycling. The gp45 clamp spontaneously dissociatesfrom DNA after the template has been fully replicated,suggesting that active clamp removal by the gp44/62clamp loader may not be necessary as illustrated inFig. 8B, step III. This result could be anticipated fromthe instability of gp45 clamps on DNA in the absence ofpolymerase, and the fact that T4 polymerase rapidlydissociates from DNA upon completing a template(Hacker & Alberts 1994b). However, the gel filtrationtechnique utilized in this report requires � 15 min at4 8C. This is a long time relative to the speed of forkmovement. If gp45 were bound to DNA for a fractionof this time it may still require assistance for efficientremoval from DNA. Hence, dissociation of gp45 clampsfrom DNA assisted by the gp44/62 complex in vivo cannot be rigorously excluded.

Experimental Procedures

Sources

Radioactive chemicals, DuPont-New England Nuclear;unlabelled ATP Pharmacia LKB; Bio-Gel A-15 m, Bio-Rad;DNA modification enzymes and the T4 DNA polymerase, NewEngland BioLabs (NEB) (specific activity = 3 units/�g where 1unit supports incorporation of 10 nmol dNTP in 30 min at 37 8C)and GIBCO BRL; gp32, Boehringer Mannheim. (Indianapolis,IN). Other proteins were purified as described: � (Kong et al.1992), �PK (Stukenberg et al. 1994), complex (Onrust et al.1995), E. coli single-stranded DNA binding protein (SSB)(Weiner et al. 1975), PCNA (Kelman 1995), PCNA with anN-terminal kinase site (Kelman et al. 1995c) and EBNA-1 DNAbinding domain (Kelman et al. 1995a). RF-C and Pol � werepurified from HeLa cells as described (Lee et al. 1991b). 3H-PCNA (520 cpm/fmol) and 3H-gp45 (75 cpm/fmol) wereprepared by reductive methylation using formaldehyde and [3H]-NaBH4 (Kelman et al. 1995a).

3H-gp45 was within 90% theactivity of wild-type gp45 in replication of gp32-coated primed

N. Yao et al.


ssDNA using gp44/62 and gp43. Modified versions of �(Stukenberg et al. 1994) and PCNA (Kelman et al. 1995c)containing protein kinase recognition motifs at the C- and N-terminus, respectively, were labelled with -32P-ATP usingcAMP-dependent protein kinase to specific activities of 65-387cpm/fmol and 225 cpm/fmol, respectively, as described(Kelman et al. 1995a). 3H-PCNA and 32P-PCNA were within50% and 99%, respectively, the activity of wild-type PCNA inreplication of gp32 coated poly(dA)5000: oligo(dT)20 and theelongation of singly primed M13 DNA using RF-C and pol �.3H-� and 32P-bPK were within 95% the activityof wild-type � inreplication of SSB-coated primed M13mp18 ssDNA usingpol III*.

Buffers

Buffer A is 20 mM Tris-HCl (pH 7.5), 0.5 mM EDTA (pH 7.5),2 mM dithiothreitol (DTT) and 20% glycerol. Buffer B is 10 mMsodium acetate (pH 7.5), 20% glycerol, 0.5 mM EDTA and 2 mMDTT. Buffer C is 50 mM sodium acetate, 0.3 M NaCl, 10 mM zincsulphate. Buffer D is 20 mM Tris-HCl (pH 7.5), 0.5 mM EDTA,100 mM NaCl and 10% glycerol. Buffer E is 20 mM Tris-HCl (pH7.5), 0.1 mM EDTA, 40�g/mL bovine serum albumin (BSA),5 mM DTT, 8 mM MgCl2, 4% glycerol.

Purification of gp45 and gp44/62 complex

The plasmid, pTL151W containing genes 45, 44 and 62, undercontrol of a PL promoter, was the gift of Dr William Konigsberg,Yale University. HB101 cells (24 L) harboring pTL151W weregrown in VAT (per litre: 10 g tryptone, 4 g yeast extract, 1 gKH2PO4, and 10 g K2HPO4) supplemented with glucose andampicillin at 30 8C and induced at 42 8C as described (Rush et al.1989) Each litre was grown to OD = 0.8 and then brought to42 8C rapidly upon addition of 250 mL of VATat 62 8C followedby continued incubation at 42 8C for 3 h. The followingprocedures were performed at 4 8C. Cells were harvested bycentrifugation and lysed with lysozyme by heat-lysis as described(Wickner 1974). The clarified lysate was dialysed against buffer A(3 g of protein in 250 mL) and then applied to a 150 mL fast-flowQ-Sepharose column equilibrated in buffer A and eluted with a2.1 L gradient of 0-0.5 M NaCl in buffer A. Presence of theproteins was followed in 10% SDS-polyacrylamide gels stainedwith Coomassie blue. The gp44/62 complex and gp45 co-eluted(at about 0.25 M NaCl) and were pooled (12 mL), dialysed againstbuffer B, and applied to a 100 mL heparin-Sepharose columnequilibrated in 10 mM sodium acetate (pH 7.5). gp45 wascollected in the flow through and gp44/62 complex was elutedwith 2 M NaCl in 10 mM sodium acetate (300 mg). The flowthrough fraction, containing gp45 was slowly adjusted to pH 5.5with 10 mM glacial acetic acid and then applied to a 100 mLheparin-Sepharose column equilibrated in buffer B (pH 5.5), andthen eluted with a 1.4 L linear gradient of 0-0.5 M NaCl in BufferB. Fractions containing gp45 (fractions 25-45, 150 mg in 180 mL)were pooled and precipitated upon addition of ammoniumsulphate to 70% saturation. After centrifugation, the pellet was

resuspended in buffer A [with Tris-HCl (pH 8.8)] and dialysedagainst 2� 4 L buffer A [with Tris-HCl (pH 8.8)] overnight at4 8C. The solution (108 mg in 25 mL) was applied to an 8 mLmono Q column equilibrated in buffer A (but at pH 8.8), andeluted with a 160 mL gradient of 25-250 mM NaCl in buffer A(pH 8.8). Fractions 7-66 containing > 95% pure gp45 werepooled (120 mg in 62 mL), dialysed against 4 L buffer Aovernight, aliquoted and stored at -70 8C.

The gp44/62 complex was further purified upon resuspendingthe ammonium sulphate pellet in 250 mL buffer B (pH 7.5),dialysed against 2 changes of 4 L each of buffer B and then appliedto a 100 mL heparin-Sepharose column. The gp44/62 was elutedwith a 1.4 L linear gradient of 0-0.5 M NaCl gradient in buffer B.Fractions 76-105 were pooled (100 mg in 250 mL) andprecipitated upon addition of ammonium sulphate to 70%saturation. Protein was dissolved in buffer A, dialysed against 4 Lof buffer A overnight, and applied to an 8 mL MonoQ columnequilibrated in buffer A. The gp44/62 complex was eluted with a160 mL linear gradient from 25 to 250 mM NaCl in buffer A.Fractions 17-36 containing gp44/62 complex were pooled(61.7 mg, 45 mL), dialysed against buffer A and stored at -70 8C.

DNA substrates

Plasmid DNAs [pET-b16 (a pET3c vector containing the 1 kbdnaN gene) (5.7 kb), pGEMoriP (5 kb) (Frappier & O’Donnell1991) and pBluescript SK+ (Stratagene, LaJolla, CA) (2.96 kb)]were purified through Qiagen columns (Qiagen Inc., Chats-worth, CA), and then were further purified by ultracentrifuga-tion in cesium chloride as described (Maniatis et al. 1982). NickedDNA was prepared by treating 24.2�g of supercoiled (RFI)plasmid with 60 units of S1-nuclease in 400�L of buffer C at37 8C until 50% of the RFI DNA was converted to RFII DNA (7min) and then was purified by phenol extraction. Plasmid DNAwith a ssDNA gap was made using 390�g of pBluescript SK+

plasmid DNA followed by treatment with gpII protein asdescribed (Meyer & Geider 1979) to produce a site specificnick in 60% of the plasmid. The nicked DNA was then treatedwith 1000 units exonuclease III at 37 8C for 10 min. A gap size of1 kb was estimated by analysis of 3�g in an alkaline agarose gel.

Replication assays

M13mp18 ssDNA was purified as described (Turner &O’Donnell 1995b) and then uniquely primed with a syntheticDNA 30-mer oligonucleotide (M13mp18 map position 6816-6847) as described (Stukenberg et al. 1991). Assays contained80 ng primed ssDNA, 2.5�g gp32, 10 ng gp44/62, 270 ng gp45or 3H-gp45, 0.1-0.3 units of gp43, 60�M dCTP and 60�MdGTP in 23�L of buffer E. Reactions were incubated at 37 8Cfor 1 min., and then 60�M dATP and 20�M [-32P]TTP wereadded to initiate a 30-s pulse of replication before quenching thereaction by spotting onto DE81 paper and quantitating theamount of DNA synthesis by scintillation counting.

Replication assays of PCNA contained 0.125�g (dA)4500.oli-

go(dT)12-18 annealed at nucleotide ratio of 20:1, 40 mM creatine



phosphate, 0.03 mg/mL creatine phosphate kinase, 1�g gp32,33.3�M [-32P]dTTP, 0-0.1�g PCNA, 0.09 unit RF-C and 0.2units of pol � in 30�L of 40 mM Tris-HCl (pH 7.8), 7 mM MgCl2,1 mM DTT, 4 mM ATP and 0.15 mg/mL BSA. The reaction wasincubated at 37 8C for 1 h before quenching the reaction byspotting on DE81 paper and quantitating the amount of DNAsynthesis by scintillation counting.

Gel filtration of PCNA, � and gp45

Radiolabelled proteins (amounts indicated in legends to thefigures) were incubated in 100�L of buffer D at 37 8C for 1.5 h.The protein mixture was analysed by gel filtration on a FastProtein Liquid Chromatography FPLC HR 10/30 Superose 12column (Pharmacia-LKB) equilibrated in buffer D at 4 8C. Afterthe first 6 mL, fractions of 150�L were collected and 100�L ofthe indicated fractions were analysed in a 12% SDS polyacryla-mide gel either stained with Coomassie Brilliant Blue orsubjected to fluorography using EN3HANCE (NEN, Boston,MA) and then exposed to X-ray film.

Gel filtration of PCNA, � and gp45 on DNA

Proteins and DNA (amounts are specified in the figure legends)were incubated in 100�L of buffer E + 1 mM ATP at 37 8C for theindicated time before applying to columns of agarose Bio-GelA15 m (5 mL bed volume) equilibrated in buffer E at 4 8C.Fractions of 180�L were collected and the amount of clamp ineach fraction was quantifyd by liquid scintillation. When the T4DNA polymerase (gp43) was present, 60�M each of dCTP anddGTP was added to the column buffer to prevent removal of theDNA primer by the proofreading 30–50 exonuclease of gp43.

AcknowledgementsWe are grateful to Dr William Konigsberg for providing us withthe pTL151 W plasmid. This work was supported by a grant fromthe National Institutes of Health, GM38839.

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Received: 28 September 1995Accepted: 16 October 1995



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