Ordered ATP Hydrolysis in the gamma  Complex Clamp Loader AAA+ Machine*

Aaron JohnsonDagger § and Mike O'DonnellDagger

From the  Howard Hughes Medical Institute and the Dagger  Rockefeller University, New York, New York 10021

Received for publication, December 13, 2002, and in revised form, February 6, 2003

    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

The gamma  complex couples ATP hydrolysis to the loading of beta  sliding clamps onto DNA for processive replication. The gamma  complex structure shows that the clamp loader subunits are arranged as a circular heteropentamer. The three gamma  motor subunits bind ATP, the delta  wrench opens the beta  ring, and the delta ' stator modulates the delta -beta interaction. Neither delta  nor delta ' bind ATP. This report demonstrates that the delta ' stator contributes a catalytic arginine for hydrolysis of ATP bound to the adjacent gamma 1 subunit. Thus, the delta ' stator contributes to the motor function of the gamma  trimer. Mutation of arginine 169 of gamma , which removes the catalytic arginines from only the gamma 2 and gamma 3 ATP sites, abolishes ATPase activity even though ATP site 1 is intact and all three sites are filled. This result implies that hydrolysis of the three ATP molecules occurs in a particular order, the reverse of ATP binding, where ATP in site 1 is not hydrolyzed until ATP in sites 2 and/or 3 is hydrolyzed. Implications of these results to clamp loaders of other systems are discussed.

    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

DNA polymerase III holoenzyme, the chromosomal replicase of Escherichia coli, contains a clamp-loading machine within its multi-component structure (reviewed in Ref. 1). The clamp loader couples ATP hydrolysis to the assembly of circular beta  clamps onto primed DNA sites. The circular beta  clamp, formed from two crescent-shaped protomers, binds to the DNA polymerase III core (alpha epsilon theta ), tethering it to template DNA for highly processive synthesis. There are at least two molecules of DNA polymerase III core within the holoenzyme architecture, held together by one clamp loader.

The gamma  complex clamp loader consists of several different subunits, i.e. three gamma  (tau ) subunits and one each of delta , delta ', chi , and psi  (2-4). The chi  and psi  subunits play roles in the primase-to-polymerase switching process (5), and they also interact with SSB1 (6, 7) but are not essential for clamp loading. The crystal structure of the minimal clamp loader, gamma 3 delta 1 delta '1, shows that the five subunits are arranged as a circular heteropentamer (4). In order for this clamp loader to bind two molecules of DNA polymerase III core, two of the gamma  subunits are replaced by two tau  subunits. tau  and gamma  are encoded by the same gene (dnaX); tau  is the full-length product, and gamma  is truncated by a translational frameshift (8-10). The N-terminal 47 kDa of tau  contains the sequence of the gamma  subunit, thus explaining how gamma  and tau  can replace one another in clamp loading action with delta  and delta ' (11). The extra 24 kDa of C-terminal sequence unique to tau  is responsible for binding DNA polymerase III core (12, 13); these sequences also bind the DnaB helicase (14, 15). Thus, a single clamp loader cross links two DNA polymerases and holds the hexameric helicase into the replisome (reviewed in Ref. 6).

The gamma  (tau ) subunits of the gamma  complex constitute the motor of the clamp loading machine, as they are the only subunits that hydrolyze ATP; neither delta  nor delta ' bind or hydrolyze ATP (1). The delta  subunit forms the main attachment to the beta  clamp and can open the beta  ring single-handedly (16-18). delta  is sometimes referred to as the wrench or crowbar of the clamp loader because it can open beta  on its own (19). The delta ' subunit modulates the ability of delta  to bind beta  (16-18). In the absence of ATP, delta ' obscures the delta  subunit within the gamma  complex from binding to beta  (4, 16). However, with ATP bound to the gamma  subunits, delta  is pulled away from delta ', allowing delta  to bind and open the beta  ring (4, 20). In this state, with beta  and ATP bound to the gamma  complex, a tight affinity for DNA is established (21-22). Upon recognizing a primed site, the ATP is hydrolyzed, resulting in the dissociation of the gamma  complex from beta , leaving beta  to close around the DNA, whereupon it may associate with the polymerase component of the holoenzyme.

beta also interacts with several other proteins besides the DNA polymerase III core. These include ligase, MutS, UvrB, DNA polymerases I, II, IV and V, and possibly many other proteins involved in DNA repair (23-26). These additional roles of beta  in other processes besides replication may account for the presence of the gamma  complex in E. coli that lacks tau  altogether, presumably freeing it for action at sites distinct from the replication fork.

The structure of gamma 3delta delta ' reveals that the ATP sites of the gamma  subunits are located at subunit interfaces (see Fig. 1) (4). Site 1 is at the delta '/gamma 1 interface, site 2 is at the gamma 1/gamma 2 interface, and site 3 is at the gamma 2/gamma 3 interface. The site 3 gamma 3 subunit is located directly next to the delta  wrench. The numbering of these sites is thought to reflect the order in which they become filled with ATP (4). The major pentameric contacts occur via the C-terminal domains of the five subunits. The ATP binding sites of gamma  are located in the N-terminal domains. The N-terminal domain of delta  is also where the beta  interactive element is located (19), although the proximity of delta ' to delta  blocks access of delta  to beta  (4). Combining several biochemical findings with the structure of gamma 3delta delta ' suggests that, as the ATP sites fill, conformation changes in gamma  are propagated around the pentamer to pull the delta  wrench away from the delta ' stator so that delta  can bind to beta  for clamp opening. The apparent rigidity of delta ', compared with gamma  and delta , which have a flexible joint for motion in clamp loading, has earned delta ' the term "stator". ATP hydrolysis presumably reverses the conformational changes in gamma  and delta  induced by ATP binding to gamma , thus bringing the N-terminal domain of delta  back into proximity to the delta ' stator. This effectively pushes beta  off of delta , allowing the beta  ring to close around DNA.

The subunits of gamma 3delta delta ' are members of the large AAA+ family (27). As their name implies (ATPases associated with a variety of cellular activities), these proteins are generally ATPases, and they function in a wide diversity of cellular processes. The structure of the homohexameric AAA+ proteins NSF and p97 (membrane fusion), RuvB (branch migration), and HslU (proteosome) reveals an Arg residue that reaches over the interface to the ATP site of the neighboring subunit (28-31). This Arg is thought to be analogous to the "arginine finger" of GAP, which plays a catalytic role in hydrolysis of GTP bound to Ras by stabilizing the accumulating negative charge in the transition state (32). It is proposed that the use of this Arg residue in catalysis provides a means of intersubunit communication that coordinates nucleotide hydrolysis around the ring.

The gamma  complex has many similarities to the homohexameric AAA+ proteins but also has several important differences. The largest differences are its heterooligomeric composition, use of five subunits instead of six, and the presence of two subunits that do not bind ATP. Like the homohexamers, the gamma  complex subunits are arranged in a ring, and the gamma  subunit ATP sites are located at interfaces where the Arg of one subunit is in proximity to ATP modeled into the subunit adjacent to it (Fig. 1). This Arg residue is embedded in an SRC motif that is conserved in clamp-loading subunits of T4 phage, eubacteria, archaea, and eukaryotes. The delta ' subunit also contains an SRC motif, and the Arg residue is proximal to ATP site 1 of gamma 1. In each case, the Arg needs to move a few angstroms to be near enough to exert an influence on the bound ATP.


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Fig. 1.   Architecture of gamma  complex ATP sites. A, orthogonal views of the gamma  ATP binding domain. Arg-169 (R169) is located far away from the phosphate binding loop (P-loop) on the same polypeptide chain, preventing formation of a functional intramolecular ATP site. B, schematic of the circular pentamer of the gamma  complex looking down the center of the ring from the C terminus. Three ATP sites are created at subunit interfaces. Each site is highlighted by the P-loop of a gamma  subunit and the sensor 1 Arg in the SRC motif from the adjacent delta ' (ATP site 1) or gamma  (sites 2 and 3) subunit. C, ATP site 1, a magnified view of the delta '/gamma 1 N-terminal domain interface with an ATP molecule modeled against the P-loop of gamma 1 based on the NSF D2 crystal structure (31). The delta ' sensor 1 Arg-158 (green residue) is in close proximity to the gamma -phosphate of ATP, suggesting a catalytic function. In ATP sites 2 and 3 the gamma 1/gamma 2 and gamma 2/gamma 3 interfaces, respectively, have a similar architecture to those in ATP site 1. The gamma  sensor 1 Arg-169 is positioned proximal to the ATP molecule modeled into the neighboring gamma . It should be noted that ATP modeled into site 2 clashes with some residues of gamma 1 and, thus, a conformation change is needed to make this site accessible to ATP.

The contribution of these potential arginine fingers to ATP binding, hydrolysis, and clamp loading is one subject of this report. The results demonstrate that these SRC motif arginine residues in both gamma  and delta ' are not required for ATP binding; however, they are important to catalysis. The finding that delta ' contributes a catalytic arginine residue to an ATP site in the gamma  trimer motor demonstrates that the delta ' stator also functions as a component of the motor of the clamp loader. This conclusion has implications for analogies between the gamma  complex and clamp loaders of other systems (see "Discussion"). Furthermore, the findings indicate that there is an ordered sequence to hydrolysis. Mutation of the arginine in gamma  removes the catalytic arginine in sites 2 and 3 but not the arginine contributed by delta ' to site 1. However, this mutation prevents hydrolysis of ATP in all three sites. This result indicates there is a sequential order to hydrolysis of the three ATP molecules, with ATP in sites 2 and/or 3 being hydrolyzed before ATP in site 1.

    EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Materials-- Unlabeled deoxyribonucleoside triphosphates were from Amersham Biosciences, and radioactive nucleoside triphosphates were supplied by PerkinElmer Life Sciences. Proteins were purified as described; alpha , epsilon , gamma , tau  (33), beta  (34), delta  and delta ' (35), chi  and psi  (36), theta  (37), and SSB (38). Core polymerase (37), and gamma  complex (11) were reconstituted from pure subunits and purified as described. Mutant subunits were purified by the same methods as wild-type proteins. The gamma  complex containing mutant subunits was reconstituted and purified using the same procedure as wild-type gamma  complex. Samples of purified complex were analyzed on a 14% SDS-polyacrylamide gel stained with Coomassie Brilliant Blue G-250, and each lane was scanned by laser densitometer (Amersham Biosciences). beta PK is beta  containing a six-residue C-terminal kinase recognition site (39) and was labeled to a specific activity of 10 dpm/fmol with [gamma -32P]ATP using the recombinant catalytic subunit of cAMP-dependent protein kinase produced in E. coli (a gift from Dr. Susan Taylor, University of California at San Diego). The following oligonucleotides were synthesized and purified by Integrated DNA Technologies: 79-mer, 5'-GGG TAG CAT ATG CTT CCC GAA TTC ACT GGC CGT CGT TTT ACA ACG TCG TGA CTG GGA AAA CCC TGG CGT TAC CCA ACT T-3'; and 45-mer, 5'-GGG TTT TCC CAG TCA CGA CGT TGT AAA ACG ACG GCC AGT GAA TTC-3'. To form the synthetic primed template, the oligonucleotides were mixed in 50 µl of 5 mM Tris-HCl, 150 mM NaCl, 15 mM sodium citrate (final, pH 8.5), then incubated in a 95 °C water bath that was allowed to cool to room temperature over a 30-min interval. M13mp18 ssDNA was purified as described (40) and primed with a 30-mer DNA oligonucleotide as described (33). Bio-gel A-15m and P6 resins were purchased from Bio-Rad.

Buffers-- Buffer A is 20 mM Tris-HCl (pH 7.5), 0.5 mM EDTA, 2 mM DTT, and 10% glycerol (v/v). Gel filtration buffer is 20 mM Tris-HCl (pH 7.5), 0.5 mM EDTA, 2 mM DTT, 10 mM MgCl2, 100 µg/ml bovine serum albumin, and 4% glycerol (v/v). Reaction buffer is 20 mM Tris-HCl (pH 7.5), 0.1 mM EDTA, 5 mM DTT, 4% glycerol (v/v), and 40 µg/ml bovine serum albumin. ATPase Buffer is 20 mM Tris-HCl (pH 7.5), 0.1 mM EDTA, 5 mM DTT, and 10% glycerol (v/v).

Equilibrium Gel Filtration-- Analysis of ATP binding to wild-type and mutant gamma  complexes was performed by equilibrium gel filtration as described (41). Wild-type and either the gamma  (R169A) gamma  complex or the delta ' (R158A) gamma  complex (8.3 µM gamma  complex in 60 µl; 6.6 µM gamma  (R169A) complex in 150 µl; 6.6 µM delta ' (R158A) gamma  complex in 150 µl) were incubated in gel filtration buffer plus 100 mM NaCl containing [alpha -32P]ATP at the indicated concentration (0.1-10 µM) for 15 min. at 25 °C. Samples were then applied to a 5-ml Biogel P-6 column (Bio-Rad) at 25 °C pre-equilibrated in gel filtration buffer plus 100 mM NaCl having the same concentration of [alpha -32P]ATP as the respective sample. Thirty-five fractions of 240 µl each were collected, and 100 µl of each fraction was analyzed by liquid scintillation to determine the total amount of ATP ([ATP]TOTAL, see below). 50 µl of the peak fractions were also analyzed for total protein concentration by the Bradford assay (Bio-Rad) using gamma  complex as a standard. Scatchard analysis from equilibrium gel filtration data was as described (42).

Gel Filtration Analysis of gamma  Complex·beta Interaction-- The ability of the wild-type and mutant gamma  complexes to associate with beta  was analyzed by gel filtration on a fast protein liquid chromatography (FPLC) Superose 12 column (Amersham Biosciences). beta  (30 µM, 480 µg) was incubated alone or with the gamma  complex (25 µM, 1.25 mg) for 15 min. at 15 °C in 200 µl of Buffer A plus 100 mM NaCl containing 1 mM ATP and 10 mM MgCl2. The mixture was then injected onto a 24-ml Superose 12 column equilibrated in the same buffer at 4 °C. After collecting the first 5.8 ml (void volume), fractions of 155 µl were collected and analyzed in a 14% SDS-polyacrylamide gel.

ATPase Assays-- Wild-type and mutant gamma  complexes were tested for ATPase activity in the presence of the synthetic primed template with or without beta . ATPase assays contained 50 nM gamma  complex, 1 mM [alpha -32P]ATP, 200 nM beta  dimer (when present), and 500 nM synthetic primed template DNA in a final volume of 60 µl of ATPase buffer. The synthetic primer/template DNA is linear and thus allows beta  to slide off the ends after it is loaded. Thus, beta  is continuously recycled during these assays as demonstrated previously (43). Reactions were brought to 37 °C and initiated upon the addition of the gamma  complex. Aliquots of 5 µl each were removed at intervals (0-10 min) and quenched with an equal volume of 0.5 M EDTA (pH 7.5). One microliter of each quenched aliquot was spotted on a polyethyleneimine cellulose TLC sheet (EM Science) and developed in 0.6 M potassium phosphate buffer (pH 3.4). The TLC sheet was dried and [alpha -32P]ATP and [alpha -32P]ADP were quantitated using a PhosphorImager (Amersham Biosciences).

Clamp Loading-- Clamp loading was measured by separating 32P-beta PK on DNA from free 32P-beta PK using BioGel A15m, a large pore resin that excludes large DNA substrates but includes protein. 32P-beta PK (13.3 nM as dimer) was incubated for 10 min. at 37 °C either alone or with mutant or wild-type gamma  complex (10.7 nM) in 75 µl of reaction buffer containing primed M13mp18 ssDNA (13.3 nM), SSB (3.2 µM as tetramer), 1 mM ATP, and 10 mM MgCl2. The reaction was applied to a 5-ml BioGel A15m column (Bio-Rad) equilibrated in gel filtration buffer plus 50 mM NaCl at 25 °C. Thirty-five fractions of 180 µl each were collected, and 100 µl was analyzed by liquid scintillation. 32P-beta PK bound to the DNA elutes early (fractions 11-15), whereas the free 32P-beta PK elutes later (fraction 17-28). The amount of beta  in each fraction was determined from its known specific activity.

Replication Activity Assays-- The activity of wild-type and mutant gamma  complexes was assayed by the requirement to load beta  onto a primed circular M13mp18 ssDNA template in order to observe nucleotide incorporation by the core polymerase (alpha epsilon theta subunits). The reaction mixture contained core polymerase (5 nM), beta  (10 nM as dimer), SSB (420 nM tetramer), primed M13mp18 ssDNA (1.1 nM), 60 µM each of dATP, dCTP, and dGTP, 20 µM [alpha -32P]TTP, 1 mM ATP, and 10 mM MgCl2 in 25 µl of reaction buffer (final volume). Replication was initiated upon the addition of either the wild-type or the mutant gamma  complex (0-1.28 nM titration) and incubated at 37 °C for 5 min. Reactions were quenched upon the addition of 25 µl of 1% SDS and 40 mM EDTA. Quenched reactions were spotted onto DE81 (Whatman) filters and then washed and quantitated by liquid scintillation as described (33).

    RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Reconstitution of gamma  (R169A) Complex-- Arginine 169 is located in the highly conserved SRC motif of the gamma  subunit. To assess the importance of gamma  arginine 169 to clamp-loading activity, we mutated it to alanine and purified the gamma  (R169A) protein from an overproducing strain of E. coli. To study the effect of this mutation on clamp loader activity, we reconstituted the gamma  complex using gamma  (R169A) with delta , delta ', chi , and psi . Our previous studies have shown that a fully assembled gamma  complex is stable to ion exchange chromatography on an fast protein liquid chromatography MonoQ column, where it elutes much later than the free subunits (11). The gamma  (R169A) mutant was mixed with an excess of delta , delta ', chi , and psi , incubated for 30 min at 15 °C, and then applied to a MonoQ column followed with a gradient of NaCl. The result, in Fig. 2A, demonstrates that the gamma  (R169A) mutant forms a "gamma (R169A) complex" in which all five subunits co-elute in fractions 41-49, whereas the excess subunits elute much earlier. As demonstrated later in this report (Fig. 4), the gamma  (R169A) complex also remains intact during analysis on a gel filtration column. The subunit ratio of the gamma  (R169A) complex is comparable with that of the wild-type gamma  complex as observed in the Coomassie Blue-stained SDS polyacrylamide gel of Fig. 2B. This ability of gamma  (R169A) to assemble into a multisubunit complex with delta , delta ', chi , and psi  demonstrates that the gamma  (R169A) mutant is properly folded. It also provides reconstituted gamma  (R169A) complex for the studies to follow.


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Fig. 2.   Reconstitution of the gamma  (R169A) complex. A, gradient elution of reconstituted gamma  (R169A) complex from a Mono-Q column. Note the excess free chi psi (fractions 17-21)and delta delta '(fractions 31-33) subcomplexes that elute early in the gradient compared with the full gamma  (R169A) complex (fractions 41-49) made with limiting the gamma  (R169A) subunit. B, reconstituted wild-type gamma  complex and gamma  (R169A) complex have comparable subunit stoichiometries as analyzed in a 14% SDS-polyacrylamide gel stained with Coomassie Blue.

The gamma  (R169A) Complex Binds Three Molecules of ATP with Similar Affinity as the Wild-type-- Next, we determined whether gamma  (R169A) complex binds ATP and, if so, whether it binds ATP with similar stoichiometry and affinity compared with the wild-type gamma  complex. To address these issues, we used the equilibrium gel filtration technique. In this analysis, a gel filtration column is equilibrated with a known concentration of [32P]ATP. The gamma  complex is incubated with the same concentration of [32P]ATP present in the column buffer and is then applied to the column. Fractions are collected, and the amount of protein and [32P]ATP in each fraction is determined. Protein-bound [32P]ATP is carried around the beads, resulting in a peak of [32P]ATP that elutes early and is followed later by a trough that has less [32P]ATP than the column buffer due to its displacement from the buffer by the protein. This information can be used to calculate the Kd value for ATP binding to the complex. However, a more accurate assessment of the Kd value can be obtained by repeating the experiment at a variety of ATP concentrations (the column is equilibrated at different ATP concentrations) followed by plotting the data as a Scatchard plot. This detailed analysis also carries the advantage of providing the stoichiometry of ATP bound to the complex.

The results, in Fig. 3, show that gamma  (R169A) complex binds three molecules of ATP with similar affinity as wild-type gamma  complex (Kd, 1-2 µM). The data for both the wild-type and the gamma  (R169A) complex fall on a relatively straight line, indicating that the three sites bind ATP with similar affinity. This conclusion is also supported by a study of a monomeric gamma  subunit (missing the C-terminal oligomerization domain) that binds ATP with a Kd value of 1.36 µM, determined by isothermal calorimetry (44). Overall, these results indicate that the two ATP sites that carry alanine residues in place of arginine 169 (sites 2 and 3) still bind ATP and that this arginine residue contributes little, if any, to the binding affinity of ATP to the gamma  complex.


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Fig. 3.   The gamma  (R169A) complex binds three ATP molecules with similar affinity as the wild-type gamma  complex. Wild-type gamma  complex (A) and gamma  (R169A) complex (B) were analyzed for ATP binding affinity and stoichiometry by equilibrium gel filtration as described under "Experimental Procedures." The concentration of ATP (diamonds) and gamma  complex (black area curve) were measured in column fractions for a series of experiments performed at different concentrations of ATP. The Scatchard plot at the top is derived from the series of column analyses (each data point is one column analysis). The 10 µM ATP analysis is included in the Scatchard plot but is not shown in the profile below the plots. The line is the least squares fit to the data. A, wild-type gamma  complex analysis yields a Kd of 0.9 µM and stoichiometry of 3.2 ATP/gamma complex. B, gamma  (R169A) complex analysis yields a Kd value for ATP binding of 2.10 µM with a stoichiometry of 3.2 ATP/gamma (R169A) complex.

gamma (R169A) Complex Binds beta -- Previous studies have demonstrated that ATP binding to the gamma  complex induces the conformation change that leads to the binding of the beta  subunit (21). This predicts that the gamma  (R169A) complex, which binds ATP, should be capable of binding to beta . To test this prediction, we analyzed a mixture of the gamma  (R169A) complex and beta  for complex formation on a Superose 12 sizing column equilibrated with buffer containing ATP. beta  alone migrates in fractions 37-43 (Fig. 4A). Analysis of a mixture of wild-type gamma  complex and beta  is shown in Fig. 4B; the beta  subunit co-elutes with the large gamma  complex in fractions 22-31 and resolves from unbound beta , which elutes in the later fractions. A similar analysis using gamma  (R169A) complex, shown in Fig. 4C, demonstrates that the mutant gamma  complex is also capable of associating with beta . The amount of beta  that comigrates with the gamma  (R169A) complex is nearly the same compared with that of the wild-type gamma  complex, indicating that it is capable of binding beta , although its affinity for beta  may be somewhat decreased by the mutation. ATP binding, not hydrolysis, powers all the steps of clamp loading except the final stage of dissociating from the beta ·DNA complex, allowing beta  to close around the DNA (21). This last step requires ATP hydrolysis. Next, the mutant gamma  complex was studied for its ability to hydrolyze ATP.


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Fig. 4.   ATP promotes beta  binding in the gamma  (R169A) complex. The interaction between beta  and the gamma  complex was analyzed by gel filtration on a Superose 12 column to separate free beta  (fractions 37-43) from beta  bound to the gamma  complex (fractions 22-31). A, beta  alone in the presence of ATP. B, beta  and wild-type gamma  complex with ATP. C, beta  and the gamma  (R169A) complex with ATP.

The gamma  (R169) Is Essential to Catalysis-- We next analyzed the gamma  (R169A) complex in three different assays, each of which require ATP hydrolysis. The first activity to be tested was DNA-dependent ATPase activity of the gamma  complex followed by clamp loading and, finally, beta -dependent DNA synthesis by core DNA polymerase.

The gamma  complex requires the presence of DNA for significant ATPase activity (45). The beta  subunit stimulates gamma  complex ATPase activity provided a primed DNA, not ssDNA, is present (45). In the experiments of Fig. 5A, we examined the gamma  complex ATPase activity using primed DNA with and without beta . Whereas the wild-type gamma  complex hydrolyzes ~217 molecules of ATP per minute in the presence of the primed template, the gamma  (R169A) complex shows no detectable ATPase activity (i.e. detection limit is ~5 ATP hydrolyzed/min/gamma complex). The beta  subunit stimulates the ATPase activity of the wild-type gamma  complex in the presence of primed DNA as illustrated in Fig. 5A. However, beta  does not provide detectable ATPase activity by the gamma  (R169A) complex in the presence of the primed template (Fig. 5A), even when a very large excess of beta  is present (2 µM; data not shown).


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Fig. 5.   Analysis of catalytic activity by gamma  (R169A) complex. Wild-type and gamma  (R169A) complex clamp loaders were tested in three assays that each required hydrolysis of ATP. A, steady-state ATPase assays in the presence of synthetic primed template with (+) and without (-) beta . Squares and diamonds represent the wild-type gamma  complex plus and minus beta , respectively. Circles and open triangles represent the gamma  (R169A) complex plus and minus beta , respectively. B, in the clamp loading assays, 32P-beta was incubated with the wild-type gamma  complex (triangles) or the gamma  (R169A) complex (squares) in the presence of SSB-coated singly primed M13mp18 ssDNA, and then DNA-associated 32P-beta was separated (fractions 11-16) from free 32P-beta (fractions 17-33) on a BioGel A15m column. The open circles are a control in which only the DNA substrate was omitted from the reaction. Above the plot is an ethidium bromide-stained neutral agarose gel analysis, which demonstrates that the large DNA substrate alone elutes in fractions 11-16. C, the wild-type gamma  complex (triangles) or the gamma  (R169A) complex (squares) was titrated into replication reactions containing beta , core, SSB-coated singly primed M13mp18 ssDNA, and 32P-dNTPs. Incorporation of 32P-dTTP indicates that the beta  clamps are loaded onto DNA for processive replication by core polymerase.

The absence of catalytic activity predicts that the mutant gamma  complex will be incapable of loading a beta  clamp onto primed DNA. To assay beta  clamp-loading activity directly, we radiolabeled beta  with 32P using a derivative of beta  that carries an N-terminal tag with a kinase site. The 32P-beta was incubated with the gamma  complex and primed M13mp18 ssDNA (SSB coated), and then the reaction was analyzed for assembly of 32P-beta onto the primed DNA by gel filtration on a BioGel A15m column. This resin has large pores that include proteins but exclude the large M13mp18 primed ssDNA (see agarose gel analysis). As a result, 32P-beta bound to DNA elutes early and resolves from free 32P-beta that is not bound to DNA. A control reaction using the wild-type gamma  complex is shown in Fig. 5B. Most of the 32P-beta elutes with the DNA in fractions 11-16. An agarose gel analysis, shown in Fig. 5B, confirms that the large SSB-coated DNA substrate alone elutes in these same fractions (11-16). However, repetition of this analysis using mutant gamma  complex did not result in the assembly of 32P-beta on the DNA, and instead the 32P-beta eluted later in fractions 17-33. A control reaction lacking the DNA template confirmed that free 32P-beta elutes in fractions 17-33 (Fig. 5B). Analysis of gamma  complex migration shows that it elutes in the included fractions in the same position as free beta , consistent with the large pore size of the A15m resin (not shown, but as in Fig. 5 of Ref. 16). Hence, the gamma  (R169A) complex is inactive for clamp loading action, consistent with its lack of ATPase activity.

Finally, the gamma  (R169A) complex and beta  were tested for the ability to support DNA synthesis by core polymerase. The core Pol III is incapable of extending a primer around an SSB-coated primed M13mp18 ssDNA template unless it is coupled to a beta  clamp and thus provides another measure of clamp-loading activity. Use of the wild-type gamma  complex yielded a strong signal in this assay but, as expected, when the mutant gamma  complex was used in this assay no activity was detected, which is consistent with the inactivity of mutant gamma  complex in ATPase and clamp-loading assays (Fig. 5C). These results support and extend those of an earlier analysis in which several conserved residues of gamma  were mutated (46). Mutation of gamma  R169 resulted in the loss of replication and ATPase activity and failed to complement a conditional lethal dnaX gene in vivo, although that study did not demonstrate that the mutant gamma  was properly folded or that it retained the ability to form a stable complex with delta  and delta ', bind ATP, and associate with beta .

The delta ' SRC Motif-- The above results strongly support an essential role of the gamma  arginine 169 in catalysis. However, the results imply something further. Mutation of gamma  arginine 169 only eliminates this catalytic residue at ATP sites 2 and 3. The putative catalytic arginine of ATP Site 1 is supplied by delta ', not gamma , and therefore this site should remain competent for ATP hydrolysis even in the gamma  (R169A) complex. Even though the gamma  (R169A) complex retains one intact ATP site, the results show that it has lost essentially all of its ATPase activity. This implies that the ATP bound to sites 2 and/or 3 must first be hydrolyzed before the ATP in site 1 is hydrolyzed. Alternatively, site 1 binds ATP but is simply not catalytic. A non-catalytic site 1 would explain why the gamma  (R169A) complex has no ATPase but would not explain why the SRC motif is broadly conserved in prokaryotic delta ' subunits. To test whether arginine 158 in the SRC motif of delta ' is important to catalysis, this arginine was mutated to alanine, and the delta ' (R158A) mutant was purified and reconstituted into the gamma  complex for analysis. If ATP site 1 is not hydrolytic, the delta ' (R158A) gamma  complex should have wild-type levels of ATPase and clamp loading activity.

The delta ' (R158A) mutant was mixed with gamma , delta , chi , and psi  to reconstitute the "delta ' (R158A) gamma  complex." The complex was stable to ion exchange chromatography, yielding a purified reconstituted delta ' (R158A) gamma  complex with a similar subunit stoichiometry as that of the wild-type gamma  complex (Fig. 6A). ATP binding analysis by equilibrium gel filtration demonstrated that the delta ' (R158A) gamma  complex retained ability to bind three ATP molecules with similar affinity as the wild-type gamma  complex (Fig. 6B; Kd, ~0.8 µM). The delta ' (R158A) gamma  complex also remained capable of forming a complex with beta  in the presence of ATP (Fig. 6C). In contrast to the gamma  (R169A) complex, the delta ' (R158A) gamma  complex retained some activity in the catalytic assays, requiring that ATP be hydrolyzed. The delta ' (R158A) gamma  complex retained ~30% of the DNA-dependent ATPase activity of the wild-type gamma  complex, although beta  no longer stimulated the ATPase as it did in the wild-type gamma  complex (Fig. 6D). Also, the DNA synthetic activity of the delta ' (R158A) gamma  complex was about 20-30% active compared with that of the wild-type gamma  complex (Fig. 6E). Hence, the delta ' (R158A) gamma  complex retains some clamp-loading activity, but is nevertheless significantly compromised compared with the wild-type gamma  complex.


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Fig. 6.   The delta ' (R158A) gamma  complex is impaired in clamp loading and ATPase activity. A reconstituted delta ' (R158A) gamma  complex was analyzed for ATP binding, beta  interaction, ATPase activity, and clamp loading. A, the reconstituted delta ' (R158A) gamma  complex has a similar ratio of subunits as the wild-type gamma  complex. B, equilibrium gel filtration demonstrates that the delta ' (R158A) gamma  complex binds 2.7 ATP molecules with a Kd value of 0.8 µM. C, the delta ' (R158A) gamma  complex binds beta  in a Superose 12 gel filtration analysis in the presence of ATP and magnesium. D, steady-state ATPase assays show that the delta ' (R158A) gamma  complex (diamonds, without (-) beta ; open squares, with (+) beta ) has reduced ATPase activity compared with the wild-type gamma  complex (~35% -beta , and ~15% +beta ). The activity of the wild-type gamma  complex is shown for comparison. (triangles, -beta ; closed circles, +beta ). E, DNA replication assays reflect the level of clamp loading activity by the delta ' (R158A) gamma  complex (diamonds) compared with the wild-type gamma  complex (triangles).

The above results demonstrate that ATP site 1 is catalytic, because the delta ' (R158A) gamma  complex is significantly less active than the wild-type gamma  complex. If site 1 was only used for ATP binding, the delta ' (R158A) gamma  complex would have been expected to be fully active in the catalytic assays. Hence arginine 158 of delta ' is important to the catalytic activity of the gamma  complex, consistent with conservation of the SRC motif among prokaryotic delta ' subunits.

    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

The delta ' Stator Contributes a Catalytic Arginine to the Clamp Loader Motor-- The gamma  complex clamp loader has been proposed to consist of three main components (4, 6), i.e. the delta  wrench (opens beta ), the gamma  trimer motor (hydrolyzes ATP), and the delta ' stator (modulates beta  interaction with delta ) (reviewed in Ref. 47). The gamma  and delta  subunits appear to have a flexible joint between the C-terminal domain (domain III) and the N-terminal domains (domains I/II). In contrast, the three domains of the delta ' stator appear to be held in a rigid conformation and, thus, the term stator, the stationary part of a machine upon which the other parts move (4, 44). The C-terminal domains of all five subunits form a tight closed circular connection, holding the subunits together. However, the N-terminal domains have an interruption between delta  and delta '. The size of this gap modulates the ability of beta  to interact with the delta  wrench. The rigid delta ' stator is proposed to function as an anvil, and when ATP is hydrolyzed the gamma  subunits move delta  close to delta ', forcing the beta  ring off the delta  wrench and allowing the beta  ring to close.

This report demonstrates that delta ', besides its role as stator, also plays an instrumental role in the motor function of the gamma  complex by supplying a catalytic arginine into ATP site 1 of the gamma  complex. A catalytic role for this arginine was suggested by its proximity to ATP modeled into ATP site 1 of the gamma  complex structure (4). Furthermore, this arginine is embedded in an "SRC" motif that is highly conserved among clamp-loading subunits of prokaryotes, eukaryotes, and archaebacteria. The E. coli delta ' subunit is a member of the AAA+ family and has the same chain fold as gamma , yet delta ' does not bind ATP (48). The P-loop of delta ' has been modified through evolution, and the N terminus blocks the nucleotide binding site. However, there are examples of prokaryotic delta ' subunits that contain a consensus P-loop (i.e. Aquifex aeolicus; Ref. 49). Whether these delta ' subunits bind ATP is not known. However, even if these delta ' subunits bind ATP they may not hydrolyze it for lack of a catalytic arginine in the neighboring delta  subunit. Perhaps noncatalytic ATP provides rigidity to these delta ' subunits without needing the extra connections between domains III and I/II observed in the E. coli delta ' stator.

The catalytic role played by delta ' in clamp-loading ATPase action may explain why the delta ' sequence is highly conserved in prokaryotes compared with the sequence of delta . The delta  subunit is the main subunit responsible for opening the beta  clamp, but it has no catalytic role (16, 19). Simple maintenance of protein-protein contacts, with no catalytic role to preserve, has apparently allowed the delta  sequence to drift considerably. The catalytic role played by delta ' may be responsible for the much greater conservation of the delta ' sequence.

An Ordered Hydrolysis Model for Clamp Loading-- This report demonstrates that the mutation of arginine 169 in gamma , which removes the arginine of the SRC motif near ATP sites 2 and 3, abolishes ATPase activity even though three ATP molecules still bind the mutant gamma  complex and the ATP site 1 remains intact. This result suggests that Site 1 cannot hydrolyze ATP until after the ATP in sites 2 and/or 3 is hydrolyzed. Conversely, if ATP must be hydrolyzed in sites 2 and/or 3 before the ATP is hydrolyzed in site 1, then an ATP site 1 mutant may not block the hydrolysis of ATP in sites 2 and 3. Indeed, this expectation is largely upheld in this study. The delta ' (R158A) gamma  complex retains significant ATPase activity, indicating that ATP in sites 2 and/or 3 can be hydrolyzed even when site 1 is missing the catalytic arginine. The delta ' (R158A) gamma  complex also retains some clamp-loading activity, allowing it to support processive DNA synthesis. Thus, the hydrolysis of ATP in sites 2 and 3 would appear to be sufficient, although not optimal, for clamp loading.

Ordered hydrolysis of ATP starting at sites 3 (or 2) and ending with site 1 is the opposite order that is predicted for ATP binding. Study of the gamma 3delta delta ' structure (4) suggested that the sites may fill starting at site 1 and ending with site 3. A model encompassing an ordered binding and a reverse order of hydrolysis, as the current study suggests, is illustrated in Fig. 7. As ATP binds to sites 1, 2, and then 3, the gap between the delta  and delta ' subunits is proposed to increase to accommodate interaction of delta  with the beta  dimer. The delta  subunit wrench then cracks one interface of the beta  ring, allowing the spring tension between the domains of the beta  ring to relax, thereby opening the ring for DNA strand passage. Upon association of a primed template through the open beta  ring, the arginines of the SRC motifs in gamma  and delta ' align for ATP hydrolysis. The current study indicates that ATP hydrolysis occurs first at site 3 (or 2) before hydrolysis at site 1. This order may be achieved via proper positioning of the catalytic Arg residues. Specifically, hydrolysis at site 3 may be required in order for the arginine in site 2 to become properly aligned, and hydrolysis in site 2 may be required for the arginine in site 1 to be aligned. The threading of a primed site through beta  may align the arginine in site 3 to start the hydrolysis cycle (discussed further, below).


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Fig. 7.   Sequential ATP binding and hydrolysis during the clamp loading operation. Diagram A, in the absence of ATP, the gamma  complex adopts a conformation that does not bind beta  because of proximity between delta  and delta '. In going from diagrams A to D, ATP binds to gamma 1, gamma 2, and then gamma 3, resulting in a conformation change that separates delta  from delta ' and creates a docking site for beta  binding to delta  and the gamma  subunits. In diagram E, primed template is bound by the gamma  complex beta -ATP. Upon sensing that the primed DNA passes through the central cavity of beta , ATP is hydrolyzed sequentially, first at site 3 (diagram F), then site 2 (diagram G) and finally site 1 (diagram H), bringing delta  back in proximity to delta ', closing the beta  ring around DNA, and ejecting the gamma  complex from beta . Upon ADP release, the gamma  complex reassumes the ground state, ready for another round of clamp loading (diagram A).

The proposed order of ATP hydrolysis in the model proceeds first from site 3, then 2, and finally to site 1. However, it is also possible that site 2 fires first. We would like to construct different gamma  complexes containing all the permutations of single and double ATP site mutants but are prevented from this strategy by the fact that sites 2 and 3 are formed solely by identical gamma  subunits.

Generalization of These Results to Other Clamp Loaders-- The studies of this report on the E. coli clamp loader have implications for subunit function in clamp loaders of other organisms. The eukaryotic RFC heteropentamer is composed of five different subunits, three of which contain both the SRC motif and consensus P-loops, suggesting that they may act similarly to the gamma  trimer motor (yeast RFC 2·3·4 and human p36·37·40) (50). Indeed, these complexes contain DNA-dependent ATPase activity (51, 52). One RFC subunit (yRFC5 and human p38) contains the SRC motif but lacks a consensus P-loop and thus is analogous to the delta ' stator and may contribute a catalytic arginine for ATP hydrolysis in the RFC2, 3, and 4 motors. The yRFC1 subunit (human p128) forms a strong attachment to the PCNA clamp (53) and thus may be analogous to the delta  wrench. Like E. coli delta , yRFC1 lacks an SRC motif, but unlike delta  it contains a consensus P-loop, suggesting that it binds ATP. Mutation of this P-loop is without significant effect on yRFC activity, indicating that this fourth ATP site in the eukaryotic clamp loader may be coupled to some other process (54).

Studies of P-loop mutants of yRFC2, 3, and 4 show that mutation of either RFC 2 or 3 greatly reduces ATPase activity and clamp-loading function of RFC, whereas mutation of RFC4 has much less of an effect on these activities (50). These results are similar to the current study of the gamma  complex; namely, the delta ' (R158A) gamma  complex is partially active, whereas the gamma  (R169A) complex is completely inactive. Thus, it seems quite possible that the RFC clamp loader may also hydrolyze ATP in an ordered sequence around the circular pentamer, as proposed here for the gamma  complex, wherein some sites must first hydrolyze ATP before ATP in other sites can be hydrolyzed.

The five subunit clamp loader of bacteriophage T4 has two different subunits, four copies of gp44 and one gp62 subunit. Biochemical studies demonstrate that ATP is hydrolyzed to load the T4 gp45 clamp onto DNA; stoichiometry measurements range from 1-4 ATP per gp45 clamp-loading event (55-58). The gp44 tetramer is an ATPase, contains both the SRC motif and P-loop, and is homologous to E. coli gamma  and delta '. The gp62 is similar to delta  in that it has neither the SRC motif nor P-loop, and its sequence has diverged from gamma /delta '. At first glance it would seem that the T4 clamp loader has done away with the stator and, indeed, it may have. However, keeping in mind that gp62 has no SRC motif, it seems likely that one gp44 subunit (i.e. adjacent to gp62) will be incapable of hydrolyzing ATP even if it binds ATP. Thus, one gp44 subunit may serve a similar role as delta ' does in modulating contact between the gp44/62 clamp loader and the gp45 clamp at the same time as it provides a catalytic arginine residue for ATP hydrolysis in the neighboring gp44 subunit.

The clamp loader of archaebacteria has been studied, but less intensively than the clamp loaders of E. coli, T4, yeast, and humans. Generally, archaebacterial clamp loaders (called RFC) consist of two subunits, RFC large and RFC small (59). The stoichiometry of these subunits is not certain, with reports ranging from 1:4, like T4, to 3:2 and even 4:2 (60-62). The crystal structure of the Pyrococcus RFC small subunit shows that its basic unit is a trimer, presumably the equivalent to the gamma  trimer motor in which each subunit contains both a nucleotide binding site and a closely juxtaposed arginine from the neighboring subunit (63). The Pyrococcus RFC large subunit contains a consensus P-loop motif and thus may bind nucleotide, but it lacks the SRC motif. The lack of a SRC motif in the RFC large subunit suggests that if there are two of these subunits in the clamp loader, then one of these will be unable to hydrolyze ATP and thus may function in an analogous fashion as the delta ' stator. Alternatively, if only one RFC large subunit is present, an adjacent RFC small subunit will not hydrolyze bound ATP, thereby acting as a stator. In this second scenario, the stator would also contain an SRC motif, like delta '.

Possible Role of the SRC Arginines in Clamp-loading Fidelity-- The gamma  complex is a very poor ATPase without a DNA effector. The crystal structure indicates that gamma  R169 and delta ' R158 are not quite close enough to function with ATP, and they must move an extra one or two angstroms to have an effect on ATP hydrolysis (4). Hence, it is tempting to speculate that misalignment of these arginines may underlie the very weak ATPase of the gamma  complex, and their proper positioning may be used as a regulatory mechanism.

DNA stimulates the gamma  complex ATPase activity and, thus, may bring the SRC motif Arg/ATP site pairs into a more favorable alignment for hydrolysis. Curiously, the beta  subunit only stimulates the gamma  complex ATPase when a primed template is used as an activator; ssDNA and duplex DNA stimulate the gamma  complex in the absence of beta  but do not give more activity when beta  is added (11). Consistent with this observation, the gamma  complex does not load beta  onto ssDNA, even though ssDNA stimulates ATPase activity (64). Furthermore, the ATPase cycle is tightly coupled to clamp loading, as only 2-3 ATPs are hydrolyzed for each beta  clamp that is loaded onto a primed template (16, 21). Finally, the head-to-tail architecture of the beta  dimer generates two distinct "front" and "back" faces, only one of which functions with the DNA polymerase and, thus, it must be oriented correctly on DNA to interface with the polymerase (34, 39). What system of checks and balances does the gamma  complex have to ensure that these criteria have been met? It seems possible that the catalytic arginines and their juxtaposition to the ATP sites may act as a fidelity mechanism to ensure that beta  is only loaded when primed DNA is threaded through beta  and only when beta  is oriented correctly for function with polymerase. Perhaps when these different criteria are met the catalytic SRC motif arginine residues are brought into register for ATP hydrolysis to propel loading of the beta  clamp onto DNA.

    FOOTNOTES

* 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.

§ To whom correspondence should be addressed: The Rockefeller University, 1230 York Ave., Box 228, New York, NY 10021-6399. Tel.: 212-327-7255; Fax: 212-327-7253; E-mail: johnsoa@mail.rockefeller.edu.

Published, JBC Papers in Press, February 10, 2003, DOI 10.1074/jbc.M212708200

    ABBREVIATIONS

The abbreviations used are: SSB, single-stranded DNA-binding protein; AAA+, ATPases associated with a variety of cellular activities; PK, protein kinase; DDT, dithiothreitol; ssDNA, single-stranded DNA; RFC, replication factor C; P-loop, phosphate binding loop.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
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