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J. Biol. Chem., Vol. 278, Issue 41, 40272-40281, October 10, 2003
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Wrench in Opening the 
Sliding Clamp*
¶
From the
Rockefeller University, Howard Hughes Medical Institute, Laboratory of DNA Replication, New York, New York 10021
Received for publication, June 3, 2003 , and in revised form, July 1, 2003.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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sliding clamp encircles DNA and tethers DNA polymerase III holoenzyme to the template for high processivity. The clamp loader, 
complex (3
'
), assembles around DNA in an ATP-fueled reaction. The subunit of the clamp loader opens the ring and is referred to as the wrench; ATP modulates contact between and among other functions. Crystal structures of · and the 3' minimal clamp loader make predictions of the clamp loader mechanism, which are tested in this report by mutagenesis. The wrench contacts at two sites. One site is at the dimer interface, where appears to distort the interface by via a steric clash between a helix on and a loop near the interface. The energy for this steric clash is thought to derive from the other site of interaction, in which binds to a hydrophobic pocket in . The current study demonstrates that rather than a simple steric clash with , specifically contacts at this site, but not through amino acid side chains, and thus is presumably mediated by peptide backbone atoms. The results also imply that the interaction of at the hydrophobic site on contributes to destabilization of the dimer interface rather than acting solely as a grip of on . Within the complex, ' is proposed to prevent from binding to in the absence of ATP. This report demonstrates that one or more subunits also contribute to this role. The results also indicate that ' acts as a backboard upon which the subunits push to attain the ATP induced change needed for the wrench to bind and open the ring. |
INTRODUCTION |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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In Escherichia coli, the -clamp is a ring-shaped dimer formed by two crescent-shaped protomers that encircle the duplex (6). Each protomer consists of three domains, each of which have the same chain fold (7). This gives the dimer a 6-fold appearance. The ring binds to the replicative DNA polymerase III core (Pol III)1 and tethers it to the template for high processivity. The clamp is opened and closed around the DNA by the complex clamp loader.
The minimal complex clamp loader machine (reviewed in Ref. 5) consists of five different subunits: , ', and three copies of (
) that are arranged as a circular heteropentamer (Fig. 1A) (8). In addition, two other subunits, and , are associated with the clamp loader, but they are not required for clamp loading in vitro (9). In order for this clamp loader to bind two molecules of DNA polymerase III core, two of the subunits are replaced by two subunits. and are encoded by the same gene (dnaX); is the full-length product, and is a truncated version produced by a translational frameshift (10-12). The unique 24-kDa C terminus of , absent in , binds DNA polymerase III core (13, 14) and DnaB helicase (15, 16), thereby acting to organize the replisome machinery.
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The () subunits of the complex are the only subunits that hydrolyze ATP (1) and therefore constitute the motor of the clamp loading machine. The subunit is referred to as the wrench of the clamp loader, since it can open the dimer at one interface on its own (17-20). The energy for ring opening is not derived from ATP (neither nor bind ATP) but from the energy of protein-protein interaction between and (17). In the absence of ATP, the complex does not bind (18). Study of the ' subunit shows that it modulates the ability of to bind even in the absence of other complex subunits (17, 19). Thus, ' is proposed to obscure the subunit within complex from binding to when ATP is not present (17, 20). However, when ATP binds to the subunits, the complex undergoes a conformational change in which it is hypothesized that a portion of ' separates from , allowing to bind and open the ring (18, 20, 22). Only one binds to the 2 dimer (18). Moreover, binds to a monomer mutant of (1) with 50-fold higher affinity than to 2 (19). This result indicates that when interacts with one member of the 2 dimer, it uses a portion of its binding energy to perform work (i.e. to part one of the dimer interfaces), thus lowering the observed affinity of to 2. Furthermore, only one subunit binds 2, suggesting that only one of the dimer interfaces is disrupted by (17-19). Opening at only one 2 interface is also consistent with the observation that 2, cross-linked at only one dimer interface, is efficiently loaded onto DNA by complex (17).
The crystal structure of in complex with a monomer mutant (·1), combined with the structure of the minimal clamp loader (3'), provides further details and allows predictions about how opens 2 (8). Of the three domains of , only the N-terminal domain (domain I) interacts with (Fig. 1B), and it contacts the clamp in two different places. The first site of the - interaction involves hydrophobic contacts between residues Leu73 and Phe74 of and a hydrophobic pocket of located between domains II and III (Fig. 1, B and C). We refer to this as "site 1." Both Leu73 and Phe74 of protrude out to form a hydrophobic plug that fits into the hydrophobic pocket on the surface of . This hydrophobic interaction is presumed to be responsible for the majority of the binding energy. Interestingly, residues Leu73 and Phe74 are the most highly conserved residues among subunits of different bacteria (20, 23). Likewise, alignment of bacterial subunits shows that residues comprising the hydrophobic pocket in to which binds are highly conserved (20).
There is a second interaction between and that involves helix 
4 and loop 1''-2''. This contact is referred to here as "site 2" (Fig. 1C). The interaction of with at this site leads to an extensive conformational change of the loop that is thought to be important for ring opening (20). This five-residue loop of (residues 274-278) is near the interface and in fact connects to the interfacial helix that contains the two residues (Ile272 and Leu273) that form the hydrophobic core of the 2 dimer interface. In contrast, the structure of the ·1 complex shows that the hydrophobic core residues of are rotated out of position, thereby precluding formation of the dimer interface (Fig. 1C). Thus, it would appear that distorts one 2 interface by altering the conformation of the 274-278 loop. This distortion at site 2 appears to be the result of a steric clash between and , which pushes on the loop, rather than being due to specific side chain contacts between and . Hence, the site 2 interaction/steric clash presumably requires an input of energy that is obtained from the binding energy at site 1.
The subunit destabilizes the interface of 2 but does not explain how a gap opens up at the interface for DNA to pass through. The shape of 1 in the ·1 structure relative to the 2 dimer suggests how the ring actually opens (20). Superposition of monomeric (i.e. of the ·1 complex) onto dimeric reveals that the shape of the monomer is less curved than in the dimer. This change in curvature is produced by rigid body motions between the three domains of . The largest rigid body motion occurs distant from the -binding sites, suggesting that the change is intrinsic to and that the closed dimer is under spring tension between domains of . After disruption of the dimer interface by the subunit, release of the spring tension results in the motions between domains that produce the gap at the broken interface (20).
The 3' structure fits nicely with biochemical data indicating that cannot bind complex in the absence of ATP and that ' participates in modulating the - interaction. The 3' structure (8) shows that each of the subunits is composed of three domains having the same chain fold and is a member of the AAA+ family. The five subunits are arranged as a circular heteropentamer (Fig. 1A). The C-terminal domains of all five subunits form a tight closed circular connection, holding the subunits together. On the contrary, the connections between the N-terminal domains contain a gap between and ' (see Fig. 1A). Docking of 2 onto shows that 2 does not fit due to steric occlusion by ' and possibly some of the subunits as well. This is consistent with the fact that ATP is not present in the structure. It is proposed that as the ATP sites fill, conformation changes in are propagated around the pentamer to increase the gap between and ', thereby allowing to bind to for clamp opening (8, 17, 18, 22). In this state, with and ATP bound to complex, a tight affinity for DNA is established (22, 24). Upon recognizing a primed site, the ATP is hydrolyzed and the subunits may move back into proximity with ', forcing the ring off the wrench and allowing the ring to close around the DNA.
The ' structure was the first clamp loader subunit to be solved (25). ' appears to be more rigid than the and subunits (8). This conclusion derives from the observation that the three domains of ' in complex are oriented nearly the same as in the ' alone. In contrast, the relative orientations of domain III relative to domains I/II of all three subunits are different in 3', and the same is true for in ·1 compared with in the 3' structure. Consistent with a rigid structure, ' has additional connections between domains compared with the few connections between the domains in either or . This rigid conformation of ' has earned it the title of stator, the stationary part of a machine upon which the other parts move (8). Perhaps the rigid ' stator serves as an anvil for the interactive element of to strike, pushing 2 off of the complex following ATP hydrolysis. A recent report demonstrates that ', besides its role as stator, also plays an instrumental role in the motor function of complex, as predicted by the structure, by supplying a catalytic arginine into the ATP site at the intersubunit junction '/1 (8, 26).
In light of the 3' and ·1 crystal structures and predictions of how these proteins function, we reexamine here the mechanism of clamp loading by the complex, in particular the - interaction, and the role of ' in rendering the interacting elements of accessible for binding the clamp. The studies contain surprises, but overall the results provide significant advancements in our understanding of how this complex machinery functions.
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EXPERIMENTAL PROCEDURES |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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, 
, , (27), (7), and ' (28), and (29), 
(30), SSB (31). Core polymerase and complex were reconstituted from wild-type and/or mutant subunits and purified as described (30). Samples of purified complex were analyzed on a 14% SDS-polyacrylamide gel stained with Coomassie Brilliant Blue G-250. M13mp18 ssDNA was purified as described (32) and primed with a 30-mer DNA oligonucleotide as described (27).
Buffers
Buffer A is 20 mM Tris-HCl (pH 7.5), 0.5 mM EDTA, 2 mM DTT, and 10% glycerol (v/v). Buffer B is Buffer A, except the pH was adjusted to 8.2. Buffer C is 10 mM sodium acetate (pH 7.5), 0.5 mM EDTA, 2 mM DTT. Buffer D is Buffer C, except the pH was adjusted to 6.1. Gel filtration buffer is Buffer A containing 100 mM NaCl, 1 mM ATP, and 10 mM MgCl2. 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. Tris-Sucrose buffer is 50 mM Tris-HCl (pH 7.5), 2 mM EDTA, and 10% sucrose.
Mutant Proteins
N62A, F65A and L73A, F74A—Two different double mutants of were constructed. In one, which we refer to here as 4, the Asn62 and Phe65 residues located on helix 4 were mutated to alanines. In the other, referred to as LF
AA, residues Leu73 and Phe74 were mutated to alanines. These mutants were constructed by DNA oligonucleotide site-directed mutagenesis of the pET expression plasmid (28) and confirmed by sequence.
Expression and purification of 4 and LFAA were performed as follows. Expression plasmids were transformed into competent BL21 (DE3) cells (Novagen). Fresh transformants were grown in 12 liters of LB containing 200 µg of ampicillin/ml to a density of A600 = 0.6 and induced with 1 mM isopropyl-1-thio--D-galactopyranoside. Cells were incubated 4 h with shaking at 37 °C, chilled to 15 °C, and incubated another 20 h with shaking at 15 °C. For cell lysis, cells were brought to a final volume of 300-400 ml with a final concentration of 30 mM spermidine, 100 mM NaCl, and 5 mM DTT in Tris-sucrose buffer. Cells were lysed by two passages through a French Press at 17,000 p.s.i. and insoluble material was removed by centrifugation at 12,000 rpm for 1 h at 4 °C in an SLA1500 rotor. The soluble cell lysate supernatant (Fraction I) was decanted and treated with 0.21 g/ml ammonium sulfate. After stirring for 30 min at 4 °C, the pellet was collected by centrifugation at 12,000 rpm for 30 min in a SLA1500 rotor. The resulting pellet was resuspended in Buffer B. The protein was diluted to a conductivity equal to 60 mM NaCl with Buffer B and then applied to a Heparin-agarose column (Bio-Rad) equilibrated in Buffer B. Protein was eluted with a 100-500 mM NaCl gradient in Buffer B. Fractions containing were pooled (Fraction II) and diluted with Buffer B to a conductivity equivalent to 100 mM NaCl. Particulate matter was removed by centrifugation at 10,000 rpm for 10 min at 4 °C in an SS34 rotor. The supernatant was applied to a MonoQ column equilibrated in Buffer B. The column was then washed with Buffer B before eluting the protein with a 100-500 mM NaCl gradient in Buffer B. Fractions containing were pooled (Fraction III, LFAA mutant: 45 ml, 1.2 mg/ml; 4 mutant: 32 ml, 1.0 mg/ml) and then stored at -80 °C.
'
N—Nucleotides encoding the N-terminal 206 residues of ' were deleted from the pET' expression plasmid (28) by PCR using the following primers: 5'-TG GCG TTG CAT ATG GGA GAT AAC TGG CAG GCT CG-3', which introduces an NdeI site (CAT ATG) and inserts an initiating ATG codon for methionine at residue 207, and 5'-TTA TTG CTC AGC GGT GGC AGC AGC CAA CTC AGC TTC CTT TCG GG-3'. The product encodes the entire C-terminal domain (domain III) of ' but eliminates domains I and II. The resulting PCR product was placed into pETIIa at the NdeI and BamHI sites to yield pET'N.
BL21 (DE3) pET'N cells were grown in 3 liters of LB containing 100 µg of ampicillin/ml to a density of A600 = 0.6 and induced with 0.8 mM isopropyl-1-thio--D-galactopyranoside. Cells were incubated for 2 h with shaking at 37 °C. For cell lysis, cells were brought to a final volume of 300-400 ml with a final concentration of 30 mM spermidine, 100 mM NaCl, and 5 mM DTT in Tris-sucrose buffer. Cells were lysed by two passages through a French press at 17,000 p.s.i., and insoluble material was removed by centrifugation at 12,000 rpm for 30 min at 4 °C in an SLA1500 rotor. The pellet was resuspended in 8 M urea and diluted to 2 M urea with buffer A. Urea was removed in the following step by application to a 40-ml Fast Flow Q-Sepharose column equilibrated in buffer A. Protein was eluted with a 400-ml, 0-500 mM NaCl gradient in Buffer A. Fractions containing 'N were pooled (108 mg) and diluted to a conductivity equivalent to 35 mM NaCl with Buffer A before being applied to a 50-ml Heparin-agarose column (Bio-Rad). Protein was eluted with a 500 ml, 50-600 mM NaCl gradient in Buffer A. Most of the protein was found in the flow-through and in the first few fractions. These were pooled together and applied to an 8-ml MonoQ column equilibrated in Buffer A. Protein was eluted using a 120-ml, 50-600 mM NaCl gradient in Buffer A. Fractions containing 'N were dialyzed against Buffer A containing 50 mM NaCl and then stored at -80 °C (15 ml, 1.4 mg/ml).
loop—An internal deletion of the dnaN gene encoding amino acids 275-278 was made in the pET expression vector by PCR using the following primers: 5'-GAT GCC GGC CAC GAT GCG TCC GGC G-3', which anneals to upstream vector sequence, and 5'-TTA TTG CTC AGC GGT GGC AGC AGC CAA CTC AGC TTC CTT TCG GG-3', which includes a SacII site at nucleotide 834 of dnaN. The resulting PCR product was digested with MfeI/SacII, and the 287-bp fragment was inserted into pET digested with the same enzymes to produce pETloop.
BL21 (DE3) pETloop cells were grown in 3 liters of LB containing 100 µg of ampicillin/ml to a density of A600 = 0.8 and induced with 0.8 mM isopropyl-1-thio--D-galactopyranoside. Cells were incubated for 2 h with shaking in fluted flasks at 37 °C. For cell lysis, cells were brought to a final volume of 300-400 ml with a final concentration of 30 mM spermidine, 100 mM NaCl, and 5 mM DTT in Tris-sucrose buffer. Cells were lysed by two passages through a French press at 17,000 p.s.i., and insoluble material was removed by centrifugation at 12,000 rpm for 30 min at 4 °C in an SLA1500 rotor. Soluble lysate was treated with 0.436 g/ml of ammonium sulfate and stirred at 4 °C for 30 min. The pellet was resuspended and dialyzed overnight against Buffer A. The protein was applied to a Fast Flow Q-Sepharose column equilibrated in buffer A and then eluted with a 0-500 mM NaCl gradient in Buffer A. Peak fractions containing the loop mutant were pooled and then dialyzed against Buffer C overnight. The protein was applied to an SP-Sepharose column equilibrated in Buffer C and eluted with a 0-500 mM NaCl gradient in the same buffer. The protein flowed through the column. The pH of the flow-through was lowered to pH 6.1 on ice by adding acetic acid, and the protein was reapplied to an SP-Sepharose column equilibrated in Buffer D. The loop mutant was eluted with 0-500 mM NaCl gradient in Buffer D. The loop mutant remained in the flow-through and was precipitated with 20% ammonium sulfate. The pellet was resuspended in Buffer A and dialyzed against Buffer A overnight. The protein was then applied to an 8-ml Mono Q column equilibrated in Buffer A. The column was washed extensively with Buffer A before eluting protein using a 160-ml gradient of 0-500 mM NaCl in Buffer A. Fractions containing the loop mutant were pooled and dialyzed overnight against Buffer A. The preparation was passed over a 10-ml ATP-agarose column equilibrated in Buffer A to remove any possible contaminant of complex that binds this column tightly. The loop protein flowed through the column and was stored at -80 °C (22 ml, 1.9 mg/ml).
Interaction of with Complex by Gel Filtration
Interaction between or the loop mutant and wild-type and mutant complex was analyzed by gel filtration on an FPLC-Superose 12 column (Amersham Biosciences). The subunit (25 or 30 µM dimer, as indicated) was incubated with 5 or 25 µM (as indicated) complex (or mutant complex) for 15 min at 15 °C in 200 µl of Buffer A containing 100 mM NaCl in the presence or absence of 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.
Clamp Loading Replication Assays
The clamp loading activity of wild-type and mutant complexes were assayed by their requirement to load onto a primed circular M13mp18 ssDNA template in order to observe nucleotide incorporation by the core polymerase ( subunits). The reaction mixture contained SSB (470 nM tetramer), primed M13mp18 ssDNA (1.3 nM), core polymerase (5 nM), 2 (10-740 nM), complex (0-0.64 nM), 60 µM each of dATP, dCTP, and dGTP, 20 µM [-32P]TTP, 1 mM ATP, and 10 mM MgCl2 in 25 µl of reaction buffer (final volume). In the complex titration assays, core polymerase (5 nM) and (10 nM as dimer) were added to the reaction mixture, and replication was initiated upon the addition of either wild-type or mutant complex (0-0.64 nM titration). In the titration assays, 0.64 nM complex was used, and DNA synthesis was initiated upon the addition of either wild-type or mutant (0-740 nM titration). Reactions were incubated at 37 °C for 5 min and quenched upon the addition of 25 µl of 1% SDS, 40 mM EDTA. Quenched reactions were spotted onto DE81 (Whatman) filters and then washed and quantitated by liquid scintillation as described (27).
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RESULTS |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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Are Essential for Strong - Interaction—The ·1 structure suggests that residues Leu73 and Phe74 of are responsible for most of the binding energy between and the hydrophobic pocket of site 1 in . In the ·1 structure, Leu73 and Phe74 stick out from a loop to form a hydrophobic plug that fits into the hydrophobic pocket on the surface of (Fig. 1B) (20). To assess the importance of these residues to clamp loading activity, we mutated them to alanines and purified the double mutant (LFAA) protein from an overproducing strain of E. coli. The complex was reconstituted using the LFAA mutant along with the ', , , and subunits. The fully assembled mutant complex was purified from excess unbound subunits on a MonoQ anion exchange column. The subunit ratio of (LFAA) complex is comparable with wild-type complex as observed in the Coomassie Brilliant Blue-stained SDS-polyacrylamide gel of Fig. 2A. The (LFAA) complex also remains intact during analysis on a gel filtration column, as shown in the middle panel of Fig. 2C. The ability of LFAA to assemble into a multisubunit complex with , ', , and indicates that the mutant subunit is properly folded. This result is also consistent with the crystal structure of 3', which shows that neither of these residues are involved in subunit-subunit interaction within complex. The major intersubunit connections within 3' occur through the C-terminal domains of each of the five subunits of the complex (8).
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Next, the reconstituted mutant complex was tested for clamp loading activity by measuring -dependent stimulation of DNA synthesis by core polymerase (). Pol III core is unable to extend a primer on an SSB-coated singly primed M13mp18 ssDNA template unless it is coupled to a clamp (1). The (LFAA) complex showed almost no clamp loading activity in this assay, whereas the wild-type complex produced a strong signal (Fig. 2B). This result is consistent with the ·1 structure, which shows that both Leu73 and Phe74 of are directly engaged in the - interaction. Therefore, the loss of activity of the (LFAA) complex may be explained by a decrease in affinity between the mutant complex and . If this is the case, the (LFAA) complex should show decreased ability to bind in a gel filtration analysis, and the addition of more may rescue the activity of (LFAA) complex.
To examine interaction between (LFAA) complex and , the proteins were mixed and analyzed for complex formation on a Superose 12 sizing column equilibrated with buffer containing ATP. Previous studies have shown that ATP is required to promote binding of the subunit to complex (18). As a control, alone migrates in fractions 33-43 (Fig. 2C, bottom panel). Analysis of a mixture of wild-type complex and is shown in Fig. 2C (top panel). The subunit binds to the large complex and therefore co-elutes with it in fractions 15-29 and resolves from excess unbound , which elutes in the later fractions. A similar analysis using the (LFAA) complex mutant shows that the mutant is unable to associate with under these conditions (Fig. 2C, middle panel). The (LFAA) complex elutes in fractions 21-29, but the clamp does not co-migrate with it; instead, the unbound migrates in the later fractions 33-43, as observed for alone (Fig. 2C, bottom panel). The experiment was repeated using a 5-fold higher concentration of (LFAA) complex, but it still did not bind to (Fig. 2C, right panels). These results indicate that the (LFAA) complex- interaction is affected by mutation of the two hydrophobic residues of in the mutant complex. Analysis of the polyacrylamide gels of Fig. 2C by laser densitometry showed no detectable in the peak fractions containing the mutant complex. Assuming that this method would have detected as little as 10% the amount of that comigrates with wild-type complex, the affinity of for the mutant complex is at least 10-fold weaker than for wild-type complex. Furthermore, study of LFAA mixed with showed no interaction between them (data not shown). However, the gel filtration analysis and clamp loading replication assays are performed using different protein concentrations. During gel filtration, protein complexes are not at equilibrium, requiring use of a relatively high concentration of protein (i.e. micromolar), and only strong complexes with relatively slow dissociation rates are observed. On the other hand, during the clamp loading replication assay, components exist in equilibrium, and even transient weak complexes can produce a signal. Therefore, it remains possible that binds transiently to (LFAA) complex. In fact, the replication assays described below indicate that they interact, albeit with much less affinity.
The above results indicate that the defect in activity of the (LFAA) complex is due to the lower affinity of for the mutant clamp loader. In this case, a high concentration of may rescue the replication activity. The result of a titration into the replication assay demonstrates that high concentrations of indeed bring the activity of the (LFAA) complex back to wild-type levels (Fig. 2D). Thus, we conclude that the primary effect of the mutation is on the strength of the - interaction.
The Residues Asn62 and Phe65 Do Not Interact with —The C-terminal portion of the 4 helix of contacts the loop near the interface of the ring and is thought to do so via a steric clash between the proteins (Fig. 1B) (20). If there were specific contact between amino acid side chains of and at site 2, residues Asn62 and Phe65 of (on the 4 helix) are the only side chains of that are close enough to interact with . If these residues contact , replacement of Asn62 and Phe65 with alanines (4) should affect the strength of - interaction. To test this possibility, these two residues were mutated to alanines, and the resulting "4" mutant was purified and then reconstituted with , ', , and to form (4) complex. As illustrated in Fig. 3A, the subunit stoichiometry of (4) complex appears similar to that of wild-type complex in an SDS-polyacrylamide gel.
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Next, we studied the (4) complex for the ability to bind and to load onto DNA. In contrast to (LFAA) complex, the interaction with does not appear to be compromised (Fig. 3B). The mutant complex remains competent to associate with as indicated by their coelution in fractions 17-29 (Fig. 3B, middle panel). Densitometric analysis of the polyacrylamide gels shows 
70% the level of comigrating with (4) complex compared with wild-type complex, indicating that if (4) complex binds less tightly than wild-type complex, the difference in affinity is probably less than 2-fold. Furthermore, the (4) complex is fully active with in stimulating DNA synthesis by the Pol III core (Fig. 3C). Although, these results support our earlier prediction that the 4 helix of is involved in a nonspecific clash with to destabilize the interface, this proposal is further tested below. The results indicate that the interaction at site 2 is not a simple steric clash between proteins and that some modification of the proposal is needed.
Analysis of the Loop near the Interface—The current model for action at the interface is that helix 4 collides sterically with to push on a loop near the interface, leading to distortion of the interface and ring opening. The loop in consists of residues 274-278 and is located adjacent to the 1'' helix of at the dimer interface. This 1'' helix is strained at one end into a 4-10 helix in the dimer structure. The ·1 structure suggests that the push on the loop results in removing the strained portion of the 1'' helix (Fig. 1C). The two hydrophobic core residues of the dimer interface are contained on this 1'' helix, and when the strain is released these two hydrophobic residues are rotated out of the dimer interface so that they no longer participate in dimer formation.
A prediction of this model is that deletion of the loop should not only render inactive and unable to open but should also increase the affinity of for , since it would no longer need to expend binding energy to push on the loop. To test this prediction, we deleted 4 residues (residues 275-278) of the loop and purified the mutant (loop) from an overproducing strain of E. coli. We wished to delete the maximum number of residues but decided on only four, since one residue is needed to link the 1'' helix with its upstream secondary structure element. Hence, a more extensive deletion may have affected the structure of and led to monomerization of the dimer.
To examine whether the loop mutant retained its dimeric status, we analyzed the size of the loop mutant by gel filtration. Fig. 4A shows the SDS-polyacrylamide gel of the gel filtration column fractions for wild-type dimer, the loop mutant, and a mutant that behaves as a monomer (mon) (19). The loop mutant elutes in fractions 33-42, the same fractions as the wild-type dimer (Fig. 4A, bottom and top panel, respectively), whereas the monomer elutes much later, in fractions 39-48 (Fig. 4A, middle panel). Hence, the loop mutant retains its dimeric structure. However, the loop mutant was inactive in the clamp loading assay using complex and core polymerase on primed M13mp18 ssDNA, consistent with the loop being important for activity (Fig. 4B).
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The loop mutation should result in tighter binding between complex (or ) and loop, since we have proposed that pushes on this loop to distort the interface. However, analysis of the loop- complex interaction by gel filtration showed that instead of binding tighter, the interaction between them was compromised. In Fig. 4C, either wild-type (top panel) or the loop mutant (middle panel) was mixed with complex and then analyzed by gel filtration in the presence of ATP. Analysis of the column fractions on an SDS-polyacrylamide gel shows that wild-type clamp co-elutes with the five subunits of the complex in fractions 12-30 (Fig. 4C, left, top panel), whereas the loop mutant does not interact, or at best only weakly associates, with the complex in fractions 21-27 (Fig. 4C, left, middle panel). This result demonstrates that the strength of the - interaction is decreased by the loop deletion, not increased as we had expected. Hence, there must be a positive interaction between and at site 2, not just a nonspecific clash. Next, the gel filtration analysis was repeated using a 5-fold greater concentration of complex (Fig. 4C, right panels). Under these conditions of elevated protein, the loop- complex interaction is evident. This result is consistent with the loop mediating a positive contact between and and with the fact that without it, a greater concentration of protein is required in order to detect an interaction between the loop mutant and complex. The fact that interaction of complex with the loop mutant is restored with a 5-fold elevation in the concentration of the complex suggests that the affinity of loop for complex is reduced only about 5-fold or less relative to wild-type . The interactions between and at site 2 are probably not as strong as site 1, since the (LFAA) complex displayed no interaction with even at elevated concentration.
To determine whether the defect in replication activity of the loop mutant is compensated by use of a higher concentration of in the assay, as observed in the experiment of Fig. 2D where additional restored activity to the (LFAA) complex, we performed clamp loading replication assays over a range of concentration. Although some activity (10-20%) was restored upon titrating more of the loop mutant into the replication assay with core and complex (Fig. 4D), full replication could not be restored as had been observed by titrating into site 1-mutated (LFAA) complex. Hence, the loop is important to its ability to be loaded onto DNA, and activity is only partially restored by adding loop in large excess. The residual activity of loop when added in excess may be explained if the site 1 interaction contributes to destabilization of the interface, leading to the low clamp loading activity observed here for the loop mutant. Alternatively, the single residue of the loop that remains in the loop mutant is sufficient to provide some activity deriving from - interaction at site 2.
In summary, the results are consistent with predictions from the structure, except the decrease in affinity of complex for the loop mutant indicates that a positive interaction at site 2 is lost. This implies that - interaction at this site is not just a steric clash between and at site 2. The affinity at this site is probably mediated via backbone atoms instead of side chains, as implied by the retention of binding and clamp loading activity by the (4) complex, as well as by the lack of homology between prokaryotic clamps in the position of the loop.
Role of the ' Stator—The N-terminal domain (domain I) of ' is thought to block interaction of with in the complex in the absence of ATP (17, 20). ATP promotes complex- interaction, presumably due to a conformation change in complex that pulls domain I of away from domain I of ', allowing to bind and open the ring (18, 20). Based on these findings, one may predict that a complex mutant containing a ' deletion that lacks domain I would bind to even in the absence of ATP. To test this prediction, we deleted the first 206 residues of '('N), corresponding to domains I and II, and cloned, overexpressed, and purified the C-terminal domain III of ' (referred to here as 'N).
The 'N mutant was first tested for its ability to assemble into a complex with the other subunits. The ('N) complex is indeed formed upon mixing all of the subunits together, and the mutant ('N) complex is also stable to the ion exchange purification step (Fig. 5A). The ('N) complex also remains intact during analysis by gel filtration (Fig. 5C), as previously reported (33). Reconstituted ('N) complex retained 10-15% clamp loading activity compared with wild-type complex in the -dependent stimulation of pol III core assay (Fig. 5B). Further, the addition of wild-type ' (0-16 ng) into a replication mixture containing 2 ng of ('N) complex did not affect the DNA replication activity (data not shown).
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The ('N) complex lacks ' domain I and thus may be expected to bind even in the absence of ATP, since should no longer need to be pulled away from domain I of
