©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Assembly of a Chromosomal Replication Machine: Two DNA Polymerases, a Clamp Loader, and Sliding Clamps in One Holoenzyme Particle
IV. ATP-BINDING SITE MUTANTS IDENTIFY THE CLAMP LOADER (*)

Hui Xiao (1), Vytautas Naktinis (1)(§), Mike O'Donnell (1) (2)(¶)

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

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
REFERENCES

ABSTRACT

The complex (`) and complex (`) clamp loaders require ATP hydrolysis to load sliding clamps onto DNA. The sliding clamp tethers the polymerase (Pol) III* replicase to DNA for processive synthesis. Pol III* contains both and , but only one each of the , `, , and subunits. Hence, there is ambiguity with respect to which clamp loader, the or complex, exists in the Pol III* replicase structure. In this study, ATP-binding site mutants of and have been prepared, and these mutants, when assembled into either the or complex, are inactive in clamp loading. These mutants have been used as a tool to determine the identity of the clamp loader in Pol III*. The nine-subunit Pol III* has been assembled using either mutant or in place of wild-type or . The results show that mutation of inactivates Pol III* activity, but mutation of does not, indicating that the complex (and not the complex) is the clamp loader of Pol III*. The subunit carries the task of dimerizing the core polymerase, and it is this association of with core that appears to direct the single copy subunits away from and onto .


INTRODUCTION

DNA polymerase III holoenzyme (holoenzyme) is tethered to DNA by the ring-shaped clamp for high processivity while replicating the chromosome (1, 2) . The clamp is assembled onto DNA by a clamp loading activity inherent in the holoenzyme structure (3, 4, 5) . The first report of this series showed that a clamp loader can be assembled from the , `, , and subunits and from a dimer of either or to produce a complex (`) or a complex (`), respectively (6) . Use of either or in forming a clamp loader complex with the , `, , and subunits is not surprising as the and subunits are encoded by the same gene (7, 8, 9) . The subunit is produced by a translational frameshift such that (47 kDa) is the N-terminal 430 amino acids of (71 kDa) plus one unique C-terminal residue of glutamic acid. Hence, since both and bind the , `, , and subunits, their binding sites must be located within the N-terminal two-thirds of . Unique to the subunit is the ability to bind tightly to the core polymerase (10, 11) . In the holoenzyme, is a dimer, and it dimerizes core, presumably for coordinated replication of the leading and lagging strands of a duplex chromosome (12, 13) . The C-terminal sequence unique to is essential for the strong interaction between and core, and thus, the core-binding site is probably located within the C-terminal third of (11) .

The holoenzyme has both and subunits and therefore may contain both clamp loaders (14) . However, structural studies in the third report of this series showed that polymerase (Pol)() III* contains only one copy of the , `, , and subunits, and therefore, it can have either a or complex, but not both (13) . Assembly studies showed that ` had to be on either or for Pol III* assembly to occur, but if ` was present on both and , Pol III* assembly was prevented (13) . These results further support the presence of only one clamp loader in the holoenzyme. However, since both and are present in the holoenzyme, the exact location of the single copy subunits, whether they are on or , remains ambiguous.

Whether the clamp loader within the holoenzyme is the or complex is the subject of this report. It has been presumed that the subunit is associated with the , `, , and subunits in the holoenzyme because a complex can be purified from Escherichia coli cell lysates (15) . Furthermore, a Pol III` assembly, consisting of two core polymerases bound to a dimer of , can also be purified from cell lysates, but it does not contain the , `, , or subunit (10) . These observations suggest that the holoenzyme contains the complex, not the complex. If the , `, , and subunits are located on either or , then there must exist a mechanism of selectivity that targets them to one subunit rather than another. In this study, we have used and subunits that carry one amino acid replacement in the ATP-binding site that inactivates their clamp loading activity. These mutants are used in combination with wild-type and to assemble Pol III* containing either mutant or (or both) as a means to establishing the location of , `, , and within Pol III*.


EXPERIMENTAL PROCEDURES

Materials and Methods

All sources and procedures not described here are described in the first report of this series (6) .

Construction of the and ATP-binding Site Mutants

The dnaX gene was amplified by the polymerase chain reaction from pZT3 (7) using as upstream primer, 5`-CCCTCTAGAAGGAGATATAAATATGAGTTA-3`, hybridized over the initiating ATG of dnaX, and as downstream primer, 5`-ACTGGTGGATCCTCAAATGGGGCGGATACT-3`, hybridized over the termination codon of dnaX. Amplification was for 30 cycles using TaqI polymerase in the following sequence: 1 min at 94 °C, 2 min at 60 °C, and 2 min at 72 °C, according to the manufacturer's instructions (Perkin-Elmer). The amplified 1.9-kilobase product was purified by phenol extraction in 2% SDS followed by digestion with XbaI and BamHI (cleavage sites underlined in the upstream and downstream primers, respectively) and ligated into pET-3c, cut with the same restriction enzymes, to yield pET3dnaX. Sequence analysis of the entire dnaX gene confirmed that no errors had been introduced during polymerase chain reaction amplification. The XbaI/BamHI fragment containing dnaX was excised from pET3dnaX and subcloned into M13mp18 (digested with XbaI and BamHI), and oligonucleotide-directed mutagenesis (16) was performed at the ATP-binding site. The mutagenic oligonucleotide (5`-CCCGTGGCGTCGGAGCGACCTCTATCGCCC-3`) contained a 3-base pair mismatch (underlined) to produce a lysine to alanine replacement at amino acid 51 of the and proteins. Mutation of the dnaX gene was confirmed by sequence analysis and then excised from M13 by XbaI/BamHI digestion and subcloned into pET-11a (digested with XbaI and BamHI) to yield pET11dnaX(Ala).

Purification of and

Eight liters of BL21(DE3) pET11dnaX(Ala) cells were grown at 37 °C in LB medium containing 100 µg/ml ampicillin. Upon growth to an A of 0.6, isopropyl-1-thio--D-galactopyranoside was added to 0.4 mM. After 3 h, the cells (32 g) were collected by centrifugation; resuspended in 32 ml of ice-cold 50 mM Tris-HCl (pH 7.5), 10% sucrose; and frozen at -70 °C. The subsequent purification steps were performed at 4 °C, and the mutant and proteins were followed on an SDS-polyacrylamide gel. The cells were lysed; the cell debris was pelleted; and the soluble lysate (2.2 g of protein) was fractionated using ammonium sulfate as described (17) . The ammonium sulfate pellet was dissolved in buffer A (275 mg of protein in 50 ml), dialyzed against buffer A to a conductivity equal to 40 mM NaCl, loaded onto a 60-ml Q-Sepharose Fast Flow column equilibrated with buffer A, and eluted with a 600-ml linear gradient of 0-0.5 M NaCl in buffer A. Seventy-two fractions were collected. Fractions 41-49 containing and (232 mg in 75 ml) were pooled, dialyzed against buffer A to a conductivity equal to 20 mM NaCl, loaded onto a 60-ml heparin-Affi-Gel column equilibrated with buffer A, and then eluted with a 600-ml linear gradient of 0-0.5 M NaCl in buffer A. Seventy-two fractions were collected, and fractions 18-30 containing and fractions 40-50 containing were pooled separately and dialyzed against buffer A to a conductivity equal to 28 mM NaCl. (180 mg in 108 ml) and (50 mg in 92 ml) were passed through separate 16-ml N-6-linked ATP-agarose columns (Sigma) equilibrated with buffer A. Mutant and flow through this column, but wild-type and bind tightly. The mutant (162 mg) and (48 mg) preparations were aliquoted and stored at -70 °C.

Wild-type and were purified from 18 liters of BL21(DE3) pLysS pET3dnaX cell culture as described for mutant and , except they bind the ATP-agarose column. The ATP-agarose columns were eluted using a 160-ml linear gradient of 0-2 M NaCl in buffer A; fractions containing and were pooled and dialyzed against buffer A; and then (230 mg) and (44 mg) were aliquoted and stored at -70 °C.

ATPase Assays

The complex was constituted from its five subunits (, `, , , and either wild-type or mutant ) and then purified from excess proteins as described in the first report of this series (6) . ATPase assays were performed in 20 µl of 20 mM Tris-HCl (pH 7.5), 8 mM MgCl, 11 µg of M13mp18 ssDNA, 1 mM [-P]ATP, and one of the following: 0.64 µg of wild-type complex, 3.84 µg of mutant complex, 0.65 µg of wild-type , or 3.84 µg of mutant . The reaction was incubated at 37 °C, and aliquots of the reaction were quenched with 25 mM EDTA (final concentration) at different time points from 2 min to 3 h. The aliquots were analyzed by spotting them (0.5 µl each) onto a TLC sheet coated with polyethyleneimine-cellulose MN300 (Brinkmann Instruments). TLC sheets were developed in 0.5 M lithium chloride, 1 M formic acid. Autoradiograms of TLC sheets were used to visualize P at the solvent front and ATP near the origin, which were then cut from the TLC sheets and quantitated by liquid scintillation counting. The extent of ATP hydrolyzed was plotted, and the initial rate of hydrolysis was used to calculate the moles of P released per mole of protein/minute. One mole of assumed the mass of a dimer, and 1 mol of complex assumed a stoichiometry of `.

Replication Assays

The complex was constituted upon mixing 7.8 µg of (83 pmol as dimer), 7.0 µg of (181 pmol), 4.8 µg of ` (130 pmol), 3.0 µg of (181 pmol), and 2.5 µg of (164 pmol) in 50 µl of buffer A and incubated at 15 °C for 30 min. The complex was constituted as the complex, except 11.0 µg of (77 pmol as dimer) was added in place of . The assay consisted of 66 ng of M13mp18 ssDNA (27.5 fmol as circles) uniquely primed with a DNA 30-mer (1) and coated with 800 ng of SSB, 30 ng of (0.37 pmol as dimer), 55 ng of core (0.35 pmol), and different amounts of constituted or complex in a final volume of 25 µl of 20 mM Tris-HCl (pH 7.5), 8 mM MgCl, 5 mM dithiothreitol, 4% glycerol, 40 µg/ml bovine serum albumin, and 60 µM each dCTP and dGTP. The reaction was incubated at 37 °C for 5 min, and then 60 µM dATP and 20 µM [-P]TTP were added to initiate a 30-s pulse of replication before quenching the reaction by spotting onto DE81 paper and quantitating the amount of DNA synthesis as described (6) . Replication assays of Pol III* were performed as described above, except instead of adding core and the complex, the constituted forms of Pol III* were titrated into the assay, and 40 mM NaCl was included in the reaction.

Constitution of Pol III*

Pol III* containing 1) wild-type and , 2) wild-type and mutant , or 3) mutant and wild-type was constituted as follows (all incubations were in buffer A at 15 °C for 30 min). The complex was formed upon incubating 174 µg of and 107 µg of in 467 µl (urea from the preparation was at a final concentration of 0.1 M); ` was formed upon incubating 610 µg of and 260 µg of ` in 573 µl; and core was formed upon incubating 455 µg of , 145 µg of , and 67 µg of in 723 µl. Either wild-type or mutant (247 µg) and either wild-type or mutant (124 µg) were added to core and incubated for an additional 60 min at 15 °C. In a separate tube, the complex was mixed with ` and incubated for an additional 30 min. Then the mixtures of ` and of were concentrated by spin dialysis using a Centricon-30 apparatus to a combined volume of <200 µl. The two mixtures were then combined and incubated at 15 °C for 5 min before injection onto a Superose 6 gel filtration column developed as described in the first report of this series (6) . Column fractions were analyzed by 15% SDS-polyacrylamide gel electrophoresis, and fractions 16-22 containing Pol III* were pooled. Protein concentration was measured as described in the first report of this series (6) , and Pol III* was aliquoted and stored at -70 °C. The Pol III* containing ATP-binding site mutants of both and was constituted and purified by Superose 6 gel filtration column using Method 1 of the third report of this series (13) ; the same amounts of protein subunits were used as described above.

Surface Plasmon Resonance

Surface plasmon resonance (SPR) was performed as described in the first report of this series (6) . The final levels of immobilized ` and complex were 3325 and 2475 response units, respectively (for , was 2095 response units, and the addition of resulted in another 380 response units). A 30-µl solution of 1 µM or (both as dimer) in SPR buffer was injected over the immobilized ` or complex at a flow rate of 15 µl/min. Then a 30-µl solution of SPR buffer was injected at a flow rate of 15 µl/min. The surface of the ` chip was regenerated by injection of 20 µl of 0.1 M glycine (pH 9.5). The surface of the chip was regenerated by consecutive injection of 20 µl of 6 M urea and 10 µl of 2 M urea in SPR buffer. The subunit was reloaded onto immobilized each time after regeneration; the final response unit obtained was highly reproducible (±20 response units).

The effect of core on the binding kinetics of was determined by forming the core- complex (1 µM (as dimer) and 3 µM core (as ) were incubated in 30 µl of SPR buffer for 30 min at 15 °C) prior to injections. Controls for the effect of core on were performed by preincubating 3 µM core with 1 µM (as dimer) as described above for . Response signals were normalized to the molecular weight of the protein in the mobile phase: , 94 10; , 142 10; and core-, 472.2 10. Apparent association and dissociation rates were determined using the nonlinear curve fitting Pharmacia Biosensor BIAevaluation 2.0 software. The sections of the kinetic traces used to determine the rates were those that yielded a low value (<2) and typically covered a range of 50-80 s.


RESULTS

ATP-binding Site Mutants of and

Consistent with the known ability of and to bind equally well to ATP, the sequence of the dnaX gene has a match with the consensus sequence of an ATP-binding site (18) in the region encoding both and (Fig. 1A). For several proteins with this consensus sequence, the Lys residue has been shown to be important for function with ATP (see Refs. 19-22). Hence, we replaced this Lys codon in dnaX with an Ala codon, and then mutant and were both expressed using the pET system and purified.


Figure 1: ATP-binding site mutants of and are inactive. A, the lysine residue (position 51 in and ) in the consensus sequence of the ATP-binding site was mutated to alanine. The conserved residues of the site are underlined. B, shown are ATPase assays of the complex constituted using either mutant (mut) or wild-type (wt) . ATPase assays of mutant and wild-type are also shown. The DNA effector in the assay is M13mp18 ssDNA. C, and complexes were constituted using either wild-type or mutant and subunits and then assayed for the ability to assemble onto DNA as determined by the ability to confer onto core the ability to replicate a singly primed SSB-coated M13mp18 ssDNA template in 30 s. Clamp loader complexes were constituted using , `, , , and or (either wild-type () or mutant ()).



In Fig. 1B, the mutant proteins were tested for DNA-dependent ATPase activity using M13mp18 ssDNA as effector. The subunit is a known DNA-dependent ATPase (23-25), and as expected, mutant was inactive. The subunit has very low DNA-dependent ATPase activity alone; it requires the and ` subunits for significant activity and is most active as a complex of all five subunits (25) . The complex was constituted from its five subunits using either wild-type or mutant , and then it was assayed for DNA-dependent ATPase activity. The results show that the complex constituted using wild-type is active, but the complex constituted using mutant is inactive. In the event that mutant and bind ATP weakly but have the same maximal velocity of hydrolysis, we have repeated these assays over a large range of ATP concentrations, but little if any activity was observed.() Hence, the replacement of Lys with Ala was effective in blocking ATP hydrolysis of the and complexes. The subunit stimulates the DNA-dependent ATPase activity of the complex (25) , but the addition of the subunit did not provide the mutant or complex with ATPase activity (data not shown).

Are these ATP-binding site mutants inactive in loading clamps onto DNA? We tested this by investigating whether clamp loaders, constituted using , `, , , and either mutant or mutant , could clamp the subunit to DNA for processive synthesis with the core polymerase on singly primed M13mp18 ssDNA coated with SSB (Fig. 1C). In this assay, the core polymerase does not give a detectable signal unless the clamp is assembled onto DNA (1, 5) . The results show that use of wild-type or gives nearly equal and complete synthesis of the DNA template. However, use of mutant or gives no detectable replication. Hence, the mutant and complexes are inactive in assembling clamps onto primed DNA, a result consistent with their loss of ATPase activity. Furthermore, we have directly followed clamp assembly onto DNA using [H] and found that the mutant complex cannot assemble [H] onto DNA (data not shown), consistent with conclusions drawn from its inactivity in replication assays. The absence of ATPase and replicative activities is not due to a loss in native conformation of these mutant proteins as they are fully capable of assembling into multiprotein complexes (i.e. we have constituted the complex (e.g. used in Fig. 1B) and Pol III* from them (e.g. as in Fig. 2)).


Figure 2: Pol III* constituted using mutant is active, but using mutant is inactive. A, scheme for assembly of Pol III* from individual subunits; B, Coomassie Blue-stained 15% SDS-polyacrylamide gel analysis of the four forms of Pol III* constituted using wild-type and (lane1), wild-type and mutant (lane2), mutant and wild-type (lane3), and mutant and (lane4); C, replication activity assays of four different forms of Pol III*. , wild-type and ; , wild-type and mutant ; , mutant and wild-type ; , mutant and .



Constitution of Pol III* Using Mutant and/or

The mutant and subunits were used along with wild-type proteins to constitute the nine-subunit Pol III* containing mutant , mutant , or both mutant and . If the single copy subunits (, `, , and ) associate randomly with either or , then two populations of Pol III* may form, one with these subunits on (i.e. a complex) and one with these subunits on (i.e. a complex). In this event, use of one mutant subunit (either or ) should inactivate Pol III* by 50%. If the , `, , and subunits associate preferentially with one subunit (for example, ), then use of mutant would result in complete inactivation of Pol III*, but use of mutant would not inactivate Pol III*. In either case, double mutant Pol III* is expected to be inactive since association of the single copy subunits with either or would not result in a productive clamp loader.

In the assembly scheme shown in Fig. 2A, the and subunits were mixed with the subunits of core and incubated, and then the , `, , and subunits were added. After a further incubation, Pol III* was separated from excess proteins and free subassemblies by gel filtration. Fig. 2B shows a Coomassie Blue-stained 15% SDS-polyacrylamide gel analysis of four forms of Pol III* prepared using wild-type and (lane1), wild-type and mutant (lane2), mutant and wild-type (lane3), and mutant and (lane4). Densitometry analysis shows that all four forms of Pol III* have a similar subunit stoichiometry (within 15%) consistent with the subunit stoichiometry of Pol III* shown in the first report of this series (`) (6). Hence, mutant and are efficiently assembled into the Pol III* structure, and use of ATP-binding site mutants of and does not affect the assembly process.

These four forms of Pol III* were assayed in replication assays using and singly primed M13mp18 ssDNA coated with SSB (Fig. 2C). Double mutant Pol III* (mutant and ) was inactive in the assay as expected. Of the two forms of single mutant Pol III*, one appeared as active as Pol III* constituted using all wild-type subunits, and the other was inactive, suggesting nonrandom assortment of the , `, , and subunits. The active single mutant Pol III* was the one containing mutant ; the inactive Pol III* was the one constituted using mutant . Hence, it appears that the single copy subunits prefer to associate with over .

Speed of Association of ` and with and

Both and associate with the , `, , and subunits to form active clamp loader complexes (26) . Then why in Pol III* do these subunits seem to preferentially associate with ? Perhaps they are more stabile on , and thus even if they associate randomly with either or , they eventually redistribute to their most stabile occupant. Alternatively, they may associate with more rapidly than with . In Fig. 3, the surface plasmon resonance technique was used to measure the association and dissociation kinetics of these subunits with and . The first report of this series showed that of the , `, , and subunits, only the ` and subunits show significant interaction with and (6) . Hence, we measured the rates of association of and with the ` subunit and with the complex ( was used since by itself is insoluble). The complex and ` were immobilized on separate sensor chips, and then solutions of either or were passed across the surface in the mobile phase. The results show that and have similar kinetics of association with the immobilized subunits. Furthermore, and also have similar kinetics of dissociation from the immobilized complex and from the ` subunit. The observed values of k and k and the K values calculated from them are shown in . Hence, the binding kinetics did not reveal a significant difference in the interaction of and with these subunits.


Figure 3: Effect of the core polymerase on the binding kinetics of ` and with and . The influence of core on the kinetics of interaction between either or with ` and was examined by SPR analysis. A, the complex was immobilized on the sensor chip, and 1 µM solutions (as dimer) of either (rightpanel) or (leftpanel) were passed over the immobilized complex. B, the ` subunit was immobilized on the sensor chip, and 1 µM solutions (as dimer) of either (rightpanel) or (leftpanel) were passed over the immobilized ` subunit. In both A and B, the binding kinetics of core- and of a mixture of and core are also shown. Openarrowheads denote the start of protein injection, and closedarrowheads mark the start of the buffer wash. RU, response units



The subunit is known to bind tightly to the subunit of the core polymerase, whereas the subunit does not bind (or core) (11) . Perhaps the addition of a bulky group, such as core, may affect the binding kinetics of with ` and the complex. This was tested using SPR by forming the core- complex prior to passing it over the immobilized complex or ` subunit (Fig. 3). The results show that the apparent dissociation constant is elevated 5-fold for and 10-fold for `. As a control, core was mixed with and passed over the immobilized complex and ` subunit. The results show that core has little influence on the binding kinetics of with these subunits, as expected from the lack of interaction between and core.


DISCUSSION

The Subunit Is the Site of ATP Action in the Complex Matchmaker

The complex couples ATP hydrolysis to the assembly of a clamp onto DNA. It has been assumed in the past that was the site of ATP action since is known to bind ATP (24) , and the amino acid sequence of contains a consensus ATP-binding site sequence. However, the subunit has also been reported to bind ATP (27), and within the sequence, there is a close match with an ATP-binding site (28) .() The complex, constituted using the mutant, was inactive in ATPase activity and in replication assays, thus confirming that is the subunit of the complex that couples ATP hydrolysis to the clamp loading task. We have also constructed a mutant in which the Lys residue of the putative ATP-binding site is replaced with Ala. The complex, constituted using the mutant, was as active as the wild-type complex in ATPase assays and in replication assays.() Hence, if interacts with ATP, the interaction is not needed in the assembly reaction (or the putative site was not correctly identified).

The Complex Versus Complex in Pol III*

The subunit contains the amino acid sequence of and an extra 213 residues at the C terminus. In fact, a five-subunit `` complex'' (`) can be constituted that is within 2-fold of the activity of the complex (6) . However, it is important to note that a complex has yet to be purified from E. coli cells. Furthermore, the , `, , and subunits are not present in Pol III` (core-) (10) . The third report of this series demonstrates that only one each of the , `, , and subunits assembles into Pol III*, and therefore, these subunits must reside on either or , but not both (13) .

The ATP-binding site mutant and subunits were used to identify which subunit, or , is harnessed by the , `, , and subunits to form the clamp loader in Pol III*. Only Pol III* constituted using mutant was active; mutation of led to inactive enzyme. Hence, the complex appears to be the clamp loader in Pol III*. Consistent with this result, SPR measurements show that the and subunits have similar binding kinetics with ` and , but interaction of with core significantly decreases the efficiency of binding to both ` and . Presumably, core partially blocks the binding sites on for ` and (Fig. 4). Alternatively, core causes a conformational change in such that interaction with ` and is weakened.


Figure 4: Core hinders the binding of ` and to . The two core polymerases on may sterically prevent access of ` and to their respective binding sites on (as shown in the figure). Alternatively, interaction of core with may result in a conformational change that reduces the affinity of ` and for (negative cooperativity).



What is the function of the ATP-binding site of ? The subunit is a DNA-dependent ATPase, but the function of this ATPase is still not clear. The results of this study suggest that its use will be downstream of clamp assembly. Perhaps it is needed during elongation or for interaction with other replication proteins such as the helicase or priming apparatus.

Does Function as a ``Glue Protein''?

It seems strange that the holoenzyme contains two proteins of similar structure and function in one macromolecular particle. Furthermore, these proteins carry out very different functions, one being recruited as a clamp loader and the other as a glue protein that not only holds two core polymerases together, but also holds the complex to the holoenzyme structure. One hypothesis regarding the origin of this arrangement is that was the first to evolve and that a tetramer carried out all these tasks, one dimer to cross-link two cores and another dimer to function with , `, , and as a clamp loader, with both dimers being held to each other by an oligomerization domain (still present on both and ).

Consistent with this notion, the addition of all the subunits of Pol III* except results in a ``-less Pol III*'' that is as active as Pol III* containing (29) . In fact, E. coli survives the genetic knockout of (by mutating the frameshift site in dnaX), suggesting the -less Pol III* can replicate the E. coli chromosome (29) . Study of the -less Pol III* indicates that it has a tetramer of and at least two core polymerases and that the , `, , and subunits are present in single copy (13) .

If can substitute for in replication of the chromosome, then why has E. coli evolved a translational frameshift to produce (or if evolved before , then why has not evolution removed )? One possibility is that the arrangement of both and in the same structure carries an advantage that has not been detected yet. Alternatively, the complex may be recruited as a stabile entity outside of the holoenzyme structure for use in other areas of DNA metabolism such as repair or recombination. Production of only would likely impose the use of the core polymerase with since the core and clamp loading activity would be physically linked. Production of would provide a means of separating the clamp loading activity from the core polymerase, thus freeing it to load clamps for use with other enzymes.

Note Added in Proof-Neither the complex nor complex could load onto primed DNA using ATPS in place of ATP. However, in replication assays containing core, ATPS supported the activity of the and complexes to 24 and 28%, respectively, relative to the level of DNA synthesis observed using ATP (dAMP-PNP was used in place of dATP).

  
Table: Effect of core on interaction of with ` and

The apparent kinetic constants of and interaction with ` and in the presence or absence of the core polymerase are listed below. The values of k and k were the average of five independent experiments. The apparent K was calculated from the apparent rates (k/k). Standard errors are shown in parentheses.



FOOTNOTES

*
This work was supported by National Institutes of Health Grant GM38839 and National Science Foundation Grant MCB-9303921. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

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

To whom correspondence should be addressed: Howard Hughes Medical Inst., Cornell University Medical College, 1300 York Ave., New York, NY 10021. Tel.: 212-746-6512; Fax: 212-746-8587.

The abbreviations used are: Pol, polymerase; ssDNA, single-stranded DNA; SPR, surface plasmon resonance; SSB, single-stranded DNA-binding protein of E. coli; ATPS, adenosine 5`-O-(thiotriphosphate); AMP-PNP, adenyl-5`-yl imidodiphosphate.

The K of mutant and for ATP, as measured using equilibrium gel filtration, was 1 mM. We have also replaced the Lys residue in the ATP-binding site with Arg and have purified and studied these Arg mutants of and . They have nearly undetectable ATPase activity, but in equilibrium gel filtration, they still bind ATP with a K of 63 µM.

The sequence within that most closely resembles an ATP-binding site consensus sequence is AXGKS at residues 220-226. The ATP-binding site consensus sequences are G/AXGKT/S, G/AXGKT/S, and G/AXGXGKT/S (18).

H. Xiao and M. O'Donnell, unpublished data.


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