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J. Biol. Chem., Vol. 276, Issue 37, 35217-35222, September 14, 2001

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Assembly of DNA Polymerase III Holoenzyme

CO-ASSEMBLY OF  AND  IS INHIBITED BY DnaX COMPLEX ACCESSORY PROTEINS BUT STIMULATED BY DNA POLYMERASE III CORE^*

Arthur E. Pritchard and Charles S. McHenry

From the Department of Biochemistry and Molecular Genetics and the Program in Molecular Biology, University of Colorado Health Sciences Center, Denver, Colorado 80262

Received for publication, March 27, 2001, and in revised form, June 21, 2001

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Although the two alternative Escherichia coli dnaX gene products,  and , are found co-assembled in purified DNA polymerase III holoenzyme, the pathway of assembly is not well understood. When the 10 subunits of holoenzyme are simultaneously mixed, they rapidly form a nine-subunit assembly containing  but not . We developed a new assay based on the binding of complexes containing biotin-tagged  to streptavidin-coated agarose beads to investigate the effects of various DNA polymerase III holoenzyme subunits on the kinetics of co-assembly of  and  into the same complex. Auxiliary proteins in combination with ' almost completely blocked co-assembly, whereas or ' alone slowed the association only moderately compared with the interaction of  with  alone. In contrast, DNA polymerase III core, in the absence of ' and , accelerated the co-assembly of  and , suggesting a role for DNA polymerase III' [₂(pol III core)₂] in the assembly pathway of holoenzyme.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

The Escherichia coli chromosome is replicated by the DNA polymerase III holoenzyme,¹ which contains three functional subassemblies: pol III core, the  sliding clamp processivity factor, and the DnaX complex. The pol III core contains the , , and  subunits and provides the polymerase function. The multiprotein DnaX complex recognizes the primer terminus, loads  onto DNA in an ATP-dependent manner, and functions as a communications and organizational node for the various replication and primosomal proteins at the replication fork (1-9).

The DnaX complex contains the ATPases  and , which are the alternative frameshift products of the dnaX gene, plus the auxiliary subunits , ', , and . The  subunit, but not the shorter translation product , dimerizes the pol III core through interactions between structural domain V of  and the  subunit to coordinate leading and lagging strand synthesis (6, 10-12). There is also a -mediated interaction between holoenzyme and DnaB that is essential for coupling the replicase and the primosomal apparatus at the replication fork (4, 7, 9, 12). In the elongation complex,  protects ₂ from removal by exogenous  complex, increasing the processivity of the replicase (12). The  subunit also is a bridge between  and a  single-stranded DNA-binding protein interaction, strengthening the holoenzyme interactions with the protein that coats the lagging strand at the replication fork (13, 14).

Within the DnaX complex, ' and  bind directly to ;  binds ', and  binds  (5, 15). The DnaX-' and DnaX- interactions occur through structural domain III, which is common to both  and  (8). It is also known that  and ' form a 1:1 complex and together with DnaX load  onto primed templates. (16, 17). The  and  subunits also form a 1:1 complex and increase the affinity of DnaX for  and ' such that a functional DnaX complex can be assembled at physiological subunit concentrations (8, 13, 15, 18).

Various forms of the clamp-loading complex have been characterized including a  complex (₃'), a  complex (₃'), and two different mixed DnaX complexes (₁₂' and ₂₁') (15, 17, 19-21). A novel assembly mechanism for the DnaX complex has been discovered recently; free DnaX is a tetramer in equilibrium with a free monomer (K_D = 170 nM), but the DnaX₄ stoichiometry is altered upon ' association, leading to the formation of a DnaX₃' complex (21).

Both  and  are found co-assembled in purified holoenzyme and in pol III*, a subassembly of holoenzyme that lacks only  (22, 23), but the assembly mechanism is not well understood. When the 10 subunits of holoenzyme are mixed simultaneously, they rapidly form a nine-subunit assembly containing  but not  (15). An alternative pathway through pol III', an isolable subassembly comprised of ₂(pol III core)₂ (10), was also investigated, but the  complex and pol III' did not associate upon mixing (17). If the entire complement of DnaX proteins is overexpressed from a single operon, mixed DnaX complexes are formed and can be purified by SP-Sepharose chromatography (21). Also, two in vitro protocols have been developed that produce a pol III* that contains both  and  in the same complex (17).

In view of the important roles for the DnaX complex in replication and its unusual mechanism of assembly, we investigated the effects of various accessory proteins on the time course of the co-assembly of  and . We hoped to discover the factors required for the assembly of  and  into the same complex and to eventually dissect the steps in the assembly pathway. In addition, efficient in vitro assembly of a proper mixed complex will be useful in future studies on the roles of specific proteins and their interactions. We have determined that association proceeds slowly and the presence of the DnaX complex auxiliary proteins impedes this association. We looked at the assembly of  and  into pol III* and discovered that pol III core, in the absence of DnaX complex accessory proteins, stimulates co-assembly, suggesting that the holoenzyme assembly pathway proceeds through the initial formation of pol III'.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Buffers-- The buffers used were: buffer SP (50 mM Tris (pH 7.5), 10% (w/v) glycerol, 5 mM DTT); buffer 25T5 (25 mM Tris (pH 7.5), 5% (v/v) glycerol, 5 mM DTT); buffer G (20 mM Tris (pH 7.5), 25 mM NaCl, 0.1 mM EDTA, 20% (w/v) glycerol, 5 mM DTT); buffer SW (20 mM Tris (pH 7.5), 200 mM NaCl, 0.02% Nonidet P-40, 20% (w/v) glycerol, 5 mM DTT); and buffer T2 (20 mM Tris (pH 7.5), 20% (w/v) glycerol, 5 mM DTT).

Protein Purification-- C(O) is the  subunit with a C-terminal fusion peptide that includes a short 13-amino acid biotinylation sequence, a hexahistidine sequence, and a thrombin cleavage site. The activity of C(O) is nearly identical to that of  in holoenzyme reconstitution assays (6). A fusion protein-overproducing strain was obtained by transforming the plasmid P_A1-C(0) (6) into the E. coli B strain AVB101 (hsdRlon11su1A1, purchased from Avidity, Inc., Denver, CO), which also harbors a plasmid, pBirAcm, with an isopropyl--D-thiogalactoside-inducible birA gene. The E. coli B strain AVB101 was used in hopes of increasing the C(O) biotinylation density. Although this expectation was not realized, lower quantities of C(O) degradation products were obtained from strain AVB101 compared with strain BL21(DE3) for unknown reasons.

Cells were grown at 37 °C in F medium (24) containing 100 µg/ml ampicillin and 10 µg/ml chloramphenicol to an A_600 nm of 1.0 followed by the addition of isopropyl--D-thiogalactoside to 1 mM and D-biotin to 50 µM and harvesting 2 h post induction. The cells were lysed in the presence of lysozyme (3 mg/g of cells), 5 mM EDTA, 5 mM benzamidine, and 0.5 mM phenylmethylsulfonyl fluoride for 2 h on ice followed by a 4-min incubation at 37 °C (24). After lysis, protein was precipitated by adding 0.226 g of ammonium sulfate for each ml of lysate supernatant. The ammonium sulfate pellet was redissolved in sufficient volumes of buffer 25T5 to achieve a conductivity equal to that of buffer SP + 50 mM NaCl and applied to an SP-Sepharose column equilibrated with the latter buffer. After a 1-column volume wash, C(O) was eluted with an 8-column volume 50-400 mM NaCl gradient in buffer SP. From 17 g of cells, 11 mg of purified C(O) was obtained. SDS-polyacrylamide gels showed a purity of >90%.

Purified Holoenzyme Subunits-- Purifications of  and  (25), (purified as a dimer because  alone is insoluble) (18),  and ' (26), and pol III core (27) have already been described.

Kinetics of Association Reactions-- The kinetics of association of C(O) and  to form a mixed complex at 15 °C was determined in the presence and absence of various combinations of the DnaX complex accessory proteins, ', and pol III core. With the exception of the pol III core reaction, each association reaction was initiated by the addition of C(O) (12.7 µg, 0.17 nmol as monomer, 5.3 µM, for each time point) to a solution pre-equilibrated at 15 °C, containing the  subunit (16.8 µg, 0.35 nmol as monomer, 11 µM) and, if present,  (13.2 µg, 0.34 nmol, 11 µM), ' (12.4 µg, 0.35 nmol, 11 µM), and (11.2 µg, 0.36 nmol, 11 µM) complexes. For pol III core reactions, C(O) (0.17 nmol) and pol III core (0.32 nmol) were incubated first in a volume of 20.4 µl for 10 min at 15 °C followed by the addition of  (0.35 nmol) to initiate the association reaction. All reactions were adjusted to a final total volume of 32 µl/time point by the addition of buffer G. For each kinetic series a reaction pot was made, and 32-µl volumes were withdrawn at selected time points and then quenched by mixing with a 17-µl volume on ice that contained any missing DnaX complex subunits needed to comprise the complete ' complex. After a further 2-min incubation on ice, the mixtures were flash-frozen by immersion in liquid nitrogen and stored at 70 °C until the streptavidin bead procedure was performed.

Streptavidin Bead Procedure-- For each time point, 80 µl of a streptavidin agarose bead slurry containing ~40 µl of streptavidin agarose beads (Molecular Probes, Eugene, OR) was washed twice with 0.8 ml of buffer SW in an Eppendorf tube by mixing, briefly spinning in a microcentrifuge, and removing the supernatant. Each quenched kinetic reaction aliquot was thawed on ice, adjusted to 200 mM NaCl/0.02% Nonidet P-40, and added to the washed beads on ice. The bead mixture was vortexed for 10 min at 4 °C and spun in a microcentrifuge for 1.5 min, and the supernatant was removed. The remaining unbound protein was removed by adding 0.8 ml of buffer SW, vortexing for 10 min at 4 °C, spinning for 1.5 min in a microcentrifuge, and removing the supernatant. The wash was repeated two more times. Bound protein was eluted from the washed beads by adding 40 µl of 0.09 M Tris (pH 6.8), 15% sucrose, 3% SDS, 90 mM DTT, and .03% bromphenol blue, boiling for 5 min, and briefly spinning. The supernatant was removed and subsequently loaded onto an SDS-polyacrylamide gel (0.75-mm thick, either 7.5-17.5% or 10% acrylamide). After staining for ~2 h in 0.05% (w/v) Coomassie Brilliant Blue R-250 (Bio-Rad), 45% methanol, and 10% acetic acid, the gels were destained overnight in at least two changes of 20% methanol and 5% acetic acid.

The amount of C(O) that binds to the streptavidin beads (in the absence of auxiliary DnaX subunits) was estimated by using the binding, washing, and elution procedures described in the preceding paragraph followed by gel quantification. 10-30% of the C(O) that was applied to the beads was bound. The binding capacity of the beads had not been exceeded, because the total amount of C(O) bound increased approximately linearly with increasing amounts of protein applied. At the concentrations used in this bead-binding protocol, more than 90% of C(O) exists as a tetramer (21). Because any tetramer with between one and four biotinylated C(O) monomers will bind, it can be calculated, assuming a binomial distribution of biotinylated and nonbiotinylated monomers, that 10-30% of total C(O) binding to beads corresponds to 3-8% biotinylation of the C(O) monomers.

Quantification-- Gel scans for quantification were obtained using a Molecular Dynamics laser densitometer. Stain intensities were measured as integrated volumes of boxes drawn around subunits with appropriate background subtraction. The / stain intensity ratios were converted to molar ratios by using a molecular weight ratio conversion factor. We know from previous studies that for / ratios, this method is nearly as accurate as having purified subunit standards on the same gel (21). The  +  association kinetics are depicted as the / molar ratio versus time of incubation. For each kinetic series three controls were included. 1) An untagged control contained all of the DnaX complex subunits (and pol III core if appropriate) except  was substituted for C(O). 2) A background control contained C(O), , ', and (and pol III core if appropriate) but not . Background stain intensity at the position expected for the  subunit was measured as a percentage of the C(O) stain intensity. This value, which was caused by slight contaminants or degradation products of C(O), ranged from 5 to 10% of the C(O) signal and was subtracted (as a percentage of the C(O) intensity for a particular time point) from the measured  subunit stain intensity for each of the time points. 3) A zero-point control was accomplished by adding, on ice, C(O) to a preincubated mixture of , , ', and , which comprised the quench. Higher levels than expected of  associated with C(O) were seen in this control (/ molar ratios ranging from 0.08 to 0.18). This did not indicate that the quench was ineffective, because the / ratio in the presence of ' (i.e. the quench conditions) did not increase with time (see below). Variations in the zero-point control such as adding  to preincubated ' or adding ' to ' also gave similar / ratios. It is possible that the  seen associated with  in this control is a consequence of the streptavidin bead procedure or caused by aggregation by nonspecific binding of C(O) and  proteins. The / value of the zero point control was subtracted from all time points.

SP Chromatography-- For experiments investigating the effect of SP-Sepharose on dissociation,  (146 µg, 2.0 nmol as monomer) was incubated with  (196 µg, 4.1 nmol) in a volume of 373 µl (achieved by adding buffer G) for 1.5 h at 15 °C and then transferred to ice. After the mixed complex formation, auxiliary DnaX complex subunits, if present in the particular experiment, were added:  (83 µg, 2.1 nmol), ' (79 µg, 2.1 nmol), and (131 µg, 4.1 nmol). The conductivity of the resulting solution was adjusted to the equivalent of ~35 mM NaCl by the addition of buffer 25T5 and then injected onto a 1-ml HiTrap SP-Sepharose column (Amersham Pharmacia Biotech) that had been attached to an Amersham Pharmacia Biotech FPLC apparatus and equilibrated with buffer T2 + 20 mM NaCl. Protein was eluted with a 20-ml 20-300 mM NaCl gradient in buffer T2.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Assay to Study Kinetics of Association of  and -- The two DnaX proteins,  and , unassociated with other subunits of holoenzyme, exist as tetramers in equilibrium with monomers. If purified  and  are mixed they can associate to form a mixed tetramer, a reaction that is blocked by the presence of the DnaX complex auxiliary proteins, , ', , and  (17). Here, we refer to the co-assembly of  and  as an exchange of DnaX protomers in complexes initially homomeric in DnaX. To study the parameters affecting these exchange reactions, we developed an assay that takes advantage of a biotin tag near the C terminus of C(O), a  fusion protein.  was allowed to exchange into complexes with C(O) under varying experimental conditions, the exchange reaction was quenched at various time points by forming a complete DnaX complex (DnaX₃'), and complexes containing  associated with C(O) were purified away from unassociated  by the binding of C(O) to streptavidin beads. The purified complexes were electrophoresed on SDS-polyacrylamide gels, and the / molar ratios were determined. An example of the procedure, the exchange in the presence of ' (Fig. 1), shows that a mixed DnaX complex did form, and the relative amount of  increased with time. However, when as well as ' were present, the amount of  did not increase with time (Fig. 1B). Thus, the association of C(O) and  was blocked in the presence of ' plus , which is consistent with an earlier study showing that ' prevented co-assembly (17). In each series of experiments a control, in which wild-type  was substituted for the tagged , showed that the bead wash procedure removed nonbiotinylated proteins that were not associated with C(O) (untagged , Fig. 1B). Another control, lacking only , was used to subtract any background signal in the gel (no  lane, Fig. 1B) caused by for example minor contaminants migrating at the  position. In the zero point control (Zero Pt lane, Fig. 1B), C(O) was added to a reaction at 0 °C that was quenched already because it contained  preincubated with '. The / molar ratio for zero-point control was subtracted from all of the experimental time points.

View larger version (23K): [in this window] [in a new window]   Fig. 1.   Assay for the association of  and . A, an example of the association reaction in the presence of ' is shown schematically. Biotinylated C(O) (shown as *) was incubated with a molar excess of ', and at selected time points aliquots were removed and quenched by the addition of . -containing complexes were purified by the streptavidin bead procedure, and complexes containing * were released from the beads by boiling and then electrophoresed. B, Coomassie Blue-stained SDS 4-20% polyacrylamide gradient gels of the time point and control samples. The sample for the no  lane included all subunits except  mixed on ice; the Zero Pt sample was * added to a mixture of ' on ice, and the untagged  sample was  rather than C(O) incubated with  on ice for 15 min followed by the addition of '. The samples for time points of the * + ,' reaction and the * + ' reactions are shown in the upper and lower gels, respectively. The time points are the same for the reactions shown in each gel.

Subunits That Prevent the Association of  and -- When C(O) was added to a preformed complex of ₃' maintained at 15 °C, there was no increase in the / ratio over time (Fig. 2A); ' blocked the association of C(O) and , confirming an earlier study (17). After 120 min the / ratio was zero after the background control, and zero-point corrections were applied. In contrast, when C(O) and  were allowed to exchange in the absence of accessory proteins, the average / ratio was 1.0 after 120 min. This result demonstrates the effectiveness of the quench used in the assay. Two other combinations of subunits, ' and ', were nearly as effective in suppressing the association of C(O) and  with / ratios of 0.17 and 0.02, respectively, after 120 min. All the accessory protein combinations that exhibited the maximal kinetic inhibition contain ' (Fig. 2A).

View larger version (16K): [in this window] [in a new window]   Fig. 2.   Kinetics of association. The molar ratio of  associated with biotin-tagged C(O) in a streptavidin-binding complex is shown as a function of incubation time in the presence of various proteins. The curves are compared with the association curve, shown in all three panels, obtained with no other added proteins. There are three classes: A, those showing nearly complete inhibition of exchange; B, those exhibiting only moderate inhibition; and C, pol III accelerating exchange. The subunits in addition to C(O) that were present during the exchange reaction are indicated to the right of each curve. The curves with error bars represent the averages of either two (', pol III), three (), or four () separate experiments. Not all of the time points in the pol III curve were repeated.

Subunits That Slow the Association of  and -- Other combinations of subunits, when allowed to form complexes with , slowed but did not abrogate the association of C(O) and  into the same complex (Fig. 2B). The curves for the C(O) plus or ' complexes were overlapping and revealed slightly slower kinetics than the C(O) plus  only curve. After 80 min the average / ratio was 1.0 with no added accessory proteins, 0.7 in the presence of , and 0.66 in the presence of '. We did not observe a significant effect on the association kinetics when only  was added to the reaction (data not shown). Similarly, the curve for the association of C(O) and  in the presence of was not altered when  was also included (data not shown). Thus, any accessory protein that binds directly to DnaX in the complex retards rather than accelerates the association  and . The magnitude of the inhibition varies for the different combinations of proteins tested.

Pol III Core Accelerates the Association of  and -- The effect of another -binding protein, pol III core, on the assembly of  and  into pol III* was also examined. Because pol III core binds to  but not to , we first incubated  with a 2-fold molar excess of pol III core for 10 min before adding  to initiate the exchange reaction. The reactions were quenched at various times by the addition of '. Pol III core accelerated the exchange reaction approximately 3-fold compared with the reaction with only  and  present (Fig. 2C). After only 2 min, the / ratio was increased from 0.15 to 0.40. This rate increase is in marked contrast to the inhibitory effects of the other -binding protein. The actual measured rate increase depends on solution conditions and possibly the presence of the C-terminal tag on C(O). It is known that the K_D for the -C(O) association is in the nM range compared with a pM range for the interaction of  with the N-terminal fusion  (6), but because our experiments were conducted in the µM range, it is unlikely that the decreased - affinity caused by the C-terminal tag affected the results.

It should be noted that we did not observe  associated with pol III' in the absence of added ' and (data not shown). However, ' and were not responsible for stimulating the association kinetics, only for trapping an intermediate, because if , ', , and  were present along with pol III' when  and  were mixed, no  was found associated with . This indicates that  can interact with pol III' in a time-dependent reaction, but the association is weak and does not survive the streptavidin bead-washing procedure without forming a stable DnaX complex.

SP-Sepharose Chromatography of the Complex-- It is clear from the streptavidin bead assay with C(O) and from previous work (17) that  and  by themselves can associate in vitro to form a heterologous DnaX oligomer. Yet it has also been demonstrated that a mixture of  and , even if obtained by co-expression from a plasmid, elute from S-Sepharose as separate  and  peaks with no evidence of a mixed complex (25). This enigma was investigated in a series of SP-Sepharose chromatography experiments (Fig. 3). To form a mixed complex,  was allowed to associate with a 2-fold molar excess of  at 15 °C for 1.5 h, which is enough time to form an equilibrated mixed complex based on our streptavidin bead experiments. The resulting mixture was then chromatographed on a 1-ml SP-Sepharose column that was developed with a 20-ml 20-300 mM NaCl gradient run slowly (0.02 ml/min). The two DnaX proteins eluted separately as  and  peaks at salt concentrations equivalent to ~100 and 210 mM NaCl, respectively. There was no evidence of any mixed oligomers confirming our earlier observation (25). However, when the SP-Sepharose column gradient was developed rapidly (0.5 ml/min), in addition to the  peak several overlapping peaks comprising mixed complexes were observed eluting between 155 and 190 mM NaCl. The / ratio in the eluted complex increased with increasing salt concentration. Although the mixed complex species were not purified to homogeneity for characterization, we expect that there were three species present corresponding to ₁₃, ₂₂, and ₃₁. The SP-Sepharose method therefore promotes the dissociation of mixed oligomers, a process that was completed if the proteins were eluted at a slow flow rate but not at the faster flow rate.

View larger version (27K): [in this window] [in a new window]   Fig. 3.   oligomers without auxiliary proteins dissociate on SP-Sepharose. SP-Sepharose chromatography profiles are shown of mixed oligomers and various mixed subassemblies of DnaX complex. The mixed oligomers were formed by incubating the two proteins together for 1.5 h followed by the addition of accessory proteins, if present, and injection onto the 1-ml SP-Sepharose column. The protein was eluted in 0.25-ml fractions with a 20-ml 20-300 mM NaCl gradient run at either 0.5 ml/min (profile labeled fast) or 0.02 ml/min (profiles labeled slow). The individual profiles were aligned by the salt gradients measured for each run. The proteins present in the load of each column are indicated on the left, and Coomassie Blue-stained SDS 10% polyacrylamide gels of peak fractions, labeled on each profile, are shown on the right. The proteins  and ' co-migrate on the gels.

Can accessory DnaX complex subunits stabilize the mixed complex subassemblies and prevent dissociation on SP-Sepharose? After the association of  and  at 15 °C for 1.5 h, three combinations (', ', or ) of the auxiliary proteins in separate experiments were added to the mixture, and the resulting subassemblies were chromatographed at the slow rate of 0.02 ml/min (Fig. 3). In all three experiments, we observed subassemblies containing both  and  eluting between 170 and 210 mM NaCl. Therefore, ', ', or can stabilize mixed oligomers against dissociation on the column. However, ' was more effective than in stabilizing mixed complexes (compare the bottom panel of Fig. 3 with the two panels above it).

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

We have shown that various combinations of the auxiliary DnaX complex proteins, , ', , and , slow the co-assembly of  and  into the same complex;  and  exchange into the same complexes faster in the absence of these proteins. This result implies that if the holoenzyme assembly pathway is through the DnaX complex, the formation of a mixed oligomer occurs before any other auxiliary protein enters the complex. Because  and  are translated from the same RNA, co-translational formation of a mixed DnaX oligomer before the accessory proteins become associated is likely.

Any DnaX complex accessory protein that is known to bind to DnaX slows the exchange of  into -containing complexes. The accessory proteins interact with DnaX through its domain III (8), where the DnaX oligomerization domain is also located.² All the auxiliary protein combinations that prevent the exchange of  and  into the same complex include '. Although ', which binds very weakly to DnaX by itself, is an ineffective inhibitor, all agents that synergistically increase the apparent affinity of ' for DnaX ( and/or ) enhance the efficacy of '-mediated inhibition. We know that the binding of ' promotes a DnaX₄ to DnaX₃' transition, an event that may tighten DnaX-DnaX interactions precluding exchange.

For the association of  and  in the absence of any other proteins, the C(O) and  concentrations are 5.3 and 11 µM, respectively, and therefore, from the K_D of 170 nM (21), ~7 and 4% of the two DnaX proteins, respectively, exist in a monomeric rather than a tetrameric state. The association of  and  in vitro could occur hypothetically via either oligomeric state.

In contrast to the DnaX accessory proteins, pol III core stimulates the co-assembly of  and  into the same complex;  incubated with pol III core and then mixed with  co-assembles faster than  and  incubated alone. This result suggests a pathway for the formation of pol III* in the cell via pol III' (₂ (pol III core)₂). We know from previous work that our protocol used in the kinetic analysis, incubating C(O) with a 2-fold molar excess of pol III core, reconstitutes pol III' in vitro (11). A pathway via pol III' is particularly attractive, because the holoenzyme replicase almost certainly contains two s and one , whereas free DnaX, unassociated with any accessory protein, is a tetramer in equilibrium with a free monomer (21). Pol III core may stimulate co-assembly by dissociating the DnaX tetramer, a potential kinetic barrier in the assembly pathway (Fig. 4). The fact that pol III' mixed with  complex is a dead end in the assembly pathway (17) is because of the stability of the ₃' complex.

View larger version (16K): [in this window] [in a new window]   Fig. 4.   Model for the pathway of assembly of pol III* containing both  and . During translation of the DnaX mRNA (A), DnaX tetramers containing both  and  are formed (B). Upon association of pol III core with tetramers containing two or more s, pol III' is formed (C), which interacts with  to form an unstable intermediate (D). Finally, the DnaX complex accessory proteins associate to form pol III* (E), stabilizing DnaX-DnaX protein interactions.

Based on our observations and knowledge of the mechanism of DnaX synthesis, we favor a model in which DnaX forms heterologous oligomers by co-translational assembly from the same DnaX mRNA (Fig. 4). High local concentrations of DnaX should facilitate rapid interaction. Then, pol III is proposed to bind to -containing complexes, with those containing two or more s being favored in the formation of pol III'. Monomeric  in equilibrium with DnaX tetramers could then associate with pol III' weakly, forming a heterologous intermediate. This unstable pol III'- intermediate is then proposed to associate with ' and , which stabilize DnaX-DnaX interactions and favor the formation of stabile pol III* (Fig. 4).

	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.

Published, JBC Papers in Press, July 19, 2001, DOI 10.1074/jbc.M102735200

² Glover, B. P., Pritchard, A. E., and McHenry, C. S. (July 19, 2001) J. Biol. Chem. 10.1074/jbc.M103719200.

	ABBREVIATIONS

The abbreviations used are: holoenzyme, E. coli DNA polymerase III holoenzyme; pol III core, E. coli DNA polymerase core (); mixed DnaX complex, ₁₂' or ₂₁'; complex, ₃'; complex, ₃'; pol III*, a complex containing all of the holoenzyme components except for ; pol III', ₂(pol III core)₂; DTT, dithiothreitol.

	REFERENCES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

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