t Binds and Organizes Escherichia coli Replication Proteins through Distinct Domains

The DnaX complex of the DNA polymerase holoenzyme assembles the b 2 processivity factor onto the primed template enabling highly processive replication. The key ATPases within this complex are t and g , alternative frameshift products of the dna X gene. Of the five domains of t , I–III are shared with g In vivo , g binds the auxiliary subunits dd * and xc (Glover, B. P., and McHenry, C. S. (2000) J. Biol. Chem. 275, 3017–3020). To localize dd * and xc binding domains within g domains I–III, we measured the binding of purified biotin-tagged DnaX proteins lacking specific domains to dd * and xc by surface plasmon resonance. Fusion proteins containing either DnaX domains I–III or domains III–V bound dd * and xc subunits. A DnaX protein only containing domains I and II did not bind dd * or xc . The binding affinity of xc for DnaX domains I–III and domains III–V was the same as that of xc for full-length t , indicating that domain III contained all structural elements required for xc binding. Domain III of t also contained dd * binding sites, although the interaction between dd * and domains III–V of t was 10-fold weaker than the interaction between dd * and full length t . The presence of both d and xc strengthened the d * -C(0) t interaction by at least 15-fold. Domain III was the only domain common to all of t fusion proteins whose interaction with d * was enhanced in the presence of d and xc . Thus, domain III of the DnaX proteins not only

The dnaX gene produces two distinct proteins, and ␥, which have differential interactions with replication proteins in the cell (4,5). Results presented in the first two reports in this series demonstrate that it is the C-terminal portion of , absent in the ␥ protein, that allows the full-length DnaX gene product to interact with both the DnaB helicase and the DNA polymerase III core. These -mediated interactions impart rapid fork movement and coordinated leading and lagging strand synthesis, respectively (6 -10). The focus of this investigation is the protein sequence common to both and ␥.
Functional homomeric DnaX complexes ( complex, 3 ␦ 1 ␦Ј 1 1 1 , and ␥ complex, ␥ 3 ␦ 1 ␦Ј 1 1 1 ) can be assembled in vitro (11,12). Thus, the N-terminal 430 residues common to both and ␥ have the minimal protein sequence necessary not only to bind the auxiliary subunits ␦, ␦Ј, , and but also to load the ␤ processivity factor onto a primed template in an ATP-dependent manner. Within the DnaX complex, ␦Ј and bind directly to ␥; ␦ binds ␦Ј, and binds (13,14). ␦ and ␦Ј form a 1:1 complex and function with DnaX to load ␤ onto primed templates in an ATP-dependent manner (10,15). The and subunits also form a 1:1 complex and increase the affinity of DnaX for ␦ and ␦Ј so that a functional DnaX complex can be assembled at physiological subunit concentrations (16). The subunit also interacts with SSB, consistent with the finding that --DnaX bridges strengthen the interactions between the holoenzyme and the SSB-coated lagging strand at the replication fork (17,18). In the preceding studies, five structural domains were assigned to the protein. The focus of this report is to determine which structural domain(s) within the portion of DnaX common to both ␥ and (domains I-III) are responsible for binding the auxiliary subunits. To this end, the relative binding of ␦␦Ј and to a series of truncated DnaX proteins lacking specific structural domains was measured using surface plasmon resonance. buffer were prepared as previously described (19).
Construction of the Fusion Plasmids-Plasmid P A1 -N-⌬221 encodes the fusion protein N-⌬221. The starting material for construction of plasmid P A1 -N-⌬221 was plasmid P A1 -N-⌬1, which encodes the protein with the initiating methionine replaced by an N-terminal fusion peptide. PCR primer N-221p1 contained a PstI sequence at the noncomplementary 5Ј-region followed by a complementary sequence extending from codons 222 to 228 of dnaX (Table I). Primer N-221p2 was complimentary to a region located 102 bases downstream of the NheI site within dnaX. The resultant PCR fragment was digested with PstI and NheI and ligated into the linearized pP A1 -N-⌬1 to generate plasmid P A1 -N-⌬221.
Plasmid P A1 -C-⌬261 encodes the truncated fusion protein C-⌬261. To construct the plasmid P A1 -C-⌬261, a partial dnaX gene sequence encoding the C-terminal 261 amino acids of was deleted from the previously constructed plasmid P A1 -C(0), which encodes the C-terminal tagged full-length protein (19). PCR primer C-⌬261P1 was complementary to a region of dnaX located 430 bases upstream of the internal RsrII site. Primer C-⌬261P2 was complementary to the dnaX from codons 380 to 382 followed by a noncomplementary SpeI cloning site (Table I). After digestion with RsrII and SpeI, the resultant PCR fragment was ligated into the linearized pP A1 -C(0) to generate pP A1 -C-⌬261.
Plasmid pET11-C-⌬422, which lacked the sequence encoding the C-terminal 422 amino acids of , was constructed as follows. pET11-C(0) was digested with AflII and SpeI to delete a dnaX sequence encoding from residue 218 to the 3Ј end (residue 643) of . The annealed oligonucleotides C-422p1 and C-422p2 containing the sequence encoding amino acid residues 218 -221 were ligated into the linearized pET11-C(0) vector to generate pET11-C-⌬422 (Table I).
Protein Purification-C(0), N-⌬1, and the other holoenzyme subunits were expressed and purified as previously described (19). Induced BL21 cells containing the expression plasmids introduced in this study were lysed in the presence of lysozyme (2.5 mg/g of cells), 5 mM EDTA, 5 mM benzamidine, and 1 mM phenylmethylsulfonyl fluoride. The expressed proteins C-⌬261, N-⌬221, and C-⌬422 were precipitated from their corresponding lysate supernatants by addition of 0.226, 0.258, and 0.361 g of ammonium sulfate to each milliliter of the lysates, respectively. The fusion proteins were purified using Ni 2ϩ -NTA affinity chromatography as previously described for P A1 -N-⌬1 (19), except that the bound C-⌬261 was eluted stepwise in Buffer W containing 150 mM imidazole. For purification of C-⌬422, the imidazole concentration was 2 mM instead of 1 mM in the binding buffer and 15 mM instead of 23 mM in the washing buffer; the bound proteins were eluted stepwise as for C-⌬261. For purification of N-⌬221, the imidazole concentration was 15 mM in the washing buffer; the bound N-⌬221 was eluted with 10 column volumes of 15-100 mM imidazole gradient in buffer W. The imidazole concentration in the peak fraction of N-⌬221 was ϳ55 mM.
Surface Plasmon Resonance-A BIAcore TM instrument was used for protein-protein binding analyses. CM5 research grade sensor chips were used for all experiments. Streptavidin was coupled to the sensor chip surface by the N-hydroxysuccinimide/1-ethyl-3-[(3-dimethylamino)propyl]-carbodiimide coupling (19). Biotin-tagged fusion proteins (700 -1600 RU) were then captured onto sensor chips via streptavidin-biotin interaction.binding studies were conducted in HKGM buffer at a flow rate of 25 l/min at 20°C. -␦Ј and -␦␦Ј binding studies were performed in HKGM buffer containing 2% glycerol and 2 mM dithiothreitol at a flow rate of 10 l/min at 20°C. In these studies, ␦ and ␦Ј were preincubated for 10 min at room temperature before injecting over the derivatized sensor chips. Kinetic parameters were determined using the BIAevaluation 2.1 software unless indicated otherwise.
SDS-Polyacrylamide Gel Electrophoresis-Proteins were separated by electrophoresis at constant current (20 mA) on 12.5% SDS-polyacrylamide mini-gels. The gels were stained and destained as previously described (19).
DNA Polymerization Assays-Activities of fusion proteins were measured by their requirement for reconstitution of holoenzyme activity as previously described (19).

Expression and Purification of the Truncated DnaX Fusion
Proteins-We constructed three plasmids, each encoding specific structural domains of under control of an inducible promoter. Plasmid P A1 -N-⌬221 encoded protein N-⌬221 (domains III-V), plasmid P A1 -C-⌬261 encoded protein C-⌬261 (domains I-III), and plasmid pET11-C-⌬422 encoded protein C-⌬422 (domains I-II) (Fig. 1). Each of these proteins contained a hexahistidine sequence to facilitate purification using Ni 2ϩ -NTA metal affinity chromatography and a short biotinylation sequence to enable immobilization on streptavidincoated BIAcore sensor chips. The biotin tag also enabled detection of fusion proteins using biotin blots (21). The expression levels of C-⌬261, N-⌬221, and C-⌬422 were ϳ3%, 2, and 0.5% of the total cell protein, respectively. After one round of Ni 2ϩ -NTA chromatography, N-⌬221 and C-⌬261 preparations at greater than 80% of purity were obtained; the purity of C-⌬422 was ϳ30%, as determined by densitometric scanning of the SDS-polyacrylamide gels of the eluted proteins fractions (Fig. 1). Biotin blots verified that these fusion proteins were the only biotinylated proteins in the eluted fractions (results not shown). The biotinylated proteins are presumably the only proteins captured onto the streptavidin sensor chips. This assumption was verified for a similarly purified protein (see footnote 2 in Ref. 19). The activities of C-⌬261 through its purification were measured in holoenzyme reconstitution assays. Purified C-⌬261 had a specific activity of 5.3ϫ10 6 units/  ␦␦Јand -Binding Domain of DnaX mg, which is comparable with that of C(0) ( Table II). As expected, N-⌬221 was inactive in this same assay because its ATPase motif (domains I and II) was deleted. C-⌬422 (domains I and II) was also inactive in this assay, suggesting that domains I-III represent the minimum protein required to assemble the ␤ processivity factor onto the DNA template.
DnaX Domain III Interacts with -The abilities of the truncated fusion proteins C-⌬261, N-⌬221, and C-⌬422 to interact with were measured by using BIAcore methodology. Because the binding region is located within the N-terminal 430 amino acids of , a fusion peptide tag at the remote C terminus of would be unlikely to interfere with binding. For this reason, C(0) was used as a positive control for examination of binding to the truncated proteins. Biotin-tagged C(0) was immobilized onto a streptavidin sensor chip, and various concentrations of (25-300 nM) were passed over immobilized C(0) so that binding of free to immobilized C(0) could be measured. Representative binding curves ( Fig.  2A) indicate that bound rapidly to C(0) with an association rate of 2.4ϫ10 5 M Ϫ1 s Ϫ1 . A dissociation rate of 2.5 ϫ 10 Ϫ3 s Ϫ1 (Table III) was obtained; this rate was measured after saturating C(0) with to minimize reassociation. The resulting K d 2 (10 Ϯ 1 nM) was ϳ5-fold higher than the K d reported for the nativeinteraction. The observed difference between the K d values could be due to differences in experimental conditions between the two studies. For example, the present study used biotin-streptavidin rather than amine coupling and employed a 5-fold higher flow rate than did the other study.
The interactions between and the truncated fusion proteins N-⌬1, N-⌬221, C-⌬422, and C-⌬261 were examined under the same experimental conditions as used for the and C(0) interaction. N-⌬1, N-⌬221, and C-⌬261 bound , but C-⌬422 did not (Fig. 2B), indicating that domain III of , the only domain shared by all of the fusion proteins shown to bind , is required for binding. Similar binding stoichiometries were obtained for the interactions of -N-⌬1 and -C-⌬261 compared with that of the -C(0) interaction (Table III). The K d for the interaction between -N-⌬221 was within 2.5-fold of that measured for the -C(0) interaction. These variations are likely within range of accuracy of affinity measurements using a BIAcore and indicate that deletion of domains I and II or deletion of domains IV and V as well as the presence of the tag at the corresponding deletion end of the proteins did not decrease the affinity of theinteraction. Thus, domain III of appears to be fully responsible for binding.
No Observable Binding of ␦Ј to Individual Domains of the DnaX Protein-The interactions between ␦Ј and the full-length proteins with either an N-or C-terminal tag were characterized in binding studies utilizing the BIAcore instrumentation. 2 K d values were obtained by dividing the measured dissociation rate constant by the association rate constant of a given interaction. In most cases, the K d values determined in this study were not true equilibrium K d values but were relative values used to compare the relative affinities of -derivatives for the same analytes. a For comparison, the specific activity of C(0) of the Ni 2ϩ -NTA purified fractions is 5.7 ϫ 10 6 units/mg. , and C-⌬422 (540 RU) proteins, respectively, were captured onto sensor chips. Solution of (150 nM) was injected over each protein-immobilized sensor chip for 3 min. Control injections, over streptavidin-immobilized sensor chip, were subtracted from the curves shown. ␦␦Јand -Binding Domain of DnaX ␦Ј samples were injected over the immobilized C(0), and the binding curves are shown in Fig. 3A. The interaction between ␦Ј and N-⌬1 is characterized by weak binding similar to that observed for the ␦Ј-C(0) interaction (Table IV). Although measurable, the weak binding observed is close to the limit of BIAcore detection.
Next, the truncated DnaX proteins N-⌬221 (domains III-V), C-⌬422 (domains I-II), and C-⌬261 (domains I-III) were captured onto streptavidin sensor chips so that the binding of ␦Ј to these proteins could be measured. The K d observed for the ␦Јsubunit-C-⌬261 interaction was similar to that of the C(0)-␦Ј interaction ( Fig. 3B and Table IV). These observations confirm that the ␦Ј binding region of is entirely within its N-terminal 382 amino acid residues (domains I-III). However, ␦Ј did not bind to N-⌬221 or C-⌬422 at concentrations of ␦Ј between 0.5-5 M (Fig. 3B). Although we observed no interactions between ␦Ј and either domains I and II (C-⌬422) or domain III (N-⌬221), a lower limit for these K d values could be estimated by comparing N-⌬221 -␦Ј interaction with the C(0)-␦Ј interaction. When 5 M of ␦Ј was injected over the N-⌬221-derivatized sensor chip, no binding was observed. However, significant binding was obtained when 0.5 M of ␦Ј, a 10-fold less concentration, was injected over the C(0) derivatized sensor chip (Fig. 3); compatible amounts of C(0) and N-⌬221 were on their respective derivatized sensor chips (see legend of Fig. 3 for details). Thus, if there is an interaction between N-⌬221 and ␦Ј, the binding affinity is at least 10-fold weaker than that of the C(0)-␦Ј interaction (500 nM). Domain III of Contains the ␦␦Ј Binding Site and the Sequence Required for the ␦ Cooperativity-The ␦ subunit has a positive cooperative effect on the -␦Ј interaction (22). Because interactions between ␦Ј and domain III or domains I and II of DnaX may have been too weak to be detectable by our methodology, we re-examined binding using ␦␦Ј instead of ␦Ј. This enabled us to evaluate whether the cooperative effects of ␦ strengthen the binding of ␦Ј to the various DnaX domain constructs to detectable levels. In the following studies, the concentrations of ␦Ј in all of the ␦␦Ј samples tested were greater than the K d of ␦-␦Ј interaction, 3 and the concentrations of ␦ were 5-10 fold higher than those of ␦Ј. Thus, nearly all the ␦Ј in these experiments was bound to ␦ to form ␦␦Ј.
When injected over immobilized C(0) (Fig. 4A), ␦␦Ј bound C(0) with an association rate of 4 ϫ 10 3 M Ϫ1 s Ϫ1 . The dissociation of ␦ and ␦Ј from the immobilized C(0) was complicated because two separate dissociation events were occurring simultaneously. One process was the dissociation of ␦␦Ј from the immobilized C(0), and the other was the dissociation of ␦ from ␦Ј still bound to immobilized C (0). Thus, the total dissociation rate measured in these studies was actually reflective of contributions from these two different processes. From the association and dissociation rates, the calculated K d was 115 nM ( Table V).
Samples of ␦␦Ј at the same concentrations used for analysis of the C(0)-␦␦Ј interaction were then passed over the N-⌬221 and C-⌬422 derivatized sensor chips. No binding of C-⌬422 to ␦␦Ј was observed, but N-⌬221 bound ␦␦Ј at each concentration of ␦␦Ј that was tested (Fig. 4B). These results indicate that
10 M of the ␦ subunit alone was injected over the immobilized C(0), C-⌬261, and N-⌬221. No interaction was observed (data not shown), consistent with the report that there is no interaction between and ␦ (13). In contrast, the interactions between C(0)-␦Ј and C-⌬261-␦Ј could be easily detected under the same experimental conditions. Thus, the ability to detect the N-⌬221-␦␦Ј interaction resulted from the positive cooperative effect of ␦ on the -␦Ј interaction; the deletion of domains I and II of did not abolish this cooperativity. Thus, domain III not only binds and ␦␦Ј but also contains the elements required for the positive cooperative effect of ␦ on the ␦Ј-DnaX interaction. This is also evidenced by similar decreases in the K d values of C-⌬261-␦Ј and N-⌬1-␦Ј interactions in the presence of ␦ (Table V). An alternative explanation is that ␦ can weakly interact with DnaX; even though the interaction is too weak to be observed by itself the interaction could lead to an increase in binding of ␦Ј to DnaX because of the additivity of the binding energies.
Domain III of DnaX Is Sufficient for to Strengthen the ␦␦Ј-C(0) Interaction-The observation that increases both -␦␦Ј and ␥-␦␦Ј binding affinity indicated that the sequence required for the positive cooperative effect of is localized in the ␥ portion of DnaX (16). To identify the domain(s) responsible for this cooperativity, C(0) and N-⌬221 were captured onto streptavidin sensor chips, and their relative affinities for ␦␦Ј in the presence of were examined. A solution of (1 M) was passed over the immobilized C(0) until no further binding of was detectable. Samples of ␦␦Ј containing were then injected over the -saturated C(0), and binding was observed. Dissociation was then carried out in the presence of the same buffer containing . This ensured that the RU decrease observed during the dissociation phase was only due to dissociation of ␦␦Ј. The C(0)␦␦Ј complex formed faster and dissociated slower than did the C(0)␦␦Ј complex (Fig. 5A). In the presence of , the K d was ϳ28 nM (Table VI), 4-fold less than that of the the C(0)-␦␦Ј interaction in the absence of .
Under similar experimental conditions, ␦␦Ј samples containing were passed over the N-⌬221-derivatized sensor chip. The presence of resulted in a 5-fold reduction in the K d of N-⌬221-␦␦Ј interaction ( Fig. 5B and Table VI). This result indicates that the absence of domains I and II did not eliminate the cooperative effect of on the N-⌬221-␦␦Ј interaction. Thus, domain III of contains the sequence required formediated augmentation of the DnaX-␦␦Ј interaction. DISCUSSION The DnaX complex in E. coli functions to assemble the ␤ processivity factor onto the primed template for processive DNA replication. Within the complex, ␦Ј and bind /␥ with ␦Ј bridging the /␥-␦ interaction and bridging the /␥interaction (13). The presence of ␦ and strengthens the DnaX-␦Ј interaction (16,23). In this study, we identified the ␦Ј and binding domain of the DnaX proteins by measuring the interactions of these two subunits with truncated proteins lacking specific domains. Our results indicate that domain III (amino ␦␦Јand -Binding Domain of DnaX acid residues 222-382) shared by ␥ and binds both and ␦␦Ј. Domain III also contains the elements required for the positive cooperative assembly of the DnaX complex.
Among the truncated proteins that bound the subunit, C(0) (domains I-V), N-⌬1 (domains I-V), N-⌬221 (domains III-V), and C-⌬261 (domains I-III) showed similar affinities for , indicating that deletion of domains I and II or domains IV and V of did not decrease the strength of theinteraction. Therefore, domain III appears to be responsible forbinding.
In studies directed toward mapping the ␦Ј binding domain, ␦Ј was observed to bind C-⌬261 (domains I-III) and full-length very weakly, near the limit of detection for the BIAcore. However, binding of ␦Ј to N-⌬221 (domains III-IV) or C-⌬422 (domains I-II) could not be detected directly. Instead, we evaluated these interactions by measuring the binding of ␦␦Ј to DnaX derivatives. This exploited the increased affinity of ␦Ј for DnaX in the presence of ␦. In the presence of both ␦ and ␦Ј, N-⌬221 was observed to bind ␦␦Ј, but C-⌬422 did not, indicating that domain III of contained the ␦␦Ј binding site. The K d of the N-⌬221-␦␦Ј interaction was 10-fold greater than those of the C(0)-␦␦Ј and C-⌬261-␦␦Ј interactions (Table V). N-⌬221 interacted less strongly with ␦␦Ј than did C(0) and C-⌬261, perhaps because the N-terminal peptide tag of N-⌬221 slightly interfered with the binding of the fusion protein to ␦␦Ј. Alternatively, deletion of domains I and II may have perturbed the structure of domain III.
␦Ј shares a high sequence similarity to the N-terminal domains I-III of DnaX (24,25). Both of the DnaX proteins, and ␥, are tetramers ( 4 , ␥ 4 ) when free in solution (12,23). The DnaX complex ( 2 ␥ 1 ␦Ј 1 ␦ 1 1 1 ) contains a total of four copies of homologous subunits (, ␥, and ␦Ј) (12). It seems reasonable to assume that one DnaX protomer of the tetrameric DnaX proteins is replaced by the homologous ␦Ј subunit during the formation of the DnaX complex. It is likely that the DnaX proteins bind each other or to the homolog ␦Ј via similar mechanisms; the same portions of probably mediate its binding to other subunits and to ␥. We have shown that domain III of DnaX is involved in ␦␦Ј binding, likely through the DnaX-␦Ј interaction in the presence of ␦. Thus, domain III is also likely involved in theand -␥ interactions. That is, the sequences responsible for the tetramerization of DnaX are probably localized in domain III.
The presence of ␦ decreases the K d of the C(0)-␦Ј interaction by approximately 3-fold, as indicated by a comparison of the K d values of the C(0)-␦Ј and C(0)-␦␦Ј interactions. The presence of ␦ also strengthens the C-⌬261-␦Ј interaction 3-fold. The cooperative effect of ␦ on the N-⌬221-␦Ј interaction could not be calculated directly because the interaction of N-⌬221, and ␦Ј in the absence of ␦ was too weak to be detected. However, the lower boundary of the N-⌬221-␦Ј interaction K d was estimated to be 5 M, based upon the comparison of the concentrations of ␦Ј and the -derivatives used in examination of the C(0)-␦Ј and N-⌬221-␦Ј interactions. The effects of ␦ on the N-⌬221-␦Ј interaction can be estimated using the lower boundary K d for the N-⌬221-␦Ј interaction and the K d for the N-⌬221-␦Ј␦ interaction (Table IV). Using these values, we calculated that ␦ augments the N-⌬221-␦Ј interaction ϳ3-fold, the same degree of enhancement for the interactions between ␦Ј and proteins containing domains I-III. Therefore, domain III appears to contain all sequences required for the full cooperative effect of ␦␦Ј on their interaction(s) with DnaX.
Previous studies indicate that the presence of strengthens both -␦␦Ј and ␥-␦␦Ј interactions (16). The cooperative effect of on the DnaX-␦␦Ј was examined using BIAcore technology.
FIG. 5. Positive cooperative assembly of the DnaX complex on BIAcore sensor chips. C(0) and N-⌬221 were immobilized to streptavidin sensor chips as described under "Experimental Procedures." The binding study was conducted in HKGM buffer containing 2% glycerol, 2 mM dithiothreitol, and 1 M at 20°C. ␦Ј and ␦ diluted in the above binding buffer were preincubated for 10 min at room temperature before injection. The injection of the ␦Ј␦ samples over the immobilized C(0) and N-⌬221 took 6 min at 10 l/min.  The C(0)-␦␦Ј interaction is strengthened approximately 4-fold (Table VI) in the presence of . The absence of domains I and II did not eliminate the cooperative effect of on domain III-␦␦Ј binding. Rather, decreased the K d of the N-⌬221-␦␦Ј interaction by 5-fold (Table VI). These results indicate that domain III alone is sufficient for the positive cooperativity of on the -␦␦Ј interaction. The presence of both ␦ and strengthens the C (0)-␦ interaction at least 15-fold, indicating a cooperative assembly of the DnaX complex. Domain III of DnaX not only contains the ␦Ј and binding sites but also the sequences required for cooperative assembly of the DnaX complex. This structural arrangement suggests that the cooperativity is a result of an allosteric effect. That is, upon interactions between and the DnaX proteins, the DnaX proteins adopt a conformation with higher affinity for ␦Ј; upon the interaction between ␦ and ␦Ј, the ␦Ј subunit adopts a conformation with higher affinity for domain III of DnaX proteins. This allosteric effect is crucial for the efficient assembly of the DnaX complex in vivo. The interaction between ␥ and ␦␦Ј is weak with a K d of about 100 nM, which is greater that the 28 nM concentration of each component of the DnaX complexes in the cell (26,27). The affinity between ␥ and is ϳ10 nM, and the ␥subassembly can readily form in the cell. Because of the binding of , ␥ adopts a higher affinity for ␦␦Ј with a K d of ϳ28 nM. Thus, the ␥complex efficiently recruits the ␦␦Ј to form ␥␦␦Ј complex at physiological subunit concentrations.
The DnaX complex functions to load the ␤ processivity factor onto primed template in an ATP-dependent manner. Based on their studies of the crystal structure of the highly homologous ␦Ј subunit considered in light of structural features of several ATPases, Guenther and colleagues (25) proposed that the Nterminal three domains of ␥ would also adapt a C-shaped conformation. They also contended that this C-shaped region is likely to open and close in response to ATP binding and hydrolysis by the ␥ subunit. Experimental results also support these hypotheses. In the absence of ATP or ATP analogs, the DnaX complex does not bind ␤, presumably because the ␤ binding partner, ␦, is buried in the complex. In contrast, DnaX-␤ interactions occur in the presence of ATP or ATP analogs, indicating that conformational changes occur as ATP binds to ␥ such that ␦ subunit becomes exposed, enabling interaction with ␤ (28). Our results show that the ␦␦Ј-binding portion of DnaX lies within domain III and suggest that this domain may be involved in mediating the ATP effects on the DnaX complex and ␤ interaction. In the absence of ATP, the C-like arrangement of domains I-III of ␥ is closed, and the auxiliary subunit ␦, which is bridged to domain III through ␦Ј, is entrapped within the DnaX complex and not freely accessible to the processivity factor ␤. In contrast, ATP binding to ␥ at the interface of domains I and II causes the "C" to open such that domain III of ␥, and hence the bridged ␦␦Ј subunits are relatively exposed; ␦Ј is then free to interact with ␤. Thus, domain III serves as a "transducer" of the ATP binding signal.
Results from our studies shed light on some of the functional differences between the structurally similar subunits, and ␥.
␥ is comprised of domains I-III. Domain III of ␥ binds the auxiliary subunit ␦␦Ј, and functions as the processivity factor (␤ 2 ) assembly apparatus. contains the same N-terminal three domains, as does ␥ plus two additional C-terminal domains (domains IV and V). Domain V binds ␣ (polymerase) to form the dimeric polymerase enabling the simultaneous synthesis of the leading and lagging strands. Domain IV interacts with the DnaB helicase to coordinate the replicase and the primosome activities at the replication fork. Domain III is thought to be involved in linking to the ␥ processivity assembly apparatus. The auxiliary subunit binds SSB to form a tether between DnaX complex and the SSB-coated lagging strand (18). interacts with both the processivity assembly apparatus (via ␥) and the polymerase (via ␣). This direct processivity assembly/polymerase link bridged by DnaX strengthens the interactions between the holoenzyme and the SSBcoated lagging strand at the replication fork. Interactions mediated by domains III-V of enable this subunit to serve as a central organizer. The subunit effectively couples the processivity assembly process, SSB binding, DnaB helicase activities, and the dimeric replicase into one replicative complex at the replication fork.