tau Binds and Organizes Escherichia coli Replication Proteins through Distinct Domains

DOMAIN III, SHARED BY gamma  AND tau , BINDS delta delta ' AND chi psi *

Dexiang Gao and Charles S. McHenry

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

Received for publication, October 27, 2000



    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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



    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Processive and efficient replication of genomic DNA in both prokaryotes and eukaryotes is facilitated by three conserved functional components: a DNA polymerase, a sliding clamp processivity factor and a multiple-subunit complex that loads the processivity factor onto a primed-template. In Escherichia coli, the DNA polymerase III holoenzyme is responsible for duplication of the genome in a rapid and processive manner. The holoenzyme contains ten different types of subunits and is composed of two DNA polymerase III cores (alpha epsilon theta ): two beta 2 sliding clamp processivity factors and the clamp-loading DnaX complex. The DnaX complex contains the DnaX proteins plus auxiliary subunits delta , delta ', chi , and psi . The distributive DNA polymerase III core becomes highly processive when the DnaX complex assembles the beta  processivity factor around DNA in an ATP-dependent process (for reviews see Refs. 1-3).

The dnaX gene produces two distinct proteins, tau  and gamma , 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 tau , absent in the gamma  protein, that allows the full-length DnaX gene product to interact with both the DnaB helicase and the DNA polymerase III core. These tau -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 tau  and gamma .

Functional homomeric DnaX complexes (tau  complex, tau 3delta 1delta '1chi 1psi 1, and gamma  complex, gamma 3delta 1delta '1chi 1psi 1) can be assembled in vitro (11, 12). Thus, the N-terminal 430 residues common to both tau  and gamma  have the minimal protein sequence necessary not only to bind the auxiliary subunits delta , delta ', chi , and psi  but also to load the beta  processivity factor onto a primed template in an ATP-dependent manner. Within the DnaX complex, delta ' and psi  bind directly to gamma ; delta  binds delta ', and chi  binds psi  (13, 14). delta  and delta ' form a 1:1 complex and function with DnaX to load beta  onto primed templates in an ATP-dependent manner (10, 15). The chi  and psi  subunits also form a 1:1 complex and increase the affinity of DnaX for delta  and delta ' so that a functional DnaX complex can be assembled at physiological subunit concentrations (16). The chi  subunit also interacts with SSB, consistent with the finding that chi -psi -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 tau  protein. The focus of this report is to determine which structural domain(s) within the portion of DnaX common to both gamma  and tau  (domains I-III) are responsible for binding the auxiliary subunits. To this end, the relative binding of delta delta ' and chi psi to a series of truncated DnaX proteins lacking specific structural domains was measured using surface plasmon resonance.


    EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Strains-- E. coli strains DH5alpha and HB101 were used for initial molecular cloning procedures and plasmid propagation. E. coli strain BL21(lambda DE3) was used for protein expression.

Materials and Buffers-- CM5 sensor chips (research grade), P-20 surfactant, N-hydroxysuccinimide, 1-ethyl-3-[(3-dimethylamino)propyl]-carbodiimide, and ethanolamine hydrochloride were obtained from BIAcore Inc. Ni2+-NTA1 resin, the QIAquick Gel extraction kit, QIAquick PCR purification kit, and the plasmid preparation kit were purchased from Qiagen. SDS-polyacrylamide gel electrophoresis protein standards were from Life Technologies, Inc. d-Biotin was obtained from Sigma. Coomassie Plus protein assay reagent and Immunopure streptavidin were from Pierce. Buffer L, Buffer W, HBS buffer, and HKGM buffer were prepared as previously described (19).

Construction of the Fusion Plasmids-- Plasmid PA1-N-Delta 221tau encodes the fusion protein N-Delta 221tau . The starting material for construction of plasmid PA1-N-Delta 221tau was plasmid PA1-N-Delta 1tau , which encodes the tau  protein with the initiating methionine replaced by an N-terminal fusion peptide. PCR primer N-221p1 contained a PstI sequence at the noncomplementary 5'-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 pPA1-N-Delta 1tau to generate plasmid PA1-N-Delta 221tau .

Plasmid PA1-C-Delta 261tau encodes the truncated fusion protein C-Delta 261tau . To construct the plasmid PA1-C-Delta 261tau , a partial dnaX gene sequence encoding the C-terminal 261 amino acids of tau  was deleted from the previously constructed plasmid PA1-C(0)tau , which encodes the C-terminal tagged full-length tau  protein (19). PCR primer C-Delta 261P1 was complementary to a region of dnaX located 430 bases upstream of the internal RsrII site. Primer C-Delta 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 pPA1-C(0)tau to generate pPA1-C-Delta 261tau .

Plasmid pET11-C-Delta 422tau , which lacked the sequence encoding the C-terminal 422 amino acids of tau , was constructed as follows. pET11-C(0)tau was digested with AflII and SpeI to delete a dnaX sequence encoding from residue 218 to the 3' end (residue 643) of tau . The annealed oligonucleotides C-422p1 and C-422p2 containing the sequence encoding tau  amino acid residues 218-221 were ligated into the linearized pET11-C(0)tau vector to generate pET11-C-Delta 422tau (Table I).


                              
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Table I
Oligonucleotides used for construction of truncated tau  fusion proteins

Cell Growth and Induction-- Plasmids PA1-C-Delta 261tau , PA1-N-Delta 221tau , and pET11-C-Delta 422tau were transformed into E. coli strain BL21 (lambda  DE3) for expression of proteins C-Delta 261tau , N-Delta 221tau , and C-Delta 422tau , respectively. The transformed BL21 (lambda  DE3) cells were grown at 37 °C in 2 liters of F medium (20) containing 100 µg/ml ampicillin. Cells were induced, biotin-treated, and harvested as previously described (19).

Protein Purification-- C(0)tau , N-Delta 1tau , 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-Delta 261tau , N-Delta 221tau , and C-Delta 422tau 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 Ni2+-NTA affinity chromatography as previously described for PA1-N-Delta 1tau (19), except that the bound C-Delta 261tau was eluted stepwise in Buffer W containing 150 mM imidazole. For purification of C-Delta 422tau , 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-Delta 261tau . For purification of N-Delta 221tau , the imidazole concentration was 15 mM in the washing buffer; the bound N-Delta 221tau was eluted with 10 column volumes of 15-100 mM imidazole gradient in buffer W. The imidazole concentration in the peak fraction of N-Delta 221tau was ~55 mM.

Surface Plasmon Resonance-- A BIAcoreTM 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 tau  fusion proteins (700-1600 RU) were then captured onto sensor chips via streptavidin-biotin interaction. tau -chi psi binding studies were conducted in HKGM buffer at a flow rate of 25 µl/min at 20 °C. tau -delta ' and tau -delta delta ' binding studies were performed in HKGM buffer containing 2% glycerol and 2 mM dithiothreitol at a flow rate of 10 µl/min at 20 °C. In these studies, delta  and delta ' were preincubated for 10 min at room temperature before injecting over the tau  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 tau  fusion proteins were measured by their requirement for reconstitution of holoenzyme activity as previously described (19).


    RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Expression and Purification of the Truncated DnaX Fusion Proteins-- We constructed three plasmids, each encoding specific structural domains of tau  under control of an inducible promoter. Plasmid PA1-N-Delta 221tau encoded protein N-Delta 221tau (domains III-V), plasmid PA1-C-Delta 261tau encoded protein C-Delta 261tau (domains I-III), and plasmid pET11-C-Delta 422tau encoded protein C-Delta 422tau (domains I-II) (Fig. 1). Each of these proteins contained a hexahistidine sequence to facilitate purification using Ni2+-NTA metal affinity chromatography and a short biotinylation sequence to enable immobilization on streptavidin-coated BIAcore sensor chips. The biotin tag also enabled detection of fusion proteins using biotin blots (21). The expression levels of C-Delta 261tau , N-Delta 221tau , and C-Delta 422tau were ~3%, 2, and 0.5% of the total cell protein, respectively. After one round of Ni2+-NTA chromatography, N-Delta 221tau and C-Delta 261tau preparations at greater than 80% of purity were obtained; the purity of C-Delta 422tau was ~30%, 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-Delta 261tau through its purification were measured in holoenzyme reconstitution assays. Purified C-Delta 261tau had a specific activity of 5.3×106 units/mg, which is comparable with that of C(0)tau (Table II). As expected, N-Delta 221tau was inactive in this same assay because its ATPase motif (domains I and II) was deleted. C-Delta 422tau (domains I and II) was also inactive in this assay, suggesting that domains I-III represent the minimum protein required to assemble the beta  processivity factor onto the DNA template.



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Fig. 1.   Purified truncated tau -fusion proteins. The upper panel shows the truncated biotinylated fusion proteins of tau  used in BIAcore analysis. C-Delta 261tau contains domains I-III, N-Delta 221tau contains domains III-V, and C-Delta 422tau contains domains I and II. The rectangular box represents the biotinylated fusion peptide. The lower panel is the Coomassie Blue-stained 12% SDS-polyacrylamide gel of 1.5 µg of each purified protein after Ni2+-NTA purification. Lane 1, C(0)tau ; lane 2, N-Delta 1tau ; lane 3, C-Delta 261tau ; lane 4, N-Delta 221tau ; lane 5, C-Delta 422tau , with the arrow indicating the C-Delta 422tau protein (two bands are clustered together with the lower band is the C-Delta 422tau protein which is ~30% of the total protein of the lane; N-Delta 1tau was shown in the first paper of this series).


                              
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Table II
Purification of tau  deletion fusion proteins

DnaX Domain III Interacts with chi psi -- The abilities of the truncated tau  fusion proteins C-Delta 261tau , N-Delta 221tau , and C-Delta 422tau to interact with chi psi were measured by using BIAcore methodology. Because the chi psi binding region is located within the N-terminal 430 amino acids of tau , a fusion peptide tag at the remote C terminus of tau  would be unlikely to interfere with chi psi binding. For this reason, C(0)tau was used as a positive control for examination of chi psi binding to the truncated tau  proteins. Biotin-tagged C(0)tau was immobilized onto a streptavidin sensor chip, and various concentrations of chi psi (25-300 nM) were passed over immobilized C(0)tau so that binding of free chi psi to immobilized C(0)tau could be measured. Representative binding curves (Fig. 2A) indicate that chi psi bound rapidly to C(0)tau with an association rate of 2.4×105 M-1 s-1. A dissociation rate of 2.5 × 10-3 s-1 (Table III) was obtained; this rate was measured after saturating C(0)tau with chi psi to minimize reassociation. The resulting Kd2 (10 ± 1 nM) was ~5-fold higher than the Kd reported for the native tau -chi psi interaction. The observed difference between the Kd 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.



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Fig. 2.   The interactions of chi psi with immobilized C(0)tau , N-Delta 1tau , C-Delta 261tau , N-Delta 221tau , and C-Delta 422tau proteins. Streptavidin was chemically immobilized to a CM5 sensor chip as described under "Experimental Procedures." Biotinylated tau  proteins were attached to sensor chips via biotin-streptavidin interaction. The binding analyses of chi psi with truncated tau  proteins were conducted in HKGM buffer at a flow rate of 25 µl/min. A, Sensorgrams of C(0)tau -chi psi binding. C(0)tau (1520 RU) was attached to a sensor chip. Varying concentrations of chi psi (50, 75, and 150 nM) were injected over the C(0)tau -immobilized sensor chip for 3 min each. B, domain III of tau  contains the chi psi binding site. The N-Delta 1tau (930 RU), C-Delta 261tau (710 RU), N-Delta 221tau (650 RU), and C-Delta 422tau (540 RU) proteins, respectively, were captured onto sensor chips. Solution of chi psi (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.


                              
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Table III
Interactions of chi psi with truncated and full-length tau  fusion proteins: kinetic and equilibrium constants
Results are the averages of two separate experiments, with the error expressed as the range of two data sets.

The interactions between chi psi and the truncated fusion proteins N-Delta 1tau , N-Delta 221tau , C-Delta 422tau , and C-Delta 261tau were examined under the same experimental conditions as used for the chi psi and C(0)tau interaction. N-Delta 1tau , N-Delta 221tau , and C-Delta 261tau bound chi psi , but C-Delta 422tau did not (Fig. 2B), indicating that domain III of tau , the only domain shared by all of the fusion proteins shown to bind chi psi , is required for chi psi binding. Similar binding stoichiometries were obtained for the interactions of chi psi -N-Delta 1tau and chi psi -C-Delta 261tau compared with that of the chi psi -C(0)tau interaction (Table III). The Kd for the interaction between chi psi -N-Delta 221tau was within 2.5-fold of that measured for the chi psi -C(0)tau 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 tau  proteins did not decrease the affinity of the tau -chi psi interaction. Thus, domain III of tau  appears to be fully responsible for chi psi binding.

No Observable Binding of delta ' to Individual Domains of the DnaX Protein-- The interactions between delta ' and the full-length tau  proteins with either an N- or C-terminal tag were characterized in binding studies utilizing the BIAcore instrumentation. delta ' samples were injected over the immobilized C(0)tau , and the binding curves are shown in Fig. 3A. The interaction between delta ' and N-Delta 1tau is characterized by weak binding similar to that observed for the delta '-C(0)tau interaction (Table IV). Although measurable, the weak binding observed is close to the limit of BIAcore detection.



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Fig. 3.   Interactions of delta ' with immobilized C(0)tau , N-Delta 1tau , C-Delta 261tau , N-Delta 221tau , and C-Delta 422tau proteins. Streptavidin was chemically immobilized to a CM5 sensor chip as described under "Experimental Procedures." Biotinylated tau  proteins were attached to sensor chips via biotin-streptavidin interaction. Binding analyses were conducted in HKGM buffer containing 2% glycerol and 2 mM dithiothreitol at a flow rate of 10 µl/min. A, sensorgrams of C(0)tau -delta ' binding. C(0)tau (1500 RU) was attached to a sensor chip. Varying concentrations of the delta ' subunit (0.5, 1, 2, and 4 µM) were injected over the C(0)tau -immobilized sensor chip for 6 min each. B, delta ' binds N-Delta 1tau and C-Delta 261tau but not N-Delta 221tau or C-Delta 422tau . The N-Delta 1tau (2300 RU), C-Delta 261tau (1600 RU), N-Delta 221tau (1470 RU), and C-Delta 422tau (730 RU) proteins, respectively, were captured onto individual sensor chips. For 6 min each, solutions containing 4 µM delta ' subunit were injected over the N-Delta 1tau and C-Delta 261tau -immobilized sensor chips; solutions containing 5 µM delta ' subunit were injected over the C-Delta 422tau and N-Delta 221tau sensor chips. Values from control injections obtained via use of an streptavidin-immobilized sensor chip were subtracted from each curve shown.


                              
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Table IV
Interactions of delta ' with truncated and full-length tau  fusion proteins: kinetic and equilibrium constants
Results are the averages of two separate experiments, with the error expressed as the range of two data sets.

Next, the truncated DnaX proteins N-Delta 221tau (domains III-V), C-Delta 422tau (domains I-II), and C-Delta 261tau (domains I-III) were captured onto streptavidin sensor chips so that the binding of delta ' to these proteins could be measured. The Kd observed for the delta 'subunit-C-Delta 261tau interaction was similar to that of the C(0)tau -delta ' interaction (Fig. 3B and Table IV). These observations confirm that the delta ' binding region of tau  is entirely within its N-terminal 382 amino acid residues (domains I-III). However, delta ' did not bind to N-Delta 221tau or C-Delta 422tau at concentrations of delta ' between 0.5-5 µM (Fig. 3B). Although we observed no interactions between delta ' and either domains I and II (C-Delta 422tau ) or domain III (N-Delta 221tau ), a lower limit for these Kd values could be estimated by comparing N-Delta 221tau -delta ' interaction with the C(0)tau -delta ' interaction. When 5 µM of delta ' was injected over the N-Delta 221tau -derivatized sensor chip, no binding was observed. However, significant binding was obtained when 0.5 µM of delta ', a 10-fold less concentration, was injected over the C(0)tau derivatized sensor chip (Fig. 3); compatible amounts of C(0)tau and N-Delta 221tau were on their respective derivatized sensor chips (see legend of Fig. 3 for details). Thus, if there is an interaction between N-Delta 221tau and delta ', the binding affinity is at least 10-fold weaker than that of the C(0)tau -delta ' interaction (500 nM).

Domain III of tau  Contains the delta delta ' Binding Site and the Sequence Required for the delta  Cooperativity-- The delta  subunit has a positive cooperative effect on the tau -delta ' interaction (22). Because interactions between delta ' 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 delta delta ' instead of delta '. This enabled us to evaluate whether the cooperative effects of delta  strengthen the binding of delta ' to the various DnaX domain constructs to detectable levels. In the following studies, the concentrations of delta ' in all of the delta delta ' samples tested were greater than the Kd of delta -delta ' interaction,3 and the concentrations of delta  were 5-10 fold higher than those of delta '. Thus, nearly all the delta ' in these experiments was bound to delta  to form delta delta '.

When injected over immobilized C(0)tau (Fig. 4A), delta delta ' bound C(0)tau with an association rate of 4 × 103 M-1 s-1. The dissociation of delta  and delta ' from the immobilized C(0)tau was complicated because two separate dissociation events were occurring simultaneously. One process was the dissociation of delta delta ' from the immobilized C(0)tau , and the other was the dissociation of delta  from delta ' 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 Kd was 115 nM (Table V).



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Fig. 4.   Interactions of delta delta ' with immobilized C(0)tau , N-Delta 1tau , C-Delta 261tau , N-Delta 221tau , and C-Delta 422tau proteins. Streptavidin was chemically immobilized to a CM5 sensor chip as described under "Experimental Procedures." Binding experiments were conducted in HKGM buffer containing 2% glycerol and 2 mM dithiothreitol at a flow rate of 10 µl/min. A, sensorgrams of C(0)tau -delta delta ' binding. C(0)tau (1550 RU) was attached to a sensor chip. delta  and delta ' samples were mixed at three different concentrations (0.7 µM delta ' + 7 µM delta , 1.2 µM delta ' + 10 µM delta , and 2 µM delta ' + 10 µM delta ), incubated for 10 min at room temperature, and injected over a C(0)tau sensor chip for 6 min. B, domain III of tau  contains the delta delta ' binding site. The N-Delta 1tau (2300 RU), C-Delta 261tau (1600 RU), N-Delta 221tau (1470 RU), and C-Delta 422tau (720 RU) proteins, respectively, were captured onto the streptavidin-bearing sensor chips. A mixture of 1.2 µM delta ' + 10 µM delta  was preincubated and injected over N-Delta 1tau , N-Delta 221tau , and C-Delta 261tau -immobilized sensor chip for 6 min each. A mixture of 10 µM delta ' + 20 µM delta  was preincubated and injected over a C-Delta 422tau immobilized sensor chip. Control values, obtained by passing injections over a sensor chip containing immobilized streptavidin only, were subtracted from each curve.


                              
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Table V
Interactions of delta ' with truncated and full-length tau  fusion proteins in the presence of delta  subunit: kinetic and equilibrium constants
Results are the averages of two separate experiments, with the error expressed as the range of two data sets.

Samples of delta delta ' at the same concentrations used for analysis of the C(0)tau -delta delta ' interaction were then passed over the N-Delta 221tau and C-Delta 422tau derivatized sensor chips. No binding of C-Delta 422tau to delta delta ' was observed, but N-Delta 221tau bound delta delta ' at each concentration of delta delta ' that was tested (Fig. 4B). These results indicate that N-Delta 221tau contains the delta delta ' binding site. The Kd of delta delta '-N-Delta 221tau interaction was 1.5 µM, which is only 10-fold weaker than that of the delta delta '-C(0)tau interaction.

10 µM of the delta  subunit alone was injected over the immobilized C(0)tau , C-Delta 261tau , and N-Delta 221tau . No interaction was observed (data not shown), consistent with the report that there is no interaction between tau  and delta  (13). In contrast, the interactions between C(0)tau -delta ' and C-Delta 261tau -delta ' could be easily detected under the same experimental conditions. Thus, the ability to detect the N-Delta 221tau - delta delta ' interaction resulted from the positive cooperative effect of delta  on the tau -delta ' interaction; the deletion of domains I and II of tau  did not abolish this cooperativity. Thus, domain III not only binds chi psi and delta delta ' but also contains the elements required for the positive cooperative effect of delta  on the delta '-DnaX interaction. This is also evidenced by similar decreases in the Kd values of C-Delta 261tau -delta ' and N-Delta 1tau -delta ' interactions in the presence of delta  (Table V). An alternative explanation is that delta  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 delta ' to DnaX because of the additivity of the binding energies.

Domain III of DnaX Is Sufficient for chi psi to Strengthen the delta delta '-C(0)tau Interaction-- The observation that chi psi increases both tau -delta delta ' and gamma -delta delta ' binding affinity indicated that the sequence required for the positive cooperative effect of chi psi is localized in the gamma  portion of DnaX (16). To identify the domain(s) responsible for this cooperativity, C(0)tau and N-Delta 221tau were captured onto streptavidin sensor chips, and their relative affinities for delta delta ' in the presence of chi psi were examined. A solution of chi psi (1 µM) was passed over the immobilized C(0)tau until no further binding of chi psi was detectable. Samples of delta delta ' containing chi psi were then injected over the chi psi -saturated C(0)tau , and binding was observed. Dissociation was then carried out in the presence of the same buffer containing chi psi . This ensured that the RU decrease observed during the dissociation phase was only due to dissociation of delta delta '. The C(0)tau delta delta 'chi psi complex formed faster and dissociated slower than did the C(0)tau delta delta ' complex (Fig. 5A). In the presence of chi psi , the Kd was ~28 nM (Table VI), 4-fold less than that of the the C(0)tau -delta delta ' interaction in the absence of chi psi .



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Fig. 5.   Positive cooperative assembly of the DnaX complex on BIAcore sensor chips. C(0)tau and N-Delta 221tau 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 µM chi psi at 20 °C. delta ' and delta  diluted in the above binding buffer were preincubated for 10 min at room temperature before injection. The injection of the delta 'delta samples over the immobilized C(0)tau and N-Delta 221tau took 6 min at 10 µl/min. A, sensorgram overlays of delta ' (1.2 µM) + delta  (10 µM) and delta ' (1.2 µM) + delta  (10 µM) + chi psi (1 µM) injected over immobilized C(0)tau are shown. B, sensorgram overlays of delta ' (1.2 µM) + delta  (10 µM) and delta ' (1.2 µM) + delta  (10 µM) + chi psi (1 µM) injected over immobilized N-Delta 221tau . The control injections of the above samples over the streptavidin-derivatized sensor chip were conducted and subtracted from the curves shown.


                              
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Table VI
Interactions of delta ' with C(0)tau and N-Delta 221tau in the presence of delta delta ' + chi psi subunits: kinetic and equilibrium constants
Results are the averages of two separate experiments, with the error expressed as the range of two data sets.

Under similar experimental conditions, delta delta ' samples containing chi psi were passed over the N-Delta 221tau -derivatized sensor chip. The presence of chi psi resulted in a 5-fold reduction in the Kd of N-Delta 221tau -delta delta ' interaction (Fig. 5B and Table VI). This result indicates that the absence of domains I and II did not eliminate the cooperative effect of chi psi on the N-Delta 221tau -delta delta ' interaction. Thus, domain III of tau  contains the sequence required for chi psi -mediated augmentation of the DnaX-delta delta ' interaction.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

The DnaX complex in E. coli functions to assemble the beta  processivity factor onto the primed template for processive DNA replication. Within the complex, delta ' and psi  bind tau /gamma with delta ' bridging the tau /gamma -delta interaction and psi  bridging the tau /gamma -chi interaction (13). The presence of delta  and chi psi strengthens the DnaX-delta ' interaction (16, 23). In this study, we identified the delta ' and chi psi binding domain of the DnaX proteins by measuring the interactions of these two subunits with truncated tau  proteins lacking specific domains. Our results indicate that domain III (amino acid residues 222-382) shared by gamma  and tau  binds both chi psi and delta delta '. Domain III also contains the elements required for the positive cooperative assembly of the DnaX complex.

Among the truncated tau  proteins that bound the chi psi subunit, C(0)tau (domains I-V), N-Delta 1tau (domains I-V), N-Delta 221tau (domains III-V), and C-Delta 261tau (domains I-III) showed similar affinities for chi psi , indicating that deletion of domains I and II or domains IV and V of tau  did not decrease the strength of the tau -chi psi interaction. Therefore, domain III appears to be responsible for tau -chi psi binding.

In studies directed toward mapping the delta ' binding domain, delta ' was observed to bind C-Delta 261tau (domains I-III) and full-length tau  very weakly, near the limit of detection for the BIAcore. However, binding of delta ' to N-Delta 221tau (domains III-IV) or C-Delta 422tau (domains I-II) could not be detected directly. Instead, we evaluated these interactions by measuring the binding of delta delta ' to DnaX derivatives. This exploited the increased affinity of delta ' for DnaX in the presence of delta . In the presence of both delta  and delta ', N-Delta 221tau was observed to bind delta delta ', but C-Delta 422tau did not, indicating that domain III of tau  contained the delta delta ' binding site. The Kd of the N-Delta 221tau -delta delta ' interaction was 10-fold greater than those of the C(0)tau -delta delta ' and C-Delta 261tau -delta delta ' interactions (Table V). N-Delta 221tau interacted less strongly with delta delta ' than did C(0)tau and C-Delta 261tau , perhaps because the N-terminal peptide tag of N-Delta 221tau slightly interfered with the binding of the fusion protein to delta delta '. Alternatively, deletion of domains I and II may have perturbed the structure of domain III.

delta ' shares a high sequence similarity to the N-terminal domains I-III of DnaX (24, 25). Both of the DnaX proteins, tau  and gamma , are tetramers (tau 4, gamma 4) when free in solution (12, 23). The DnaX complex (tau 2gamma 1delta '1delta 1chi 1psi 1) contains a total of four copies of homologous subunits (tau , gamma , and delta ') (12). It seems reasonable to assume that one DnaX protomer of the tetrameric DnaX proteins is replaced by the homologous delta ' subunit during the formation of the DnaX complex. It is likely that the DnaX proteins bind each other or to the homolog delta ' via similar mechanisms; the same portions of tau  probably mediate its binding to other tau  subunits and to gamma . We have shown that domain III of DnaX is involved in delta delta ' binding, likely through the DnaX-delta ' interaction in the presence of delta . Thus, domain III is also likely involved in the tau -tau and tau -gamma interactions. That is, the sequences responsible for the tetramerization of DnaX are probably localized in domain III.

The presence of delta  decreases the Kd of the C(0)tau -delta ' interaction by approximately 3-fold, as indicated by a comparison of the Kd values of the C(0)tau -delta ' and C(0)tau -delta delta ' interactions. The presence of delta  also strengthens the C-Delta 261tau -delta ' interaction 3-fold. The cooperative effect of delta  on the N-Delta 221tau -delta ' interaction could not be calculated directly because the interaction of N-Delta 221tau , and delta ' in the absence of delta  was too weak to be detected. However, the lower boundary of the N-Delta 221tau -delta ' interaction Kd was estimated to be 5 µM, based upon the comparison of the concentrations of delta ' and the tau -derivatives used in examination of the C(0)tau -delta ' and N-Delta 221tau -delta ' interactions. The effects of delta  on the N-Delta 221tau -delta ' interaction can be estimated using the lower boundary Kd for the N-Delta 221tau -delta ' interaction and the Kd for the N-Delta 221tau -delta 'delta interaction (Table IV). Using these values, we calculated that delta  augments the N-Delta 221tau -delta ' interaction ~3-fold, the same degree of enhancement for the interactions between delta ' and tau  proteins containing domains I-III. Therefore, domain III appears to contain all sequences required for the full cooperative effect of delta delta ' on their interaction(s) with DnaX.

Previous studies indicate that the presence of chi psi strengthens both tau -delta delta ' and gamma -delta delta ' interactions (16). The cooperative effect of chi psi on the DnaX-delta delta ' was examined using BIAcore technology. The C(0)tau -delta delta ' interaction is strengthened approximately 4-fold (Table VI) in the presence of chi psi . The absence of domains I and II did not eliminate the cooperative effect of chi psi on domain III-delta delta ' binding. Rather, chi psi decreased the Kd of the N-Delta 221tau -delta delta ' interaction by 5-fold (Table VI). These results indicate that domain III alone is sufficient for the positive cooperativity of chi psi on the tau -delta delta ' interaction. The presence of both delta  and chi psi strengthens the C (0)-delta interaction at least 15-fold, indicating a cooperative assembly of the DnaX complex.

Domain III of DnaX not only contains the delta ' and chi psi 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 chi psi and the DnaX proteins, the DnaX proteins adopt a conformation with higher affinity for delta '; upon the interaction between delta  and delta ', the delta ' 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 gamma  and delta delta ' is weak with a Kd 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 gamma  and chi psi is ~10 nM, and the gamma -chi psi subassembly can readily form in the cell. Because of the binding of chi psi , gamma  adopts a higher affinity for delta delta ' with a Kd of ~28 nM. Thus, the gamma -chi psi complex efficiently recruits the delta delta ' to form gamma delta delta 'chi psi complex at physiological subunit concentrations.

The DnaX complex functions to load the beta  processivity factor onto primed template in an ATP-dependent manner. Based on their studies of the crystal structure of the highly homologous delta ' subunit considered in light of structural features of several ATPases, Guenther and colleagues (25) proposed that the N-terminal three domains of gamma  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 gamma  subunit. Experimental results also support these hypotheses. In the absence of ATP or ATP analogs, the DnaX complex does not bind beta , presumably because the beta  binding partner, delta , is buried in the complex. In contrast, DnaX-beta interactions occur in the presence of ATP or ATP analogs, indicating that conformational changes occur as ATP binds to gamma  such that delta  subunit becomes exposed, enabling interaction with beta  (28). Our results show that the delta delta '-binding portion of DnaX lies within tau  domain III and suggest that this domain may be involved in mediating the ATP effects on the DnaX complex and beta  interaction. In the absence of ATP, the C-like arrangement of domains I-III of gamma  is closed, and the auxiliary subunit delta , which is bridged to domain III through delta ', is entrapped within the DnaX complex and not freely accessible to the processivity factor beta . In contrast, ATP binding to gamma  at the interface of domains I and II causes the "C" to open such that domain III of gamma , and hence the bridged delta delta ' subunits are relatively exposed; delta ' is then free to interact with beta . 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, tau  and gamma . gamma  is comprised of domains I-III. Domain III of gamma  binds the auxiliary subunit delta delta ', and chi psi functions as the processivity factor (beta 2) assembly apparatus. tau  contains the same N-terminal three domains, as does gamma  plus two additional C-terminal domains (domains IV and V). Domain V binds alpha  (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 tau  to the gamma  processivity assembly apparatus. The auxiliary subunit chi  binds SSB to form a tether between DnaX complex and the SSB-coated lagging strand (18). tau  interacts with both the processivity assembly apparatus (via gamma ) and the polymerase (via alpha ). This direct processivity assembly/polymerase link bridged by DnaX strengthens the interactions between the holoenzyme and the SSB-coated lagging strand at the replication fork. Interactions mediated by domains III-V of tau  enable this subunit to serve as a central organizer. The tau  subunit effectively couples the processivity assembly process, SSB binding, DnaB helicase activities, and the dimeric replicase into one replicative complex at the replication fork.


    FOOTNOTES

* This work was supported by Grant GM35695 from the National Institutes of Health.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.

Published, JBC Papers in Press, November 14, 2000, DOI 10.1074/jbc.M009827200

2 Kd values were obtained by dividing the measured dissociation rate constant by the association rate constant of a given interaction. In most cases, the Kd values determined in this study were not true equilibrium Kd values but were relative values used to compare the relative affinities of tau -derivatives for the same analytes.

3 M. Song, H. G. Dallmann, P. Pham, R. Schaaper, and C. S. McHenry, manuscript in preparation.


    ABBREVIATIONS

The abbreviations used are: NTA, nitrilotriacetic acid; SSB, single-stranded DNA-binding protein; PCR, polymerase chain reaction; RU, resonance unit.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES


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