tau Binds and Organizes Escherichia coli Replication Proteins through Distinct Domains

DOMAIN IV, LOCATED WITHIN THE UNIQUE C TERMINUS OF tau , BINDS THE REPLICATION FORK HELICASE, DnaB*

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

Interaction between the tau  subunit of the DNA polymerase III holoenzyme and the DnaB helicase is critical for coupling the replicase and the primosomal apparatus at the replication fork (Kim, S., Dallmann, H. G., McHenry, C. S., and Marians, K. J. (1996) Cell 84, 643-650). In the preceding manuscript, we reported the identification of five putative structural domains within the tau  subunit (Gao, D., and McHenry, C. (2000) J. Biol. Chem. 275, 4433-4440). As part of our systematic effort to assign functions to each of these domains, we expressed a series of truncated, biotin-tagged tau  fusion proteins and determined their ability to bind DnaB by surface plasmon resonance on streptavidin-coated surfaces. Only tau  fusion proteins containing domain IV bound DnaB. The DnaB-binding region was further limited to a highly basic 66-amino acid residue stretch within domain IV. Unlike the binding of immobilized tau 4 to the DnaB hexamer, the binding of monomeric domain IV to DnaB6 was dependent upon the density of immobilized domain IV, indicating that DnaB6 is bound by more than one tau  protomer. This observation implies that both the leading and lagging strand polymerases are tethered to the DnaB helicase via dimeric tau . These double tethers of the leading and lagging strand polymerases proceeding through the tau -tau link and an additional tau -DnaB link are likely important for the dynamic activities of the replication fork.



    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

The DNA polymerase III holoenzyme is responsible for the replication of the Escherichia coli chromosome. Like other replicases from eukaryotes and prokaryotes, the holoenzyme contains three functional subassemblies (for reviews see Refs. 1-3): the DNA polymerase III (alpha epsilon theta ) core, the beta  sliding clamp processivity factor, and the DnaX complex, a clamp assembly apparatus. The DNA polymerase III core contains the alpha , epsilon , and theta  subunits and provides the polymerase function. The DnaX complex (tau 2gamma delta delta 'chi ,psi ) is a multiprotein ATPase that recognizes the primer terminus and loads the beta  processivity factor onto DNA.

The tau  and gamma  subunits are different translation products of the dnaX gene (4-7). The tau  subunit plays central roles in the structure and function of the holoenzyme. It interacts with the core polymerase to coordinate leading and lagging strand synthesis (8, 9). tau  also interacts with DnaB helicase to couple the replicase with the primosome and mediate rapid replication fork movement (10, 11). These two important functions of tau  reside in C-tau , a proteolytic fragment consisting of its unique C-terminal 213 amino acid residues. tau  binds tightly to the alpha  subunit; the shorter translation product gamma  does not. C-tau is a monomer and binds alpha  with a 1:1 stoichiometry as determined by sedimentation equilibrium analyses (12). Results from a recent study indicated that C-tau binds DnaB, can partially replace full-length tau  in reconstituted rolling circle replication reactions, and effectively couples the leading strand polymerase with DnaB helicase at the replication fork (12). DnaB helicase is composed of six identical subunits and is a stable hexamer over a wide range of concentration in the presence of magnesium ions (13, 14).

In the preceding manuscript, we reported that tau  comprises five potential structural domains (15). Domains I, II, and III are common to both gamma  and tau . Domain IV includes 66 amino acid residues of the C-tau sequence and the C-terminal 17 residues of gamma . Domain V corresponds to the 147 C-terminal residues of the tau  subunit. Based on these assignments, biotin-hexahistidine-tagged tau  proteins lacking specific domains were produced. Results from binding studies employing these truncated fusion proteins indicated that the binding site of tau  for alpha  subunit lies within its C-terminal 147 amino acid residues (domain V).

The objective of this study was to determine the domain(s) of the tau  subunit involved in binding DnaB. Biotin-hexahistidine-tagged tau  proteins lacking specific domains were expressed and purified. Analysis of DnaB binding to these truncated tau  proteins by surface plasmon resonance permitted the assignment of the DnaB-binding domain of tau .


    EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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

Buffers-- Buffer L, Buffer W and HKGM Buffer were prepared as previously described (15).

Chemicals and Reagents-- SDS-polyacrylamide gel electrophoresis protein standards were obtained from Amersham Pharmacia Biotech, and prestained molecular mass markers were from Bio-Rad or Life Technologies, Inc. Ni2+-NTA1 resin, QIAquick Gel extraction kits, QIAquick PCR purification kits, and plasmid preparation kits were purchased from Qiagen. The Coomassie Plus protein assay reagent and ImmunoPure Streptavidin were from Pierce. 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.

Proteins-- Three biotin-tagged tau  proteins C(0)tau , C-Delta 147tau , and N-Delta 413tau as well as holoenzyme subunits were prepared as previously described (15).

Construction of the Fusion Plasmids-- Plasmids PA1-C-Delta 213tau and pET11-N-Delta 430tau were constructed to express the fusion proteins C-Delta 213tau and N-Delta 430tau , respectively. Fusion protein C-Delta 213tau corresponds to gamma , the shorter of the two potential dnaX products. In this construct, the 213 C-terminal residues found exclusively in the tau  subunit are replaced by a peptide tag, which includes hexahistidine and a 13-amino acid residue biotinylation sequence. N-Delta 430tau corresponds to C-tau ; the N-terminal 430 amino acids found in both tau  and gamma  are replaced by the hexahistidine/biotinylation tag. PA1 is a semi-synthetic E. coli RNA polymerase-dependent promoter containing two lac operators (16). The pET11 vector is under the control of the T7 promoter.

The starting material for construction of plasmid PA1-C-Delta 213tau was PA1-C(0)tau , which encodes the C-terminal tagged full-length tau  protein (15). PCR primer C-213P1 (Table I) is complementary to a 110-nucleotide stretch upstream of the RsrII site within dnaX. Primer C-213P2 (Table I) corresponded to dnaX codons 423-430 preceded by a noncomplementary SpeI restriction site. PA1-C(0)tau was digested with RsrII and SpeI. The PCR product generated by use of primers C-213P1/C-213P2 was cleaved with RsrII and SpeI and then ligated into the linearized vector to generate plasmid PA1-C-Delta 213tau .


                              
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Table I
Oligonucleotides used for construction of truncated tau  fusion proteins

Primers N-430p1 and N-430p2 (Table I) and plasmid PA1-N-Delta 1tau , which encodes an N-terminal tagged tau  protein (15), were used to generate a PCR product for the construction of pET11-N-Delta 430tau . The resultant PCR product consisted of a PstI restriction site within the noncomplementary 5' region followed by dnaX codons 431-436 and a KpnI site near the 3' end. The KpnI site located downstream of the natural dnaX stop codon. After digestion with PstI and KpnI, the resultant 929-base pair fragment was used to replace the dnaE gene of vector pET11-N0 (16) to produce plasmid pET11-N-Delta 430tau .

Growth and Induction of Expressing E. coli Strains-- E. coli strain BL21 (lambda  DE3) containing the expression plasmids pET11-N-Delta 430tau or PA1-C-Delta 213tau was grown at 37 °C in 2 and 6 liters, respectively, of F medium (17) containing 100 µg/ml ampicillin. Cells were induced with isopropyl-beta -D-thio-galactoside, biotin-treated, and harvested as described (15).

Protein Purification-- The procedures for purification of C-Delta 213tau and N-Delta 430tau were similar to those described for other truncated tau  fusion proteins (15). Briefly, induced cells (14 g for C-Delta 213tau or 22 g for N-Delta 430tau ) were lysed in the presence of lysozyme (2.5 mg/ml), EDTA (5 mM), benzamidine (5 mM), and phenylmethylsulfonyl fluoride (1 mM) for 2 h at 4 °C and 6 min at 37 °C. For purification of C-Delta 213tau , 0.226 g of ammonium sulfate was added to each milliliter of the resulting supernatant, and the precipitate was collected by centrifugation at 23,300 × g at 4 °C for 1 h. The pellets were then resuspended in Buffer L. Each suspension was mixed with 1 ml of Ni2+-NTA resin and pre-equilibrated with Buffer L, and the slurries were then packed into 1-ml columns. Columns were washed with ~30 column volumes of buffer W containing 23 mM imidazole. Bound proteins were then eluted with buffer W containing 150 mM imidazole in a single step. 13 mg of C-Delta 213tau were obtained in the preparation used for these studies. The purification of N-Delta 430tau was as that for C-Delta 213tau except that: 1) the supernatant proteins were precipitated with 65% ammonium sulfate, 2) 3 ml of pre-equilibrated Ni2+-NTA resin were used, 3) the columns were washed with buffer W containing 10 mM imidazole, and 4) elutions were effected by a 10-100 mM imidazole gradient in buffer W. 61 mg of purified N-Delta 430tau were obtained in the preparation used for these studies.

Surface Plasmon Resonance-- A BIAcoreTM instrument was used for protein-protein binding studies. Research grade CM5 sensor chips were used in all experiments. Streptavidin was captured onto sensor chips by N-hydroxysuccinimide/1-ethyl-3-[(3-dimethylamino)propyl)] carbodiimide coupling as previously described (15). The biotinylated tau  proteins were then injected over the immobilized streptavidin sensor chip. Binding analyses of tau  to DnaB (0.025-1 µM) were performed in HKGM buffer at 20 °C. Kinetic parameters were determined using the BIAevaluationTM 2.1 software.

Other Procedures-- DNA polymerization assays, protein determinations, and SDS-polyacrylamide gel electrophoresis were performed as described in the preceding paper (15).


    RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Expression and Purification of the Truncated tau  Fusion Proteins-- The tau  subunit binds to DnaB helicase and is the only subunit within the holoenzyme shown to interact with DnaB (10). The unique C terminus of tau  (C-tau ) bound DnaB in a coupled immunoblotting method (12). To confirm this observation and more precisely map the DnaB binding region of tau , a series of truncated tau  proteins lacking specific domains were produced, and their interactions with DnaB helicase were quantified using BIAcore methodology. The tau  fusion proteins employed in this study included C(0)tau (domains I-V), C-Delta 147tau (domains I-IV), C-Delta 213tau , which was equivalent to gamma  (domains I-III plus 17 amino acids of domain IV), N-Delta 413tau (domains IV and V), and N-Delta 430tau , which was equivalent to C-tau (the C-terminal 66 residues of domain IV plus domain V in its entirety). The truncated terminus of each fusion protein was tagged with a peptide containing both a hexahistidine sequence to aid in purification as well as a short biotinylation sequence. The biotinylation sequence enabled oriented immobilization of the fusion proteins onto BIAcore sensor chips via biotin-streptavidin binding. C(0)tau , C-Delta 147tau , and N-Delta 413tau were expressed and purified as previously described (15). C-Delta 213tau and N-Delta 430tau were expressed in the BL21 (lambda  DE3) strain by induction with isopropyl-beta -D-thio-galactoside and reached similar expression levels (2-5% of total cell proteins). Both C-Delta 213tau and N-Delta 430tau were purified by Ni2+-NTA affinity chromatography. After Ni2+-NTA purification, C-Delta 213tau was obtained at 80% purity, and N-Delta 430tau at 90% purity as determined by SDS-polyacrylamide gel electrophoresis analysis (Fig. 1). The activities of the fusion proteins were ascertained by their ability to replace gamma  or tau  in DNA polymerase III reconstitution assays (15). The specific activity of C-Delta 213tau was 5.5 × 106 units/mg, similar to that of full-length C(0)tau (5.7 × 106 units/mg). As expected, no holoenzyme reconstitution activity was detected for N-Delta 430tau , which lacks the gamma  sequence required for assembly of the beta  processivity factor on DNA.



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Fig. 1.   Purified truncated tau  fusion proteins. The upper panel shows the truncated fusion proteins of tau  used in this study: C(0)tau (domain I-V); C-Delta 147tau (domains I-IV); C-Delta 213tau (domains I-III + 17 amino acids of domain IV), which is equivalent to the gamma  protein plus a C-terminal tag; N-Delta 413tau (domains IV and V); and N-Delta 430tau contains the intact domain V and the majority of domain IV lacking its N-terminal 17-amino acid sequence. The rectangular box represents the 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 chromatography (C-Delta 147tau and N-Delta 413tau were shown in the preceding paper).

DnaB Binding to tau  Proteins Containing Domain IV-- The interaction between DnaB and C(0)tau was first characterized via use of BIAcore technology. C(0)tau (2025 RU) was immobilized onto a streptavidin sensor chip. DnaB solutions of varying concentrations were passed over the immobilized C(0)tau , and binding activity was monitored (Fig. 2A). Attempts to fit the dissociation phase to a single first-order dissociation equation were unsuccessful, suggesting that a more complex mechanism was operative. To simplify the kinetic analysis, a limited interval (35-125 s following the starting point of dissociation) was analyzed from each binding curve and fit to a model in which two simultaneous independent dissociation processes occur. The two apparent dissociation rate constants koff major and koff minor (Table II) corresponded to 70-80% and 20-30% of the dissociating species, respectively. koff major was used to calculate the apparent association rate (kon). The apparent Kd was calculated from kon and koff. The interaction between DnaB and C(0)tau had an apparent Kd of 4 nM (Table II). Under the conditions employed in these studies, DnaB is known to exist as a hexamer (14), and C(0)tau is a tetramer (18). The binding ratio of the DnaB hexamer (DnaB6) to the C(0)tau tetramer [C(0)tau 4] was 0.72, indicating that these multimers likely interact with a 1(DnaB6): 1[C(0)tau 4] stoichiometry.



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Fig. 2.   DnaB interacts with C(0)tau and C-Delta 147tau but not C-Delta 213tau . Streptavidin was chemically immobilized onto sensor chip as described under "Experimental Procedures." tau  fusion proteins were captured onto the streptavidin sensor chip via biotin-streptavidin interaction. DnaB diluted in HKGM to the indicated concentrations was injected over immobilized tau  for 6 min at 5 µl/min. Following injection, buffer was passed over the sensor chips for 30 min to permit dissociation of the bound DnaB protein from immobilized tau  derivatives. A, DnaB interacts with C(0)tau . 2025 RU of C(0)tau were captured on the sensor chip, and varying concentrations of DnaB were passed over it. Control injections over a streptavidin sensor chip were performed and subtracted from the data shown. B, DnaB does not interact with C-Delta 213tau . 3390 RU of C-Delta 213tau were captured on the sensor chip, and DnaB at 1 µM was injected over the immobilized C-Delta 213tau . The control injection over a streptavidin sensor chip was also shown. C, DnaB interacts with C-Delta 147tau . 2860 RU of C-Delta 147tau were captured onto the streptavidin sensor chip varying concentrations of DnaB were passed over it. Control injections over a streptavidin sensor chip were performed and subtracted from the data shown.


                              
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Table II
Binding of tau  derivatives to DnaB

C-Delta 213tau , equivalent to C-terminally tagged gamma , was captured onto the streptavidin-derivatized sensor chip (3400 RU), followed by injection of DnaB (1 µM). No interaction between C-Delta 213tau and DnaB was detected (Fig. 2B), consistent with the previous finding that gamma  does not interact with DnaB helicase (10).

Next, DnaB samples (0.05-0.5 µM) were injected over immobilized C-Delta 147tau (2860 RU) (Fig. 2C). An apparent Kd of 5 nM was obtained, which is similar to that of the C(0)tau -DnaB interaction (Table II). This suggests that C-Delta 147tau contains elements sufficient for binding to DnaB at the same level observed for the intact tau  subunit. C-Delta 147tau (domains I-IV) bound DnaB, but C-Delta 213tau (domains I-III) did not, localizing the region required for DnaB binding to somewhere within domain IV.

DnaB Recognizes a 66-Amino Acid Sequence within Domain IV-- To confirm that domain IV was the DnaB-binding domain, N-Delta 430tau (1200 RU) was captured onto a BIAcore sensor chip, and its interaction with DnaB was assessed (Fig. 3A). The dissociation phase did not fit to a single first-order dissociation equation, so the binding data were fit to the model that assumes two parallel dissociation processes. The apparent Kd was about 8 nM, which was similar to that of the interaction between DnaB and C(0)tau (Table II). The sum of these results indicates that the DnaB binding site is located within the unique C-terminal 66 residues of the tau  subunit.



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Fig. 3.   N-Delta 430tau and N-Delta 413tau bind DnaB. Streptavidin was chemically coupled onto the BIAcore sensor chips as described under "Experimental Procedures." 1200 RU of N-Delta 430tau (A) and 1293 RU of N-Delta 413tau (B) were captured onto streptavidin-derivatized sensor chips, and DnaB diluted in HKGM buffer at the indicated concentrations was injected over the chips bearing immobilized N-Delta 430tau and N-Delta 413tau , respectively. Control injections over a streptavidin derivatized sensor chip were performed and subtracted from the data shown.

The C-terminal 17 amino acid residues of domain IV are lacking in N-Delta 430tau . To investigate whether these 17 residues provide additional binding energy for the tau -DnaB interaction, DnaB binding studies using fusion protein N-Delta 413tau were performed. However, the DnaB/N-Delta 413tau interaction was characterized by an apparent Kd of 5 nM, which is similar to that observed for the interaction of DnaB with N-Delta 430tau (Fig. 3B and Table II). Thus, it is unlikely that the C-terminal 17 residues of domain IV contribute significantly to DnaB binding interactions.

More than One tau  Protomer Binds a DnaB Hexamer-- In the preceding experiment, the binding ratio of DnaB6 to the monomeric N-Delta 430tau was less than 0.1. This value is significantly different from 0.72, the observed ratio for the interaction between DnaB6 and C(0)tau 4. One potential underlying cause of the low binding ratio for the former interaction is the binding of DnaB6 to more than one immobilized N-Delta 430tau molecule. To test this hypothesis, we examined the interactions of DnaB (1 µM) with sensor chips bearing differing amounts of N-Delta 430tau (146 RU-2700 RU). The corresponding densities of the six different levels of N-Delta 430tau tested are shown in Table III. Binding of DnaB to immobilized N-Delta 430tau at 146 RU was not observed. Increased binding ratios of DnaB to N-Delta 430tau were observed for surfaces bearing greater densities of N-Delta 430tau (Fig. 4). If we assume that each DnaB6 binds two (N-Delta 430tau )1 molecules, then the binding ratio of DnaB to N-Delta 430tau is increased from 0.04 to 0.24 within the range of the amount of immobilized N-Delta 430tau tested (Table III). The same apparent dissociation and association rate constants for the DnaB and N-Delta 430tau interaction were obtained at different N-Delta 430tau density as reported in Table II. These results are consistent with the multivalent binding of DnaB and N-Delta 430tau . The observed Kd is the product of the individual Kd values for single site binding interactions. No binding was observed at low N-Delta 430tau density, suggesting that the monomeric tau -DnaB6 interaction is too weak to be observed with the BIAcore methodology. The apparent Kd values of the DnaB-N-Delta 430tau interaction and DnaB-C(0)tau interaction were the same, suggesting that the interaction between DnaB and C(0)tau is also multivalent; more than one C(0)tau monomer binds each DnaB6. The binding ratio between DnaB and N-Delta 413tau was also N-Delta 413tau density-dependent and increased with increased immobilized N-Delta 413tau density (data not shown).


                              
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Table III
Density dependence of N-Delta 430tau binding to DnaB



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Fig. 4.   N-Delta 430tau density dependence of binding of DnaB. The left panel shows the interactions of DnaB with six N-Delta 430tau derivatized sensor chips: A, 146 RU; B, 368 RU; C, 720 RU; D, 1156 RU; E, 1998 RU; F, 2700 RU, respectively. A streptavidin-derivatized sensor chip lacking the bound N-Delta 430tau provided a blank control, and the subtracted data are shown. DnaB at 1 µM diluted in HKGM buffer was injected over the six N-Delta 430tau immobilized sensor chips for 4 min at 5 µl/min. The schematic at right indicates that if the N-Delta 430tau density on a sensor chip is so low that only one N-Delta 413tau molecule binds each DnaB6 molecule, the resultant interaction is too weak to be observed using this methodology. However, at higher densities of immobilized N-Delta 430tau , multiple N-Delta 413tau molecules bind each DnaB6 molecule, and the resultant interaction is strong enough to be detected.

To ensure that the observed binding ratio of the hexameric DnaB to the tetrameric C(0)tau was not density-dependent, the binding ratio of DnaB6 to C(0)tau was examined at an increased density (4262 RU) of C(0)tau on a sensor chip. In a previous experiment, 2025 RU of C(0)tau was used (Fig. 2A), and a binding ratio of 0.72 DnaB6 to C(0)tau 4 was observed. The C(0)tau concentrations in these two different experiments corresponded to 568 and 270 µM of C(0)tau as monomer, within the density range of N-Delta 430tau used in the density dependence experiment (Table III). The observed binding ratio of DnaB6 to C(0)tau 4 was 0.69, which was not significantly different from the ratio obtained when using with 2025 RU of C(0)tau (Table III).


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

In the preceding paper, we detailed our use of limited proteolysis studies to identify five putative structural domains of the tau  protein (15). Domains I-III are common to both tau  and gamma . Domain IV is composed of 17 amino acid residues from the C-terminal end of gamma  plus 66 amino acids from the unique C terminus of tau . Domain V is located at the C-terminal end of tau . One function of tau  is to bind DnaB, coupling the holoenzyme with the primosome at the replication fork. C-tau , the unique C terminus of tau , bound DnaB in a coupled immunoblotting method (12). In reconstituted rolling circle replication reactions, C-tau can partially replace full-length tau  in coupling the leading strand polymerase with the DnaB helicase at the replication fork (12).

This study further defined the DnaB binding domain of tau  by analyzing the interactions of DnaB with several truncated tau  proteins. N-Delta 413tau , N-Delta 430tau , C-Delta 147tau , and C(0)tau bound DnaB with similar apparent Kd values. Because complicated binding kinetics were operative, the apparent Kd values we obtained in this study were not the true constants. However, the resulting apparent Kd values presumably contain the same systematic errors and therefore permit a quantitative comparison of relative affinities. The relative binding affinities of the different tau  fusion proteins for DnaB indicate that tau  amino acid residues 431-496 are sufficient for DnaB binding. This 66-residue stretch corresponds to the C-terminal portion of tau  domain IV.

Although similar apparent Kd values were obtained for the interactions of DnaB6-C(0)tau 4 and DnaB6-(N-Delta 430tau )1, the binding ratios for the DnaB6-(N-Delta 430tau )1 was density-dependent. We conclude that more than one N-Delta 430tau monomer is required to bind DnaB6 and that the interactions between DnaB and tau  involved multivalent binding. Thus, the true microscopic Kd for binding of DnaB6 to a single N-Delta 430tau was too weak to observe using a BIAcore. At higher N-Delta 430tau densities, binding was observed between DnaB6 to two or more N-Delta 430tau molecules; the observed macroscopic Kd is roughly equal to the product of each of the constituent microscopic Kd values.2

Consistent with this interpretation, the number of DnaB6 binding to a BIAcore chip surface increases with the density of immobilized N-Delta 430tau . Increases in N-Delta 430tau density would result in increased numbers of N-Delta 430tau molecules becoming located within each DnaB6 binding sphere. The DnaB6 binding sphere is a function of the diameter of the distance between two binding sites within each DnaB6 molecule. Within each binding sphere, a certain number (n) of N-Delta 430tau molecules can be accommodated; n is equal to the maximum potential binding stoichiometry of N-Delta 430tau to DnaB6.

Recently, a model for quantifying the principal aspects of multivalent binding was developed (19). We used this model to estimate the probability of more than one N-Delta 430tau molecules binding DnaB simultaneously. The proportion of spheres containing a given number of N-Delta 430tau molecules was calculated assuming a binomial distribution. The DnaB-binding sphere was defined as the volume within which binding of the DnaB by two N-Delta 430tau molecules can occur, and it was calculated using the following equation: VS = 4/3*pi *D3, where D is the distance between two binding sites on DnaB. The DnaB hexamer is a cyclic structure and contains six chemically identical subunits (14, 20, 21). Based on hydrodynamic and electron microscopic studies, the cyclic structure of the DnaB hexamer has an outside diameter of ~140 Å and an inner channel of ~40 Å. The expected DnaB binding sphere would be in the range from 4/3*pi *(40 Å)3 to 4/3*pi *(140 Å)3 (268-11480 nm3, respectively). If we assume that the interaction between tau  and DnaB involves two N-Delta 430tau molecules, the calculated DnaB binding sphere is 2500 nm3, within the possible range for an interaction between two tau  protomers and DnaB6.

The notion that two tau  protomors bind each DnaB hexamer is consistent with the presence of a tau  dimer at the replication fork (Fig. 5). tau 2 functions to dimerize the DNA polymerase III core to enable simultaneous synthesis of leading and lagging strands. We already know that the leading strand polymerase is tethered to DnaB (12). The findings presented in this report indicate that the same DnaB molecule couples both of the leading and lagging strand polymerases. Thus, a double tethers exists between the leading and lagging strand polymerase, one is through the tau -tau link and the additional one through the tau -DnaB link. This second tether might help keep the lagging strand associated with the replication fork and may serve to help retarget the dissociated lagging strand polymerase to the next primer synthesized at the replication fork (Fig. 5). Our mapping results demonstrate that the DnaB helicase binds tau  domain IV and that the polymerase alpha  subunit binds domain V (15). These findings indicate important roles that the C terminus tau  plays in DNA synthesis.



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Fig. 5.   Both the leading and lagging strand polymerases couple the DnaB helicase via tau  at the replication fork. The dimeric tau  protein binds the leading and lagging strand polymerses through domain IV. The two domain IVs of the dimeric tau  protein also bind a hexameric DnaB molecule at the replication fork. This double polymerase tether proceeding through tau -tau and tau -DnaB interactions helps keep the lagging strand associated with the replication fork and may serve to help retarget the dissociated lagging strand polymerase to the next primer synthesized at the replication fork.



    FOOTNOTES

* This work was supported by Grant GM36255 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.M009830200

2 Assuming that the 8 nM apparent Kd resulted from (N-Delta 430tau )2-DnaB6 interactions, the affinity between DnaB and each monomeric N-Delta 430tau would be in the 900 µM range provided that there was no cooperativity involved ((8 nM)1/2 = 900 µM). This low (900 µM) affinity range is consistent with the lack of detected interaction.


    ABBREVIATIONS

The abbreviations used are: NTA, nitrilotriacetic acid; PCR, polymerase chain reaction; RU, resonance unit.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES


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