 |
INTRODUCTION |
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 (

) core, the
sliding clamp processivity
factor, and the DnaX complex, a clamp assembly apparatus. The DNA
polymerase III core contains the
,
, and
subunits and
provides the polymerase function. The DnaX complex
(
2

'
,
) is a multiprotein ATPase that
recognizes the primer terminus and loads the
processivity factor
onto DNA.
The
and
subunits are different translation products of the
dnaX gene (4-7). The
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).
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
reside in C-
, a proteolytic fragment
consisting of its unique C-terminal 213 amino acid residues.
binds
tightly to the
subunit; the shorter translation product
does
not. C-
is a monomer and binds
with a 1:1 stoichiometry as
determined by sedimentation equilibrium analyses (12). Results from a
recent study indicated that C-
binds DnaB, can partially replace
full-length
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
comprises five
potential structural domains (15). Domains I, II, and III are common to
both
and
. Domain IV includes 66 amino acid residues of the
C-
sequence and the C-terminal 17 residues of
. Domain V
corresponds to the 147 C-terminal residues of the
subunit. Based on
these assignments, biotin-hexahistidine-tagged
proteins lacking
specific domains were produced. Results from binding studies employing
these truncated fusion proteins indicated that the binding site of
for
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
subunit involved in binding DnaB. Biotin-hexahistidine-tagged
proteins lacking specific domains were expressed and purified. Analysis
of DnaB binding to these truncated
proteins by surface plasmon
resonance permitted the assignment of the DnaB-binding domain of
.
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EXPERIMENTAL PROCEDURES |
Strains--
E. coli DH5
and HB101 were used for
initial molecular cloning procedures and plasmid propagation. E. coli BL21(
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
proteins C(0)
,
C-
147
, and N-
413
as well as holoenzyme subunits were
prepared as previously described (15).
Construction of the Fusion Plasmids--
Plasmids
PA1-C-
213
and pET11-N-
430
were constructed to
express the fusion proteins C-
213
and N-
430
, respectively. Fusion protein C-
213
corresponds to
, the shorter of the two potential dnaX products. In this construct, the 213 C-terminal residues found exclusively in the
subunit are replaced
by a peptide tag, which includes hexahistidine and a 13-amino acid residue biotinylation sequence. N-
430
corresponds to C-
; the N-terminal 430 amino acids found in both
and
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-
213
was PA1-C(0)
, which encodes
the C-terminal tagged full-length
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)
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-
213
.
Primers N-430p1 and N-430p2 (Table I) and plasmid
PA1-N-
1
, which encodes an N-terminal tagged
protein (15), were used to generate a PCR product for the construction
of pET11-N-
430
. 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-
430
.
Growth and Induction of Expressing E. coli Strains--
E.
coli strain BL21 (
DE3) containing the expression plasmids
pET11-N-
430
or PA1-C-
213
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-
-D-thio-galactoside, biotin-treated, and
harvested as described (15).
Protein Purification--
The procedures for purification of
C-
213
and N-
430
were similar to those described for other
truncated
fusion proteins (15). Briefly, induced cells (14 g for
C-
213
or 22 g for N-
430
) 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-
213
, 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-
213
were obtained in the preparation used for these studies.
The purification of N-
430
was as that for C-
213
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-
430
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
proteins were then injected over the immobilized streptavidin sensor chip. Binding analyses of
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 |
Expression and Purification of the Truncated
Fusion
Proteins--
The
subunit binds to DnaB helicase and is the only
subunit within the holoenzyme shown to interact with DnaB (10). The unique C terminus of
(C-
) bound DnaB in a coupled immunoblotting method (12). To confirm this observation and more precisely map the
DnaB binding region of
, a series of truncated
proteins lacking
specific domains were produced, and their interactions with DnaB
helicase were quantified using BIAcore methodology. The
fusion
proteins employed in this study included C(0)
(domains I-V),
C-
147
(domains I-IV), C-
213
, which was equivalent to
(domains I-III plus 17 amino acids of domain IV), N-
413
(domains IV and V), and N-
430
, which was equivalent to C-
(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)
, C-
147
, and
N-
413
were expressed and purified as previously described (15).
C-
213
and N-
430
were expressed in the BL21 (
DE3) strain
by induction with isopropyl-
-D-thio-galactoside and
reached similar expression levels (2-5% of total cell proteins). Both
C-
213
and N-
430
were purified by Ni2+-NTA
affinity chromatography. After Ni2+-NTA purification,
C-
213
was obtained at 80% purity, and N-
430
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
or
in DNA
polymerase III reconstitution assays (15). The specific activity of
C-
213
was 5.5 × 106 units/mg, similar to that
of full-length C(0)
(5.7 × 106 units/mg). As
expected, no holoenzyme reconstitution activity was detected for
N-
430
, which lacks the
sequence required for assembly of the
processivity factor on DNA.

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Fig. 1.
Purified truncated fusion proteins. The upper panel shows the truncated
fusion proteins of used in this study: C(0) (domain I-V);
C- 147 (domains I-IV); C- 213 (domains I-III + 17 amino
acids of domain IV), which is equivalent to the protein plus a
C-terminal tag; N- 413 (domains IV and V); and N- 430
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- 147
and N- 413 were shown in the preceding paper).
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DnaB Binding to
Proteins Containing Domain IV--
The
interaction between DnaB and C(0)
was first characterized via use of
BIAcore technology. C(0)
(2025 RU) was immobilized onto a
streptavidin sensor chip. DnaB solutions of varying concentrations were
passed over the immobilized C(0)
, 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)
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)
is a tetramer (18). The
binding ratio of the DnaB hexamer (DnaB6) to the C(0)
tetramer [C(0)
4] was 0.72, indicating that these
multimers likely interact with a 1(DnaB6):
1[C(0)
4] stoichiometry.

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Fig. 2.
DnaB interacts with C(0)
and C- 147 but
not C- 213 .
Streptavidin was chemically immobilized onto sensor chip as described
under "Experimental Procedures." 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 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 derivatives. A,
DnaB interacts with C(0) . 2025 RU of C(0) 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- 213 . 3390 RU of C- 213 were captured on the sensor
chip, and DnaB at 1 µM was injected over the immobilized
C- 213 . The control injection over a streptavidin sensor chip was
also shown. C, DnaB interacts with C- 147 . 2860 RU of
C- 147 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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C-
213
, equivalent to C-terminally tagged
, was captured onto
the streptavidin-derivatized sensor chip (3400 RU), followed by
injection of DnaB (1 µM). No interaction between
C-
213
and DnaB was detected (Fig. 2B), consistent with
the previous finding that
does not interact with DnaB helicase
(10).
Next, DnaB samples (0.05-0.5 µM) were injected over
immobilized C-
147
(2860 RU) (Fig. 2C). An apparent
Kd of 5 nM was obtained, which is
similar to that of the C(0)
-DnaB interaction (Table II). This
suggests that C-
147
contains elements sufficient for binding to
DnaB at the same level observed for the intact
subunit. C-
147
(domains I-IV) bound DnaB, but C-
213
(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-
430
(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)
(Table II). The sum of these results indicates
that the DnaB binding site is located within the unique C-terminal 66 residues of the
subunit.

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Fig. 3.
N- 430 and
N- 413 bind DnaB.
Streptavidin was chemically coupled onto the BIAcore sensor chips as
described under "Experimental Procedures." 1200 RU of N- 430
(A) and 1293 RU of N- 413 (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- 430 and N- 413 , respectively. Control
injections over a streptavidin derivatized sensor chip were performed
and subtracted from the data shown.
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The C-terminal 17 amino acid residues of domain IV are lacking in
N-
430
. To investigate whether these 17 residues provide additional binding energy for the
-DnaB interaction, DnaB binding studies using fusion protein N-
413
were performed. However, the
DnaB/N-
413
interaction was characterized by an apparent Kd of 5 nM, which is similar to that
observed for the interaction of DnaB with N-
430
(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
Protomer Binds a DnaB Hexamer--
In the
preceding experiment, the binding ratio of DnaB6 to the
monomeric N-
430
was less than 0.1. This value is significantly different from 0.72, the observed ratio for the interaction between DnaB6 and C(0)
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-
430
molecule. To test this hypothesis, we examined the interactions of DnaB
(1 µM) with sensor chips bearing differing amounts of
N-
430
(146 RU-2700 RU). The corresponding densities of the six
different levels of N-
430
tested are shown in Table
III. Binding of DnaB to immobilized
N-
430
at 146 RU was not observed. Increased binding ratios of
DnaB to N-
430
were observed for surfaces bearing greater
densities of N-
430
(Fig. 4). If we
assume that each DnaB6 binds two (N-
430
)1
molecules, then the binding ratio of DnaB to N-
430
is increased
from 0.04 to 0.24 within the range of the amount of immobilized
N-
430
tested (Table III). The same apparent dissociation and
association rate constants for the DnaB and N-
430
interaction
were obtained at different N-
430
density as reported in Table II.
These results are consistent with the multivalent binding of DnaB and
N-
430
. The observed Kd is the product of the
individual Kd values for single site binding
interactions. No binding was observed at low N-
430
density,
suggesting that the monomeric
-DnaB6 interaction is too
weak to be observed with the BIAcore methodology. The apparent
Kd values of the DnaB-N-
430
interaction and
DnaB-C(0)
interaction were the same, suggesting that the interaction
between DnaB and C(0)
is also multivalent; more than one C(0)
monomer binds each DnaB6. The binding ratio between DnaB
and N-
413
was also N-
413
density-dependent and
increased with increased immobilized N-
413
density (data not
shown).

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Fig. 4.
N- 430 density
dependence of binding of DnaB. The left panel shows the
interactions of DnaB with six N- 430 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- 430 provided a blank control, and the subtracted data are
shown. DnaB at 1 µM diluted in HKGM buffer was injected
over the six N- 430 immobilized sensor chips for 4 min at 5 µl/min. The schematic at right indicates that
if the N- 430 density on a sensor chip is so low that only one
N- 413 molecule binds each DnaB6 molecule, the
resultant interaction is too weak to be observed using this
methodology. However, at higher densities of immobilized N- 430 ,
multiple N- 413 molecules bind each DnaB6 molecule,
and the resultant interaction is strong enough to be detected.
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To ensure that the observed binding ratio of the hexameric DnaB to the
tetrameric C(0)
was not density-dependent, the binding ratio of DnaB6 to C(0)
was examined at an increased
density (4262 RU) of C(0)
on a sensor chip. In a previous
experiment, 2025 RU of C(0)
was used (Fig. 2A), and a
binding ratio of 0.72 DnaB6 to C(0)
4 was
observed. The C(0)
concentrations in these two different experiments
corresponded to 568 and 270 µM of C(0)
as monomer,
within the density range of N-
430
used in the density dependence
experiment (Table III). The observed binding ratio of DnaB6
to C(0)
4 was 0.69, which was not significantly different from the ratio obtained when using with 2025 RU of C(0)
(Table III).
 |
DISCUSSION |
In the preceding paper, we detailed our use of limited proteolysis
studies to identify five putative structural domains of the
protein
(15). Domains I-III are common to both
and
. Domain IV is
composed of 17 amino acid residues from the C-terminal end of
plus
66 amino acids from the unique C terminus of
. Domain V is located
at the C-terminal end of
. One function of
is to bind DnaB,
coupling the holoenzyme with the primosome at the replication fork.
C-
, the unique C terminus of
, bound DnaB in a coupled
immunoblotting method (12). In reconstituted rolling circle replication
reactions, C-
can partially replace full-length
in coupling the
leading strand polymerase with the DnaB helicase at the replication
fork (12).
This study further defined the DnaB binding domain of
by
analyzing the interactions of DnaB with several truncated
proteins. N-
413
, N-
430
, C-
147
, and C(0)
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
fusion proteins for DnaB indicate that
amino acid
residues 431-496 are sufficient for DnaB binding. This 66-residue
stretch corresponds to the C-terminal portion of
domain IV.
Although similar apparent Kd values were obtained
for the interactions of DnaB6-C(0)
4 and
DnaB6-(N-
430
)1, the binding ratios for
the DnaB6-(N-
430
)1 was
density-dependent. We conclude that more than one
N-
430
monomer is required to bind DnaB6 and that the
interactions between DnaB and
involved multivalent binding. Thus,
the true microscopic Kd for binding of
DnaB6 to a single N-
430
was too weak to observe using a BIAcore. At higher N-
430
densities, binding was observed
between DnaB6 to two or more N-
430
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-
430
. Increases in N-
430
density would result
in increased numbers of N-
430
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-
430
molecules can be accommodated; n is equal to the maximum potential binding stoichiometry of N-
430
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-
430
molecules binding DnaB
simultaneously. The proportion of spheres containing a given number of
N-
430
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-
430
molecules can occur, and it was
calculated using the following equation: VS = 4/3*
*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*
*(40 Å)3 to 4/3*
*(140
Å)3 (268-11480 nm3, respectively). If we
assume that the interaction between
and DnaB involves two
N-
430
molecules, the calculated DnaB binding sphere is 2500 nm3, within the possible range for an interaction between
two
protomers and DnaB6.
The notion that two
protomors bind each DnaB hexamer is consistent
with the presence of a
dimer at the replication fork (Fig.
5).
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
-
link and the additional one through the
-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
domain IV and that the polymerase
subunit binds domain V
(15). These findings indicate important roles that the C terminus
plays in DNA synthesis.

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Fig. 5.
Both the leading and lagging strand
polymerases couple the DnaB helicase via at
the replication fork. The dimeric protein binds the leading
and lagging strand polymerses through domain IV. The two domain IVs of
the dimeric protein also bind a hexameric DnaB molecule at the
replication fork. This double polymerase tether proceeding through
- and -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.
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