From the Department of Biochemistry and Molecular
Biology, University of Florida, Gainesville, Florida 32610-0245,
Department of Biological Sciences and Chemistry,
Hedco Molecular Biology Laboratories, University of Southern
California, Los Angeles, California 90089-1340, and
Rockefeller University and Howard Hughes Medical
Institute, New York, New York 10021
Received for publication, November 18, 2002, and in revised form, January 2, 2003
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The Escherichia coli DNA
polymerase III In the absence of processivity proteins, synthesis by DNA
polymerases is relatively inefficient because of repeated DNA
dissociation and rebinding events. Sliding clamps alleviate this
problem by encircling DNA and tethering polymerases to the DNA
templates to dramatically enhance the processivity of synthesis. The
Escherichia coli The E. coli clamp loader is composed of five different
subunits, three copies of the dnaX gene product ( This paper addresses a key question regarding the E. coli
clamp-loading reaction. How does the E. coli clamp loader
target the Enzymes--
All of DNA polymerase III proteins were purified,
and Oligonucleotides--
Synthetic oligonucleotides were made on an
ABI 392 DNA synthesizer using standard
Amino modifiers were incorporated into this template to allow for
site-specific labeling with X-rhodamine isothiocyanate (catalog number
X-491, Molecular Probes) as described previously (19, 20). These amino
modifiers were positioned at three separate sites for control
experiments to demonstrate that binding results were not affected by
the position of the X-rhodamine (RhX) probe. Two modifiers were located
on T at positions 26 and 39 (amino modifier C2dT, Glen Research)
indicated by lowercase letters, and the third modifier was located on
the 5'-terminal hydroxyl (amino modifier C6, Glen Research).
Three 30-nucleotide primers that are complementary to different
sites on this 105-mer (see Fig. 1) were annealed separately by
incubating 1.2 equivalents of primer and with 1 equivalent of template
in 20 mM Tris·HCl, pH 7.5, and 50 mM NaCl at
80 °C for 5 min and then slowly cooling to room temperature.
Annealed p/ts were used without further purification because we
previously demonstrated that a small excess of primer had no effect on
loading reactions (16).
Steady-state Fluorescence Anisotropy
Measurements--
Steady-state anisotropy measurements were taken
using a QuantaMaster QM-1 fluorometer (Photon Technology International,
London, Ontario, Canada) as described previously (16). Titration
experiments were performed by the addition of a constant volume of Competition Binding Assays--
The binding of Pre-steady-state Fluorescence Anisotropy
Measurements--
Assays were performed using a Biologic SFM-4
stopped-flow (Molecular Kinetics, Pullman, WA) equipped with four
independently driven reagent syringes and a 30-µl cuvette (model
FC-15) with a 1.5-mm path length as described previously (16). One
stopped flow syringe was loaded with 480 nM Pre-steady-state ATPase Assays--
E. coli
phosphate-binding protein covalently labeled at Cys-197 with
N-[2-(1-maleimidyl)ethyl]-7-(diethylamino)coumarin-3-carboxamide was used to quantitate inorganic phosphate released by ATP hydrolysis in real time as described previously (17). An Applied Photophysics SX.18MV stopped-flow was used. A "three-syringe" experiment was performed by loading one syringe with 0.97 µM Combined Analysis of DNA Binding and ATP Hydrolysis
Kinetics--
Kinetics of ATP hydrolysis and DNA binding were measured
separately in reactions containing 260 nM
These reaction kinetics were simulated by the model shown in Fig.
5C using KINSIM (22). The same model and associated rate constants were used to simulate both data sets and output the concentration of PBP-MDCC bound to Pi and the anisotropy of
RhX on DNA. The observed anisotropy (robs) at
any time (t) was calculated using Equation 1 where
rf and rb represent the anisotropy of free and bound DNA, respectively, and
xf and xb represent the fraction
of free and bound DNA as a function of reaction time.
The experimentally determined anisotropy for free DNA
(rf = 0.19) was used. An anisotropy of 0.32 was used
for bound DNA (rb). This value represents the
increase in anisotropy observed when DNA is saturated with either Binding of
When Competition Binding Assays--
Competition binding assays were
used to measure the ability of each of the p/t substrates to compete
with a single-stranded DNA substrate for Kinetics of
In contrast, time courses for binding reactions with the
elongation-deficient 3'-blunt p/t (Fig. 3C,
This dynamic interaction with elongation-proficient DNA substrates is
not an artifact of performing assays in the absence of the Kinetics of ATP Hydrolysis in the Presence of Different DNA
Substrates--
The anisotropy data show that DNA substrates suitable
for polymerase extension induce a change in
DNA substrates capable of supporting synthesis by a DNA polymerase gave
different time courses for ATP hydrolysis than those that could not. In
assays without the
The addition of Combined Analysis of ATP Hydrolysis and DNA Binding and Release
Kinetics--
To determine whether the burst of ATP hydrolysis seen in
the reaction with the center p/t was associated with the release of
DNA, pre-steady-state kinetics of DNA binding and ATP hydrolysis were
measured in assays under identical reaction conditions. The center
rather than 5'-blunt p/t DNA was chosen for purely technical reasons to
more closely examine the relationship of ATP hydrolysis to the
Time courses for ATP hydrolysis and DNA binding showed similar features
as those in Figs. 3 and 4. Rapid biphasic hydrolysis of ATP followed by
a slower steady-state hydrolysis was observed in the ATPase assay. A
concomitant rapid increase in anisotropy followed by a decrease to the
level of free DNA was observed in the anisotropy binding assay (Fig.
5, A and B). A
combined analysis of these two data sets allows us to determine the
timing of ATP hydrolysis relative to DNA binding and release. DNA
binding occurs prior to ATP hydrolysis, but ATP hydrolysis occurs prior
to DNA release. A kinetic model for the binding of
The rapid biphasic release of Pi by
The kinetic simulation starts with an equilibrium mixture of
These assays (Fig. 5) provide information regarding the kinetics of the
first turnover but less about subsequent turnovers where the rates of
ADP release from The E. coli clamp loader accomplishes the mechanical
task of assembling the ring-shaped complex clamp loader assembles the ring-shaped
sliding clamp onto DNA. The core polymerase is tethered to the template
by
, enabling processive replication of the genome. Here we
investigate the DNA substrate specificity of the clamp-loading reaction
by measuring the pre-steady-state kinetics of DNA binding and ATP
hydrolysis using elongation-proficient and deficient primer/template
DNA. The ATP-bound clamp loader binds both elongation-proficient and
deficient DNA substrates either in the presence or absence of
.
However, elongation-proficient DNA preferentially triggers
complex
to release
onto DNA with concomitant hydrolysis of ATP. Binding to
elongation-proficient DNA converts the
complex from a high affinity
ATP-bound state to an ADP-bound state having a 105-fold
lower affinity for DNA. Steady-state binding assays are misleading,
suggesting that
complex binds much more avidly to non-extendable
primer/template DNA because recycling to the high affinity binding
state is rate-limiting. Pre-steady-state rotational anisotropy data
reveal a dynamic association-dissociation of
complex with
extendable primer/templates leading to the diametrically opposite
conclusion. The strongly favored dynamic recognition of extendable DNA
does not require the presence of
. Thus, the
complex uses ATP
binding and hydrolysis as a mechanism for modulating its interaction
with DNA in which the ATP-bound form binds with high affinity to DNA
but elongation-proficient DNA substrates preferentially trigger
hydrolysis of ATP and conversion to a low affinity state.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
sliding clamp is composed of two
identical crescent-shaped subunits that form a ring in solution (1, 2).
The clamp does not spontaneously load onto DNA but requires the
activity of a clamp loader for assembly. Clamp loaders perform the
mechanical task of opening clamps and depositing them on DNA templates
(3). The binding and hydrolysis of ATP drive this assembly reaction.
and/or
),
,
',
, and
(4-7). The dnaX gene produces
two polypeptides, a full-length gene product,
, and a truncated gene
product,
, approximately two-thirds the length of
(8-10). The
C-terminal region of
interacts with the
subunit of the
polymerase and supports the formation of a DNA polymerase III
holoenzyme complex in vivo that contains two core
polymerases and a clamp loader consisting of
2
'
(11-13). Both
and
contain ATP
binding sites and are capable of functioning in fully active
clamp-loading complexes referred to as the
(
3
'
) and
(
3
'
)
complexes, respectively (5, 14). ATP binding by the clamp loader
induces a conformational change that exposes the binding sites for the
clamp (15) and DNA (16). Hydrolysis of ATP is coupled to the
release of the clamp on DNA and most probably produces a conformational
change that masks binding sites for
and DNA (17, 18). Thus, ATP binding and hydrolysis modulate
complex-
and
complex-DNA interactions during the clamp-loading cycle.
clamps to the correct sites on DNA? Ideally, DNA
replication would be most efficient if the clamp loader assembled
clamps onto primed templates at primer 3' ends. This DNA substrate
specificity could be achieved if the clamp loader had a high affinity
for ss/ds1 junctions at
primer 3' ends. However, the DNA polymerase must also bind to these
ss/ds junctions so the clamp loader would compete with the polymerase
for binding and reduce the efficiency of DNA synthesis. This
competition would be particularly detrimental on the lagging strand
where clamps must be loaded for every one- to two-kilobase Okazaki
fragment that is synthesized. The clamp loader does in fact have a high
affinity for ss/ds DNA junctions at primer 3' ends, but these sites
trigger a change in the
complex causing it to release DNA (16). The
clamp loader then exists in a state with reduced affinity for DNA. This
DNA-induced decrease in affinity provides a mechanism that prevents the
clamp loader from competing with the polymerase for loaded clamps. It
also could provide a dynamic mechanism for recognition of sites for loading
where only the appropriate sites trigger the loading reaction. In this paper, we investigate the DNA structural features required to trigger
complex to release the clamp and DNA and show
that the presence of an elongation-proficient p/t DNA substrate is
absolutely necessary to trigger
complex to hydrolyze ATP and
release the clamp.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
complex was reconstituted as described previously (1, 5) and stored in 20 mM Tris·HCl, pH 7.5, 2 mM DTT,
0.5 mM EDTA, and 10% glycerol. Assay buffers for all of
the experiments contained 8 mM MgCl2 in 20 mM Tris·HCl, pH 7.5, 50 mM NaCl, 5 mM DTT, and 40 µg/ml BSA.
-cyanoethylphosphoramidite
chemistry and reagents from Glen Research (Sterling, VA). DNA was
purified by denaturing polyacrylamide gel electrophoresis. A
105-nucleotide template with the sequence shown as follows was
used in all of the experiments: 5'-GAG CGT CAA AAT GTA GGT ATT TCC
AtG AGC GTT TTT CCt GTT GCA ATG GCT GGC GGT AAT
ATT GTT CTG GAT ATT ACC AGC AAG GCC GAT AGT TTG AGT TCT
TCT-3'.
complex solution to a cuvette containing a solution of RhX-labeled DNA and assay buffer. Assays contained variable amounts of
complex and
50 nM RhX-labeled DNA and 0.5 mM ATP in assay
buffer. Binding experiments were performed with RhX probes located at
three different sites, and the results were not influenced by the site
of labeling (data not shown).
complex to a
mixture of labeled and unlabeled DNA was initiated by the addition of
ATP as described previously (16). After the addition of ATP, solutions
contained 50 nM RhX-labeled ss 50-mer, 400 nM
complex, 0-500 nM unlabeled competitor DNA, and 0.5 mM ATP in assay buffer.
complex,
0.5 mM ATP, and 1 µM
dimer (when present)
in assay buffer. The second syringe was loaded with 100 nM
DNA and 0.5 mM ATP in assay buffer. Reactions were
initiated by mixing 80 µl of
complex solution with 80 µl of DNA
solution at a flow rate of 10 ml/s at 20 °C. The reaction dead time
under these mixing conditions was 3.7 ± 0.7 ms as determined using the method described by Peterman (21). Vertically and horizontally polarized emission intensities were measured at 1-ms intervals, and 16-24 stopped flow runs were signal-averaged. Raw anisotropy data were fit to sums of exponentials and used in kinetics simulations. Data shown in Fig. 3 have been smoothed over 3 data points. As a control, pre-steady-state experiments with the 3' center
p/t were done with the probe at three different sites to demonstrate
that the results were not influenced by the site of labeling (data not shown).
complex
and 1.2 µM
dimer (when present), a second syringe
with 20 µM MDCC-PBP and 800 µM ATP, and a
third syringe with 0.6 µM unlabeled DNA. The contents of
the first two syringes were mixed to pre-incubate
complex and
dimer when present with ATP for 1 s prior to adding DNA
from the third syringe. The computer-calculated dead time was 1.5 ms.
complex and
450 nM DNA in assay buffer. ATPase assays contained 200 µM ATP and 5 µM MDCC-PBP, and anisotropy
binding assays contained 200 µM ATP. Both experiments were done in the Applied Photophysics stopped-flow using a mixing scheme where
complex was pre-incubated with ATP for 1 s prior to adding DNA (and MDCC-PBP in ATPase assays).
(Eq. 1)
and
complex or with single-stranded binding protein.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
Complex to DNA Substrates with Primers
Located at Different Sites--
The anisotropy of RhX covalently
attached to DNA substrates was used to "report" on binding
interactions with the
complex. In these experiments, the increase
in anisotropy of RhX in the presence of increasing concentrations of
complex was measured under steady-state binding conditions. Three
partially duplex DNA substrates were made by annealing three separate
primers, 30 nucleotides in length, to different sites on a 105-nt-long template (see Fig. 1). Each of these DNAs
were covalently labeled with RhX at position 5 of a template T located
39 nucleotides from the 5' end. Two substrates, the 5'-blunt p/t and
center p/t, have 5'-template overhangs and support primer extension by
a DNA polymerase (elongation-proficient). The third substrate, 3'-blunt p/t, positions the primer 3' end at a blunt duplex end and cannot be
extended by a DNA polymerase (elongation-deficient).
View larger version (16K):
[in a new window]
Fig. 1.
Steady-state anisotropy binding assays
measuring the affinity of the clamp loader for DNA substrates primed at
different sites. Each of the DNA substrates (50 nM)
containing ss/ds junctions was titrated with complex (0-1600
nM). The increase in anisotropy for the RhX probe on DNA is
plotted as a function of
complex concentration for the 5'-blunt p/t
substrate (filled triangles), center p/t substrate
(open squares), and 3'-blunt substrate (filled
circles). The DNA template is labeled with RhX on T at position
39. Assay buffer contained 500 µM ATP and 8 mM MgCl2 in 20 mM Tris·HCl, pH
7.5, 50 mM NaCl, 40 µg/ml BSA, and 5 mM
DTT.
complex (0-1600 nM) was titrated into a solution
containing 50 nM DNA substrate and 500 µM
ATP, a relatively large increase in anisotropy was observed for the
3'-blunt p/t substrate, whereas smaller increases were observed for the
5'-blunt and center p/t substrates (Fig. 1). These results seem to
indicate that the apparent affinity of
complex is greater for the
3'-blunt p/t substrate that is not extendable by DNA
polymerases. However, earlier work (16) with the center p/t
substrate revealed that
complex does in fact bind with high
affinity to this substrate under pre-steady-state conditions. This
interaction with the center p/t substrate converts
complex from a
high affinity to a low affinity binding state so that only a small
population of DNA is bound at steady state.
complex binding.
Increasing concentrations of unlabeled p/t competitor were added to a
solution of
complex (400 nM) and ss 50-mer DNA (50 nM) covalently labeled with RhX. As the concentration of
competitor DNA was increased, the fraction of
complex bound to the
ss 50-mer decreased in each case (Fig. 2). However, the 5'-blunt and center p/ts
more effectively competed with the ss 50-mer for
complex than the
3'-blunt p/t substrate. These results apparently contradict those of
direct binding assays (Fig. 1), which suggested that
complex binds
the 5'-blunt and center p/ts more weakly. Together, these steady-state
binding assays indicate that the
complex interacts with the
5'-blunt and center p/t substrates in the similar manner but interacts differently with the 3'-blunt substrate.
View larger version (15K):
[in a new window]
Fig. 2.
Ability of partially duplex DNA substrates to
compete with a single-stranded 50-mer for complex binding. The binding of
complex (400 nM)
to a ss 50-mer (50 nM) that was 5'-end-labeled with RhX was
measured in the presence of increasing concentrations (0-400
nM) of unlabeled competitor DNA. The fraction of labeled
50-mer that remained bound by
complex was plotted as a function of
concentration of unlabeled 5'-blunt p/t (filled triangles),
center p/t (open squares), and 3'-blunt p/t (filled
circles) competitors. Assay buffer contained 500 µM
ATP and 8 mM MgCl2 in 20 mM
Tris·HCl, pH 7.5, 50 mM NaCl, 40 µg/ml BSA, and 5 mM DTT.
Complex Binding to Different DNA
Substrates--
Pre-steady-state anisotropy measurements were made to
examine the dynamic nature of the binding interactions between
complex and various p/t DNA substrates including ss DNA. In the
absence of
clamp, initial rapid increases in
anisotropy indicated that
-complex bound rapidly to each of the four
DNA substrates. However, two different types of binding kinetics were
observed for elongation-proficient and deficient substrates. For the
elongation-proficient 5'-blunt p/t (Fig.
3A,
) and
center p/t (Fig. 3B,
) substrates, a rapid increase in anisotropy over the first 50 ms was followed by a slower
decrease in anisotropy over approximately the next 250 ms. At steady
state in the absence of the
clamp, the anisotropy was barely above
that of free DNA (light gray), indicating that only an
extremely small fraction of DNA was bound. These binding kinetics are
consistent with a reaction cycle in which an activated
complex
rapidly binds DNA; the interaction with DNA inactivates
complex,
converting it to a state with reduced affinity for DNA; and the
-complex slowly reverts back to the activated DNA binding state
(16).
View larger version (67K):
[in a new window]
Fig. 3.
Kinetics of complex
binding to DNA and loading
clamps on
DNA. Solutions of
complex were pre-incubated with ATP in the
presence or absence of the
clamp prior to the addition of each of
the three p/t DNA substrates and a ss 105-mer substrate. Binding
kinetics are plotted in separate graphs for the 5'-blunt p/t
(A), the center p/t (B), the 3'-blunt p/t
(C), and the ss 105-mer (D) where each graph
shows three plots of anisotropy as a function of time. In each case,
the dark gray plot showing the greatest increase in
anisotropy is the kinetics of loading of
onto DNA, the dark
gray plot showing a small increase in anisotropy is the kinetics
complex binding DNA, and the light gray plot showing no
change in anisotropy is free a DNA control generated by the addition of
buffer only. Smooth black lines through the data represent
empirical fits to the sums of exponentials. Final concentrations were
240 nM
complex, 50 nM DNA, 500 µM ATP, 8 mM MgCl2, and 500 nM
clamp when present in 20 mM Tris·HCl,
pH 7.5, 50 mM NaCl, 40 µg/ml BSA, and 5 mM
DTT. The three p/t substrates (A-C) were labeled with RhX
on the template on T at position 26, and the ss 105-mer was
5'-end-labeled with RhX.
) and ss 105-mer DNA (Fig. 3D,
) showed a rapid increase in anisotropy but little or
no decrease in anisotropy. The reaction with the 3'-blunt p/t showed a
slight decrease in anisotropy but not nearly the magnitude observed for
the 5'-blunt or center p/t DNA. The higher anisotropy values seen in
the steady-state regime of these reactions indicate that a greater
fraction of DNA was bound by
complex. These DNA substrates do not
seem to efficiently trigger the
complex to release DNA and convert
to a low affinity DNA binding state.
clamp but part of the clamp-loading reaction. Similar results
were obtained in loading reactions where the
clamp was present. In
assays containing
and the elongation-proficient 5'-blunt (Fig.
3A, +
) and center (Fig. 3B,
+
) p/t DNA, a rapid increase in anisotropy attributed to
·
complex binding followed by a slower decrease attributed to
the release of the clamp on DNA and dissociation of
complex was
observed. Because the clamp has been loaded onto DNA, anisotropy values
remain higher at steady state than in reactions without the clamp. In
reactions with the 3'-blunt p/t (Fig. 3C, +
)
and ss 105-mer (Fig. 3D, +
), a rapid increase
in anisotropy to a constant value was observed, which is consistent
with an equilibrium binding interaction of the
·
complex with
these DNA substrates rather than DNA-triggered release of the clamp on
DNA. Time courses for reactions containing elongation-deficient DNA
substrates and ATP (Fig. 3, C and D) resemble
those done previously with elongation-proficient DNA substrates in the
presence of non-hydrolyzable ATP
S where clamp release does not occur
(23). The magnitude of the increase in anisotropy was greater in
reactions with
than without
. This difference in
amplitudes is consistent with the model presented below where an
equilibrium population of approximately 40% of the
complex is
present in a conformation that is active for DNA binding in the absence
of
.
complex that reduces
its affinity for DNA, whereas elongation-deficient DNA substrates do
not. The kinetics of ATP hydrolysis in assays with each of the four DNA
substrates were measured to determine whether they also differed for
extendable DNA substrates compared with non-extendable substrates. A
real time fluorescence-based assay was used to quantitate the
concentration of inorganic phosphate released on hydrolysis of ATP
(24). This assay uses E. coli phosphate-binding protein (PBP), which avidly binds inorganic phosphate. When PBP is covalently labeled with a coumarin fluorophore (MDCC-PBP), phosphate binding produces an increase in fluorescence of the probe.
clamp, the first turnover of ATP was rapid
relative to the steady-state turnover for the extendable 5'-blunt and
center p/ts (Fig. 4A). The
first turnover was biphasic with the combined amplitudes indicating
that three ATP molecules were hydrolyzed for every molecule of
complex present. The kinetics of ATP hydrolysis in assays with the
non-extendable 3'-blunt p/t and ss 105-mer differed significantly. They
lacked a "burst" of ATP hydrolysis and showed only a linear
increase in Pi release at a rate of 0.92 µM/s.
View larger version (16K):
[in a new window]
Fig. 4.
Kinetics of ATP hydrolysis by
complex in the presence and absence of the
clamp. The concentration of MDCC-PBP bound to
inorganic phosphate released by hydrolysis of ATP is plotted as a
function of time. A, reactions with
complex alone.
B, reactions with
complex in the presence of
. The
dotted gray line in each graph represents the concentration
of Pi expected for the hydrolysis of three molecules of ATP
per molecule of
complex. Reactions done in both the presence and
absence of
show a rapid increase in Pi release followed
by a slow steady-state release for 5'-blunt (black) and
center (gray) p/t substrates, whereas reactions with the
3'-blunt p/t (black) and ss DNA (gray) show a
linear steady-state release of Pi. Assays were performed by
pre-incubating a solution of
complex (240 nM), ATP (200 µM), MgCl2 (8 mM), and
(300 nM, when present) for 1 s prior to adding a solution
of DNA (300 nM) and MDCC-PBP (5 µM) to give
final concentrations indicated in 20 mM Tris·HCl, pH 7.5, 50 mM NaCl, 40 µg/ml BSA, and 5 mM DTT.
to assays containing the extendable 5'-blunt and
center p/ts increased the overall rate of the first ATP turnover by
complex (Fig. 4B). Three molecules of ATP (3.2 based on
amplitudes) per molecule of
complex were hydrolyzed in a single
rapid phase taking approximately 150-200 ms to complete compared with
600-700 ms in the absence of
(Fig. 4A). The increased rate was attributable primarily to the disappearance of the slower second pre-steady-state phase seen in assays with
complex only. No
burst and a linear increase in Pi release was observed at
rates of 1.2 and 1.0 µM/s for the elongation-deficient
3'-blunt p/t and ss DNA, respectively, in assays with
complex and
. The presence of
did not significantly increase the rate of ATP
hydrolysis for non-extendable substrates, suggesting that hydrolysis is
not coupled to a productive loading reaction (17).
complex-binding and release reactions. This center p/t is typically
used in all of our assays and allows us to compare these results with
those of other studies. These experiments were performed with an excess
of DNA over
complex so that every clamp loader could bind DNA and
hydrolyze ATP in the first turnover.
complex,
hydrolysis of ATP, and release of the extendable p/t DNA including rate
constants for each reaction (Fig. 5C) was used to simulate
pre-steady-state anisotropy binding and ATP hydrolysis data (Fig. 5,
A and B). The solid lines through the
data points were obtained from simulation of this model using KINSIM
(Fig. 5C) (22).
View larger version (25K):
[in a new window]
Fig. 5.
Correlation between hydrolysis of ATP and
release of DNA by complex. ATPase and
DNA binding assays were performed by pre-incubating
complex and ATP
for 1 s prior to adding DNA (and MDCC-PBP in ATPase assays). Final
concentrations were 0.26 µM
complex, 0.45 µM center p/t DNA (5'-end-labeled with RhX on the
template), 200 µM ATP, and 8 mM
MgCl2 and 5 µM MDCC-PBP (in ATPase assays
only) in assay buffer. The concentration of MDCC-PBP bound to inorganic
phosphate (left axis) and the anisotropy of RhX on DNA
(right axis) are plotted as a function of time. Data over a
time range of 1.5 s are shown in A, and data on an
expanded scale of 0.6 s are shown in B. Solid
lines through the data were obtained from simulation of the
kinetic model shown in C. In this model,
c·T·T·T represents
complex bound to 3 molecules of ATP (T) that is inactive for binding DNA,
c*·T·T·T represents
complex bound to three
ATPs that is active for DNA binding, N represents DNA;
D represents ADP, and P represents inorganic
phosphate. Rate constants used in the simulation are indicated for each
step. Experimentally determined rate constants for inorganic phosphate
binding to and dissociating from PBP-MDCC (24) were included.
complex could be
because of the presence of non-equivalent sites where individual
subunits hydrolyze ATP at two different rates. Alternatively, the
biphasic kinetics could result from two populations of
complex in
equilibrium where one population exists in a conformation that is
active for DNA binding and rapidly hydrolyzes all three molecules of
ATP at the same rate. The second population is initially inactive for
DNA binding and ATP hydrolysis but can slowly convert to the active
form, which gives rise to the slow phase of Pi release. Our
model assumes the second case in which
complex exists as an
equilibrium mixture of two different
conformational states as supported by unpublished
data.2 Following ATP hydrolysis, the ADP-bound form
of
complex rapidly releases DNA.
complex that is active (40%
c*·T·T·T) and
inactive for DNA binding (60%
c·T·T·T).2 The exact nature of these
two species is not defined by this experiment, and it is assumed that
they both contain three molecules of ATP. However, it is also formally
possible that binding one or more ATP molecules converts the inactive
state to active state. Under these experimental conditions where
saturating ATP concentrations were used, we cannot distinguish between
these two possibilities. The active state of
complex
(
c*·T·T·T) binds DNA and hydrolyzes all three
molecules of ATP sequentially at the same rate before rapidly
dissociating from DNA. The inactive form of
complex slowly (2.7 s
1) converts to the active form where it binds DNA
and hydrolyzes its ATP prior to releasing DNA.
complex and ATP binding come in to play. For this
reason, we have combined these steps into one first-order reaction
where the ADP-bound form of
complex is converted to the ATP-bound
form and used apparent rate constants to model the steady-state portion
of the reaction. This first-order approximation is a reasonable
approach because the ATP concentration is saturating and <1% ATP is
converted to ADP over the time course of the reaction so that the ATP
concentration effectively remains constant. Finally, because
Pi release could not be observed until it bound MDCC-PBP,
we included rate constants for this Pi binding step (24) in
our model.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
clamp onto DNA. Our results show that the clamp loader uses a dynamic mechanism to target clamps specifically to DNA substrates that can serve as templates for synthesis by DNA polymerases (Fig. 6).
Primed template DNA substrates that contain a ss/ds junction with the
correct polarity for extension by a DNA polymerase, which is 3'-primer
end with a 5'-template overhang, efficiently trigger hydrolysis of ATP
and release of DNA by the clamp loader (Figs. 3, A and
B, and 4, A and B). The
complex is
also able to bind non-extendable 3'-blunt-end p/t DNA and ss DNA in
either the presence or absence of
but, in contrast, fails to
release
on the DNA (Fig. 3, C and D) and fails to exhibit a pre-steady-state burst of ATP hydrolysis (Fig. 4,
A and B). These data are completely consistent
with our previous data showing that ATP hydrolysis is also associated
with release of the clamp (17). Thus, the appropriate sites on DNA for
clamp assembly trigger the clamp loader to hydrolyze ATP and release the clamp on DNA. These studies provide a mechanistic explanation for
earlier observations that
complex selectively loads clamps at ss/ds
junctions with a 5'-ss overhang (25).
View larger version (16K):
[in a new window]
Fig. 6.
Complex uses a dynamic
mechanism for targeting clamps to sites where DNA synthesis is slated
to begin. In both the presence and absence of the
clamp,
ATP-bound
complex binds with high affinity to extendable and
non-extendable DNA substrates. However, extendable DNA substrates
trigger
complex to hydrolyze ATP and release the clamp on DNA.
Thus,
complex uses a dynamic mechanism for recognition of
extendable primers where those sites preferentially trigger the loading
reaction.
A key result is that steady-state measurements of direct binding
interactions between complex and different DNA substrates can be
misleading by apparently demonstrating that the
complex binds with
greater affinity to DNA substrates that are not extendable by DNA polymerase. Real time anisotropy binding assays were key to
uncovering the dynamic nature of the interaction of
complex with
DNA constructs that can serve as substrates for DNA polymerases. These
assays show a rapid increase in anisotropy followed by a slower
decrease when
complex binds to extendable DNA substrates containing
ss/ds junctions with a 5'-ss overhang (Fig. 3, A and B). A "simple" equilibrium-binding reaction of
complex to DNA would have shown an increase in anisotropy to a value
that represented the equilibrium population of DNA bound by
complex
(i.e. a simple exponential rise) as observed in assays with
ss DNA alone and 3'-blunt p/t DNA containing
(Fig. 3, C
and D). The decrease in anisotropy in assays with
elongation-proficient p/t substrates having a 5'-ss overhang indicated
that a new state of
complex with reduced affinity for DNA forms
during the binding reaction. Because the rate-limiting step in the
steady-state reaction is recycling of this low affinity DNA binding
state to the high affinity state (Fig. 5C), very little DNA
is bound, giving the appearance that the affinity of
complex for
elongation-proficient DNA is low. DNA p/ts that are not extendable do
not efficiently trigger the conversion of
complex to the low
affinity state and thus give the appearance that they are bound with
greater affinity in steady-state assays. It is likely that this low
affinity DNA binding state is an ADP-bound form of
complex based on
the results from ATPase assays. This dynamic DNA binding interaction is
also likely to occur with the eukaryotic clamp loader
(replication factor C). Stronger binding interactions with an
extendable p/t were seen in steady-state assays with non-hydrolyzable
ATP
S than in assays with ATP (26).
The complex has long been known to have DNA-dependent
ATPase activity. More recently, the ATP requirements for individual steps in the clamp-loading reaction have been defined. ATP binding but
not hydrolysis is required for DNA binding activity (16, 20) and clamp
binding activity (15, 27) of the
complex. A conformational change
in the clamp loader that exposes sites for binding both DNA and the
clamp is most probably produced by ATP binding. The hydrolysis of ATP
is required for the release of the clamp on DNA (17, 18, 23). Here we
show that hydrolysis of ATP is dependent on the structural features of
the DNA substrate. DNA structures that can be extended by a DNA
polymerase, the 5'-blunt p/t, and center p/t efficiently trigger a
pre-steady-state burst of ATP hydrolysis, whereas those that cannot be
extended, the 3'-blunt and ss DNA, do not (Fig. 4).
A combined analysis of DNA binding and ATP hydrolysis assays done under
identical conditions in the absence of the clamp revealed that
complex initially binds DNA prior to ATP hydrolysis and subsequently
hydrolyzes ATP before releasing DNA. This same sequence of events
occurs in assays containing
that result in a productive loading
reaction (17). Our results are consistent with a model (Fig.
5C) in which the
-complex cycles through a reaction where
it has a high affinity for DNA, binds DNA, and then converts to a lower
affinity state and releases DNA. The high affinity DNA binding state is
an ATP-bound form of
complex, and the low affinity state is an
ADP-bound form. The kinetic parameters derived from this model suggest
that the ATP-bound form of
complex binds DNA with an affinity that
is 105 times greater (2 nM) than the ADP-bound
form (100 µM). Thus, the clamp loader uses ATP binding
and hydrolysis as a means for modulating its interaction with DNA so
that it has a high affinity before clamp loading and a low affinity afterward.
Taken together, these results demonstrate the complex uses a
dynamic mechanism for targeting the
sliding clamp to template sites
where DNA synthesis is slated to begin. The clamp loader binds DNA with
high affinity, and those sites with an ss/ds junction of the correct
polarity to be extended by a DNA polymerase efficiently trigger the
clamp loader to hydrolyze ATP and release the clamp on DNA (Fig. 6).
DNA-triggered hydrolysis of ATP converts the
complex to a state
most probably ADP-bound with lower affinity for DNA, thus providing a
mechanism for targeting the clamp to the proper sites on DNA and for
preventing the clamp loader from competing with the polymerase for
primer/template ends. Coordination between the clamp loader and the
polymerase is critical for DNA synthesis on the lagging strand where a
clamp must be loaded for each of the 1-2-kilobase Okazaki fragments
synthesized every 1-2 s by the polymerase. Pre-steady-state kinetic
data show that the
complex-catalyzed
clamp loading and release
reactions occur rapidly (~12 s
1) (20) compared with the
time scale for Okazaki fragment synthesis and, therefore, are not
rate-limiting for Okazaki fragment synthesis. ATP binding and
hydrolysis modulate the affinity of the clamp loader for DNA and the
clamp so that it has a high affinity for both before the clamp is
loaded and a low affinity for both after the clamp is loaded. This
prevents competition between the clamp loader and core polymerase for
clamps that have just been loaded on DNA. The dynamic nature of
complex-DNA interactions is probably a common theme in DNA replication
where many enzymes are required to work at the replication fork and
each must have access to the DNA at the appropriate time.
![]() |
FOOTNOTES |
---|
* This work was supported by National Institutes of Health Grants GM55596 (to L. B. B.), GM21422 (to M. F. G.), and GM38839 (to M. O.) and National Science Foundation Training Grant DBI-9602258.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.
§ Present address: Dept. of Biochemistry, University of Wisconsin, Madison, WI 53706.
¶ Dept. of Pharmacology and Therapeutics, University of Florida, Gainesville, FL 32610.
** Depts. of Molecular Biology and Biochemistry, Wesleyan University, Middletown, CT 06459.
§§ To whom correspondence should be addressed: Dept. of Biochemistry and Molecular Biology, 1600 S. W. Archer Rd., JHMHC Rm. R3-234, University of Florida, Gainesville, FL 32610-0245. Tel.: 352-392-8708; Fax: 352-392-6511; E-mail: lbloom@ufl.edu.
Published, JBC Papers in Press, January 8, 2003, DOI 10.1074/jbc.M211741200
2
C. R. Williams, A. Johnson, M. F. Goodman,
M. O'Donnell, and L. B. Bloom, manuscript in preparation. The
"two-state model" for the biphasic kinetics of ATP hydrolysis by
complex in the absence of
provides a reasonable explanation for
the effects of
on the kinetics of DNA binding and ATP hydrolysis.
Our model assumes that
, like DNA, only binds with high affinity to
the "active" state of
complex. Pre-incubation of
with
complex and ATP would "trap" nearly all of the
complex in the
active DNA binding conformation by forming a
·
complex. This
active
·
complex then rapidly binds DNA and hydrolyzes ATP.
Because nearly all of the
complex is present in the active
·
complex, the amplitude of the anisotropy rise in reactions
with
increases relative to reactions without
of which a
fraction (60%) of
complex is initially in the inactive
conformation. For the same reasons, reactions with
result in a
single rapid phase of Pi release.
![]() |
ABBREVIATIONS |
---|
The abbreviations used are:
ss/ds, single-stranded/double-stranded;
p/t(s), primer/template(s);
PBP, E. coli phosphate-binding protein;
MDCC-PBP, PBP covalently
labeled at Cys-197 with
N-[2-(1-maleimidyl)ethyl]-7-(diethylamino)coumarin-3-carboxamide;
RhX, X-rhodamine;
ATPS, adenosine
5'-O-(thiotriphosphate);
DTT, dithiothreitol;
BSA, bovine
serum albumin.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1. | Kong, X.-P., Onrust, R., O'Donnell, M., and Kuriyan, J. (1992) Cell 69, 425-437[Medline] [Order article via Infotrieve]. |
2. |
Yao, N.,
Turner, J.,
Kelman, Z.,
Stukenberg, P. T.,
Dean, F.,
Shechter, D.,
Pan, Z.-Q.,
Hurwitz, J.,
and O'Donnell, M.
(1996)
Genes Cells
1,
101-113 |
3. | Bloom, L. B., and Goodman, M. F. (2001) Nat. Struct. Biol. 8, 829-831[CrossRef][Medline] [Order article via Infotrieve] |
4. |
Maki, S.,
and Kornberg, K.
(1988)
J. Biol. Chem.
263,
6555-6560 |
5. |
Onrust, R.,
Finkelstein, J.,
Naktinis, V.,
Turner, J.,
Fang, L.,
and O'Donnell, M.
(1995)
J. Biol. Chem.
270,
13348-13357 |
6. |
Pritchard, A. E.,
Dallman, H. G.,
Glover, B. P.,
and McHenry, C. S.
(2000)
EMBO J.
19,
6536-6545 |
7. | Jeruzalmi, D., O'Donnell, M., and Kuriyan, J. (2001) Cell 106, 429-441[Medline] [Order article via Infotrieve] |
8. | Blinkowa, A. L., and Walker, J. R. (1990) Nucleic Acids Res. 18, 1725-1729[Abstract] |
9. | Tsuchihashi, Z., and Kornberg, A. (1990) Proc. Natl. Acad. Sci. U. S. A. 87, 2516-2520[Abstract] |
10. | Flower, A. M., and McHenry, C. S. (1990) Proc. Natl. Acad. Sci. U. S. A. 87, 3713-3717[Abstract] |
11. |
Studwell-Vaughan, P. S.,
and O'Donnell, M.
(1991)
J. Biol. Chem.
266,
19833-19841 |
12. |
Onrust, R.,
Finkelstein, J.,
Turner, J.,
Naktinis, V.,
and O'Donnell, M.
(1995)
J. Biol. Chem.
270,
13366-13377 |
13. |
Dallmann, H. G.,
Kim, S.,
Pritchard, A. E.,
Marians, K. J.,
and McHenry, C. S.
(2000)
J. Biol. Chem.
275,
15512-15519 |
14. |
Dallmann, H. G.,
and McHenry, C. S.
(1995)
J. Biol. Chem.
270,
29563-29569 |
15. |
Naktinis, V.,
Onrust, R.,
Fang, F.,
and O'Donnell, M.
(1995)
J. Biol. Chem.
270,
13358-13365 |
16. |
Ason, B.,
Bertram, J. G.,
Hingorani, M. M.,
Beechem, J. M.,
O'Donnell, M.,
Goodman, M. F.,
and Bloom, L. B.
(2000)
J. Biol. Chem.
275,
3006-3015 |
17. |
Bertram, J. G.,
Bloom, L. B.,
Hingorani, M. M.,
Beechem, J. M.,
O'Donnell, M.,
and Goodman, M. F.
(2000)
J. Biol. Chem.
275,
28413-28420 |
18. |
Hingorani, M. M.,
Bloom, L. B.,
Goodman, M. F.,
and O'Donnell, M.
(1999)
EMBO J.
18,
5131-5144 |
19. | Perez-Howard, G. M., Weil, P. A., and Beechem, J. M. (1995) Biochemistry 34, 8005-8017[Medline] [Order article via Infotrieve] |
20. |
Bloom, L. B.,
Turner, J.,
Kelman, Z.,
Beechem, J. M.,
O'Donnell, M.,
and Goodman, M. F.
(1996)
J. Biol. Chem.
271,
30699-30708 |
21. | Peterman, B. F. (1979) Anal. Biochem. 93, 442-444[Medline] [Order article via Infotrieve] |
22. | Barshop, B. S., Wrenn, R. F., and Frieden, C. (1983) Anal. Biochem. 130, 134-145[Medline] [Order article via Infotrieve] |
23. |
Bertram, J. G.,
Bloom, L. B.,
Turner, J.,
O'Donnell, M.,
Beechem, J. M.,
and Goodman, M. F.
(1998)
J. Biol. Chem.
273,
24564-24574 |
24. | Brune, M., Hunter, J. L., Corrie, J. E. T., and Webb, M. R. (1994) Biochemistry 33, 8262-8271[Medline] [Order article via Infotrieve] |
25. |
Yao, N.,
Leu, F. P.,
Anjelkovic, J.,
Turner, J.,
and O'Donnell, M.
(2000)
J. Biol. Chem.
275,
11440-11450 |
26. |
Gomes, X. V.,
and Burgers, P. M.
(2001)
J. Biol. Chem.
276,
34768-34775 |
27. |
Hingorani, M. M.,
and O'Donnell, M.
(1998)
J. Biol. Chem.
273,
24550-24563 |