From the
The Escherichia coli replicase, DNA polymerase III
holoenzyme, derives its processivity from the
The cellular replicases of prokaryotes and eukaryotes attain
high processivity by use of circular proteins that behave as DNA
sliding clamps
(1, 2) . There is an increasing number of
proteins that encircle DNA. For example, Escherichia coli topoisomerase I (
Little is known about the mechanism by which circular
proteins are assembled onto DNA. As a model system, we have studied the
E. coli
The goal of this study was
initially to identify which subunit(s) of the
We have also studied individual subunits of the
We
demonstrated the effect of ATP in two ways, gel filtration and SPR. In
Fig. 5
,
Next, the
Activation of the
Dissociation of the
Observed on and off rates
were determined by surface plasmon resonance as described under
``Experimental Procedures.''
We are grateful to Dr. Susan S. Taylor for the
catalytic subunit of cAMP-dependent protein kinase.
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
subunit sliding
clamp that encircles DNA and tethers the replicase to the template. The
dimer is assembled around DNA by the
complex clamp loader
in an ATP-dependent reaction. In this report, the essential contact
between the clamp loader and
is identified as mediated through
the
subunit of the
complex. The
subunit appears to
contact the face of the
dimer ring that contains the two C
termini. Surprisingly, ATP is required for the
complex to bind
, but not for
to bind
. This indicates that
is
buried in the
complex and suggests a role for ATP in exposing
for interaction with
. A protease protection assay has been
developed to specifically probe the
subunit within the
complex. The results of the assay are consistent with an ATP-induced
conformational change in the
complex that alters the state of the
subunit within it. The implication of these key features to the
clamp loading mechanism of the
complex is discussed.
protein) has a cavity through which DNA
strands are passed
(3) , and the RuvB recombination protein
appears to encircle two DNA molecules during Holiday junction
movement
(4) . The DNA polymerase and recombination clamps
require accessory protein clamp loaders (also called molecular
matchmakers), which couple ATP hydrolysis to the assembly of the clamp
onto DNA.
complex (
`
), the
clamp loader of DNA polymerase III holoenzyme that assembles
clamps onto DNA. Studies of individual subunits of the
complex
have shown that no one subunit can assemble
onto DNA. Minimally,
both
and
are required
(5) , although
` is needed
for an efficient reaction at low ionic strength
(6) , and the
and
subunits are also needed at physiological ionic
strength
(5) . The multiple protein requirement suggests that the
assembly of a protein ring around DNA is a multistep process.
Consistent with the concept of a multistep reaction, the clamp loaders
of the T4 bacteriophage (gene 44/62 protein) and eukaryotic (RF-C,
activator-1) systems are also composed of multiple proteins and require
ATP for efficient assembly of their respective clamps onto DNA (gene 45
protein and PCNA,
respectively)
(7, 8, 9, 10) . The T4 gene
44/62 protein complex contains five subunits, four protomers of gene 44
protein and one copy of gene 62 protein. The eukaryotic RF-C
(activator-1) clamp loader consists of five different subunits, like
the E. coli
complex. The amino acid homology between
some of the subunits of these clamp loaders from prokaryotic,
eukaryotic, and viral systems suggests that their basic mechanism of
action may be the same
(11) .
complex bind to
to further the understanding of the overall structure of DNA
polymerase III holoenzyme. This goal was quickly reached: the
subunit forms the major (if not sole) contact between the
complex
and
. But as the study progressed, we found that the
complex
required ATP to bind
, whereas
, at a comparable
concentration to the
complex, did not require ATP. This
observation indicates that
is masked from binding
by other
subunits of the
complex. In the presence of ATP, the masking is
relieved, and
is exposed for interaction with
. This
complex-
interaction is likely an early intermediate on the
mechanistic path of assembly of the protein ring around DNA.
Materials and Methods
All sources and procedures
not described here are described in the first report of this
series
(12) . Streptomyces griseus Pronase and the
serine protease inhibitor Pefabloc SC were from Boehringer Mannheim;
the autoradiography enhancer ENHANCE was from DuPont NEN.
The protein kinase (EC 2.7.1.37, ATP:protein phosphotransferase)
catalytic subunit of murine cAMP-dependent protein kinase (recombinant
expressed in E. coli(13) ) was kindly provided by Dr.
Susan S. Taylor (Department of Chemistry, University of California at
San Diego, La Jolla, CA).
Preparative Constitution of the
The
Complex
subunit (5.64 mg, 340 nmol as monomer) was
incubated with
(3.9 mg, 257 nmol as monomer) in 3 ml of buffer A
(urea was present at 0.5 M due to the need for urea in the
purification of
as described
(14, 15) ) for 60 min
at 15 °C, at which time
(8.0 mg, 85 nmol as dimer) was added.
This mixture was further incubated for 60 min at 15 °C in a final
volume of 7 ml of buffer A containing 100 mM NaCl. This
mixture was loaded onto a 8-ml Mono Q HR 10/10 column (Pharmacia
Biotech Inc.) and eluted with a 180-ml linear gradient of 0-0.4
M NaCl in buffer A. Eighty fractions of 2.5 ml each were
collected and analyzed on an SDS-polyacrylamide gel stained with
Coomassie Blue. Fractions 44-48 containing
were
pooled (7.7 mg, 11.5 ml) and dialyzed against buffer A to a
conductivity equal to 29 mM NaCl.
Preparative Constitution of the
The `
Complex
` complex was constituted from pure
,
, and
` as described previously
(14) .
Preparative Constitution of the
The `
Complex
`
complex was assembled in two
steps. First, the
complex (1.5 mg, 52.5 nmol) was
constituted as described above and then was incubated with
` (3.89
mg, 105 nmol) in 3.06 ml of buffer A, followed by chromatography on a
Mono Q HR 5/5 column equilibrated with buffer A. The
`
complex was eluted from the column with a 32-ml
linear gradient of 0-0.4 M NaCl in buffer A. The
`
complex eluted last at
0.28 M
NaCl. Column fractions containing
`
were pooled
(4.2 mg in 3 ml) and stored at -70 °C. Preparative Constitution of the
[
H]
`
Complex-The
subunit was tritiated by reductive
methylation as described
(17) to a specific activity of 12.5
10
cpm/µg
(24) . The
complex was
constituted from pure subunits using [
H]
in
place of
and was purified from free subunits as described in the
first report of this series
(12) . The specific activity of the
[
H]
`
complex was 3.3
10
cpm/µg, and its activity was >90% of the
activity of the unlabeled
complex in replication assays with
and the core polymerase.
Kinase Protection Assays
The six-residue protein
kinase recognition sequence was engineered onto the C terminus of
as described
(17, 24) . Then 30 pmol (as dimer) of the
derivative was incubated either with 1) 240 pmol (9.3 µg) of
and 18.7 µg of bovine serum albumin or with 2) 30.5 µg of
bovine serum albumin in 13 µl of buffer A at 15 °C for 30 min.
Each mixture was then adjusted to a final volume of 30 µl in 20
mM Tris-HCl (pH 7.5), 2 mM dithiothreitol, 100
mM NaCl, 12 mM MgCl
, 10 mM NaF,
60 µM ATP, and 5 µCi of
[
-
P]ATP. The reaction was incubated at 37
°C, and phosphotransfer was initiated upon the addition of 0.0015
units of protein kinase. Aliquots of 3 µl were withdrawn and
brought to a final concentration of 5 mM ATP and 50
mM EDTA in 10 µl, followed by analysis on a 12%
SDS-polyacrylamide gel. The gel was dried and exposed to x-ray film,
and the autoradiogram was quantitated using a PhosphorImager (Molecular
Dynamics, Inc.).
Protease Protection Assays
Limited proteolytic
digestion of 0.4 µg of [H]
or 1.5 µg
of
complex constituted using the [
H]
subunit was performed in 25 µl of buffer A containing 100
mM NaCl and, when present, 1 mM ATP and 10
mM MgCl
. To equalize the total amount of protein
in each assay, 1.1 µg of bovine serum albumin was added to the
reaction containing only [
H]
. Proteolytic
digestion was initiated upon adding Pronase to a concentration of 5.6
µg/ml on ice, and then the reaction was shifted to 25 °C for 10
min. Digestion was quenched upon adding 2 µl of buffer A containing
10 mg/ml protease inhibitor (Pefabloc SC) and 2% SDS, followed
immediately by boiling for 5 min. Samples were analyzed by
electrophoresis on a 15% SDS-polyacrylamide gel. The gel was
equilibrated with EN
HANCE according to the
manufacturer's protocol and fluorographed on Biomax MR x-ray film
at -70 °C for 60 h.
Surface Plasmon Resonance
Methods of
immobilization of and SPR
(
)
experiments
were as described in the first report of this series
(12) . For
analysis of the
-
binding kinetics, the chip contained 1185
response units of immobilized
, and the mobile phase (SPR buffer)
contained
at the following concentrations: 5, 10, 20, 40, 80, and
160 nM (as dimer). When 0.5 mM ATP and 10 mM
MgCl
were present in the mobile phase, the concentrations
of
were 25, 100, and 250 nM. After the initial
immobilization and between injections, noncovalently bound proteins
were removed with 10 µl of 0.1 M glycine (pH 9.5). For
analysis of the
complex-
binding kinetics, the chip
contained 810 response units of immobilized
, and then the
complex was reconstituted into immobilized
by injecting 35 µl
of 4 µM
`
complex in SPR buffer
followed by 35 µl of 1 µM
`
and
finally 35 µl of 20 nM
`
. The use of
20 nM
`
in the mobile phase resulted in
a stabile signal of 660 response units (over the initial 810 response
units) and was included in the mobile phase in experiments using 1280
and 5120 nM
(use of 5, 20, 80, and 320 nM
produced insignificant signals). For analysis in the presence of 0.5
mM ATP and 10 mM MgCl
, the mobile phase
also included 20 nM
`
and the following
concentrations of
: 20, 40, 80, and 160 nM. Treatment
with 0.1 M glycine was not required between injections in
experiments using the immobilized
complex as the base line
returned to the initial level during the dissociation phase. The
k
and k
values were
determined from the result of each injection using the nonlinear curve
fitting BIAevaluation 2.0 software (based on Ref. 18).
The
Initially, we used gel filtration to
study the interaction of Subunit Is the Major Contact Point in the
Complex for
with the
complex.During
gel filtration, components are not at equilibrium, and therefore, only
strong complexes with relatively slow dissociation rates are observed.
In Fig. 1A, the
complex was mixed with
and
then gel-filtered. Analysis of the column fractions on an
SDS-polyacrylamide gel shows that
comigrates with the five
subunits of the
complex in fractions 21-27, much earlier
than the position at which
elutes alone (Fig. 1D,
fractions 35-39). Hence,
binds to the
complex with
high enough affinity to survive gel filtration. To determine which
subunit(s) of the
complex interact with
, we studied two
subassemblies of the
complex. Analysis of a mixture of
with
the
` complex (Fig. 1B) shows that
comigrates with
`, indicating that
and
are
not essential for interaction with
. This is substantiated in
Fig. 1C, where analysis of a mixture of
with the
complex showed no comigration of
with
.
Figure 1:
The subunit interacts with the
complex. The
subunit was incubated with
`
(A),
`
(B), or
(C) or alone (D)
and then gel-filtered on Superose 12. A, mixture of
(36 µg, 0.45 nmol as dimer) and constituted
`
(360 µg, 1.8 nmol); B,
mixture of
(36 µg, 0.45 nmol as dimer) and 3.2 nmol of
` complex constituted by mixing 300 µg of
(3.2
nmol as dimer), 70 µg of
(1.8 nmol as monomer), and 66.7
µg of
` (1.8 nmol as monomer); C, mixture of
(36 µg, 0.45 nmol as dimer) and 1.8 nmol of
complex constituted by mixing 169 µg of
(1.8 nmol as dimer),
37.4 µg of
(2.25 nmol as monomer), and 30 µg of
(2.0 nmol as monomer); D, the
subunit alone (36 µg,
0.45 nmol as dimer). 50-µl aliquots of column fractions were
analyzed. Fraction (Frx) numbers are indicated above each gel.
The first lane of each gel contains molecular weight standards
(MW), and their weights are indicated to the left. The
,
,
`,
,
, and
subunits are identified to the
right.
Which subunit(s) of the ` complex
bind
? In Fig. 2, the
,
, and
` subunits were
analyzed individually for interaction with
. In
Fig. 2A, a mixture of
and
showed no
interaction (
and
did not comigrate), but instead migrated
in the same position as when analyzed alone. Likewise, in
Fig. 2B, a mixture of
` and
showed no
interaction. However, in Fig. 2C, an interaction was
observed between
and
; the two proteins comigrated in
fractions 29-35, significantly earlier than
alone
(fractions 35-41), indicating that they form a
complex. The
dimer is 81 kDa, and
is a monomer of 38.7 kDa.
Thus, these two proteins should have resolved from each other if they
did not interact. Consistent with
complex formation,
alone elutes much later (Fig. 2D). Scans of the
Coomassie Blue-stained SDS-polyacrylamide gel of Fig. 2C were performed to estimate the stoichiometry of the
complex. However, the ratio of
to
increased continuously
from fractions 29 to 35, indicating that the
complex
dissociates during gel filtration, thus precluding an accurate
determination of their molar ratio by this technique.
Figure 2:
The
subunit interacts with the
subunit of the
complex.
The
subunit was incubated with
,
`, or
and then
gel-filtered (Superose 12) as described under ``Experimental
Procedures.'' A, mixture of
(36 µg, 0.45 nmol
as dimer) and
(169 µg, 1.8 nmol as dimer); B,
mixture of
(144 µg, 1.8 nmol as dimer) and
` (16.6
µg, 0.45 nmol as monomer); C, mixture of
(91 µg,
1.1 nmol as dimer) and
(293.5 µg, 7.6 nmol as monomer);
D, the
subunit alone (293.5 µg, 7.6 nmol as
monomer). 50-µl aliquots of column fractions were analyzed. Column
fractions are indicated above each gel. Positions of molecular weight
standards (MW) and their weights are indicated to the left.
The
,
,
`, and
subunits are identified to the
right.
The
subunit is the only
complex subunit to form a gel-filterable
complex with
, indicating that
is the major contact point
between the
complex and
. Consistent with this, a
`
complex showed no interaction with
in a
gel filtration analysis (data not shown). This result rules out the
existence of multiple weak interactions that add up to a gel-filterable
interaction between
and two or more subunits of the
complex
(other than
).
complex for weak interaction with
using the SPR technique.
In these experiments, the
dimer was immobilized on the sensor
chip, and 1 µM solutions of individual subunits of the
complex were serially passed over the top. No interaction was
observed between
and the
,
`,
, or
complex (data not shown). The
-
interaction was observed as
shown below in Fig. 6and was also utilized as a control in our
previous report on interaction between PCNA and the p21 cyclin kinase
inhibitor
(19) .
Figure 6:
SPR
analysis of the effect of ATP on interaction of with
and
the
complex. The influence of ATP on the kinetics of interaction
between either
or the
complex with
was examined by
SPR analysis as described under ``Experimental Procedures,''
except that in these experiments,
was at 80 nM tested in
all four panels. Openarrowheads denote the start of
injection, and closedarrowheads mark the start
of the buffer wash. In A and B, the
subunit was
immobilized on the sensor chip. In C and D, the
complex was reconstituted on the sensor chip, and the mobile phase was
supplemented with 20 nM
`
. In B and D, the buffer contained both ATP and
MgCl
; in A and C, the buffer contained
neither ATP nor MgCl
. RU, response
units.
The minimum molecular mass of the
complex was estimated to be 125 kDa by gel filtration upon comparing
its volume of elution relative to that of size standards
(Fig. 3). This mass is consistent with a
complex (81 + 38.7 kDa =
119.7 kDa), although in light of the apparent dissociation of the
complex during gel filtration, a
complex cannot be excluded. Several
attempts have been made to assess the exact stoichiometry of the
complex, but the complex lacks sufficient stability for an
accurate determination. However, the
complex binds to the
dimer and contains one monomer of
. Thus, it appears that the
association of one
subunit with a
dimer is sufficient for
function during clamp loading by the
complex.
Figure 3:
Size
estimate of the complex. The arrow shows the
elution position of the
complex, relative to protein
standards of known molecular mass, from a Superose 12 gel filtration
column. Also shown are the elution positions of
and
alone.
Thy, bovine thyroglobulin (670 kDa);
-G, bovine
-globulin (158 kDa); Ova, chicken ovalbumin (44 kDa);
Myo, horse myoglobulin (17 kDa); B-12, vitamin B-12
(1.35 kDa).
The Kinase Protection Assay Reveals That
A six-amino acid
recognition sequence for cAMP-dependent protein kinase was engineered
onto the C terminus of Interacts
with the C-terminal Face of the
Ring
for the purpose of radiolabeling
with
P
(17, 24) . Both C termini of the
dimer protrude from one face of the
ring as shown in
Fig. 4A. In the experiment of Fig. 4B,
this
derivative was incubated with
and then treated with
protein kinase and [
-
P]ATP. At the times
indicated, the reaction was quenched, and the extent of phosphorylation
was determined by autoradiography of an SDS-polyacrylamide gel
(Fig. 4B). In the absence of
, the
derivative
is phosphorylated within 1 min, but in the presence of
,
phosphorylation is nearly completely blocked.
(
)
These results suggest that
interacts with the
C-terminal face of the
dimer.
Figure 4:
Kinase protection assay of
interacting with the
clamp. The
dimer structure is shown in
A. The view on the left shows the central cavity through which
the DNA fits. On the right, the
dimer is turned 90° to show
the two C termini, which extrude out from the same side of the ring.
The boxes placed on the C termini denote the location of the
protein kinase recognition sequence engineered into
(denoted as
). In B,
was treated with protein kinase and
[
-
P]ATP either in the presence or absence
of
(see scheme), and aliquots were withdrawn at the indicated
times, followed by analysis on an SDS-polyacrylamide gel and
autoradiography. The autoradiogram at the top shows the time course of
phosphorylation in the absence of
,
and the bottom autoradiogram is in the presence of
. Quantitation
of the autoradiograms is shown below.
, absence of
;
,
presence of
.
ATP Is Required for Interaction of the
In the course of these
studies, we noticed that ATP and magnesium were required in the gel
filtration buffer to observe the interaction between the Complex with
, but Not for
with
complex
and
(ATP and MgCl
were present in the experiments of
Fig. 1
and Fig. 2). However, in experiments with
, ATP
and MgCl
were not needed to observe the
-
interaction. Furthermore, study of the effect of ATP and MgCl
on the interaction of the
complex (and
) with
showed that without ATP and MgCl
, the
complex-
interaction is much weaker than the
-
interaction, while with
ATP and MgCl
, the
complex-
interaction is
comparable in affinity to the
-
interaction.
was incubated with a substoichiometric amount of
either the
complex or
and then gel-filtered in either the
presence or absence of ATP and MgCl
in the column buffer.
PanelsB and E are analyses of
and the
complex, respectively, with
performed in the presence of
ATP and MgCl
. PanelsA and F are
analyses of
and the
complex, respectively, with
performed in the absence of ATP and MgCl
. It is apparent
that in the presence of ATP and MgCl
,
comigrates with
both the
complex (fractions 22-30; panel E) and
(fractions 36-38; panel B) (see panels C and G for elution position of
alone and panelD for
alone). In the absence of ATP,
interacts only with
(fractions 34-38; panel A),
but essentially no interaction is observed with the
complex
(panel F). An experiment described below (see Fig. 8)
shows that both ATP and MgCl
are required to observe the
interaction between the
complex and
.
Figure 5:
ATP is required for the
complex-
interaction, but not for the
-
interaction. The
dimer (4.8 nmol) was incubated with 1.2 nmol of either
or
complex in 200 µl of column buffer (with or without 1
mM ATP and 10 mM MgCl
) at 15 °C for
30 min. Mixtures were gel-filtered on Superose 12 as described in the
first report of this series (12) (ATP and MgCl
, when
present in the incubation mixture, were included in the column buffer
as well). Aliquots (50 µl) of column fractions were analyzed on 12%
SDS-polyacrylamide gels as described (12). A,
and
without ATP and MgCl
; B,
and
with ATP
and MgCl
; C and G,
alone;
D,
alone; E, the
complex and
with
ATP and MgCl
; F, the
complex and
without ATP and MgCl
. Fraction numbers are at the top of
the gels; molecular mass standards are in the first lane with their
respective masses (in kilodaltons) shown to the left of each gel; and
subunits of the
complex and
are indicated to the right of
each gel.
Figure 8:
Metal and nucleotide requirements for the
complex-
interaction. The [
H]
subunit (52 pmol as dimer) was incubated with 104 pmol of
complex
in 100 µl of column buffer for 5 min at 37 °C before injection
into a Superose 6 column equilibrated with column buffer and developed
at 37 °C. After the first 2.8 ml, fractions of 200 µl were
collected and analyzed for [
H]
by
scintillation counting. Present in the incubation and gel filtration
buffer during the analysis were the following: A, 0.2
mM ATP and 8 mM MgCl
; B, no
addition; C, 8 mM MgCl
; D, 0.2
mM ATP and 8 mM MgCl
; E, 0.2
mM dATP and 8 MgCl
; F, 0.2 mM
ATP
S and 8 mM MgCl
; G, 0.2
mM AMP-PNP and 8 mM MgCl
; H, 0.2
mM ADP and 8 mM MgCl
; I, 0.2
mM dTTP and 8 mM
MgCl
.
In Fig. 6,
the binding kinetics of with
and of
with the
complex were compared by the surface plasmon resonance technique. In
panels A and B, the
subunit was immobilized on
the sensor chip, and then
was passed over immobilized
either in the absence (panelA) or in the presence
(panelB) of ATP and MgCl
. After 3 min of
observation of the association phase, buffer either lacking or
containing ATP and MgCl
was passed over the chip to observe
the dissociation rate (panelsA and B,
respectively). The results show that
binds
whether ATP and
MgCl
are present or not. These experiments were repeated
using different concentrations of the
subunit in the buffer, and
the apparent kinetic parameters (k
and
k
) and the apparent dissociation constant
(K
) were calculated from these data.
These values are listed in . The apparent
K
value for interaction of
with
was 7-10 nM with or without ATP.
complex was immobilized on a sensor chip, and the experiments
were repeated. To immobilize the
complex, the
`
complex was passed over immobilized
to
reconstitute the
complex, and then
was passed over the
immobilized complex. In the absence of ATP and MgCl
, very
little interaction of
with the
complex was observed
(Fig. 6C), but with ATP and MgCl
present in
the buffer, a clear signal was obtained (Fig. 6D).
Similar experiments were repeated using different concentrations of
in the buffer, and the kinetic and equilibrium constants for the
complex-
interactions were calculated as described above for
the
complex (). The results show that
associates very slowly with the
complex in the absence of ATP and
MgCl
(250-fold slower than with
), but in the presence
of ATP and MgCl
,
associates with the
complex as
fast as with
. In contrast, the off rates of
from the
complex were affected only 3.5-fold by the presence of ATP and
MgCl
. The calculated K
value
for the
complex-
interaction in the presence of ATP and
MgCl
was similar to that of the
complex, but in
the absence of ATP and MgCl
, the K
value was increased 1000-fold.
A Protease Protection Assay Reveals a Conformational
Change in the
The ATP requirement
for the Complex Induced by ATP
complex to bind
, but not for
to bind
,
could be interpreted as
being buried within the
complex and
ATP inducing the
complex to expose
for interaction with
. If ATP induces the
complex to expose
, then
should become more susceptible to proteolysis. To test this,
was
H-labeled and reconstituted into the
complex. The
[
H]
`
complex was
treated with Pronase in the presence or absence of ATP (and
MgCl
), and the partial digestion pattern of
[
H]
was analyzed on an SDS-polyacrylamide
gel (Fig. 7).
Figure 7:
ATP
influences the protease digestion pattern of
[H]
within the
complex. The
[
H]
subunit (lanes2 and
3) and the
complex constituted using
[
H]
(lanes5 and
6) were treated with Pronase in the presence or absence of 1
mM ATP and 10 mM MgCl
as specified at the
top. After treatment, reactions were analyzed on an SDS-polyacrylamide
gel, followed by fluorography as described under ``Experimental
Procedures.'' Untreated samples of [
H]
and the
complex containing [
H]
were
analyzed in lanes1 and 4, respectively. For
the
complex, arrows indicate new or enhanced cleavages
in the presence of ATP (lane6) relative to in the
absence of ATP (lane5). Circles indicate
cleavages present in the absence of ATP (lane5)
relative to in the presence of ATP (lane6).
Fig. 7
shows the
[H]
subunit alone, either untreated
(lane1) or treated with Pronase without ATP
(lane2) or with ATP (lane3). As
expected, ATP had no detectable influence on the digestion pattern of
the isolated [
H]
subunit. Study of
[
H]
assembled into the
complex is
shown in lanes 4-6. Lane4 is the
complex untreated with Pronase. Lanes5 and
6 are the
complex treated with Pronase in the absence or
presence of ATP, respectively. Several ATP-dependent changes in the
digestion pattern of [
H]
within the
complex are observed, and they assort into two categories, enhanced
cleavages (arrows) and decreased cleavage (circles).
Hence, ATP induces several changes in the digestion pattern of
[
H]
within the
complex, consistent
with ATP inducing a conformational change. These Pronase digestion
experiments were performed in the absence of
, and therefore,
is not required for the ATP-induced conformational change in the
complex.
Metal and Nucleotide Requirements for the
A study of the nucleotide requirements
for induction of the
Complex-
Interaction
complex-
interaction is presented in
Fig. 8
. In these experiments, [
H]
was
used to follow its position of elution during gel filtration.
Nucleotide and MgCl
, when present, were in the reaction and
the column buffer. PanelA shows the elution of
[
H]
bound to the
complex, and
panelB shows the elution of
[
H]
alone (no
complex added). Both
MgCl
and ATP are needed for the
complex-
interaction as [
H]
is not bound to the
complex if either ATP or MgCl
is omitted
(panelsC and D). PanelE shows that dATP induces the
complex-
interaction,
consistent with the ability of dATP to support the
complex-catalyzed assembly of
onto DNA. The nonhydrolyzable
analog AMP-PNP is not an effector of the
complex-
interaction (panelG), nor are ADP and TTP
(panelsH and I, respectively). With
ATP
S, only one-half of the [
H]
was
bound to the
complex (panelF). This result did
not change with longer incubation times. Furthermore, the ATP
S was
present at a saturating level as lower concentrations of ATP
S did
not result in a decrease in the extent of complex formation between
[
H]
and the
complex (data not shown).
Mechanism of the
This study has identified the Complex in Assembly of
Clamps onto DNA
subunit of
the
complex as the main (if not sole) contact point with
.
Using individual
and
subunits, the observed
K
value for the
-
interaction
is 7-10 nM. However, when
is assembled into the
complex, the K
value for
interaction with
is increased
350-fold
(K
3 µM) and requires
ATP and MgCl
to achieve the high affinity interaction with
(K
3 nM). A simple
interpretation of these data is that
is sequestered within the
complex, and ATP induces a conformational change that presents
for interaction with
(as in Fig. 9). Consistent with
this hypothesis, the Pronase digestion pattern of
in the
complex is changed by the addition of ATP. We propose this
ATP-activated interaction of the
complex with
is an early
step in the assembly of the
dimer onto a primed template.
Downstream events include recognition of the primed template and
assembly of
around the DNA.
Figure 9:
Activation of the complex by ATP.
The diagram of the
complex is consistent with the stoichiometry
of subunits and the intermolecular contacts between them (12). In the
first diagram, the interface of the
subunit that interacts with
is shown as being partially buried within the
complex to
explain its inability to bind
in the absence of ATP. In the
presence of ATP, a conformational change exposes the
subunit
(second diagram) for binding the
dimer (third diagram), followed
by transfer of
onto primed DNA (fourth
diagram).
These studies in the E. coli system can be compared with studies in the phage T4 replication
system. In the T4 system, the clamp loader is a complex of two
proteins, the gene 44/62 protein complex. Laser cross-linking studies
on the gene 44/62 protein complex and the gene 45 protein clamp suggest
that the clamp loader undergoes a large conformational change during
the process of loading the clamp onto primed DNA
(20) .
When Does the
Although the Ring Open?
dimer is very stabile, it is conceivable that the
ring is opened
in this ATP-activated complex (as hypothesized in
Fig. 9
).
(
)
It has been reported that the
holoenzyme can act processively in the absence of ATP, provided
is present at a high concentration
(21, 22) . Perhaps a
high concentration of
circumvents the need for ATP by simply
driving the unfavorable interaction of
with the
complex in
the absence of ATP. Processive synthesis in the absence of ATP implies
that ATP is not essential to open the
ring; perhaps the binding
energy between
and
opens the
ring. However, it is
important to note that the templates were linear in the earlier studies
on holoenzyme activity in the absence of ATP, and therefore, the
ring may have loaded onto DNA independent of the
complex by
threading over DNA ends (i.e. without opening at the
dimer interface).
The Activated
Whether the activated
state of the Complex
complex (ATP-induced conformational change) requires
ATP binding or hydrolysis is still uncertain. Further studies using
ATP-binding site mutants of
constituted into the
complex
are in progress to distinguish the roles of ATP binding and hydrolysis.
In this report, the use of the nonhydrolyzable ATP analog AMP-PNP in
place of ATP did not activate the
complex for interaction with
, indicating that hydrolysis may be necessary to achieve the
activated state. However, the
complex is partially activated for
interaction with
by ATP
S, suggesting that ATP hydrolysis is
not necessary as ATP
S is generally nonhydrolyzable. However, the
holoenzyme has been shown to be capable of hydrolyzing
ATP
S
(23) . The extent of
complex activation by
ATP
S is approximately half that observed using ATP. Previous
studies have shown that ATP
S results in one-half the amount of
holoenzyme clamped to DNA relative to the use of ATP, and it was
suggested that there were two populations of the holoenzyme, one that
can hydrolyze ATP
S and another that cannot
(23) . The
results of this report suggest that the observed value of one-half may
be rooted entirely in the
complex. Perhaps there are two
populations of the
complex, one that can be activated by
ATP
S and another that cannot. The results of this report suggest
yet another explanation. Perhaps the
complex hydrolyzes ATP
S
slower than ATP, thus giving time between binding and hydrolysis of ATP
during which one-half of the activated
complex re-laxes back to
the form in which
is unavailable for interaction with
.
complex by ATP may explain how the
complex achieves its catalytic capability in assembling multiple
clamps onto DNA. In the ATP-activated state,
is exposed for
interaction with
, and upon relaxing from the activated state, the
subunit is resequestered into the
complex, thus severing
the
-
interaction. Hence, after loading the
clamp onto
DNA, the relaxation of the
complex would sever the
-
contact, resulting in a loss of affinity of the
complex for the
ring on DNA. This loss of affinity of the
complex for
would promote
complex dissociation from DNA, thus freeing it to
load other
clamps onto DNA.
complex
from
after assembling it onto DNA is also essential for yet
another function. The core polymerase must interact with the
ring
on DNA to achieve high processivity. We observe that point mutants in
the C terminus of
bind neither
(or the
complex) nor
core.
(
)
Hence, core and the
complex have
overlapping binding sites on
, and they both interact with the C
terminus of
. Both core and the
complex recognize a primed
template single-stranded/double-stranded DNA junction for their action.
Hence, it seems likely that the
ring is positioned on the duplex
portion of a primed template such that its C-terminal face points
toward the 3` terminus, where the
complex acts to assemble it.
Loss of the activated state of the
complex, such that its tie to
is broken, would clear
from the C termini of
for the
next interaction, namely that of tethering core to DNA for highly
processive synthesis.
Table:
Apparent kinetic constants for binding of
to
and of
to the
complex
S, adenosine 5`-O-(thiotriphosphate).
preparation since the same kinase recognition
sequence has been engineered onto the C terminus of the EBNA1 protein,
and
does not inhibit its phosphorylation, nor is phosphorylation
of the EBNA1 derivative inhibited in the presence of
and the
derivative.
complex acts by cutting DNA and threading it through the
ring.
This possibility would be similar to the action of topoisomerases in
cutting DNA followed by strand passage through the cut.
inactivates
in replication assays and prevents
interaction of
with
and core (V. Naktinis and M.
O'Donnell, manuscript in preparation).
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.