From The Rockefeller University and ** Howard Hughes
Medical Institute, Laboratory of DNA Replication, New York, New York
10021 and ¶ Department of Microbiology, Cornell University Medical
College, New York, New York 10021
Received for publication, January 22, 2001
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ABSTRACT |
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The The DNA polymerase III (Pol
III)1 holoenzyme is primarily
responsible for replicating the 4.4-megabase Escherichia
coli genome (1, 2). Pol III holoenzyme performs this task with
high speed and accuracy with the help of ten component subunits. These are Rapid and processive DNA synthesis by Pol III holoenzyme is dependent
on the interaction between the The crystal structure of The Although all three subunits, Study of the Nucleotides, DNAs, and Buffers--
Radioactive nucleotides were
purchased from PerkinElmer Life Sciences. Unlabeled nucleotides were
purchased from Amersham Pharmacia Biotech. M13mp18 ssDNA was prepared
by phenol extraction of purified M13mp18 phage that had been banded
twice in CsCl gradients (45) and primed with a 30-nucleotide primer
(Life Technologies, Inc.) as described (46). Buffer A contained 20 mM Tris-HCl (pH 7.5), 0.5 mM EDTA (pH 8.0), 100 mM NaCl, and 10% glycerol. DNA replication buffer
contained 20 mM Tris-HCl (pH 7.5), 0.1 mM EDTA, 40 µg/ml bovine serum albumin, 5 mM dithiothreitol, 8 mM MgCl2, 4% glycerol, 0.5 mM ATP,
60 µM dGTP, and 60 µM dCTP. Surface plasmon resonance (SPR) buffer contained 10 mM Hepes-NaOH (pH 7.4),
150 mM NaCl, 3.4 mM EDTA, and 0.005% Tween 20.
Proteins--
Proteins were purified as described: Gel Filtration Analysis of
Interaction between DNA Replication Assays--
Singly primed M13mp18 ssDNA (20 fmol), 0.8 µg of SSB, 75 fmol of Pol III*, and 750 fmol of SPR Analysis of
The kinetic constants for interaction between Protomer Exchange Assay--
The two
Spontaneous protomer exchange was measured (i.e. no other
proteins besides
To measure the effect of Nickel Column Affinity Assay for
As a prerequisite for an experiment of this type, it is important that
the 32P-
Next, we examined how the
The results of this experiment (Fig. 2, lane 1)
demonstrate that very little heterodimeric
In the study of Fig. 3 we designed
another experiment to examine the oligomeric state of
The results of this experiment, shown in Fig. 3, illustrate that
similar amounts of heterodimer are formed within 2 h in the presence or absence of How Does
To form a stable monomer of
In Fig. 4C, wild type
It has long been presumed that the circular structure of the
The gel filtration analysis revealed that
Kinetic analysis of the SPR data yielded the association
(kon) and dissociation
(koff) rate constants, from which the
equilibrium dissociation constant for the The
The The Critical Role of Hydrophobic Interface Contacts in the
This study confirms an important role of the hydrophobic residues at
the interface in maintaining a stable The
The amount of work to open one interface can be calculated to be ~2.4
kcal, assuming a difference of 57-fold in the equilibrium binding
constants for The
The scheme in Fig. 7 illustrates our
current view of the
Upon interaction of
Events in proceeding from diagram C to
D, where the Comparison with the Eukaryotic PCNA Clamp and RFC Clamp
Loader--
The eukaryotic clamp, the PCNA ring, has essentially the
same shape and structure as sliding clamp encircles the
primer-template and tethers DNA polymerase III holoenzyme to DNA for
processive replication of the Escherichia coli genome. The
clamp is formed via hydrophobic and ionic interactions between two
semicircular
monomers. This report demonstrates that the
dimer
is a stable closed ring and is not monomerized when the
complex
clamp loader
(
3
1
1
1
1) assembles the
ring around DNA.
is the subunit of the
complex that binds
and opens the ring; it also does not appear to
monomerize
. Point mutations were introduced at the
dimer
interface to test its structural integrity and gain insight into its
interaction with
. Mutation of two residues at the dimer interface
of
, I272A/L273A, yields a stable
monomer. We find that
binds the
monomer mutant at least 50-fold tighter than the
dimer. These findings suggest that when
interacts with the
clamp, it binds one
subunit with high affinity and utilizes some of
that binding energy to perform work on the dimeric clamp, probably
cracking one dimer interface open.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
(the DNA polymerase (3)),
(the proofreading 3'-5'
exonuclease (4)), and
(unknown function) that form the DNA
polymerase III core (5);
(the sliding clamp (6, 7)); and the
multisubunit DnaX complex (
'
) that functions as the
clamp loader (8-10) and contains at least two subunits of the
"organizer" that binds two core polymerases (11-13) and connects
to the DnaB helicase at the replication fork (14, 15).
subunit of the core polymerase and
the
clamp (6).
is a ring-shaped protein that encircles
double-stranded DNA and can slide freely along its length (6, 7). By
itself, core polymerase can extend a primer by only a few nucleotides
before dissociating from DNA (16). When
is bound to the polymerase
and topologicaly linked to the primer-template, it serves as a mobile
tether to keep the enzyme associated with DNA, facilitating replication
of several thousand nucleotides at a time. Similar mechanisms for
processive DNA synthesis by replicative polymerases have been
discovered in a variety of other organisms (reviewed in Refs. 2, 17, 18, and 19), including eukaryotic DNA polymerase
(tethered to DNA
by the PCNA sliding clamp (20, 21)) and bacteriophage T4 DNA
polymerase, gp43 (tethered by the gp45 sliding clamp (22)).
shows it to be a ring-shaped dimer formed
by the head-to-tail interaction of two semicircle-shaped monomers (7).
A continuous
-sheet forms a scaffold around the outer surface of the
ring that supports 12
-helices lining the inside of the ring. The
central cavity is about 35 Å in diameter, which is large enough to
encircle double-stranded DNA as well as one or two layers of water
molecules. Moreover, although the inside of the
ring is positively
charged, it lacks specific contact with DNA, allowing
to form a
stable topological link with the DNA and yet slide freely along the
duplex. At the two identical dimer interfaces, a continuous
-sheet
formed by hydrogen bonding between
strands from each monomer
stabilizes the ring structure in addition to a small hydrophobic core
formed by packing of Ile272 and Leu273 of one
monomer with Phe106 and Leu108 on the other
monomer. Charged amino acids at the interface are also in position to
form six ion pairs (these interactions are detailed in Fig. 4). These
numerous and potentially strong interactions between the two
subunits presumably underlie the highly stable dimeric structure of
and its ability to remain bound to DNA with a half-life of over 100 min
(23, 24). Yet the closed circular clamp must be opened frequently
during DNA replication for assembly on DNA to initiate processive
replication as well as for disassembly of the
ring from DNA when
replication is complete.
complex clamp loader (
'
) assembles
clamps on
primer-template DNA (where they can be used by the polymerase) and can
also remove clamps from DNA when necessary (23-26). The process of
clamp assembly requires that the
complex open the
clamp, guide
DNA into the central cavity, and facilitate closure of the clamp around
DNA. Crystal structure
analysis,2 and a recent
biochemical study (27) reveals that the
complex contains three
copies of
; the other subunits (
,
',
,
) are each
present in a single copy (10, 13). The
subunit of
complex binds
to
and destabilizes or opens the dimer interface (28, 29). The
subunits are the only ones that hydrolyze ATP (30-32). The
'
subunit is homologous to
and appears to play a role in modulating
the access of
to
(10, 33, 34). In the absence of ATP, the
affinity between the
complex and
is low compared with the
affinity between the
subunit and
(28). Clamp assembly initiates
when ATP binds the
subunits and induces a change in conformation of
the
complex that results in ability of
to bind
(28, 29,
32). The
' subunit appears at least partially responsible for
modulating the access of
to
, since a previous study indicated
that
' and
compete for interaction with
(29). The
ATP-induced conformational change of
complex may entail removing a
surface of
' from
, allowing
to bind and open the
clamp.
In the presence of a nonhydrolyzable ATP analogue, the clamp loader-
complex binds primer-template DNA with high affinity (32, 36).
Interaction of
complex with DNA, especially primed template,
triggers ATP hydrolysis and is stimulated by the presence of
(29,
32, 36, 37). ATP hydrolysis is coupled to closure of the clamp around
DNA and
complex turnover. The
subunit of
complex binds to
SSB and helps coordinate the switch between the primase, clamp loader, and polymerase proteins at the primer template (38, 39), and
enhances the stability of the
complex; however, these two proteins
are not absolutely essential for clamp assembly (40-43).
,
, and
', are required for
loading
onto DNA, the single
subunit appears to be the
predominant contact between
and the
complex (28). It remains
possible that weaker interactions between
and the other
complex
subunits exist.3 However, our
previous studies demonstrated that
alone can open and remove
clamps from circular DNA molecules with nearly the same efficiency as
complex
(kunloading
complex = 0.015 s
1; k
unloading = 0.011 s
1) (24). We were therefore curious as to how the
subunit generates the leverage required to part the apparently tightly
closed
dimer interfaces. Previous studies indicate that
opening
at just one interface is sufficient to allow passage of DNA into (or
out of) the central cavity (29). Experiments herein measure the
exchange of labeled
subunits as they are utilized by the
complex, and the results support the conclusion that the dimeric clamp
is not split apart into monomers but rather stays intact during clamp
assembly, presumably opening at only one interface for entry of DNA. In
the simplest possible mechanism, the clamp loader could prompt clamp
opening merely by perturbing one of the dimer interfaces and
transiently reducing its stability.
-
interaction in this report provides insight into
how the
and
complex might open the
ring. We demonstrate here that the
ring retains its dimeric structure when bound by one
subunit. Furthermore, we have mutated two hydrophobic residues in
the
dimer interface to produce a stable monomeric version of
.
Only one
subunit binds the
monomer, which is surprising, given
the one
/two
stoichiometry of the wild type
-
complex.
This suggests that the binding site of
on the
ring is located
primarily on one of the two
subunits. The affinity of
for the
monomer mutant is about 50-fold greater than for the
dimer,
implying that the binding energy of
to a single
subunit of the
dimer is harnessed to perform work, namely to force open one of the
dimer interfaces. The
subunit binds
at the carboxyl terminus,
which lies in the vicinity of the dimer interface (44). Therefore, it
is conceivable that
binding to one
protomer disrupts the
contacts in a nearby dimer interface that hold the ring closed.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
,
,
(46),
,
',
,
(33),
(47), and SSB (46).
complex and
Pol III* (a subcomplex of Pol III holoenzyme lacking the
subunit)
were reconstituted from individual subunits and purified as described in Refs. 9 and 13, respectively. Mutant
proteins were constructed using DNA oligonucleotide site-directed mutagenesis. Various N-terminal tagged versions of
(described below) were purified according to the
previously described protocol for wild type
(7). Radiolabeling of
tagged
with 32P was performed using
[
-32P]ATP and cAMP-dependent protein
kinase to a specific activity of ~100 cpm/fmol as described (48). The
catalytic subunit of cAMP-dependent protein kinase produced
in E. coli was a gift from Dr. Susan Taylor (University of
California, San Diego). 3H-
was labeled by reductive
methylation as described (48).
,
, L273A-
, and
I272A/L273A-
--
The
, L273A-
, and I272A/L273A-
proteins
(3 µM as dimer) were sized by gel filtration (at 4 °C)
on an FPLC HR 10/30 Superose 12 column (Amersham Pharmacia Biotech)
equilibrated with Buffer A. The proteins were incubated in a final
volume of 200 µl of Buffer A for 15 min at 15 °C and then applied
to the column. After collecting 6-ml, 170-µl fractions were
collected, and 25-µl aliquots of the indicated fractions were
analyzed by SDS-polyacrylamide gel electrophoresis (15% gels);
proteins were visualized by Coomassie Blue staining. For size
standards,
(130 kDa), bovine serum albumin (66 kDa), and
(39 kDa) were analyzed similarly.
and
was analyzed by incubating 9 µM
with 12.5 µM wild type
(as
dimer) or 25 µM I272A/L273A-
, (as monomer) for 15 min
at 15 °C in a final volume of 200 µl of Buffer A, followed by gel
filtration chromatography and SDS-polyacrylamide gel electrophoresis
analysis as described above.
(wild
type and mutant concentrations are calculated as monomer) were
incubated at 37 °C for 2 min in 25 µl (final volume) of DNA
replication buffer (this buffer contains ATP, dCTP, and dGTP). DNA
synthesis was initiated upon the addition of the remaining two
deoxyribonucleoside triphosphates (60 µM dATP, 20 µM dTTP (final concentrations), and 1 µCi of
[
-32P]dTTP). After 20 s, reactions were quenched
with 25 µl of 40 mM EDTA and 1% SDS. Aliquots (20 µl)
of the quenched reactions were analyzed by electrophoresis on a 1%
TBE-agarose gel, and the radiolabeled DNA was visualized on a
PhosphorImager (Molecular Dynamics, Inc., Sunnyvale, CA). Synthesis was
quantitated by spotting 20-µl aliquots of the reaction on DE81
filters, followed by liquid scintillation counting as described
(49).
-
Interaction--
The
subunit (10 µl of 0.6 µM) was immobilized on a carboxymethylated
dextran matrix-coated sensor chip (CM5; Biacore) by carbodiimide
coupling in 10 mM sodium acetate (pH 5.5). SPR analysis was
performed at 23 °C by injecting 15 µl of
or I272A/L273A
(0.25 and 1.23 µM; concentrations for both are given as
monomer) in SPR buffer, at a flow rate of 5 µl/s. After each analysis
was complete, the chip surface was regenerated by injecting 10 µl of
0.1 M glycine (pH 9.5) over the chip, which releases
bound
with no significant effect on the binding capacity of the
immobilized
protein.
and
were
determined by nonlinear curve fitting, using the BIAevaluation 2.1 software. The rate of dissociation (koff) was
calculated by fitting the curves to a single exponential decay
described by Equation 1,
where R0 represents the response and
t0 represents the time at the start of the
dissociation phase. The association rate (kon)
was calculated using the binding model A + B= AB and Equation 2,
(Eq. 1)
where Req is the response at steady
state, C is the concentration of
(Eq. 2)
, and
t0 is the time at the start of the association phase. The dissociation constant (Kd) for
interaction between
and
was calculated as
koff/kon.
mutants for this assay
were constructed by placing the
gene into either the pHKEp vector
or the pHKEpmut vector (50). Both of these vectors place a
34-amino acid tag onto the N terminus of the protein. The tags contain
a protein kinase site (to label the protein with 32P) and
either a functional (pHKEp) or a nonfunctional (pHKEpmut)
hemagglutinin (HA) epitope. The nonfunctional epitope was formed by
replacing two amino acids; YPYDVPDYA was changed to
YPYDVPAAA. After expression and purification, one
contains a functional HA epitope (ha
2) and
the other
contains a nonfunctional HA epitope, which we use in this
report in the phosphorylated form and refer to as
32P-
2. The
with the mutated HA-epitope
was labeled with 32P (32P-
) as described
(48). Titrations of these
variants showed that they were as active
as wild type
in replication assays with Pol III* on SSB-coated
M13mp18 ssDNA primed with a single oligonucleotide. Monoclonal antibody
to the HA epitope was purchased from BabCo, and Protein A-Sepharose 4B
was from Zymed Laboratories Inc. The HA antibody was
conjugated to Protein A beads by incubation for 15 min at 25 °C in
400 µl of 20 mM Hepes (pH 7.4), 150 mM NaCl,
0.1% Triton X-100, 10% glycerol.
) in 50-µl reactions containing 2 pmol of
32P-
2 and 2 pmol of
ha
2 in 20 mM Hepes (pH 7.5), 150 mM NaCl, and 10% glycerol. Reactions were incubated at
37 °C for 0, 1, 2, 4, 6, or 8 h before the addition of 50 µl
of HA antibody-conjugated beads and placed at 4 °C for a further 30 min. Beads were pelleted, washed three times with 1 ml of 50 mM Tris-HCl (pH 8.0), 500 mM NaCl, 5 mM EDTA, 0.1% SDS, and 0.1% Triton X-100; resuspended in
Eco-Lume (ICN); and counted in a scintillation counter. Control
experiments were performed similarly except that either no antibody was
conjugated to the beads or the ha
2 was not
added to the reaction.
complex on
protomer exchange during
clamp assembly onto DNA, 250 fmol each of
ha
2 and 32P-
2
were incubated for 5 min at 37 °C with 500 fmol of
complex and
1.8 pmol of nicked pBS DNA in 70 µl of 20 nM Tris-HCl
(pH. 7.5), 0.1 mM EDTA, 4% glycerol, and 8 mM
MgCl2. The reaction was then applied to a 5-ml A15
M gel filtration column equilibrated with the same buffer
plus 0.15 M NaCl. Fractions of six drops each were
collected, and those containing
on DNA were identified by
scintillation counting and pooled (420 µl), and then the DNA was
linearized upon treatment with 700 units of BamHI for 3 min at 37 °C to release
. To confirm that linearization was complete within this time, an aliquot (20 µl) was removed, quenched with 20 µl of 1% SDS, 40 mM EDTA, and then analyzed in a native
agarose gel. Then 50 µl of HA antibody beads were added to the
reaction, and incubation was continued for a further 30 min at 4 °C.
The beads were pelleted; washed three times with 1 ml of 50 mM Tris-HCl (pH 8.0), 500 mM NaCl, 5 mM EDTA, 0.1% SDS, and 0.1% Triton X-100; resuspended in
Eco-Lume (ICN); and counted in a scintillation counter. In a control
experiment, the above procedure was repeated except that heterodimeric
was used in the assembly reaction by first preincubating 250 fmol
of each
in one tube for 5 h at 37 °C before adding to the
reaction containing
complex and DNA.
-
2
Complex--
Reactions contained 67.5 pmol of
3H-
2 (wild type
labeled by reductive
methylation), 1.7 nmol of his
2, which
contained a six-residue histidine tag on a 23-residue N-terminal leader
(
was cloned into the pHK vector in Ref. 50), and 6.6 nmol of
(where present) in 200 µl of 20 mM Tris-HCl (pH 7.5),
10% glycerol, 8 mM MgCl2, and 100 mM NaCl. A control reaction utilized 1.7 nmol of unlabeled
wild type
2 in placed of the
his
2 derivative. Reactions were assembled on
ice and then shifted to 37 °C, and aliquots of 20 µl were removed
at 2 and 24 h of incubation. Upon removal of an aliquot, NaCl was
added to a final concentration of 0.5 M, and the reaction
was applied to a 1-ml nickel chelate column (HiTrap; Amersham Pharmacia
Biotech) equilibrated in 20 mM Tris-HCl (pH 7.9), 5 mM imidazole, 8 mM MgCl2, and 10% glycerol. The column was washed with 5 ml of the same buffer and then
eluted with 3 ml of 20 mM Tris-HCl (pH 7.9), 1 M imidazole, 8 mM MgCl2, and 10%
glycerol. Fractions of 1 ml were collected. The flow-through (wash) and
bound (elution) fractions were analyzed by liquid scintillation
counting and analyzed in a 10% SDS-polyacrylamide gel to confirm the
presence of
with
in the bound fractions. The typical yield of
3H-
2 off the column was greater than
85%.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
Is Not Monomerized during Assembly onto DNA--
We have shown
previously that the
clamp is a tight dimer and remains a dimer even
when diluted to a concentration of 50 nM (23). Nonetheless,
it is possible that
complex dissociates the
dimer into monomers
using the energy of ATP hydrolysis and then reassembles the
dimer
onto DNA in a second step. To test this possibility, we constructed two
chemically distinct
mutants; one was phosphorylated and contained a
protein kinase tag (32P-
pk), and the other
had a hemagglutinin epitope tag (ha
). If the
complex
monomerizes
dimers and reassembles them onto DNA, then it should
act upon a mixture of 32P-
2 and
ha
2 to form
32P-
-ha
heterodimers on DNA.
2 and ha
2
mixture does not undergo spontaneous protomer exchange to form heterodimers during the time of the experiment. The time course for
spontaneous heterodimer formation was measured in the experiment of
Fig. 1 by mixing equal amounts of
32P-
2 and ha
2,
followed by removal of aliquots at time intervals and
immunoprecipitation of the mixture using Protein A beads to which an
antibody to hemagglutinin is attached. Initially, 32P-
will not be precipitated, since it lacks the epitope. But as protomer
exchange occurs, the 32P-
-ha
heterodimer
will be formed, which should result in the appearance of radioactivity
in the pellet. The result, shown in Fig. 1, demonstrates that the time
scale of spontaneous subunit exchange is on the order of hours
(t1/2 ~ 2 h). As the clamp-loading reaction
only requires 5 min, spontaneous protomer exchange during the reaction
should be nearly negligible. Control reactions not shown here have been
performed that demonstrate requirements for both the antibody and the
presence of the ha
to detect radioactivity attached to
the beads in the pellet. In both controls, the pellets lacked
radioactivity above background levels (0.5 fmol of
32P-
2).
View larger version (20K):
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Fig. 1.
Time course of protomer exchange. Two chemically distinct species of
,
one containing a hemagglutinin epitope and one labeled with
32P, were mixed together to initiate formation of
heterodimers as indicated. After various times of incubation, aliquots
were withdrawn, and beads to which antibody to the hemagglutinin tag
were attached were added. Heterodimeric
consists of a
32P-
protomer attached to a ha
protomer
that should be trapped by the hemagglutinin beads. Radioactivity in the
pellet, representing heterodimeric
, is plotted with respect
to time. I.P., immunoprecipitation.
complex loads a mixture of these two
variants onto DNA to determine whether it catalyzes protomer exchange
during the clamp assembly process (i.e. whether
complex breaks
dimers apart and reassembles them onto DNA as illustrated in
the scheme of Fig. 2). To test this
possible action, 32P-
2 and
ha
2 were mixed together, and
complex was
added immediately along with ATP and circular plasmid DNA containing a
single nick to initiate clamp assembly. After 5 min, the reaction was
applied to a gel filtration column to separate clamps that had been
assembled on DNA from those remaining in solution. Following this, the
isolated
-DNA complex was treated with BamHI to rapidly
linearize the DNA, allowing the clamps to slide off DNA into solution.
Then the reaction was analyzed for heterodimer formation by
immunoprecipitation using the hemagglutinin antibody beads (see the
scheme in Fig. 2).
View larger version (29K):
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Fig. 2.
complex does not
monomerize
during clamp assembly onto
DNA. The
complex and ATP were used to assemble a mixture of
32P-
2 and ha
2 onto nicked
circular plasmid DNA. As indicted, the
-DNA complex was isolated
from free
and then linearized to release
into solution.
Hemagglutinin antibody bound to beads was used to quantitate
heterodimers that were assembled onto DNA by the
complex.
Lane 1, 32P-
2 and
ha
2 were mixed immediately prior to their
transfer to DNA by
complex; lane 2,
32P-
2 and ha
2
were premixed and incubated 5 h to form heterodimers before being
transferred to DNA by
complex.
is formed in the
reaction, indicating that
complex does not catalyze
monomerization during clamp loading. In control reactions not shown
here, we confirmed that
complex loads approximately equal amounts
of 32P-
2 and
32P-ha
2 on DNA, and both
variants of
were as active as wild type
in replication assays
with PolIII*. In another control experiment 32P-
2 and ha
2
were premixed for 6 h to form the
32P-
-ha
heterodimer prior to use by
complex in assembly onto DNA. The result, shown in Fig. 2,
lane 2, demonstrates that the experimental strategy is functional in detecting heterodimers that are assembled on
DNA. Thus, it would appear that
complex does not monomerize
but
probably only opens one interface of the ring during the clamp opening
process. This conclusion is consistent with a previous finding that
showed that
complex was capable of assembling a
dimer onto DNA
that was cross-linked at one interface by a disulfide bond
(i.e. indicating that
complex does not need to open both interfaces to assemble
onto DNA (29)).
during clamp
assembly, this time while it is in complex with
, the clamp-opening
subunit of the
complex. Previous studies indicated that one
monomer binds to the
dimer, consistent with the single copy of
in
complex (28). The
subunit is capable of removing
rings
from circular DNA (24, 29) and thus must either destabilize one
interface or perhaps transiently dissociate
into monomers. In
either case, one may expect
to accelerate the rate of protomer
exchange. We examined these possibilities in a variation of the
protomer exchange assay. The assay utilized a hexahistidine-tagged
2 (his
2) and tritiated wild
type
2 (3H-
2). The
3H-
2 was mixed with a 25-fold molar excess
of his
2 in the presence or absence of a
4-fold molar excess of
(over total
), and then the mixture was
analyzed at either 2 or 24 h for heterodimer formation by nickel
chelate chromatography. Homodimeric 3H-
2
should not bind to the column (flow-through fraction), and heterodimeric 3H-
-his
should be retained
(bound fraction) and detected by elution from the nickel chelate
column, followed by scintillation counting.
View larger version (45K):
[in a new window]
Fig. 3.
does not monomerize
2. A mixture of
3H-
2 and a histidine-tagged
(his
) were mixed in the presence or absence of a 4-fold
excess of
subunit (over total
). At either 2 h
(lanes 2 and 3) or 24 h
(lanes 5 and 6), aliquots were removed
and loaded onto nickel-chelate columns. After washing the columns,
bound protein was eluted with buffer containing 1 M
imidazole. Since 3H-
lacks a His tag, 3H-
in the bound fraction represents 3H-
-his
heterodimers. Controls in which wild type
2 was
substituted for his
are shown in lanes
1 and 4.
, indicating that
does not appreciably speed up protomer interchange. Also, the fact that 3H-
is retained on the column in the presence of
supports the
1-
2 stoichiometry, since if
monomerized
2, heterodimer would not be present for
retention on the column. As a control, wild type
2
was substituted for his
2, which
should form a 3H-
-
wt heterodimer, but
should not bind the nickel chelate column. The result of this control
showed that 3H-
was not retained on the column, as
expected (not shown).
Open One Interface of the
Ring?--
The experiments described above demonstrate that the
complex does not catalyze the exchange of
protomers during the
clamp loading operation. The results also demonstrate that
does not monomerize the
dimer. These results support and extend earlier studies that indicate that only one interface of the
dimer ring is
cracked open during assembly onto DNA. The
subunit is the clamp-opening subunit of
complex. How does
open an interface of
the
ring? To gain insight into how
performs its ring opening task, we mutated
to form a stable
monomer. Initially, we set out to determine whether
mainly binds only one protomer of the
dimer, in which case
should still bind a
monomer about as well
as a
dimer. Alternatively,
may need to associate with elements
on both protomers of
2 in order to establish a firm grip
on the
ring. The results of this line of investigation were
unexpected and provided significant insight into the clamp opening
function of
.
, we utilized the crystal structure to
design site-specific mutations that would destabilize the dimer
interface. The crystal structure of the dimeric
clamp revealed a
small interface between the two
subunits that, despite its size,
has an abundance of potentially strong interactions (see the
diagrams in Fig. 4,
A and B) (7). These interactions facilitate the
formation of a highly stable circular clamp that maintains its dimeric
structure even at low nanomolar concentrations. In particular, a small
hydrophobic core of four amino acid residues (Phe106,
Leu108, Ile272, and Leu273) at the
dimer interface appears to play an important role in the stability of
the clamp structure. Initially, we constructed three single residue
mutants in which Ala was substituted in place of either
Phe106, Leu108, or Leu273 (we could
not obtain the I272A mutant). Each of these point mutants migrated as a
dimer in gel filtration analysis and retained 70-100% activity
with PolIII* (not shown). However, a double mutant, I272A/I273A, behaved as a monomer and lacked replication activity (explained below).
The experiments to follow focus on the double mutant and compare it
with wild type
.
View larger version (27K):
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Fig. 4.
Mutation of the interface to form a stable
monomer. A, the crystal structure of the
dimer shows that it is a ring-shaped structure, with a 35-Å central
hole, wide enough to encircle double-stranded DNA (7). The
dimer
interface, in particular the amino acid residues forming the
hydrophobic core, is highlighted on the right. B,
the dimer interface consists of two
sheets,
8 and
'4 (one from each subunit), that form an antiparallel
sheet across the dimer interface and neighboring amino acid contacts
that stabilize the clamp structure. The lines connect amino
acid residues predicted to form six ion pairs across the interface. The
hydrophobic core residues, Phe106, Leu108,
Ile272, and Leu273, are indicated, and
gray circles highlight Ile272 and
Leu273, which were mutated to Ala in this study.
C, analysis of wild type
and mutants of
on a sizing
column followed by SDS-polyacrylamide gel electrophoresis. The results
show that wild type
(top panel) and
L273A
(middle panel) elute as dimers (81.2 kDa). In contrast, the I272A/L273A-
(bottom
panel) elutes as a smaller, monomeric protein (40.6 kDa),
indicating that the double mutation severely disrupts the
dimer
interface. D, quantitation of DNA synthesis by Pol III* on
SSB-coated singly primed M13mp18 ssDNA in the presence of either no
, I272A/L273A-
, or wild type
.
and the
mutants, L273A-
and
I272A/L273A-
, were examined by gel filtration to determine their
oligomeric state. Fig. 4C shows the SDS-polyacrylamide gel
electrophoresis analysis of column fractions from the gel filtration
analysis of wild type
, L273A-
, and I272A/L273A-
, in the
top, middle, and bottom
panel, respectively. Wild type
elutes as a dimer in peak
fraction 21, as does the L273A-
mutant (calculated mass = 81.2 kDa). In contrast, the double mutant I272A/L273A-
migrates more
slowly through the column, indicative of a smaller size, and elutes as
a monomer (calculated mass = 40.6 kDa). The gel filtration
experiments were performed with 3 µM
(as dimer). Therefore, even at high protein concentration, I272A/L273A-
is unable to form a stable dimer.
dimer
is required for its action as a DNA polymerase processivity factor.
There are, however, single subunit processivity factors that do not
appear to encircle DNA, particularly the herpes simplex virus UL42
protein, which in fact is structurally similar to the eukaryotic PCNA
clamp but does not oligomerize into a ring (51). To determine if a
monomeric form of
can serve as a processivity factor, the monomeric
I272A/L273A-
mutant was tested for DNA replication activity with
PolIII* using primed M13mp18 ssDNA as substrate. The result, in Fig.
4D, demonstrates that the monomeric
mutant is inactive
with PolIII*. The dimeric single mutants (L273A, L108A, and F106A)
retained 70-100% the activity of wild type
(not shown). Thus, a
monomer that does not form a circular clamp is not capable of
tethering Pol III* to DNA for processive DNA replication.
Binds the
Monomer with Higher Affinity than the
Dimer--
Only one copy of the
subunit is present in the
complex, consistent with the stoichiometry of one
to two
in the
-
complex. The stoichiometry of only one
subunit per
dimer invokes the question of whether
interacts with both
protomers or can stably attach to one
protomer, perhaps somehow
preventing a second
from binding the other
protomer
(e.g. by steric occlusion). Interaction of
with the
monomer was tested in Fig. 5 by mixing
with an excess of either wild type
2 or the momeric
1 mutant, followed by gel filtration analysis on a
sizing column. The elution profiles of the proteins were analyzed by
SDS-polyacrylamide gel electrophoresis. As expected from previous
studies, Fig. 5A shows that
and wild type
2 form a stable complex with an apparent molecular mass
of 111 kDa, consistent with the
1
2
complex observed in our previous study (38.7-kDa
+ 2 × 40.6-kDa
= 119.9 kDa) (28). Fig. 5B shows that
and the I272A/L273A-
mutant also interact, forming a smaller
1
1 complex that migrates at an
intermediate position between the
1
2
complex (Figs. 5A) and the free I272A/L273A-
monomer
(Fig. 5E). This result reveals that the binding site for
on the
clamp resides within one monomer and demonstrates that
need not bind both subunits of the dimer to form a stable contact with
the clamp.
View larger version (64K):
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Fig. 5.
binds the
monomer mutant.
-
interaction was analyzed by gel
filtration on a sizing column, and the proteins were visualized in
column fractions by SDS-polyacrylamide gel electrophoresis and
Coomassie staining. The complex of the wild type
dimer with
(A) elutes faster than free
(C) and free
(D), indicative of its large size (81.2 + 38.7-kDa
1
2 complex). B, the monomeric
I272A/L273A-
protein also interacts stably with
, forming a
1
1 complex that elutes in an intermediate
position between the
1
2 complex
(A) and either
(C) or free
monomer
(E). The elution positions of molecular weight standards are
shown at the bottom of the gel.
can bind a single
protomer, but the possibility remained that the affinity of
for
may be affected by disruption of the dimeric structure. In particular,
we noticed that during gel filtration
trails as free protein from a
complex with wild type
(fractions 24-31 in Fig. 5A),
whereas in Fig. 5B most of the
appears in complex with
the
monomer, suggesting that
may bind
1 tighter
than
2. Next, we used the SPR technique to
examine more closely the relative affinity between
and the
dimer versus the I272A/L273A-
monomer mutant (Fig.
6). The
subunit was immobilized on a
sensor chip, and a solution of
in buffer (at different
concentrations) was passed over it. The increase in mass (response
units) resulting from interaction between
and
was measured over
time; this is the association phase from which the association rate
(kon) can be calculated. Next, buffer lacking
was passed over the
-
complex on the chip, and the resulting
decrease in mass over time provides information from which the
dissociation rate (koff) can be calculated. Fig.
6, A and B, shows sensorgrams of the interaction between
and two different concentrations of
and
I272A/L273A-
, respectively.
View larger version (20K):
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Fig. 6.
binds the
monomer tighter than the
dimer.
The sensorgrams of wild type
(A), and I272A/L273A-
(B) were obtained by measuring the increase in response
units when a 0.25 or 1.23 µM solution of either
(as
dimer for wild type
, as monomer for mutant
) was passed over
immobilized on a sensor chip. The sensorgrams were analyzed for kinetic
and equilibrium parameters of the
-
interaction as described
under "Experimental Procedures" (summarized in Table I). The
Kd values indicate that
binds I272A/L273A-
with ~80-fold higher affinity than the wild type
dimer.
-
interaction could be
calculated (Kd = koff/kon). The
parameters, summarized in Table I, reveal
that
binds the
monomer mutant with substantially higher
affinity than the wild type
protein. The average
Kd value for interaction between
and
is
about 0.46 µM (average of values determined at 0.25 and
1.23 µM
concentrations), which is ~57-fold higher
than the Kd for the interaction between
and
I272A/L273A-
(average Kd = 0.0075 µM). The tighter interaction between
and the
monomer is particularly striking because the
monomer has only one
potential
binding site, in contrast to the
dimer.
Kinetic and equilibrium constants describing the interaction between
and
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
Subunit Remains Dimeric during Clamp Loading--
This
study has examined whether
complex monomerizes
during the clamp
loading operation but could detect no evidence for splitting of
dimers during their assembly onto DNA. Consistent with retention of the
dimeric state, the
subunit does not appear to monomerize
2 or to significantly increase the rate of protomer
exchange among
dimers. Hence, it seems likely that the
subunit
opens only one interface of the
dimer during clamp loading,
consistent with the ability of
complex to load a
dimer onto DNA
that has been cross-linked via a disulfide bond across one of the two
interfaces (29).
subunit can open
and remove it from DNA but cannot load
onto DNA. The
and
' subunits of the
complex, along with
,
may orient DNA inside the open ring. The
' subunit, possibly also
assisted by
, must also sever the
-to-
contact, allowing the
ring to close around DNA. Release of the
complex and closure of the
clamp around the DNA are tied to ATP hydrolysis and are probably
coordinated with sensing the appropriate structure of DNA.
Clamp Structure--
Two
monomers contact each other in a
head-to-tail fashion at two small, identical interfaces to form the
ring-shaped, dimeric
clamp (Ref. 7; see also Fig. 4). A central
feature of the
clamp structure is the continuous layer of sheet
around the entire molecule, including the dimer interfaces. Further,
particular hydrophobic amino acid side chains, contributed by each
monomer, pack to form a small hydrophobic core within each interface.
There are also six potential ion pairs formed at the interface, which may further strengthen the dimer. Earlier studies demonstrated that the
clamp retains its dimeric structure even when it is highly dilute
(23). It is possible that the
clamp may "breathe" by alternate
opening and closing of one or the other interface. However, the
observed long lifetime of
on circular DNA when topologically linked
to it (23, 24) indicates that if there is breathing at the interfaces,
opening a wide distance (i.e. to slip off DNA) is a rare occurrence.
clamp structure. Although
retained its dimeric status upon mutation of only one hydrophobic
residue at the interface,4
mutation of two of the amino acids at the hydrophobic core,
Ile272 and Leu273, to Ala destabilizes the two
interfaces to such an extent that the
dimer exists as a stable
monomer in solution.
-to-
Binding Energy Opens the
Clamp--
In this
study, we show that
binds the
monomer about 50-fold tighter
than the
dimer. Moreover, the tight
-
monomer complex has a
1:1 stoichiometry, indicating that
has a binding site for only one
protomer. Thus, in the
1-
2 complex,
probably binds only one of the
subunits. The apparent higher
affinity of
for the
monomer mutant compared with wild type
also indicates that some of the binding energy of
to a
protomer
is put into performing work on the
dimer, thus lowering the
observed affinity. Given that upon binding of
to
, the dimer
opens, the work of the "lost" binding energy is probably utilized
to part one of the
dimer interfaces.
binding to either
1 or to
2 (since
does not monomerize, there is no entropy
component, and the free energy represents work). It is interesting to
note that
remains a dimer well below 50 nM (23), and
thus the free energy for dissociation to monomers is in excess of 10 kcal. These calculated free energies imply that the amount of work
required to open one interface (i.e. ~2.4
kcal) is far less than the free energy to open the second interface
(i.e. the full 10 kcal needed for
to monomerize). These
results imply that the
dimer is constructed in such a way as to
ensure preservation of a dimeric structure, even after one interface
has been pried open.
Complex Mechanism--
The high stability of the wild type
dimeric clamp explains the need for a clamp loader/unloader protein
during DNA replication. The
complex serves this function by binding
and opening the
clamp when it must be loaded onto primer-template
DNA or unloaded from a newly replicated duplex (25, 45). The
complex utilizes energy from ATP binding and hydrolysis to perform its
function. However, to our surprise, we found in earlier studies that
complex opens the clamp simply on binding ATP and that energy from
ATP hydrolysis is not necessary to crack the
dimer interface open (29, 32). The ATP binding energy is not utilized for opening the clamp
but rather to expose the
subunit in the
complex (28, 29), which
then opens one interface of the
dimer, prior to ATP hydrolysis. In
fact, free
protein appears to open the clamp almost as well as the
ATP-using
complex, as evidenced by the fairly similar rates at
which they catalyze unloading of
from DNA
(k
complex unloading = 0.015 s
1;
k
unloading = 0.0115 s
1) (24). These results are consistent with the above
conclusions, that the binding energy of the interaction between
and
is sufficient to open the clamp.
complex mechanism. Only the
,
, and
'
subunits are shown, since previous studies have demonstrated that the
and
subunits of the
complex are not essential for clamp
loader action (40). The stoichiometry of
in the
complex has
recently been demonstrated by crystal structure analysis of
'
to be three per complex,2 which is also consistent with the
conclusions of a recent biochemical study (27).
and
' are each
present in single copy (10, 13). The
' subunit is composed of three
domains organized in a C-shape (52). The crystal structure shows that
the
subunits are three domain proteins like
', consistent with
their known homology to
' (10, 33, 34). Although the
subunit
shares no recognizable homology to
and
', the crystal structure
of
has recently been solved, and it has a domain structure similar to that of
'.5 The
affinity of the
complex for
is quite reduced in comparison with
the affinity of
for
, indicating that
is sequestered when it
is in the
complex. But in the presence of ATP, the affinity of
complex for
is enhanced, suggesting that
becomes more available
to bind
. This is illustrated in Fig. 7, going from diagram A to B, as a conformational
change that increases the exposure of
for
. The illustration is
consistent with previous studies that indicate that ATP induces a
conformation change in the
complex (28, 32) and that
' competes
with
for
(29). These earlier observations indicate that
'
binding to
may partially occlude
in the
complex and that,
upon binding ATP,
may relieve this occlusion via a conformational
change. Hence, diagram B shows a separation
between
' and
, due to an ATP-induced conformation change in
.
View larger version (39K):
[in a new window]
Fig. 7.
Scheme of complex
action. The five subunits of
complex needed for clamp loading,
3
1
'1, are shown as related
C-shaped proteins. A, The area on
that binds
is in
contact with
' to indicate that it is blocked for
binding.
B, ATP binding to
subunits induces a conformational
change pulling
' from
, so that
can bind to
.
C, the
subunit contacts one protomer of
, and the
binding energy of this interaction is placed into wedging open one
interface of the
ring. D,
complex/
locate a
proper DNA structure for loading
, which triggers ATP hydrolysis,
leading to dissociation of the
complex and leaving
to close
around DNA.
with
(Fig. 7, diagram
C), the ring opens. This report demonstrates that
binds
only one protomer of the
dimer in performing this ring-opening
action. Further, as described above,
binds the
monomer tighter
than the
dimer, indicating that the binding energy between
and
one
protomer is used to perform work on the dimer, to open or
destabilize one interface. The remaining single interface of an open
dimer is stronger when
is in the open conformation, thereby
preventing decay to monomers.
ring is closed around DNA, are relatively
unknown. Presumably, primed template is recognized and positioned
within the open ring, at which time the ATP is hydrolyzed. Hydrolysis
is stimulated by
and primed template and is associated with
dissociation of
complex from DNA, leaving the
ring closed
around the duplex (29, 32, 37). At this time, we propose that the
energy of ATP hydrolysis is utilized to pull
off of
, allowing
to close. Particular roles of
complex subunits in DNA
recognition, orientation of DNA inside
, and ring closure await
further study.
, except that each monomer is composed of only two domains, and therefore PCNA trimerizes to form a six-domain ring (20, 21). PCNA, like
, is highly stable on DNA, exhibiting a
half-life of ~24 min for spontaneous dissociation from circular DNA
at 37 °C (23). The eukaryotic clamp loader, RFC, is composed of five
different subunits, but each are homologous to
/
' and thus are
probably shaped and arranged like the five subunits of the E. coli
3
' clamp loader (34, 53). Given these
striking similarities, it seems likely that the internal workings of
RFC and the mechanism by which it opens PCNA will be quite similar to
the E. coli
complex and
. Thus, one subunit of RFC
may contact one protomer of PCNA and through the energy of this
protein-protein interaction may force the ring open. Multiple RFC
subunits appear to bind PCNA, making it seem different from the
complex. However, we have recently determined that the
and
subunits of
complex bind
, albeit much weaker than
.3 Perhaps these other
and PCNA interactive subunits
function in positioning
on DNA, aid
in ring opening, or
function in the ring closure step. These and many other possible
functions for additional
interactive subunits must await future studies.
![]() |
ACKNOWLEDGEMENTS |
---|
We are grateful to Drs. David Jeruzalmi and
John Kuriyan for information on crystal structures in advance of
publication and for the diagram of the interface in Fig.
4A.
![]() |
FOOTNOTES |
---|
* This work was supported by National Institutes of Health Grant GM38839.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: Molecular Biology and Biochemistry Dept., Wesleyan University, Middletown, CT 06459.
Present address: Center for Advanced Research in
Biotechnology, 9600 Gedelesky Dr., Rockville, MD 20850.
To whom correspondence should be addressed: The Rockefeller
University and Howard Hughes Medical Inst., Laboratory of DNA Replication, 1230 York Ave., New York, NY 10021.
Published, JBC Papers in Press, March 6, 2001, DOI 10.1074/jbc.M100592200
2
The crystal structure of ' complex has
been solved (D. Jeruzalmi and J. Kuriyan, personal communication). The
stoichiometry is
3
1
'1, and
the five subunits form a pentameric ring.
3
Weak interaction between and
and between
and
can be detected by surface plasmon resonance (A. Yuzhakov
and M. O'Donnell, unpublished observations).
4 J. Stewart and M. O'Donnell, unpublished data.
5
The crystal structure of in complex with a
monomer of
has been solved (D. Jeruzalmi and J. Kuriyan, personal
communication).
has the folding pattern of
'.
![]() |
ABBREVIATIONS |
---|
The abbreviations used are:
Pol III, DNA
polymerase III;
PCNA, proliferating cell nuclear antigen;
ssDNA, single-stranded DNA;
SPR, surface plasmon resonance;
HA, hemagglutinin;
3H- and 32P-, 3H- and
32P-labeled
subunit, respectively;
ha
and
pk, subunit
with HA epitope
tag and protein kinase tag, respectively;
RFC, replication factor
C.
![]() |
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