From the ¶ Howard Hughes Medical Institute and the
Rockefeller University, New York, New York 10021
Received for publication, December 13, 2002, and in revised form, February 6, 2003
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ABSTRACT |
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The DNA polymerase III holoenzyme, the chromosomal replicase of
Escherichia coli, contains a clamp-loading machine within
its multi-component structure (reviewed in Ref. 1). The clamp loader couples ATP hydrolysis to the assembly of circular The The The structure of The subunits of The complex couples ATP hydrolysis to the
loading of
sliding clamps onto DNA for processive replication. The
complex structure shows that the clamp loader subunits are arranged
as a circular heteropentamer. The three
motor subunits bind ATP, the
wrench opens the
ring, and the
' stator modulates the
-
interaction. Neither
nor
' bind ATP. This report
demonstrates that the
' stator contributes a catalytic arginine for
hydrolysis of ATP bound to the adjacent
1 subunit.
Thus, the
' stator contributes to the motor function of the
trimer. Mutation of arginine 169 of
, which removes the catalytic
arginines from only the
2 and
3 ATP
sites, abolishes ATPase activity even though ATP site 1 is intact and
all three sites are filled. This result implies that hydrolysis of the
three ATP molecules occurs in a particular order, the reverse of ATP
binding, where ATP in site 1 is not hydrolyzed until ATP in sites 2 and/or 3 is hydrolyzed. Implications of these results to clamp loaders
of other systems are discussed.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
clamps onto primed DNA sites. The circular
clamp, formed from two
crescent-shaped protomers, binds to the DNA polymerase III core
(
), tethering it to template DNA for highly processive
synthesis. There are at least two molecules of DNA polymerase III core
within the holoenzyme architecture, held together by one clamp loader.
complex clamp loader consists of several different subunits,
i.e. three
(
) subunits and one each of
,
',
, and
(2-4). The
and
subunits play roles in the
primase-to-polymerase switching process (5), and they also interact
with SSB1 (6, 7) but are not
essential for clamp loading. The crystal structure of the minimal clamp
loader,
3
1
'1, shows that
the five subunits are arranged as a circular heteropentamer (4). In
order for this clamp loader to bind two molecules of DNA polymerase III
core, two of the
subunits are replaced by two
subunits.
and
are encoded by the same gene (dnaX);
is the
full-length product, and
is truncated by a translational frameshift
(8-10). The N-terminal 47 kDa of
contains the sequence of the
subunit, thus explaining how
and
can replace one another in
clamp loading action with
and
' (11). The extra 24 kDa of
C-terminal sequence unique to
is responsible for binding DNA
polymerase III core (12, 13); these sequences also bind the DnaB
helicase (14, 15). Thus, a single clamp loader cross links two DNA
polymerases and holds the hexameric helicase into the replisome
(reviewed in Ref. 6).
(
) subunits of the
complex constitute the motor of the
clamp loading machine, as they are the only subunits that hydrolyze
ATP; neither
nor
' bind or hydrolyze ATP (1). The
subunit
forms the main attachment to the
clamp and can open the
ring
single-handedly (16-18).
is sometimes referred to as the wrench or
crowbar of the clamp loader because it can open
on its own (19).
The
' subunit modulates the ability of
to bind
(16-18). In
the absence of ATP,
' obscures the
subunit within the
complex from binding to
(4, 16). However, with ATP bound to the
subunits,
is pulled away from
', allowing
to bind and open
the
ring (4, 20). In this state, with
and ATP bound to the
complex, a tight affinity for DNA is established (21-22). Upon
recognizing a primed site, the ATP is hydrolyzed, resulting in the
dissociation of the
complex from
, leaving
to close around
the DNA, whereupon it may associate with the polymerase component of
the holoenzyme.
also interacts with several other proteins besides the DNA
polymerase III core. These include ligase, MutS, UvrB, DNA polymerases I, II, IV and V, and possibly many other proteins involved in DNA
repair (23-26). These additional roles of
in other processes besides replication may account for the presence of the
complex in
E. coli that lacks
altogether, presumably freeing it for action at sites distinct from the replication fork.
3
' reveals that the ATP sites of
the
subunits are located at subunit interfaces (see Fig. 1) (4). Site 1 is at the
'/
1 interface, site 2 is at the
1/
2 interface, and site 3 is at the
2/
3 interface. The site 3
3 subunit is located directly next to the
wrench.
The numbering of these sites is thought to reflect the order in which
they become filled with ATP (4). The major pentameric contacts occur
via the C-terminal domains of the five subunits. The ATP binding sites
of
are located in the N-terminal domains. The N-terminal domain of
is also where the
interactive element is located (19), although
the proximity of
' to
blocks access of
to
(4). Combining several biochemical findings with the structure of
3
' suggests that, as the ATP sites fill,
conformation changes in
are propagated around the pentamer to pull
the
wrench away from the
' stator so that
can bind to
for clamp opening. The apparent rigidity of
', compared with
and
, which have a flexible joint for motion in clamp loading, has
earned
' the term "stator". ATP hydrolysis presumably reverses
the conformational changes in
and
induced by ATP binding to
, thus bringing the N-terminal domain of
back into proximity to
the
' stator. This effectively pushes
off of
, allowing the
ring to close around DNA.
3
' are members of the large AAA+
family (27). As their name implies (ATPases
associated with a variety of cellular
activities), these proteins are generally ATPases, and they
function in a wide diversity of cellular processes. The structure of
the homohexameric AAA+ proteins NSF and p97 (membrane fusion), RuvB
(branch migration), and HslU (proteosome) reveals an Arg residue that
reaches over the interface to the ATP site of the neighboring subunit
(28-31). This Arg is thought to be analogous to the "arginine
finger" of GAP, which plays a catalytic role in hydrolysis of GTP
bound to Ras by stabilizing the accumulating negative charge in the
transition state (32). It is proposed that the use of this Arg residue
in catalysis provides a means of intersubunit communication that
coordinates nucleotide hydrolysis around the ring.
complex has many similarities to the homohexameric AAA+
proteins but also has several important differences. The largest differences are its heterooligomeric composition, use of five subunits
instead of six, and the presence of two subunits that do not bind ATP.
Like the homohexamers, the
complex subunits are arranged in a ring,
and the
subunit ATP sites are located at interfaces where the Arg
of one subunit is in proximity to ATP modeled into the subunit adjacent
to it (Fig. 1). This Arg residue is
embedded in an SRC motif that is conserved in clamp-loading subunits of
T4 phage, eubacteria, archaea, and eukaryotes. The
' subunit also
contains an SRC motif, and the Arg residue is proximal to ATP site 1 of
1. In each case, the Arg needs to move a few angstroms
to be near enough to exert an influence on the bound ATP.
View larger version (48K):
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Fig. 1.
Architecture of complex ATP sites. A, orthogonal views of
the
ATP binding domain. Arg-169 (R169) is located far
away from the phosphate binding loop (P-loop) on the same polypeptide
chain, preventing formation of a functional intramolecular ATP site.
B, schematic of the circular pentamer of the
complex
looking down the center of the ring from the C terminus. Three ATP
sites are created at subunit interfaces. Each site is highlighted by
the P-loop of a
subunit and the sensor 1 Arg in the SRC motif from
the adjacent
' (ATP site 1) or
(sites 2 and 3) subunit.
C, ATP site 1, a magnified view of the
'/
1
N-terminal domain interface with an ATP molecule modeled against the
P-loop of
1 based on the NSF D2 crystal structure (31).
The
' sensor 1 Arg-158 (green residue) is in close
proximity to the
-phosphate of ATP, suggesting a catalytic function.
In ATP sites 2 and 3 the
1/
2 and
2/
3 interfaces, respectively, have a
similar architecture to those in ATP site 1. The
sensor 1 Arg-169
is positioned proximal to the ATP molecule modeled into the neighboring
. It should be noted that ATP modeled into site 2 clashes with some
residues of
1 and, thus, a conformation change is needed
to make this site accessible to ATP.
The contribution of these potential arginine fingers to ATP binding,
hydrolysis, and clamp loading is one subject of this report. The
results demonstrate that these SRC motif arginine residues in both and
' are not required for ATP binding; however, they are important
to catalysis. The finding that
' contributes a catalytic arginine
residue to an ATP site in the
trimer motor demonstrates that the
' stator also functions as a component of the motor of the clamp
loader. This conclusion has implications for analogies between the
complex and clamp loaders of other systems (see "Discussion").
Furthermore, the findings indicate that there is an ordered sequence to
hydrolysis. Mutation of the arginine in
removes the catalytic
arginine in sites 2 and 3 but not the arginine contributed by
' to
site 1. However, this mutation prevents hydrolysis of ATP in all three
sites. This result indicates there is a sequential order to hydrolysis
of the three ATP molecules, with ATP in sites 2 and/or 3 being
hydrolyzed before ATP in site 1.
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EXPERIMENTAL PROCEDURES |
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Materials--
Unlabeled deoxyribonucleoside triphosphates were
from Amersham Biosciences, and radioactive nucleoside triphosphates
were supplied by PerkinElmer Life Sciences. Proteins were
purified as described; ,
,
,
(33),
(34),
and
'
(35),
and
(36),
(37), and SSB (38). Core polymerase (37), and
complex (11) were reconstituted from pure subunits and purified
as described. Mutant subunits were purified by the same methods as
wild-type proteins. The
complex containing mutant subunits was
reconstituted and purified using the same procedure as wild-type
complex. Samples of purified complex were analyzed on a 14%
SDS-polyacrylamide gel stained with Coomassie Brilliant Blue G-250, and
each lane was scanned by laser densitometer (Amersham Biosciences).
PK is
containing a six-residue
C-terminal kinase recognition site (39) and was labeled to a specific
activity of 10 dpm/fmol with [
-32P]ATP using the
recombinant catalytic subunit of cAMP-dependent protein
kinase produced in E. coli (a gift from Dr. Susan Taylor, University of California at San Diego). The following oligonucleotides were synthesized and purified by Integrated DNA Technologies: 79-mer,
5'-GGG TAG CAT ATG CTT CCC GAA TTC ACT GGC CGT CGT TTT ACA ACG TCG TGA
CTG GGA AAA CCC TGG CGT TAC CCA ACT T-3'; and 45-mer, 5'-GGG TTT TCC
CAG TCA CGA CGT TGT AAA ACG ACG GCC AGT GAA TTC-3'. To form the
synthetic primed template, the oligonucleotides were mixed in 50 µl
of 5 mM Tris-HCl, 150 mM NaCl, 15 mM sodium citrate (final, pH 8.5), then incubated in a
95 °C water bath that was allowed to cool to room temperature over a
30-min interval. M13mp18 ssDNA was purified as described (40) and
primed with a 30-mer DNA oligonucleotide as described (33). Bio-gel
A-15m and P6 resins were purchased from Bio-Rad.
Buffers-- Buffer A is 20 mM Tris-HCl (pH 7.5), 0.5 mM EDTA, 2 mM DTT, and 10% glycerol (v/v). Gel filtration buffer is 20 mM Tris-HCl (pH 7.5), 0.5 mM EDTA, 2 mM DTT, 10 mM MgCl2, 100 µg/ml bovine serum albumin, and 4% glycerol (v/v). Reaction buffer is 20 mM Tris-HCl (pH 7.5), 0.1 mM EDTA, 5 mM DTT, 4% glycerol (v/v), and 40 µg/ml bovine serum albumin. ATPase Buffer is 20 mM Tris-HCl (pH 7.5), 0.1 mM EDTA, 5 mM DTT, and 10% glycerol (v/v).
Equilibrium Gel Filtration--
Analysis of ATP binding to
wild-type and mutant complexes was performed by equilibrium gel
filtration as described (41). Wild-type and either the
(R169A)
complex or the
' (R158A)
complex (8.3 µM
complex in 60 µl; 6.6 µM
(R169A) complex in 150 µl; 6.6 µM
' (R158A)
complex in 150 µl) were
incubated in gel filtration buffer plus 100 mM NaCl
containing [
-32P]ATP at the indicated concentration
(0.1-10 µM) for 15 min. at 25 °C. Samples were then
applied to a 5-ml Biogel P-6 column (Bio-Rad) at 25 °C
pre-equilibrated in gel filtration buffer plus 100 mM NaCl
having the same concentration of [
-32P]ATP as the
respective sample. Thirty-five fractions of 240 µl each were
collected, and 100 µl of each fraction was analyzed by liquid
scintillation to determine the total amount of ATP
([ATP]TOTAL, see below). 50 µl of the peak fractions
were also analyzed for total protein concentration by the Bradford
assay (Bio-Rad) using
complex as a standard. Scatchard analysis
from equilibrium gel filtration data was as described (42).
Gel Filtration Analysis of Complex·
Interaction--
The ability of the wild-type and mutant
complexes
to associate with
was analyzed by gel filtration on a fast protein
liquid chromatography (FPLC) Superose 12 column (Amersham Biosciences).
(30 µM, 480 µg) was incubated alone or with the
complex (25 µM, 1.25 mg) for 15 min. at 15 °C in 200 µl of Buffer A plus 100 mM NaCl containing 1 mM ATP and 10 mM MgCl2. The mixture
was then injected onto a 24-ml Superose 12 column equilibrated in the
same buffer at 4 °C. After collecting the first 5.8 ml (void
volume), fractions of 155 µl were collected and analyzed in a 14%
SDS-polyacrylamide gel.
ATPase Assays--
Wild-type and mutant complexes were
tested for ATPase activity in the presence of the synthetic primed
template with or without
. ATPase assays contained 50 nM
complex, 1 mM [
-32P]ATP, 200 nM
dimer (when present), and 500 nM
synthetic primed template DNA in a final volume of 60 µl of ATPase
buffer. The synthetic primer/template DNA is linear and thus allows
to slide off the ends after it is loaded. Thus,
is continuously
recycled during these assays as demonstrated previously (43). Reactions were brought to 37 °C and initiated upon the addition of the
complex. Aliquots of 5 µl each were removed at intervals (0-10 min)
and quenched with an equal volume of 0.5 M EDTA (pH 7.5). One microliter of each quenched aliquot was spotted on a
polyethyleneimine cellulose TLC sheet (EM Science) and developed in 0.6 M potassium phosphate buffer (pH 3.4). The TLC sheet was
dried and [
-32P]ATP and [
-32P]ADP
were quantitated using a PhosphorImager (Amersham Biosciences).
Clamp Loading--
Clamp loading was measured by separating
32P-PK on DNA from free
32P-
PK using BioGel A15m, a large pore resin
that excludes large DNA substrates but includes protein.
32P-
PK (13.3 nM as dimer) was
incubated for 10 min. at 37 °C either alone or with mutant or
wild-type
complex (10.7 nM) in 75 µl of reaction
buffer containing primed M13mp18 ssDNA (13.3 nM), SSB (3.2 µM as tetramer), 1 mM ATP, and 10 mM MgCl2. The reaction was applied to a 5-ml
BioGel A15m column (Bio-Rad) equilibrated in gel filtration buffer plus
50 mM NaCl at 25 °C. Thirty-five fractions of 180 µl
each were collected, and 100 µl was analyzed by liquid scintillation.
32P-
PK bound to the DNA elutes early
(fractions 11-15), whereas the free 32P-
PK
elutes later (fraction 17-28). The amount of
in each fraction was
determined from its known specific activity.
Replication Activity Assays--
The activity of wild-type and
mutant complexes was assayed by the requirement to load
onto a
primed circular M13mp18 ssDNA template in order to observe nucleotide
incorporation by the core polymerase (
subunits). The reaction
mixture contained core polymerase (5 nM),
(10 nM as dimer), SSB (420 nM tetramer), primed
M13mp18 ssDNA (1.1 nM), 60 µM each of dATP,
dCTP, and dGTP, 20 µM [
-32P]TTP, 1 mM ATP, and 10 mM MgCl2 in 25 µl
of reaction buffer (final volume). Replication was initiated upon the
addition of either the wild-type or the mutant
complex (0-1.28
nM titration) and incubated at 37 °C for 5 min.
Reactions were quenched upon the addition of 25 µl of 1% SDS and 40 mM EDTA. Quenched reactions were spotted onto DE81
(Whatman) filters and then washed and quantitated by liquid
scintillation as described (33).
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RESULTS |
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Reconstitution of (R169A) Complex--
Arginine 169 is located
in the highly conserved SRC motif of the
subunit. To assess the
importance of
arginine 169 to clamp-loading activity, we mutated it
to alanine and purified the
(R169A) protein from an overproducing
strain of E. coli. To study the effect of this mutation on
clamp loader activity, we reconstituted the
complex using
(R169A) with
,
',
, and
. Our previous studies have shown
that a fully assembled
complex is stable to ion exchange
chromatography on an fast protein liquid chromatography MonoQ column,
where it elutes much later than the free subunits (11). The
(R169A)
mutant was mixed with an excess of
,
',
, and
, incubated
for 30 min at 15 °C, and then applied to a MonoQ column followed
with a gradient of NaCl. The result, in Fig.
2A, demonstrates that the
(R169A) mutant forms a "
(R169A) complex" in which all five
subunits co-elute in fractions 41-49, whereas the excess subunits
elute much earlier. As demonstrated later in this report (Fig. 4), the
(R169A) complex also remains intact during analysis on a gel filtration column. The subunit ratio of the
(R169A) complex is
comparable with that of the wild-type
complex as observed in the
Coomassie Blue-stained SDS polyacrylamide gel of Fig. 2B. This ability of
(R169A) to assemble into a multisubunit complex with
,
',
, and
demonstrates that the
(R169A) mutant
is properly folded. It also provides reconstituted
(R169A) complex for the studies to follow.
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The (R169A) Complex Binds Three Molecules of ATP with Similar
Affinity as the Wild-type--
Next, we determined whether
(R169A)
complex binds ATP and, if so, whether it binds ATP with similar
stoichiometry and affinity compared with the wild-type
complex. To
address these issues, we used the equilibrium gel filtration technique.
In this analysis, a gel filtration column is equilibrated with a known
concentration of [32P]ATP. The
complex is incubated
with the same concentration of [32P]ATP present in the
column buffer and is then applied to the column. Fractions are
collected, and the amount of protein and [32P]ATP in each
fraction is determined. Protein-bound [32P]ATP is carried
around the beads, resulting in a peak of [32P]ATP that
elutes early and is followed later by a trough that has less
[32P]ATP than the column buffer due to its displacement
from the buffer by the protein. This information can be used to
calculate the Kd value for ATP binding to the
complex. However, a more accurate assessment of the
Kd value can be obtained by repeating the experiment
at a variety of ATP concentrations (the column is equilibrated at
different ATP concentrations) followed by plotting the data as a
Scatchard plot. This detailed analysis also carries the advantage of
providing the stoichiometry of ATP bound to the complex.
The results, in Fig. 3, show that (R169A) complex binds three molecules of ATP with similar affinity as
wild-type
complex (Kd, 1-2 µM).
The data for both the wild-type and the
(R169A) complex fall on a
relatively straight line, indicating that the three sites bind ATP with
similar affinity. This conclusion is also supported by a study of a
monomeric
subunit (missing the C-terminal oligomerization domain)
that binds ATP with a Kd value of 1.36 µM, determined by isothermal calorimetry (44). Overall,
these results indicate that the two ATP sites that carry alanine
residues in place of arginine 169 (sites 2 and 3) still bind ATP and
that this arginine residue contributes little, if any, to the binding
affinity of ATP to the
complex.
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(R169A) Complex Binds
--
Previous studies have
demonstrated that ATP binding to the
complex induces the
conformation change that leads to the binding of the
subunit (21).
This predicts that the
(R169A) complex, which binds ATP, should be
capable of binding to
. To test this prediction, we analyzed a
mixture of the
(R169A) complex and
for complex formation on a
Superose 12 sizing column equilibrated with buffer containing ATP.
alone migrates in fractions 37-43 (Fig.
4A). Analysis of a mixture of
wild-type
complex and
is shown in Fig. 4B; the
subunit co-elutes with the large
complex in fractions 22-31 and
resolves from unbound
, which elutes in the later fractions. A
similar analysis using
(R169A) complex, shown in Fig.
4C, demonstrates that the mutant
complex is also capable
of associating with
. The amount of
that comigrates with the
(R169A) complex is nearly the same compared with that of the wild-type
complex, indicating that it is capable of binding
, although its
affinity for
may be somewhat decreased by the mutation. ATP
binding, not hydrolysis, powers all the steps of clamp loading except
the final stage of dissociating from the
·DNA complex, allowing
to close around the DNA (21). This last step requires ATP
hydrolysis. Next, the mutant
complex was studied for its ability to
hydrolyze ATP.
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The (R169) Is Essential to Catalysis--
We next analyzed the
(R169A) complex in three different assays, each of which require
ATP hydrolysis. The first activity to be tested was
DNA-dependent ATPase activity of the
complex followed
by clamp loading and, finally,
-dependent DNA synthesis by core DNA polymerase.
The complex requires the presence of DNA for significant ATPase
activity (45). The
subunit stimulates
complex ATPase activity
provided a primed DNA, not ssDNA, is present (45). In the experiments
of Fig. 5A, we examined the
complex ATPase activity using primed DNA with and without
.
Whereas the wild-type
complex hydrolyzes ~217 molecules of ATP
per minute in the presence of the primed template, the
(R169A)
complex shows no detectable ATPase activity (i.e. detection
limit is ~5 ATP hydrolyzed/min/
complex). The
subunit
stimulates the ATPase activity of the wild-type
complex in the
presence of primed DNA as illustrated in Fig. 5A. However,
does not provide detectable ATPase activity by the
(R169A)
complex in the presence of the primed template (Fig. 5A),
even when a very large excess of
is present (2 µM; data not shown).
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The absence of catalytic activity predicts that the mutant complex
will be incapable of loading a
clamp onto primed DNA. To assay
clamp-loading activity directly, we radiolabeled
with
32P using a derivative of
that carries an N-terminal
tag with a kinase site. The 32P-
was incubated with the
complex and primed M13mp18 ssDNA (SSB coated), and then the
reaction was analyzed for assembly of 32P-
onto the
primed DNA by gel filtration on a BioGel A15m column. This resin has
large pores that include proteins but exclude the large M13mp18 primed
ssDNA (see agarose gel analysis). As a result, 32P-
bound to DNA elutes early and resolves from free 32P-
that is not bound to DNA. A control reaction using the wild-type
complex is shown in Fig. 5B. Most of the 32P-
elutes with the DNA in fractions 11-16. An agarose gel analysis, shown
in Fig. 5B, confirms that the large SSB-coated DNA substrate alone elutes in these same fractions (11-16). However, repetition of
this analysis using mutant
complex did not result in the assembly
of 32P-
on the DNA, and instead the 32P-
eluted later in fractions 17-33. A control reaction lacking the DNA
template confirmed that free 32P-
elutes in fractions
17-33 (Fig. 5B). Analysis of
complex migration shows
that it elutes in the included fractions in the same position as free
, consistent with the large pore size of the A15m resin (not shown,
but as in Fig. 5 of Ref. 16). Hence, the
(R169A) complex is
inactive for clamp loading action, consistent with its lack of ATPase activity.
Finally, the (R169A) complex and
were tested for the ability to
support DNA synthesis by core polymerase. The core Pol III is incapable
of extending a primer around an SSB-coated primed M13mp18 ssDNA
template unless it is coupled to a
clamp and thus provides another
measure of clamp-loading activity. Use of the wild-type
complex
yielded a strong signal in this assay but, as expected, when the mutant
complex was used in this assay no activity was detected, which is
consistent with the inactivity of mutant
complex in ATPase and
clamp-loading assays (Fig. 5C). These results support and
extend those of an earlier analysis in which several conserved residues
of
were mutated (46). Mutation of
R169 resulted in the loss of
replication and ATPase activity and failed to complement a conditional
lethal dnaX gene in vivo, although that study did
not demonstrate that the mutant
was properly folded or that it
retained the ability to form a stable complex with
and
', bind
ATP, and associate with
.
The ' SRC Motif--
The above results strongly support an
essential role of the
arginine 169 in catalysis. However, the
results imply something further. Mutation of
arginine 169 only
eliminates this catalytic residue at ATP sites 2 and 3. The putative
catalytic arginine of ATP Site 1 is supplied by
', not
, and
therefore this site should remain competent for ATP hydrolysis even in
the
(R169A) complex. Even though the
(R169A) complex retains
one intact ATP site, the results show that it has lost essentially all
of its ATPase activity. This implies that the ATP bound to sites 2 and/or 3 must first be hydrolyzed before the ATP in site 1 is hydrolyzed. Alternatively, site 1 binds ATP but is simply not catalytic. A non-catalytic site 1 would explain why the
(R169A) complex has no ATPase but would not explain why the SRC motif is
broadly conserved in prokaryotic
' subunits. To test whether arginine 158 in the SRC motif of
' is important to catalysis, this
arginine was mutated to alanine, and the
' (R158A) mutant was
purified and reconstituted into the
complex for analysis. If ATP
site 1 is not hydrolytic, the
' (R158A)
complex should have
wild-type levels of ATPase and clamp loading activity.
The ' (R158A) mutant was mixed with
,
,
, and
to
reconstitute the "
' (R158A)
complex." The complex was stable
to ion exchange chromatography, yielding a purified reconstituted
'
(R158A)
complex with a similar subunit stoichiometry as that of the
wild-type
complex (Fig.
6A). ATP binding analysis by equilibrium gel filtration demonstrated that the
' (R158A)
complex retained ability to bind three ATP molecules with similar affinity as the wild-type
complex (Fig. 6B;
Kd, ~0.8 µM). The
' (R158A)
complex also remained capable of forming a complex with
in the
presence of ATP (Fig. 6C). In contrast to the
(R169A)
complex, the
' (R158A)
complex retained some activity in the
catalytic assays, requiring that ATP be hydrolyzed. The
' (R158A)
complex retained ~30% of the DNA-dependent ATPase activity of the wild-type
complex, although
no longer
stimulated the ATPase as it did in the wild-type
complex (Fig.
6D). Also, the DNA synthetic activity of the
' (R158A)
complex was about 20-30% active compared with that of the
wild-type
complex (Fig. 6E). Hence, the
' (R158A)
complex retains some clamp-loading activity, but is nevertheless
significantly compromised compared with the wild-type
complex.
|
The above results demonstrate that ATP site 1 is catalytic, because the
' (R158A)
complex is significantly less active than the
wild-type
complex. If site 1 was only used for ATP binding, the
' (R158A)
complex would have been expected to be fully active in
the catalytic assays. Hence arginine 158 of
' is important to the
catalytic activity of the
complex, consistent with conservation of
the SRC motif among prokaryotic
' subunits.
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DISCUSSION |
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The ' Stator Contributes a Catalytic Arginine to the Clamp
Loader Motor--
The
complex clamp loader has been proposed to
consist of three main components (4, 6), i.e. the
wrench
(opens
), the
trimer motor (hydrolyzes ATP), and the
' stator
(modulates
interaction with
) (reviewed in Ref. 47). The
and
subunits appear to have a flexible joint between the C-terminal
domain (domain III) and the N-terminal domains (domains I/II). In
contrast, the three domains of the
' stator appear to be held in a
rigid conformation and, thus, the term stator, the stationary part of a
machine upon which the other parts move (4, 44). The C-terminal domains
of all five subunits form a tight closed circular connection, holding
the subunits together. However, the N-terminal domains have an
interruption between
and
'. The size of this gap modulates the
ability of
to interact with the
wrench. The rigid
' stator is proposed to function as an anvil, and when ATP is hydrolyzed the
subunits move
close to
', forcing the
ring off the
wrench and allowing the
ring to close.
This report demonstrates that ', besides its role as stator, also
plays an instrumental role in the motor function of the
complex by
supplying a catalytic arginine into ATP site 1 of the
complex. A
catalytic role for this arginine was suggested by its proximity to ATP
modeled into ATP site 1 of the
complex structure (4). Furthermore,
this arginine is embedded in an "SRC" motif that is highly
conserved among clamp-loading subunits of prokaryotes, eukaryotes, and
archaebacteria. The E. coli
' subunit is a member of the
AAA+ family and has the same chain fold as
, yet
' does not bind
ATP (48). The P-loop of
' has been modified through evolution, and
the N terminus blocks the nucleotide binding site. However, there are
examples of prokaryotic
' subunits that contain a consensus P-loop
(i.e. Aquifex aeolicus; Ref. 49). Whether these
' subunits bind ATP is not known. However, even if these
'
subunits bind ATP they may not hydrolyze it for lack of a catalytic
arginine in the neighboring
subunit. Perhaps noncatalytic ATP
provides rigidity to these
' subunits without needing the extra
connections between domains III and I/II observed in the E. coli
' stator.
The catalytic role played by ' in clamp-loading ATPase action may
explain why the
' sequence is highly conserved in prokaryotes compared with the sequence of
. The
subunit is the main subunit responsible for opening the
clamp, but it has no catalytic role (16, 19). Simple maintenance of protein-protein contacts, with no
catalytic role to preserve, has apparently allowed the
sequence to
drift considerably. The catalytic role played by
' may be
responsible for the much greater conservation of the
' sequence.
An Ordered Hydrolysis Model for Clamp Loading--
This report
demonstrates that the mutation of arginine 169 in , which removes
the arginine of the SRC motif near ATP sites 2 and 3, abolishes ATPase
activity even though three ATP molecules still bind the mutant
complex and the ATP site 1 remains intact. This result suggests that
Site 1 cannot hydrolyze ATP until after the ATP in sites 2 and/or 3 is
hydrolyzed. Conversely, if ATP must be hydrolyzed in sites 2 and/or 3 before the ATP is hydrolyzed in site 1, then an ATP site 1 mutant may
not block the hydrolysis of ATP in sites 2 and 3. Indeed, this
expectation is largely upheld in this study. The
' (R158A)
complex retains significant ATPase activity, indicating that ATP in
sites 2 and/or 3 can be hydrolyzed even when site 1 is missing the
catalytic arginine. The
' (R158A)
complex also retains some
clamp-loading activity, allowing it to support processive DNA
synthesis. Thus, the hydrolysis of ATP in sites 2 and 3 would appear to
be sufficient, although not optimal, for clamp loading.
Ordered hydrolysis of ATP starting at sites 3 (or 2) and ending with
site 1 is the opposite order that is predicted for ATP binding. Study
of the 3
' structure (4) suggested that the sites
may fill starting at site 1 and ending with site 3. A model encompassing an ordered binding and a reverse order of hydrolysis, as
the current study suggests, is illustrated in Fig.
7. As ATP binds to sites 1, 2, and then
3, the gap between the
and
' subunits is proposed to increase to
accommodate interaction of
with the
dimer. The
subunit
wrench then cracks one interface of the
ring, allowing the spring
tension between the domains of the
ring to relax, thereby opening
the ring for DNA strand passage. Upon association of a primed template
through the open
ring, the arginines of the SRC motifs in
and
' align for ATP hydrolysis. The current study indicates that ATP
hydrolysis occurs first at site 3 (or 2) before hydrolysis at site 1. This order may be achieved via proper positioning of the catalytic Arg
residues. Specifically, hydrolysis at site 3 may be required in order
for the arginine in site 2 to become properly aligned, and hydrolysis
in site 2 may be required for the arginine in site 1 to be aligned. The
threading of a primed site through
may align the arginine in site 3 to start the hydrolysis cycle (discussed further, below).
|
The proposed order of ATP hydrolysis in the model proceeds first from
site 3, then 2, and finally to site 1. However, it is also possible
that site 2 fires first. We would like to construct different complexes containing all the permutations of single and double ATP site
mutants but are prevented from this strategy by the fact that sites 2 and 3 are formed solely by identical
subunits.
Generalization of These Results to Other Clamp Loaders--
The
studies of this report on the E. coli clamp loader have
implications for subunit function in clamp loaders of other organisms. The eukaryotic RFC heteropentamer is composed of five different subunits, three of which contain both the SRC motif and consensus P-loops, suggesting that they may act similarly to the trimer motor
(yeast RFC 2·3·4 and human p36·37·40) (50). Indeed, these complexes contain DNA-dependent ATPase activity (51, 52). One RFC subunit (yRFC5 and human p38) contains the SRC motif but lacks
a consensus P-loop and thus is analogous to the
' stator and may
contribute a catalytic arginine for ATP hydrolysis in the RFC2, 3, and
4 motors. The yRFC1 subunit (human p128) forms a strong attachment to
the PCNA clamp (53) and thus may be analogous to the
wrench. Like
E. coli
, yRFC1 lacks an SRC motif, but unlike
it
contains a consensus P-loop, suggesting that it binds ATP. Mutation of
this P-loop is without significant effect on yRFC activity, indicating
that this fourth ATP site in the eukaryotic clamp loader may be coupled
to some other process (54).
Studies of P-loop mutants of yRFC2, 3, and 4 show that mutation of
either RFC 2 or 3 greatly reduces ATPase activity and clamp-loading function of RFC, whereas mutation of RFC4 has much less of an effect on
these activities (50). These results are similar to the current study
of the complex; namely, the
' (R158A)
complex is partially
active, whereas the
(R169A) complex is completely inactive. Thus,
it seems quite possible that the RFC clamp loader may also hydrolyze
ATP in an ordered sequence around the circular pentamer, as proposed
here for the
complex, wherein some sites must first hydrolyze ATP
before ATP in other sites can be hydrolyzed.
The five subunit clamp loader of bacteriophage T4 has two different
subunits, four copies of gp44 and one gp62 subunit. Biochemical studies
demonstrate that ATP is hydrolyzed to load the T4 gp45 clamp onto DNA;
stoichiometry measurements range from 1-4 ATP per gp45 clamp-loading
event (55-58). The gp44 tetramer is an ATPase, contains both the SRC
motif and P-loop, and is homologous to E. coli and
'.
The gp62 is similar to
in that it has neither the SRC motif nor
P-loop, and its sequence has diverged from
/
'. At first glance it
would seem that the T4 clamp loader has done away with the stator and,
indeed, it may have. However, keeping in mind that gp62 has no SRC
motif, it seems likely that one gp44 subunit (i.e. adjacent
to gp62) will be incapable of hydrolyzing ATP even if it binds ATP.
Thus, one gp44 subunit may serve a similar role as
' does in
modulating contact between the gp44/62 clamp loader and the gp45 clamp
at the same time as it provides a catalytic arginine residue for ATP
hydrolysis in the neighboring gp44 subunit.
The clamp loader of archaebacteria has been studied, but less
intensively than the clamp loaders of E. coli, T4, yeast,
and humans. Generally, archaebacterial clamp loaders (called RFC) consist of two subunits, RFC large and RFC small (59). The
stoichiometry of these subunits is not certain, with reports ranging
from 1:4, like T4, to 3:2 and even 4:2 (60-62). The crystal structure
of the Pyrococcus RFC small subunit shows that its basic
unit is a trimer, presumably the equivalent to the trimer motor in
which each subunit contains both a nucleotide binding site and a
closely juxtaposed arginine from the neighboring subunit (63). The
Pyrococcus RFC large subunit contains a consensus P-loop
motif and thus may bind nucleotide, but it lacks the SRC motif. The
lack of a SRC motif in the RFC large subunit suggests that if there are
two of these subunits in the clamp loader, then one of these will be
unable to hydrolyze ATP and thus may function in an analogous fashion
as the
' stator. Alternatively, if only one RFC large subunit is
present, an adjacent RFC small subunit will not hydrolyze bound ATP,
thereby acting as a stator. In this second scenario, the stator would
also contain an SRC motif, like
'.
Possible Role of the SRC Arginines in Clamp-loading
Fidelity--
The complex is a very poor ATPase without a DNA
effector. The crystal structure indicates that
R169 and
' R158
are not quite close enough to function with ATP, and they must move an extra one or two angstroms to have an effect on ATP hydrolysis (4).
Hence, it is tempting to speculate that misalignment of these arginines
may underlie the very weak ATPase of the
complex, and their proper
positioning may be used as a regulatory mechanism.
DNA stimulates the complex ATPase activity and, thus, may bring the
SRC motif Arg/ATP site pairs into a more favorable alignment for
hydrolysis. Curiously, the
subunit only stimulates the
complex
ATPase when a primed template is used as an activator; ssDNA and duplex
DNA stimulate the
complex in the absence of
but do not give
more activity when
is added (11). Consistent with this observation,
the
complex does not load
onto ssDNA, even though ssDNA
stimulates ATPase activity (64). Furthermore, the ATPase cycle is
tightly coupled to clamp loading, as only 2-3 ATPs are hydrolyzed for
each
clamp that is loaded onto a primed template (16, 21). Finally,
the head-to-tail architecture of the
dimer generates two distinct
"front" and "back" faces, only one of which functions with the
DNA polymerase and, thus, it must be oriented correctly on DNA to
interface with the polymerase (34, 39). What system of checks and
balances does the
complex have to ensure that these criteria have
been met? It seems possible that the catalytic arginines and their
juxtaposition to the ATP sites may act as a fidelity mechanism to
ensure that
is only loaded when primed DNA is threaded through
and only when
is oriented correctly for function with polymerase.
Perhaps when these different criteria are met the catalytic SRC motif
arginine residues are brought into register for ATP hydrolysis
to propel loading of the
clamp onto DNA.
![]() |
FOOTNOTES |
---|
* 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.
§ To whom correspondence should be addressed: The Rockefeller University, 1230 York Ave., Box 228, New York, NY 10021-6399. Tel.: 212-327-7255; Fax: 212-327-7253; E-mail: johnsoa@mail.rockefeller.edu.
Published, JBC Papers in Press, February 10, 2003, DOI 10.1074/jbc.M212708200
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ABBREVIATIONS |
---|
The abbreviations used are: SSB, single-stranded DNA-binding protein; AAA+, ATPases associated with a variety of cellular activities; PK, protein kinase; DDT, dithiothreitol; ssDNA, single-stranded DNA; RFC, replication factor C; P-loop, phosphate binding loop.
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