High-speed replication of chromosomal DNA requires
the DNA polymerase to be attached to a sliding clamp
(known as proliferating cell nuclear antigen, or PCNA, in
eukaryotes) that prevents the polymerase from falling off
DNA [1,2]. In all cells and in some viruses, the clamp is a
ring-shaped protein complex that encircles DNA, forming
a sliding platform on which DNA polymerases and other
proteins that move along DNA are assembled. Sliding
clamps play a part in DNA replication, DNA repair, cell
cycle control and modification of chromatin structure
[3,4], and defects in several clamp-associated factors are
associated with cancer and other disorders caused by
abnormalities in DNA replication and repair [5].
Because sliding clamps encircle DNA but do not
interact tightly with it, they can slide along the double
helix by diffusion [6-9]. Sliding clamps from different
branches of life have different subunit stoichiometry
(they are dimers in bacteria [10] and trimers in eukarya,
archaea and bacteriophage [11-15]) and their sequences
have diverged beyond recognition. Nevertheless, their
structures are remarkably similar. The conserved
structure is an elegant symmetrical elaboration of a
simple b-a-b motif, repeated 12 times around a circle
[10,14]. The circular geometry is broken when the clamp
is opened for loading onto DNA, but the elegance is
retained during the loading step as the clamp assumes a
helical symmetry that reflects the helical symmetry of
DNA (see below).
The increase in the processivity and speed of DNA
synthesis when DNA polymerases are engaged to sliding
clamps is very considerable. For example, in the absence
of the clamp, the polymerase subunit of the bacterial
replicase synthesizes DNA at a rate of about 10 base pairs
per second [16] and is hardly processive. In contrast, the
same polymerase subunit synthesizes 500 to 1,000
nucleotides per second when bound to the sliding clamp
[17-19]. To consider a startling analogy based on scaling
linear dimensions, if the bacterial replicase were a car, it
would travel only about 5 to 10 miles per hour without
the clamp and faster than the speed of sound with the
clamp. The bacterial replicase has a processivity of about
10 base pairs in the absence of the clamp [20], but has an
average processivity of approximately 80 kilobases when
bound to the sliding clamp in the replisome [21]. To
invoke another analogy based on scaling dimensions, if
the polymerase were a tightrope walker, without the aid
of the clamp only about 20 feet of the tightrope would be
traversed before the polymerase ‘walker’ fell off. The
clamp allows the polymerase to hold on to the DNA
‘rope’ without letting go, and now it would ‘walk’ almost
30 miles before falling off.
The enhancement in speed and processivity that the
clamp confers on the polymerase would not be possible
without the clamp loader, the less glamorous but much
more hardworking handmaiden of the sliding clamp,
which diligently loads the clamps onto primed DNA
Abstract
Clamp loaders are pentameric ATPases of the AAA+
family that operate to ensure processive DNA
replication. They do so by loading onto DNA the ringshaped
sliding clamps that tether the polymerase
to the DNA. Structural and biochemical analysis of
clamp loaders has shown how, despite differences in
composition across different branches of life, all clamp
loaders undergo the same concerted conformational
transformations, which generate a binding surface
for the open clamp and an internal spiral chamber
into which the DNA at the replication fork can slide,
triggering ATP hydrolysis, release of the clamp loader,
and closure of the clamp round the DNA. We review
here the current understanding of the clamp loader
mechanism and discuss the implications of the
differences between clamp loaders from the different
branches of life.
Clamp loader ATPases and the evolution of DNA
replication machinery
Brian A Kelch1,3,
*, Debora L Makino1,3,7
, Mike O’Donnell6
, John Kuriyan1,2,3,4,5
REVIEW	 Open Access
* Correspondence: bkelch@berkeley.edu
1
Department of Molecular and Cell Biology, University of California, Berkeley,
CA 94720, USA
Full list of author information is available at the end of the article
© 2012 Kelch et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons
Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any
medium, provided the original work is properly cited.
Kelch et al. BMC Biology 2012, 10:34
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throughout the process of DNA replication. Together, the
clamp and the clamp loader lie at the heart of the
replisome – the DNA replication machinery, which, with
the polymerases (leading and lagging strand), includes
the helicases that unwind the double-stranded DNA
ahead of the polymerase at the replication fork, the
primase that synthesizes the RNA primer required for
the initiation of DNA synthesis, and the single-strand
DNA-binding proteins that prevent the DNA from reannealing
in the wake of the helicases (Figure 1). The
clamp loader opens sliding clamps and places them on
the DNA at the site of the primer-template junction in
the correct orientation for polymerase to bind, both at
the initiation of DNA synthesis on the leading strand and
continually at the start of each Okazaki fragment on the
lagging strand. Thus, the clamp loader is critical for the
tight coupling of leading and lagging strand synthesis.
Indeed, in bacteria the clamp loader acts physically to
hold the leading and lagging strand polymerases together
[22-25] so that the two polymerases progress in tandem,
with the lagging strand wrapped around the replisome in
trombone fashion [26]. How leading and lagging strand
polymerases are coupled in eukaryotic DNA synthesis is
not known, and this is one of the major open questions
about the operation of the eukaryotic replisome.
Despite the uncertainty in the precise architectural role
of the clamp loader in the eukaryotic replisome, it is clear
that sliding clamps are centrally important. The sliding
clamp recruits the polymerase as well as other factors to
the replication fork, including the chromatin-modifying
proteins required to reassemble chromatin on the newly
synthesized DNA [27,28].
The clamp loader is a molecular switch operated by
binding and hydrolysis of ATP
Clamp loaders are members of the AAA+ (ATPases
associated with various cellular activities) family of
ATPases [29] and derive from the same evolutionary root
as helicases and other motors that work on DNA, many
of which are also AAA+ ATPases. The role of AAA+
proteins is not restricted to DNA-dependent processes,
and there is hardly an aspect of cellular function that
does not have an AAA+ machine playing an important
role. In architecture and mechanism, the AAA+ ATPases
are related distantly to the F1
-ATPase [30], and as with
that energy-transducing machine, evolution has built
AAA+ systems into the master plan of life. A
comprehensive review of AAA+ ATPases is provided by
Berger and Erzberger [31], to which the reader is referred
for a thorough discussion of these ideas.
Initially thought to be a motor [32-34], the clamp
loader is now better thought of as a timing device or
molecular switch [35], related conceptually and in
molecular mechanism to small GTPases such as Ras [36].
The clamp loader must be bound to ATP in order to bind
and open the clamp [37,38] and to bind primer-template
DNA [39-41] (Figure 2). ATP hydrolysis is, however, not
necessary for clamp opening, which is thought to depend
simply on the affinity of the ATP-bound clamp loader for
the open conformation of the clamp: in the ADP or
empty state, the clamp loader has low affinity for the
clamp [42,43]. The ATPase activity of the clamp loader is
stimulated by binding both to the clamp and to DNA
[39,40], and upon ATP hydrolysis the affinity of the clamp
loader for both clamp and DNA is greatly diminished,
leading to ejection of the clamp from the clamp loader.
This complex but fundamental mechanism is embodied
in an assembly of surprisingly diverse composition from
bacteria to eukaryotes.
The structure of the clamp loader is more
conserved than its composition
Unlike other AAA+ ATPases, which are typically
hexameric, all clamp loaders are composed of five
subunits in a circular arrangement, with a gap between
the first and the fifth subunit, at the position of the
missing sixth subunit. The individual subunits are
designated A through E, starting with the subunit at the
open interface and proceeding counter-clockwise around
the clamp loader in the standard view (Figure 3). Each
subunit has three domains. The amino-terminal domain
Figure 1. Architecture of the bacterial replication fork. The
helicase is a homohexamer that encircles the lagging strand and
binds directly to the primase synthesizing the primer RNA. The clamp
loader acts to hold the replisome together by binding directly to
the helicase as well as three polymerase subunits for simultaneous
synthesis of the leading and lagging strands. The leading strand
polymerase synthesizes DNA continuously, while the other two
polymerases presumably cycle on and off the lagging strand, which
is coated in single-strand binding protein (SSB). The polymerase
subunits are attached to circular clamps that encircle duplex DNA for
enhanced processivity and speed.
polymerase
clamp
loader
sliding
clamp
helicase
SSB
RNA primer
primase
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and its adjacent domain assume the fold of AAA+
proteins (with one exception to be explained later); the
first structure to be determined for an AAA+ fold was
one of the five Escherichia coli subunits in isolation [44].
The subunits are held in a ring by their carboxy-terminal
domains, which together form a tight pentameric collar.
The clamp loader shares an essential aspect of its
mechanism with other AAA+ complexes and other
oligomeric ATPases, such as the F1
-ATPase [30]: the
binding of ATP brings together the inter-subunit
interfaces, most notably the arginine finger residues that
are essential for hydrolysis of ATP [45,46] (so called by
analogy with the catalytic residue from the activators of
small GTPases [47]). This conformational rearrangement
also results in a spiral organization of the five
amino-terminal regions of the clamp loader. The coupling
of the suicidal binding of ATP to large-scale
conformational change drives alterations in molecular
organization that are necessary to hold the sliding clamp
open and position it on the DNA.
Because clamp loaders are so fundamental to the
replication process, it is no surprise that their structure
and mechanism turn out to be highly conserved in all
branches of life. This conservation has been somewhat
difficult to appreciate, because the extensive biochemical
analyses of bacterial, eukaryotic and bacteriophage
clamps and clamp loaders have to a great extent
proceeded independently in the past, and scientists
working on these systems have used different conventions
to identify the subunits. Adding to the confusion is the
Figure 2. Clamp loaders place sliding clamps at primer-template junctions for processive DNA replication. When bound to ATP, clamp
loaders are competent to bind and open the sliding clamp protein. This ternary complex can now bind to a primer-template junction, which
activates the ATPase activity of the clamp loader. ATP hydrolysis causes the clamp loader to dissociate from the clamp and DNA, resulting in a
loaded clamp that is competent for acting as a processivity factor for DNA polymerase. Figure adapted from [60].
clamp loader
I
II
III
5 2
1
43
6
ADP
ATP ATP
ADP
primer-template
junction
5
3
3
5
clamp
DNA polymerase
3
5
3
5
Figure 3. Composition of clamp loaders from the different branches of life. (a) Bacterial clamp loaders consist of three different proteins: δ, δ′,
and the τ or γ protein (γ, a truncation of the τ protein, is shown here). The δ protein is at the A position, with three copies of the ATPase subunits τ
or γ at the B, C and D positions. The δ′ protein sits at the E position. (b) Eukaryotic clamp loaders consist of five distinct proteins, RFC1 through RFC5.
RFC1 lies at the A position, RFC4 at B, RFC3 at C, RFC2 at D and RFC5 at the E position. Eukaryotic A subunits contain an additional domain that
bridges the gap between the A and E positions. (c) Bacteriophage clamp loaders consist of two distinct proteins. The gp62 protein, which lacks a
AAA+ module but has an A′ domain similar to that of eukaryotic clamp loaders, lies at the A position. The ATPase gp44 protein lies at the B, C, D, and
E positions. Archaeal clamp loaders have a similar composition (see text for details). Figure adapted from [60].
bacterial eukaryotic T4 bacteriophage
collar
AAA+
module
domain II
domain I
E D
C
B
A
E
D
C
B
A
E
D
C B
AA
RFC1
RFC5
A
ATP
ATP
gp44
gp62
ATP
gp44
c)a) b)
RFC2
RFC3
RFC4
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fact that, even though all clamp loaders have five essential
subunits, the protein stoichiometry is different in clamp
loaders from different branches of life. This confusion
can be alleviated by using the simple A through E scheme
for identifying the clamp loader subunits. Figure 3 is a
schematic illustration of the bacterial, eukaryotic and
bacteriophage clamp loaders showing the relationship of
the different proteins they are composed of to the
conserved subunit organization; and to help avoid
confusion as we discuss the clamp loader mechanism on
the basis of studies in different organisms, we describe
below the three major variants.
The bacterial clamp loader is formed from three
essential subunits: δ, δ′, and the τ or γ protein (Figures 3a
and 4a). The δ and δ′ proteins, which have no ATPase
activity, are present in one copy each in the clamp loader,
at the A and E positions, respectively. The B, C and D
positions in the assembly are composed of either τ or (in
E. coli) γ ATPase proteins. The γ protein is a truncated
version of the τ protein that lacks the elements necessary
for binding to the helicase or the polymerase. There is
evidence that most replisomes in vivo contain the τ
protein at the B, C and D positions, so that the replisome
has three polymerase subunits bound [48,49] (Figure 1).
Bacterial clamp loaders often have two accessory
subunits (χ and ψ) that are not members of the AAA+
family [50] and are not necessary for the clamp loading
process but couple the clamp loader to single-strand
DNA-binding protein [51-53]. Binding of the ψ protein
also induces a conformational change in the clamp loader
that increases its affinity for DNA [54,55].
The eukaryotic clamp loader, Replication Factor C or
RFC [56-58], is composed of five unique proteins, RFC1
through RFC5 (Figures 3b and 4b). The largest subunit,
RFC1, is at the A position and contains an active ATPase
site as well as an extra domain (called the A′ domain)
Figure 4. Structures of clamp loaders from different branches of life and in different bound states. The five subunits of the clamp loader – A, B, C, D
and E – are shown in different colors. (a) The structure of the clamp loader of E. coli (known as the γ-complex) in the apo form (PDB code 1JR3) [114]. This
structure illustrates the three conserved domains of clamp loader subunits. The two amino-terminal domains constitute the AAA+ module. The carboxyterminal
domains form a disc-like structure that holds the complex together as a tight pentamer. (b) Structure of the budding yeast clamp loader, replication
factor-C (RFC), bound to the sliding clamp, PCNA, and an ATP analog (PDB code 1SXJ) [59]. ATP induces a spiral arrangement of the AAA+ modules. The
clamp is not open in this structure, probably because of mutations in key interfacial residues (the arginine fingers) that disrupt the tight interactions between
adjacent AAA+ modules and prevent hydrolysis of the ATP analog. (c) Structure of the T4 bacteriophage clamp loader bound to primer-template DNA, an
open sliding clamp and ATP analog (PDB code 3U60) [60]. The duplex region of the primer-template junction is bound within the central chambers of the
clamp loader and the sliding clamp, with the 5′ single-stranded template extruded through the gap in the clamp loader where the missing sixth subunit
would be. The AAA+ modules of the clamp loader, bridged by the ATP analog, form a spiral that perfectly matches the helical symmetry of DNA. To see the
structures in upper panels a and b rotate, and a movie showing closure of the clamp in upper panel c, click on the individual images. The movies are also
available as addtional files 1,2 and 3. (Adobe Reader Version 8 or higher required).
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carboxy-terminal to the collar and that interacts with the
AAA+ module of the E subunit [59], thus bridging the
gap left by the missing sixth subunit. The other four
positions in the clamp loader are occupied by similarly
sized proteins: RFC4 at B, RFC3 at C, RFC2 at D, and the
ATPase-incompetent RFC5 protein at the E position.
The bacteriophage and archaeal clamp loaders are both
composed of two proteins each with one unique protein
occupying the A position (gp62 in T4 bacteriophage and
RFC-l in archaea) and identical ATPase subunits at the B,
C, D and E positions (the gp44 protein in T4
bacteriophage and RFC-s in archaea) [11,60-62] (Figures
3b and 4c). While the archaeal clamp loader contains an
active ATPase at the A position [63], the A subunit in the
T4 bacteriophage clamp loader does not have a AAA+
fold [60]. Like the eukaryotic clamp loader, the A subunit
of the T4 and archaeal clamp loaders contains an A′
domain [60,62].
The bacteriophage clamp loader, whose structure
seems to reflect a rather curious evolutionary history (we
return to this briefly later), has played a particularly
important part in the elucidation of the clamp loader
mechanism. A recently determined structure of the T4
bacteriophage clamp loader bound to an ATP analog and
in complex with the sliding clamp and primer template
DNA [60] revealed a state of the system that we have long
sought to visualize: an open clamp encircling DNA while
in complex with an ATP-bound clamp loader. Another
structure shows what happens when the loader
hydrolyzes a single ATP molecule. Through a
combination of the most recent T4 structures with
previous structural and biochemical data, many general
features of clamp loader structure and function can be
placed in the context of detailed structural models for
changes in conformation and the assembly of complexes.
Recognition of the clamp during loading onto DNA
In three crystal structures of ATP-bound clamp loaders,
the AAA+ modules can be seen to form a right-handed
spiral [55,59,60]. The clamp binds ‘under’ the clamp
loader in the ‘standard view’ (Figure 4b,c). Threedimensional
image reconstructions from electron
microscopy of a clamp-bound archaeal clamp loader
show that the loader holds the clamp in an open spiral
form [62]. The crystal structure of the T4 clamp loader
bound to the clamp and DNA confirms the generality of
this interaction [60] (Figure 4c). Indeed, there is some
evidence from molecular dynamics simulations that
clamp proteins are inherently biased toward a righthanded
spiral shape when opened [64,65], although not
all simulations show this right-handed bias [66].
The right-handed spiral of the open clamp roughly
matches the helical symmetry of DNA, with the clamp
tracking along the minor groove of the DNA duplex. The
right-handed spiral of the clamp can be described as a
series of rigid-body rotations of the six domains present
in the clamp (Figure 5a). The distortions of the clamp
from its planar conformation are not uniform around the
spiral. The largest distortion occurs between domains 3
and 4 of the clamp (a 13.4° rotation), which is the domain
interface directly opposite the open interface. While it
may seem counterintuitive that the domains nearest the
opening show the least perturbation, a large rotation at
the site opposite the broken interface is amplified around
the ring, thus providing the greatest leverage for clamp
opening.
The recent structures of the T4 clamp loader bound to
an open clamp and primer template DNA indicate how
loaders open a clamp [60]. The T4 clamp loader holds the
clamp at six contact points: one each from the B, C, D,
and E subunits, and two from the A subunit (the A
domain and A′ domain contact the clamp on either side
of the open interface; Figures 4c and 5b.) Three of the
contacts (from A, C and E subunits) occupy a
hydrophobic pocket between the two domains of each
subunit; this is the canonical site whereby clamps interact
with other proteins [12,32,67-69]. The other clamp loader
contacts (from B, D, and A′) occupy grooves that lie at
the interfaces between clamp subunits. In this way the
clamp loader completely occludes the face on which the
clamp binds other components of the replisome. This
occlusion explains the observation that binding of DNA
polymerase and clamp loader are mutually exclusive
[70-72].
The B, C, D and E subunits of the T4 clamp loader are
identical (see above), yet they can occupy two very
different binding surfaces on the clamp: the canonical
binding cleft or the inter-subunit crevice (Figure 5b).
These subunits bind to the clamp through relatively nonspecific
van der Waals contacts, with limited hydrogen
bonds and ionic interactions. The B, C, and D subunits of
the bacterial clamp loader (also identical proteins, g or t)
can also bind to two kinds of sites on the clamp. This
raises the question of how the T4 and bacterial clamp
loaders achieve proper alignment with the clamp, so that
the ATP-driven conformational change in the clamp
loader can be coupled appropriately to opening of the
clamp. (Note that this question does not arise for
eukaryotic clamp loaders, whose five subunits are distinct
and can be specific for their requisite binding site.)
In both the T4 and the E. coli clamp loaders, this
problem is solved by specific interactions between the
clamp and the unique A subunit (Figure 5b). (The
eukaryotic A subunit also makes specific contacts with
the open end of the clamp.) In the case of the E. coli
clamp loader, the A subunit interacts more tightly with
the clamp than do any of the other subunits [73]; it binds
through a helix and loop that insert snugly into a
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hydrophobic pocket, accompanied by specific hydrogen
bonds and ions pairs between the A subunit and clamp
[32]. Likewise, the A subunit of the T4 clamp loader has
the largest interaction surface area of all its subunits and
makes interactions that are specific for the deep
hydrophobic binding cleft [60], consistent with
biochemical studies that have shown that the A subunit is
necessary for productive binding of the clamp [74,75].
The clamp loader, as we have seen, interacts with the
clamp through the cooperative action of multiple
interactions that appear to be weak. Most other clampbinding
proteins rely primarily on one tight interaction
with the clamp. For example, most DNA polymerases
[12,67,76,77] and the cell cycle regulator p21 [68] interact
with the clamp primarily through a single, high-affinity
binding site. The weak cooperative interactions between
clamp loader and clamp are easily regulated to facilitate
clamp loader ejection after loading the clamp onto DNA
(see below). Proteins that interact primarily through one
tight binding site allow the clamp to bind multiple
partners at once, so that, as suggested in earlier studies,
the clamp can be a sliding tool belt on DNA [78-80].
While the recently determined structures show how
the clamp is held in the open state by the clamp loader,
they do not address whether the clamp loader actively
opens the clamp or simply traps a transiently open state.
The T4 clamp appears to be dynamic and can open and
close even in the absence of the loader [81-84], possibly
explaining the short lifetime of the T4 clamp on DNA
compared with cellular clamps [85]. Hence, the T4 clamp
loader may not need to force the clamp open, but may
simply trap it in the open state and close it around a
primer-template junction in the correct orientation for
polymerase action. Conversely, the E. coli clamp forms an
extremely tight, closed structure in isolation [10,37,86]
and has a long lifetime on DNA (t1/2
~ 1 h) [85]. Thus,
bacterial clamp loaders are thought to open the clamp
actively [37,86,87], probably through contacts from the A
subunit that cause a conformational change in the clamp
that destabilizes the interface between clamp subunits
[32]. Therefore, clamp loaders from different organisms
can be expected to behave differently: some clamps may
need to be actively unloaded from DNA while others will
fall off spontaneously. The same is true for clamp loaders
with different functions – for example, the RFC-Ctf18
variant clamp loader, which is involved in sister
chromatid cohesion [88,89], has been suggested to be a
dedicated clamp unloader [90].
2
1
4
3
6
5
2
1
4
3
6
5
a)
I
III
A
A
B
C
D
E
E ADCBA
ATP
I II III
b)
Figure 5. Clamp loader interactions to open the sliding clamp. (a) The clamp is held in a distorted conformation. In the top panel, displacement
vectors compared from the closed planar structure (PDB code 1CZD) [13] to that of the open form (PDB code 3U60) [60] are mapped out onto
the structure and are scaled up by a factor of 4 and color coded blue to red. In the bottom panel, the relative domain rotations from the closed
planar structure to the open conformation are mapped onto the schematic, with the vector length and width scaled by the magnitude of rotation.
Figures adapted from [60]. (b) Clamp loader contacts to the open sliding clamp. All five clamp loader subunits contact the open clamp. In the top
panel, the clamp-interacting motifs of the clamp loader are shown with the rest of the structure displayed as a wireframe for clarity. In the bottom
schematic diagram, the clamp loader AAA+ modules and the sliding clamp are shown from the side and flattened out onto the page so that all
subunits can be viewed simultaneously. The contacts between the ATPs and arginine fingers direct the clamp loader to form a spiral assembly in
which the AAA+ modules are arranged to perfectly match the symmetrically positioned binding clefts on the sliding clamp. Figure adapted from
[60].
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The clamp loader and the clamp collaborate in
specific binding of the clamp loader to primertemplate
junctions
The clamp loader must position the sliding clamp on the
DNA specifically at the primer-template junction where
the polymerase is to be recruited. How the clamp loader
recognizes the primer-template junction was first
suggested by the structure of yeast clamp loader RFC
bound to PCNA in the absence of DNA [59] (Figure 4b).
The duplex segment of primer-template DNA is
positioned in the central chamber of the clamp loader
where it is stabilized by basic residues and aminoterminal
helix dipoles lining the interior surface of the
clamp loader spiral. Because the circular collar domains
form a disc with no central cavity, it blocks the 3′ end of
the primer strand and in this way selects for DNA
structures that can bend sharply and thus leave the
interior of the clamp loader through the gap between the
A and E subunits (in bacterial clamp loaders) or the A
and A′ domains (in the other clamp loaders) (Figures 3
and 6). The single-stranded template DNA at the primed
template junction is such a flexible structure. This escape
route for the template DNA is possible only because the
clamp loader is pentameric, and not hexameric, as is
typical for AAA+ machines: it is the gap where the sixth
subunit would be that allows egress of the single-stranded
template. The model in Figure 6 also shows how the
binding of primed DNA by the clamp loader would
automatically position the duplex region of a primertemplate
junction through the ring of the sliding clamp.
The clamp loader AAA+ spiral does not conform to Bform
DNA, but matches instead the helical symmetry of
duplex A-form DNA [55,60]. It thus forces the duplex
DNA in the chamber into the A conformation. It is
perfectly adapted to bind the RNA-DNA hybrid segment
where an RNA primer is present, since RNA-DNA
hybrids assume a conformation that closely resembles Aform
DNA [91]. The principal interactions between the
Figure 6. A detailed model for clamp loading mechanism. (1) Prior to ATP binding, the clamp loader AAA+ modules are not organized in a
manner competent to bind the clamp. (2) Upon fully binding ATP, the clamp loader AAA+ modules assume a spiral shape that can hold the clamp
in an open lockwasher conformation. (3) This binary complex is competent to bind to primer template DNA. We propose that the dimensions of
the open clamp restrict DNA access to the central chamber such that only single-stranded regions or a major groove at a single-stranded/doublestranded
junction can slip through the crack in the clamp. (4) The duplex region of primer-template DNA then slides up into the clamp loader
central chamber, which activates the ATPase. (5) ATP hydrolysis or Pi
release initiates at the end of the AAA+ spiral, which allows the clamp to close.
Further hydrolysis events cause release of the clamp loader from the clamp and DNA, resulting in a loaded clamp. Figure adapted from [60].
(1)
(2)
(3)(4)
(5)
Pi
Pi
loaded
clamp
Pi
primer-
template
DNA
ATP
clamp
A A
BC
D
E
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clamp loader and double helix are restricted to the
template strand [55,60]. These features enable the clamp
loader to bind either RNA-DNA or DNA-DNA primertemplate
junctions, a versatility that is important in the
participation of clamp loaders in DNA repair, where the
primer is DNA.
For DNA to bind in the central chamber of the clamp
loader, it must enter through the clamp opening as well
as the gap in the clamp loader spiral, which in the T4
clamp loader is flanked by the A′ and A domains of the A
subunit. The structures suggest, however, that both gaps
are too narrow for either duplex DNA or a DNA-RNA
hybrid to pass through. The clamp is only open by
approximately 9  Å, and the gap between the A and A′
domains of the clamp loader is as small as 14  Å [60].
(Eukaryotic and archaeal clamp loaders, with larger A
domains, would be expected to have an even smaller gap
between the A and A′ domains.) Although the T4
structure already has DNA present, Förster resonance
energy transfer experiments of the yeast clamp bound to
the clamp loader have shown that the opening of the
clamp is not significantly changed upon DNA binding
[92].
To explain how the DNA-RNA hybrid at the primertemplate
junction gains access to the chamber of the
clamp loader, we propose that single-stranded DNA, or
possibly the major groove of duplex DNA directly
adjacent to a primer-template junction, initially enters
through the opening in the clamp (Figure 6). Once
through the opening, the duplex region could then screw
up into the interior of the clamp loader to occupy the
central chamber, where it would stimulate ATPase
activity and induce clamp loader ejection (see below).
This feature may have important functional implications
for the clamp loader mechanism at a replication fork, as
the small opening may act as a filter for regions of DNA
with single-stranded character, thereby aiding in the
search for primer-template junctions.
DNA-triggered ATP hydrolysis drives ejection of
the clamp loader
In all clamp loaders tested thus far, ATPase activity is
greatly enhanced by the binding of primer-template DNA
[40,93-95]. Hydrolysis of ATP leads to closure of the
clamp and ejection of the clamp loader from the clamp
and DNA, leaving the clamp loaded onto DNA [37,40].
The DNA-dependent hydrolysis of ATP is a key feature of
clamp loaders because it prevents futile cycles caused by
premature release of a clamp before a primer-template
junction is found.
The structural data suggest how DNA binding could
play a role in switching on the ATPase activity of the
clamp loader subunits. Earlier structural analyses of the
bacterial and eukaryotic clamp loaders showed how the
formation of tight intersubunit interfaces on ATP binding
organizes the arginine finger at the catalytic center
[55,59]. The availability of the T4 structure, along with
other structures, suggests a possible allosteric switch
mechanism, in which a DNA-binding residue (the switch
residue) undergoes a DNA-dependent conformational
change that appears to control the positioning of the
catalytic glutamate in the Walker B motif [60]. This
mechanism was suggested on the basis of sequence
covariation to function in the eukaryotic clamp loaders
[96], and is conceptually similar to the glutamate switch
mechanism that has been proposed for other AAA+
proteins, in which a conserved asparagine holds the
catalytic glutamate in an inactive conformation until
ligand binds [97]. We now propose that this mechanism
plays a role in ATPase activation in all clamp loaders. In
the absence of DNA, the conserved basic switch residue
(Arg383 in the yeast A subunit, Lys100 in E. coli B, C and
D subunits, and Lys80 in the T4 B, C, D and E subunits)
is tucked into the interior of the AAA+ module, where it
interacts with the backbone of the Walker B residues and
holds the catalytic glutamate in an inactive conformation
[59]. Upon binding DNA, the switch residue interacts
directly with the phosphate backbone of the DNA
template strand and is released from the interior of the
AAA+ module, allowing the catalytic glutamate to enter
a conformation consistent with activation of water for
hydrolysis of the g-phosphate of ATP [55,60]. Mutational
analysis of the switch residue and neighboring residues
supports the hypothesis that the switch controls the
ATPase activity [42], but experimental verification of this
mechanism is still incomplete and our analysis is based
on comparisons of structures that are quite divergent in
sequence so we cannot be sure of their functional
equivalence. Further biochemical and structural data,
particularly for the same clamp loader captured in
different states of the cycle, are necessary to test this
hypothesis.
In addition to the state fully bound by ATP, a ternary
complex of T4 clamp loader, DNA, and clamp has been
crystallized in a state in which the clamp loader B subunit
is bound to ADP while the other active sites are bound to
an ATP analog [60]. Thus, this structure, which was
obtained adventitiously, represents a state in which only
one ATPase site has hydrolyzed ATP. The release of the A
and B ATPase from the rest of the ATPase subunits, as a
consequence of this ATP hydrolysis, shows that a
conformational change within the AAA+ module caused
by ATP hydrolysis is incompatible with the symmetric
AAA+ spiral.
The AAA+ spiral probably comes apart in a cooperative
fashion, starting from one end of the spiral (Figure 6).
ATP hydrolysis is cooperative in the presence of primer
template DNA [98]. Therefore, hydrolysis at one site in
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the clamp loader will promote hydrolysis at another site.
We also suspect that the cooperative disassembly of the
AAA+ spiral will be directional, starting from the ends of
the spiral, because ATP hydrolysis requires movement of
neighboring subunits away from each other. This
movement will be favored at an end of the spiral, as this
order imposes changes to only one subunit for each
hydrolysis event. Biochemical studies [45,99] and
structural data [60] corroborate this hypothesis.
Hydrolysis from the ends has the functional benefit of
allowing the clamp to close before the clamp loader is
fully ejected, which would prevent the DNA from
slipping out from the opening of the clamp before the
loading reaction is complete. In support of this idea, the
clamp loader with ADP at the B site is bound to a closed
clamp in the structure, which is a result of the breakdown
in the symmetric matching of the clamp loader’s binding
sites with those of both the DNA and the sliding clamp
[60]. Thus, upon further ATP hydrolysis events at the C
and/or D sites, the clamp loader can no longer recognize
the symmetrically arrayed binding sites on the clamp and
DNA, and therefore ejects from both macromolecular
substrates, leaving the primer-template junction threaded
through the ring of the clamp (Figure 6).
One apparently consistent feature of clamp loaders
from different domains of life is that only three of the
ATPase subunits are catalytically active in clamp loading.
(Mutated clamp loader complexes with fewer than three
active ATPase sites can accomplish clamp loading
[46,100,101], but only with greatly diminished efficiency.)
In the case of bacterial clamp loaders, it is clear that only
the B, C, and D subunits are active ATPases. The
eukaryotic clamp loaders contain five subunits that can
bind ATP, but activity appears to be necessary only in the
B, C, and D subunits. The E site can bind nucleotide [59],
but lacks catalytic activity. The A subunit can both bind
and hydrolyze ATP, but its activity is not necessary for
clamp loading in either the eukaryotic [102] or archaeal
clamp loader [63]. Likewise, in T4, the identical B, C, D
and E subunits all have ATP binding sites, but the E
subunit appears to be catalytically inactive, because the
A′ domain does not contribute an arginine finger residue
(normally supplied in trans) to complete the catalytic
machinery [60]. However, there is some controversy
regarding the stoichiometry of ATP usage in the T4
clamp loading cycle [94,98,103-105].
The T4 bacteriophage replication system seems to
be a chimera, with functional modules borrowed
from bacteria and eukaryotes
The genome of T4 bacteriophage is a curiosity, in that it
is thought to be composed of genes derived from both
eukaryotic and bacterial sources [106], even though it
infects only bacteria. The hybrid nature of T4 is further
supported by the evidence that double-stranded DNA
bacteriophages share similarity in their capsid proteins
[107-109] as well as DNA packaging machinery [110,111]
with eukaryotic viruses such as herpesviruses. In fact,
many eukaryotic viruses seem to have a hybrid genome,
with significant horizontal transfer of bacterial and
archaeal genes [112,113]. In the case of T4, the similarity
of T4 proteins to their eukaryotic counterparts is
apparent from the sequence and structures of the clamp
and clamp loader. Both eukaryotic and T4 clamps are
homotrimers, unlike the dimeric bacterial clamps, and
the T4 clamp loader AAA+ subunit shows more sequence
homology to eukaryotic clamp loaders than to bacterial
clamp loading subunits (Figure 7a): this sequence
homology is reflected in the high structural similarity of
the B, C, D and E subunits of T4 with those of the yeast
clamp loader (Figure 7b).
One of the unique features of both the eukaryotic and
T4 clamp loaders is the A subunit. The T4 A subunit
forms an inverted U shape and reaches across the clamp
interface [60]. Therefore, the A subunit binds two clamp
subunits, with the A domain binding to the clamp I
subunit and the A′ domain binding to the clamp III
subunit (Figures 4c and 5b). We propose that the A
subunit of the eukaryotic clamp loader is also likely to
bind to both the clamp I and the clamp III subunits. The
yeast clamp loader structure shows that the A subunit
also forms an inverted U, although the A′ domain has
collapsed down onto the AAA+ module of the A subunit,
and there is no contact between the A′ domain and the
clamp [59]. This collapse probably resulted from
mutations in the clamp loader that prevented clamp
opening, or deletion of the carboxy-terminal portion of
the A′ domain, or both. Whatever the evolutionary
history, the yeast and T4 A′ domains interact with the E
subunit in similar ways and have essentially the same fold
(Figure 7c). Furthermore, electron microscopic
reconstruction of the Pyrococcus furiosus clamp loader
demonstrates that archaeal clamp loaders also have the
A′ domain and shows electron density that reaches across
the interface and touches the clamp, like the T4 loader
[60,62] (Figure 7d) but unlike the E. coli A subunit, which
binds only one protomer of the clamp and contains no
A′ domain [32,114].
These findings may bear on the question of how the
oligomeric character of the clamp influences the
structure and mechanism of its loader. In particular, the
trimeric clamps of T4 and eukaryotes are less stable [85],
and thus may require a subunit to bind both sides of the
open interface to help stabilize the open form and to keep
clamp protomers from dissociating. In contrast, bacterial
clamps are dimeric and appear to be the most stable [85],
perhaps eliminating a requirement for additional
stabilization of the open form by the A′ domain.
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The similarity between the eukaryotic and T4
bacteriophage clamp loaders may reflect a deeper shared
evolutionary history. The T4 DNA polymerase belongs to
the B family, as do the eukaryotic replicases [115],
whereas the bacterial replicases are members of the
unrelated C-family of polymerases [116,117]. In other
components of the replisome, however, there are
significant similarities between T4 components and
those of bacteria. The T4 and E. coli primases are singlesubunit
enzymes [118-121] and both have TOPRIM folds
[122-124]. In contrast, the eukaryotic primase (Pol α) is a
four subunit assembly containing both a primase activity
and a DNA polymerase that extends the RNA to make a
chimeric RNA-DNA primer [125,126]. The T4 helicase is
related to the bacterial replicative helicase (E. coli DnaB)
[122] and has the same directionality of unwinding as E.
coli DnaB, implying that these helicases encircle the
lagging strand at a replication fork [125]. By contrast, the
archaeal and eukaryotic MCM hexamers translocate in
the opposite direction [127-130], implying they surround
the leading strand. Additionally, the single-stranded
DNA binding proteins from T4 (gp32) and E. coli (SSB)
have been suggested to be related [106], although there
are significant functional differences in their mechanisms
of action. Finally, the T4 gp69 protein, which has been
suggested to assist in the initiation of DNA replication
[106], has significant homology to the E. coli DnaA
protein that initiates DNA replication [131].
Figure 7. Sequence and structural relationships between the T4 clamp loader and those from other branches of life. (a) A sequence
similarity dendrogram for the structurally characterized clamp loader subunits. The T4 clamp loader is more similar to yeast clamp loader than to
the bacterial clamp loader subunits. Tree calculated using PHYLIP [133] using the neighbor-joining bootstrap criterion. (b) The structure of the T4
clamp loader B position AAA+ module (the gp44 protein; purple) is highly similar to the yeast clamp loader B subunit AAA+ module (the RFC4
protein; salmon). The Cα
root mean square deviation (RMSD) is approximately 1.1 and 1.4 Å for the amino- and carboxy-terminal domains of the
AAA+ modules, respectively. ATP is shown in spacefilling representation. (c) The A′ domain is similar in yeast and T4 clamp loaders. The T4 and
yeast A′ domains have identical fold topology (the gp62 and RFC1 proteins in purple and salmon, respectively). Additionally, their packing against
the E subunit AAA+ module is very similar. (d) A negative stain electron microscopy reconstruction of the P. furiosus clamp loader, clamp and DNA
complex [62] reveals the presence of an A′ domain. The correspondence of the T4 clamp loader structure (fit to the electron microscopy-derived
molecular envelope) with that of the archaeal clamp loader suggests that the P. furiosus clamp loader also has an A′ domain. Figure adapted from [60].
RFC-A’T4A’domain
RFC-E
Euk.
A’ domainT4
A’ domain
gp44-E
?
c)
a) b)
d)
RFC-B
T4 gp44
A’ domain
EukaryoticT4
E subunit
(gp44)
A’ domain (gp62) A’ domain (RFC1)
E subunit
(RFC5)
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These results imply that replisomes can be pieced
together from different modules. The helicase, primase
and single-stranded binding protein of T4 are most
closely related to those of bacteria, while the clamp,
clamp loader and polymerase are similar to those of
eukaryotes. These two groups of proteins represent
different functional modules of the replisome: the
helicase and primase travel on the lagging strand as one
unit, while the polymerase and clamp are tightly
associated. These observations suggest that entire
modules may be evolutionarily exchanged more readily
than individual proteins.
Open questions
We have now reached a satisfactory state of
understanding regarding the structural basis for clamp
loader action, at least at the level of the general
mechanism, understood in broad strokes (Figure 6). We
know what these assemblies look like when they are
alone, with and without ATP bound [114,132], and how
their structure converts to a tightly integrated spiral form
upon binding ATP [55,59,60,62]. The structures have
explained how the primer-template junction is
recognized [59], and how the clamp loader can
accommodate both RNA and DNA primers [55,60]. The
recently determined structures of the T4 clamp loader
have shown how the ATP-bound clamp loader stabilizes
an open form of the clamp, and how ATP hydrolysis
might be coupled to release of the clamp onto DNA [60].
Although the inferences drawn from these structures
are compelling, it is important to recognize that we only
have one structure for some of the key steps in the clamp
loading cycle, and that these structures are for clamp
loaders that are very divergent in sequence. The structure
of the ATP-free clamp loader is for the E. coli system
[114] (Figure 4a), as is the ATP-loaded form bound to
DNA [55]. A structure of the loader in complex with a
closed clamp in the absence of DNA is for the eukaryotic
clamp loader [59] (Figure 4b), and the complex with the
open clamp and DNA is for the T4 complex [60] (Figure
4c). A more comprehensive understanding of how ATP
binding and hydrolysis is coupled to the opening of the
clamp and its loading on DNA requires that we have
structures of the same clamp loader (or, at least, very
similar clamp loaders) in different states of the clamp
loading cycle. It is hoped that the considerable
information now available about the general nature of the
conformational changes that are intrinsic to clamp loader
function will allow such structures to be obtained in the
near future. In addition, it is hoped that a clamp loader
bound to an open clamp in the absence of DNA can also
be crystallized, which will provide information about
how much the clamp is opened before it is loaded on
to DNA.
Although we have emphasized the clamp loading
aspect of the clamp loader machine, clamp loaders are
critical for the proper coordination of leading and lagging
strand synthesis. This is most clearly evident in bacteria,
where the clamp loaders are physically attached to the
polymerase subunits that replicate the leading strand
continuously and those that cycle between Okazaki
fragments on the lagging strand. The greater challenge
ahead is to understand how clamp loaders serve to
orchestrate rapid replication of chromosomal DNA, and
the bacterial replisome is likely to provide the route to
clearer structural understanding, because of the depth of
biochemical information available, as well as the ability to
purify intact complexes in reasonable amounts. We look
forward to future advances in the structural analysis of
intact replisome assemblies.
Additional material
Acknowledgements
Insightful comments and guidance provided by Miranda Robertson and
Stephen Harrison are warmly appreciated. We thank Kate Engel and Dr Tiago
Barros for help with figure preparation. This work was supported by a Ruth L
Kirschstein National Research Service Award through the National Institute of
General Medical Sciences to BAK and by grants from the NIH to JK (GM45547)
and MOD (GM38839).
Author details
1
Department of Molecular and Cell Biology, University of California, Berkeley,
CA 94720, USA. 2
Department of Chemistry, University of California, Berkeley,
CA 94720, USA. 3
California Institute for Quantitative Biosciences, University
of California, Berkeley, CA 94720, USA. 4
Howard Hughes Medical Institute,
University of California, Berkeley, CA 94720, USA. 5
Physical Biosciences Division,
Lawrence Berkeley National Laboratory, Berkeley, CA 94720, USA. 6
Howard
Hughes Medical Institute, Rockefeller University, New York, NY 10021, USA.
7
Current address: Max-Planck Institute of Biochemistry, Department of
Structural Cell Biology, D-82152 Martinsried, Germany
Published: 20 April 2012
Additional file 1
Rotating structure of the clamp loader of E. coli (known as the
γ-complex) in the apo form (PDB code 1JR3)
Click here for file
http://www.biomedcentral.com/content/supplementary/1741-7007-10-
34-s1.mov
Additional file 2
Rotating structure of the budding yeast clamp loader, replication
factor-C (RFC), bound to the sliding clamp, PCNA, and an ATP
analog (PDB code 1SXJ)
Click here for file
http://www.biomedcentral.com/content/supplementary/1741-7007-10-
34-s2.mov
Additional file 3
Movie of the T4 bacteriophage clamp loader bound to primertemplate
DNA, an ATP analog, and the sliding clamp during clamp
closure (PDB code 3U60)
Click here for file
http://www.biomedcentral.com/content/supplementary/1741-7007-10-
34-s3.mov
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Kelch et al. BMC Biology 2012, 10:34
http://www.biomedcentral.com/1741-7007/10/34
doi:10.1186/1741-7007-10-34
Cite this article as: Kelch BA, et al.: Clamp loader ATPases and the evolution
of DNA replication machinery. BMC Biology 2012, 10:34.
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