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Figure 1: Structure of the clamp loader:DNA complex (A) Schematic diagram of the clamp loader cycle. (B) Unbiased electron density for the DNA, calculated using a model at a stage prior to the inclusion of DNA and improved using density modification (Terwilliger, 2000). Contour lines at 1.2 standard deviations above the mean are shown in blue. The phosphate groups in the final DNA model are shown as spheres. The DNA interacting helices of the clamp loader are shown in yellow in this and subsequent figures. (C) The structure of wild type γ complex bound to primer-template DNA (left panel) and a schematic representation (right panel). The contacts between the clamp loader and the template strand are restricted primarily to the template strand, which is shown outlined in yellow.
Figure 2: DNA recognition
(A) Diagram showing the ATPase subunits of the clamp loader and DNA duplex. The DNA interacting helices are shown in yellow. The three rotation axes that relate the B subunit to the C subunit, the C subunit to the D subunit, and the D subunit to the E subunit are shown in blue, red and green, respectively. The three axes are nearly coincident with each other and with the axis of the DNA duplex (not shown). (B) Schematic diagram of contacts with the primer-template DNA. (C) Expanded view of a domain 1:DNA interaction, highlighting hydrogen bonding interactions between the DNA and the protein. (D) The sidechain of Tyr 316 blocks the path of the primer strand by stacking on the last nucleotide base of the primer.
Figure 3: Symmetry in the AAA+ spiral and interfacial ATP coordination
(A) The left panel shows the B, C, D, and E subunits (domain 1 only) of the
γ complex, in a view that is orthogonal to that shown in Figure 2A.
The middle panel shows a symmetrized version of the RFC clamp loader. In
this model, the A subunit is in the same position, docked on the PCNA
clamp, as in the crystal structure of the mutant RFC:PCNA complex
(Bowman et al., 2004). The B, C, D and E subunits are positioned by applying
the transformation that relates one subunit to the next one in the γ complex.
The right panel shows the actual positions of the RFC subunits in the
crystal structure of the mutant RFC:PCNA complex. (B) Coordination of the
ATP analog bound to the B subunit by the arginine finger presented by the C
subunit. Only the AAA+ modules (domains 1 and 2) are shown (left). A
schematic representation of this interaction is also shown (right). (C) An
expanded view of the coordination of ADP•BeFi3 bound to B is shown on the
left. A similar view of ATPγS bound to the A subunit of the mutant RFC
complex is shown on the right (Bowman et al., 2004). The arginine finger
in each of the subunits of the mutant RFC complex is replaced by
glutamine. A modeled arginine sidechain at the glutamine position is shown
in grey, and it is positioned to coordinate the γ-phosphate of ATP, as do
the actual arginine fingers in the γ complex. Each of the ATP binding sites
in the γ complex has essentially the same configuration of sidechains
shown here (see Figure S3). This symmetry is absent in the structure of the
mutant RFC complex, in which only the A and C sites display
tight coordination of ATP.
Figure 4: The exit channel for the template strand overhang
(A) The structure of the clamp loader is shown, with the E(δ) subunit
removed to reveal a tunnel leading through the collar, indicated by
red spheres. In the expanded view on the right, sidechains presented
by the collar domain of the A(δ) subunit and that interact with DNA are
shown. Two sidechains that line the collar tunnel are also shown. (B)
Fluorescence anisotropy data for fluorescently labeled primer-template
DNA binding to the wild type γ complex and five mutants are
shown. Mutation of residues that line the collar tunnel (R299A and
R307A; see panel A) does not affect DNA binding affinity while mutation
of residues that are in the observed exit path (R252A, R248A, and K313A)
reduces DNA binding affinity. (C) Dissociation constants of mutant clamp
loaders for DNA. The inset shows a zoomed in region of the chart.
Error bars are the standard error of the fit.
Figure 5: Recognition of RNA-DNA hybrids
(A) The DNA strand of an RNA:DNA hybrid ((Fedoroff et al., 1993);
PDB code 124D) is aligned on the template strand in the crystal
structure of the clamp loader. (B) The RNA and DNA strands of an
RNA:DNA hybrid, aligned as in (A) are shown. The RNA strand (orange)
is accommodated without steric clash because the clamp loader only
engages the template strand. (C) The structure of an RNA:DNA hybrid,
with its DNA strand aligned on the template strand in the crystal
structure as in (A) and (B), is shown. Note that the RNA strand of
the aligned hybrid duplex preserves the interaction with the
separation pin (Compare with Figure 2D).
Figure 6: Binding of the ψ-peptide to the clamp loader collar
(A) Isothermal titration calorimetry data for the binding of the ψ-peptide
to the clamp loader complex. The calorimetric titration of 100
μM wild-type ψ-peptide into 10 μM of the clamp loader complex (left)
and the ψ-peptide with Trp 17 mutated to Ser (right) are shown.
The Trp 17 mutation leads to a 55-fold decrease in binding affinity.
(B) Fluorescence anisotropy data for the binding of fluorescently
labeled DNA to the clamp loader in the absence of the clamp. DNA
binding in the presence (blue) and absence (red) of 10μM ψ-peptide
are shown. Error bars are the standard deviation of individual
readings. The values of the KD of DNA binding are 0.38±0.03μM and 18±3μM
in the presence and absence of ψ-peptide, respectively. (C) The
crystal structure of the ψ-peptide bound to the clamp loader collar.
The clamp loader collar domains are shown as surface representations.
The ψ-peptide is shown in red. (D) Close up view of the ψ-peptide
interactions with the collar domains of the B(γ), C(γ), and D(γ)
subunits, with hydrophobic sidechains shown as spheres. The C-terminal
tail of B(γ), which forms a short anti-parallel β-sheet with the
ψ-peptide, is shown as a blue ribbon.
Figure 7: ψ binding links SSB to the clamp loader and breaks symmetry in the collar
(A) The collar domain of the B(γ) subunit undergoes a conformational
change upon the binding of DNA by the clamp loader. Alignment of the
collar domains of the apo E. coli clamp loader (Jeruzalmi et al., 2001)
onto the DNA bound clamp loader reveals close overlap of the collar
domains, with the exception of the B(γ) collar domain (dark blue)
which undergoes a rotation of ~10° toward the AAA+ spiral in the DNA
bound complex (shown in light blue). (B) The collar domains of the
B(γ) and C(γ) subunits of the DNA bound complex are overlayed, revealing
a difference in the orientation of the AAA+ domains of these subunits
with respect to the collar. The AAA+ module of the B subunit (shown in
light blue) rises up towards the collar domain, forming a tight
interaction, whereas the C subunit (shown in red) is in an extended
conformation. (C) The location of the ψ peptide on the clamp loader
positions the χ:ψ assembly for interaction with SSB bound to the single
stranded template exiting the clamp loader. The χ:ψ assembly
(Gulbis et al., 2004) is positioned at the C-terminal end of the
ψ-peptide bound to the clamp loader. The γ subunit binds the
C-terminal tail of SSB. The 5’ template overhang of the DNA (green
spheres) exits the clamp loader and wraps around SSB
(Raghunathan et al., 2000).
Figure S2: The protein:DNA interactions are highly symmetric
(A) The interface between Domains 1 from the C(γ) and B(γ) subunits
is shown, along with the phosphate groups (sticks) in the template
strand backbone that these domains interact with, as well as the
nucleotide analogs bound to each subunit (spheres). (B) Three pairs
of subunits (E/D, D/C, and C/B) are shown superimposed and in the
same orientation as in (A). Note the close overlap of the
protein structures, the ATP analogs, and the phosphate groups.
Figure S3: Comparison of ATP analog coordination at the three ATP binding sites.
The coordination of ADP•BeF3 at the three interfacial binding sites in
the clamp loader is shown. An overlay of these three sites was
constructed by aligning Domain 1 from the subunits to which the
analog is bound. Note the resulting overlap in positions of the
arginine finger residues that are contributed by the adjacent subunits
in each interface. Electron density from the final model for the ATP
analog bound at the B:C interface is also shown.
Figure S4: FRET assay for clamp opening and closing by clamp loader mutants.
(A) Clamps labeled with FRET donor/acceptor pairs on either side
of a dimer interface were used to follow the open or closed state
of the clamp (See Supplemental Experimental Procedures). The
schematic diagram shows the expected decrease in FRET as the
clamp is opened by the clamp loader, followed by an increase in
FRET upon ATP hydrolysis and release of the clamp. The experiment
is done using a clamp that is labeled with donor/acceptor pairs as
indicated (Goedken et al., 2005; Goedken et al., 2004). ATP is
added to solutions containing the clamp and various forms of the
clamp loader, followed by primer-template DNA and then by excess
ADP, which resets the system. (B) Fluorescence spectra are shown
for wild type and one mutant (K313A) clamp loader. The ratio of
fluorescence at the donor and acceptor wavelengths are shown in the
insets for the four different conditions indicated. (C) The effects
of mutations on clamp opening and closing are reported by comparing
changes in FRET for mutant clamp loaders to the results obtained for
the wild type clamp loader. The decrease in FRET upon addition of
ATP is set to 100% for the wild type clamp loader. This decrease
in FRET is the same for each of the mutant proteins (shown in the
bar diagrams to the left in red), which are therefore not impaired
in their ability to bind to and open the clamp. The increase in
FRET upon addition of DNA reflects the hydrolysis of ATP and the
release of the closed clamp, and is set to 100% for the wild type
clamp loader. Mutations in the collar tunnel (R299A and R307A) have
no significant effect on DNA-stimulated clamp release (shown on the
right in green). In contrast, mutations in the observed exit channel
(R252A, R248A, and K313A) lead to a reduction in the efficiency
of clamp release.
Figure S5: Difference electron density for the ψ-peptide bound to the clamp loader:DNA complex
An unbiased difference electron density map, calculated prior to the
inclusion of the ψ-peptide in the model, is shown. Contour lines at
2.5 standard deviations above the mean value of electron denisty are
shown in green. The final model for the ψ-peptide is shown in red.
Note the clear electron density features surrounding Trp 7 (left panel)
and Trp 17 (right panel), which allowed unambiguous determination
of the sequence register of the ψ-peptide in the density.
Figure S6: Ψ binding is inconsistent with an inherent symmetry in the clamp loader collar
(A) The apo structure of the E. coli clamp loader (Jeruzalmi et al.,
2001) is shown with the AAA+ modules shown in a surface representation
and the collar domains shown as ribbons. (B) The B(γ):C(γ) collar
interaction is shown as in A (top) and the C(γ):D(γ) collar interaction
is shown in the same orientation (bottom). The B(γ):C(γ) and C(γ):D(γ)
collar interactions are overlayed (right), revealing almost perfect
overlap and the inherent symmetry in the collar domains of the γ
subunits in the apo complex. (C) Left: The ψ-peptide bound to
the clamp loader collar. Middle: An expanded view of ψ-peptide
binding to the B(γ), C(γ), and D(γ) collar domains in the DNA bound
clamp loader structure, in which the inherent symmetry in the γ collar
domains has been broken by a rotation of the B(γ) collar domain.
Right: A model of the ψ-peptide bound to a collar in which the
B(γ) collar domain (grey) has been positioned relative to the C(γ)
collar as in the apo clamp loader structure. Note the steric
clashes between the ψ-peptide and the B(γ) collar. (D) The same as
(C) except from a side view with the collar domain of the C(γ)
subunit removed.
Figure S7: Recognition of DNA structures with 3’ overhangs
(A) A schematic representation of the modeling procedure used in
this analysis. The primer-template junction was removed from the
crystal structure and rotated by 180° around an axis perpendicular
to the helical axis. This results in an inversion of the
polarity of the template strand such that the 3’ end of the
template is now at the “top.” The phosphate groups of this
template strand running in the opposite direction could now
be aligned with the positions of the phosphate groups in the
template from the structure to generate a model of the clamp
loader which interacts with a template running in the opposite
direction. (B) The phosphate groups of the template strand with
reversed polarity are shown in red, and they overlap closely
with the phosphate groups of the original template strand (green).
(C) Both strands of the DNA duplex with reversed polarity are
shown, aligned as in (B). The clamp loader subunits now track the
major groove rather than the minor groove. The "primer" strand,
shown in orange, is positioned such that it terminates within
the inner chamber of the clamp loader, near the collar subunit of
the A subunit, which has been removed for clarity. The altered
conformation of the "primer strand" is accommodated by the clamp
loader, which does not interact with it. The "template" strand
is positioned such that the 3' end is located at the gap between
the A and E subunits. A 3' overhang extending from the reversed
polarity "template" would interact with the A subunit. This
model explains why replacement of the A subunit alone is
sufficient to enable DNA with reversed polarity to be recognized
by the clamp loader (Ellison and Stillman, 2003).
Figure S8: Ψ-peptide binding induces the collar conformational change in the apo clamp loader
(A) The orientation of the B(γ) collar domains from the three
clamp loader complexes in the crystal structure of the ψ-peptide
complex in the absence of DNA (cartoon, light blue) are shown
relative to the position of the B(γ) collar domain (dark blue)
in the apo clamp loader (Jeruzalmi et al., 2001). (B) Difference
electron density for the ψ-peptide bound to one of the clamp
loaders in the asymmetric unit is shown (green contour lines
at 3.0 standard deviations above the mean). The final
ψ-peptide model from the clamp loader:DNA:ψ-peptide structure,
positioned by aligning the collar domains of the C(γ) subunits,
is shown (sticks). (C) and (D) are the same as (B) for the other
two clamp loaders in the asymmetric unit.
Table S1: Data Processing
Table S2: Data Processing
Table S3: Data Processing
Table S4: Refinement Statistics
Table S5: Data statistics for the wild-type Apo γ complex:ψ-peptide structure
Table S6: Refinement statistics for the wild-type Apo γ complex:ψ-peptide structure
