Cdc6-Induced Conformational Changes
in ORC Bound to Origin DNA Revealed
by Cryo-Electron Microscopy
Jingchuan Sun,1,5Hironori Kawakami,2,5Juergen Zech,3Christian Speck,3Bruce Stillman,2,* and Huilin Li1,4,*
1Biology Department, Brookhaven National Laboratory, Upton, NY 11973, USA
2Cold Spring Harbor Laboratory, 1 Bungtown Road, Cold Spring Harbor, NY 11724, USA
3DNA Replication Group, MRC Clinical Sciences Centre, Imperial College Faculty of Medicine, Hammersmith Hospital Campus,
Du Cane Road, London W12 0NN, UK
4Department of Biochemistry and Cell Biology, Stony Brook University, Stony Brook, NY 11794, USA
5These authors contributed equally to this work
*Correspondence: firstname.lastname@example.org (B.S.), email@example.com (H.L.)
The eukaryotic origin recognition complex (ORC)
interacts with and remodels origins of DNA replica-
tion prior to initiation in S phase. Here, we report
a single-particle cryo-EM-derived structure of the
supramolecular assembly comprising Saccharo-
myces cerevisiae ORC, the replication initiation
factor Cdc6, and double-stranded ARS1 origin DNA
in the presence of ATPgS. The six subunits of ORC
are arranged as Orc1:Orc4:Orc5:Orc2:Orc3, with
Orc6 binding to Orc2. Cdc6 binding changes the
conformation of ORC, in particular reorienting the
Orc1 N-terminal BAH domain. Segmentation of
the 3D map of ORC-Cdc6 on DNA and docking with
the crystal structure of the homologous archaeal
Orc1/Cdc6 protein suggest an origin DNA binding
model in which the DNA tracks along the interior
surface of the crescent-like ORC. Thus, ORC bends
and wraps the DNA. This model is consistent with
the ORC footprint on origin DNA from both ends.
Before a cell can divide, its genome must be duplicated (Korn-
berg and Baker, 1992). DNA polymerases, the workhorses that
synthesize the new strands of the double helix, work only at an
established replication fork in which the double-helix DNA has
already been unwound by a replicative helicase complex.
However, the close-ring, hexameric structured helicase cannot
load onto DNA by itself and needs to be actively assembled
onto DNA by the concerted actions of initiator proteins, which
comprise the eukaryotic origin recognition complex (ORC),
Cdc6, and Cdt1 in eukaryotes (Bell and Dutta, 2002; Bell and
Stillman, 1992; Bochman and Schwacha, 2008; Evrin et al.,
2009; Kawakami and Katayama, 2010; Remus and Diffley,
2009; Stillman, 2005).
MCM2-7 was found recently to be loaded onto double-
stranded DNA as a head-to-head double hexamer in vitro, with
the double-stranded DNA likely running through the center of
the barrel-shaped MCM2-7 hexamers (Evrin et al., 2009; Remus
et al., 2009). The double hexamer of MCM2-7 is loaded as an
inactive complex, with the two MCM2-7 hexamers primed for
activation to form bidirectional DNA replication forks (Botchan
and Berger, 2010). It is known that ATP binding by ORC and
Cdc6 and then hydrolysis, first by Cdc6 and then by ORC, are
required for ordered assembly of prereplication complexes
(pre-RCs), with multiple MCM2-7 hexamers loading per origin
and then for subsequent initiation of DNA replication (Bowers
et al., 2004; Klemm et al., 1997; Lee et al., 2000; Randell et al.,
2006; Speck et al., 2005; Speck and Stillman, 2007). Unwinding
of origin DNA only occurs after activation at S phase, but the
mechanism is not known. In contrast, bacterial DnaA, which
has limited similarity to some subunits of ORC and to Cdc6, is
monomeric is solution, but 10–20 of them oligomerize at origin
DNA during replication initiation and form a right-handed super-
helix when crystallized at high concentration and in the absence
of DNA (Erzberger et al., 2006; Mott and Berger, 2007). It was
proposed that bacterial origin DNA might wrap around the
superhelical proteinaceous DnaA core, forming a positively
supercoiled structure on DnaA and consequently causing the
nearby AT-rich region to unwind (Duderstadt et al., 2010; Erz-
berger et al., 2006). DnaC then helps to load the hexameric
DnaB helicase onto the melted region of DNA (Kawakami and
Katayama, 2010). The eukaryotic Orc1 subunit and the Cdc6
protein share considerable amino acid similarity with each other
and are related in sequence to the Orc1/Cdc6 initiator protein in
archaeal species, but archaea lack proteins related to Orc2,
Orc3, Orc4, Orc5, and Orc6 (Duncker et al., 2009). The crystal
structures of archaeal Orc1/Cdc6 in complex with origin DNA
reveal a bipartite origin recognition mechanism (Dueber et al.,
2007; Gaudier et al., 2007). Archaeal Orc1 binds origin DNA
with both a C-terminal winged-helix domain (WHD) and a helix-
loop-helix insertion in the N-terminal ATPase domain. The
C-shaped archaeal Orc1 acts like two claws of a lobster to
grip, bend, and deform the origin DNA (Dueber et al., 2007;
Gaudier et al., 2007; Remus and Diffley, 2009). The helix-loop-
helix insertion in the ATPase domain distinguishes the initiator
534 Structure 20, 534–544, March 7, 2012 ª2012 Elsevier Ltd All rights reserved
clade from other AAA+ proteins, and is termed the initiator-
specific motif (Iyer et al., 2004). Therefore, it is possible that
some of the eukaryotic AAA+-type ORC subunits also bind the
origin DNA with two claws.
Structural analysis of eukaryotic replication initiators lags
behind that of prokaryotic systems, likely because of the fact
that eukaryotic initiators form preexisting large multiprotein
complexes and that these complexes are flexible, rendering
crystallization of the intact complexes difficult. Single-particle
electronmicroscopy has been applied to reveal the overall archi-
tecture of ORC from Saccharomyces cerevisiae (Speck et al.,
2005) and Drosophila melanogaster (Clarey et al., 2006). The
S. cerevisiae ORC (ScORC) is roughly a two-lobed and cres-
cent-like structure. The approximate positions of the individual
yeast ORC subunits were localized by systematically fusing the
subunits at either the amino or carboxyl terminus, one subunit
and one terminus at a time, with a bacterial maltose-binding
protein (Chen et al., 2008). The D. melanogaster ORC (DmORC)
has a similarly elongated shape (Clarey et al., 2006). Interest-
ingly, it was found that hyperphosphorylation, a major regulatory
event for DmORC function, did not markedly change its overall
structure (Clarey et al., 2008).
The yeast replication initiator Cdc6, upon binding to ORC that
is bound to origin DNA, was shown to turn on an ATP hydrolysis-
dependent molecular switch in ORC (Speck et al., 2005),
although the physical basis for the switch is unknown. Further-
more, it is not known how ORC interacts with and remodels
origin DNA in preparation for loading of the replicative helicase
component MCM2-7 to form a pre-RC. Existing structural anal-
ysesof eukaryotic ORCwere donein the absence of DNAand by
negative-stain EM, which has limited resolution (Chen et al.,
2008; Clarey et al., 2006, 2008; Speck et al., 2005). Cryo-EM
methods avoid stain-associated artifacts and have the potential
to achieve better resolution. We therefore launched a significant
cryo-EM effort to overcome difficulties associated with imaging
the relatively small ORC particles. We have now determined
cryo-EM structures of S. cerevisiae ORC and ORC-Cdc6 in the
presence of origin DNA and ATPgS. Comparison of these
structures reveals a series of conformational changes in ORC
upon DNA and Cdc6 binding and suggests a model of how
ORC binds origin DNA. This work represents an important step
toward understanding the biochemistry of how the pre-RC is
Orc6 Interacts with Orc2, but Not with Orc3
ScORC is a bilobed structure (Speck et al., 2005). In a previous
study, we were able to assign Orc1:Orc4:Orc5 to the top lobe
of the ScORC and determine that the lower lobe contained
Orc2, Orc3, and Orc6 (Chen et al., 2008). However, we were
unable to distinguish between Orc2 and Orc3, and thus it was
unclear whether Orc6 bound to Orc2 or Orc3. Therefore, we
analyzed binary interactions between GST-Orc6 and each of
the other ORC subunits that were translated in vitro. In human
ORC, Orc3 associates with Orc6 in vitro (Siddiqui and Stillman,
2007) but, contrary to our expectations, only Orc2 of the
S. cerevisiae Orc1/2/3/4/5 bound to GST-Orc6 (Figure 1A). A
domain-mapping experiment showed that the N-terminal region
of Orc2 (amino acids 1–265) retained affinity for GST-Orc6 but
that the C terminus did not (Figure 1B). Further deletion of only
35–40 residues from either terminus of the 1–265 Orc2 fragment
reduced the interaction with GST-Orc6, and further deletions
from either terminus eliminated the interaction (Figure 1C,
81–265 or 1–170), suggesting that a relatively large region(s)
within the Orc2 N-terminal third (amino acids 1–265) participates
in Orc6 binding. This information enabled us to more accurately
interpret the difference observed in the negative-stain EM
images of the ORC (Orc1–6) and Orc1–5 subcomplex (missing
Orc6) (Chen et al., 2008). In the projection, the lower lobe of
ure 1D). In the Orc1–5 subcomplex, all of the three densities are
present, indicating that none of these densities can be exclu-
sively attributed to Orc6. However, the densities ε and h are
much weaker in Orc1–5. Given the information that Orc6
binds to Orc2 and the knowledge that Orc1–5 binds origin
DNA in a sequence-specific and ATP-dependent manner that
is indistinguishable from that of the intact ORC (Lee and Bell,
1997), we conclude that the densities ε and h are largely from
Orc2 and that Orc6 binds on top of Orc2. This assignment of
Orc6 is consistent with the 3D difference mapping between
ORC and Orc1–5 showing two different peaks, the larger one
region (Chen et al., 2008). Consequently, density z at the bottom
left can be assigned to Orc3, unaffected by the presence or
absence of Orc6. In conclusion, we suggest the assignment of
the overall architecture of ScORC as Orc1:Orc4:Orc5:Orc2:Orc3
with Orc6 binding to Orc2, on the top of Orc2 in this view
Cryo-EM of ORC and Complexes with Origin DNA
In order to visualize how ORC interacts with the origin DNA
and the replication initiator Cdc6, cryo-EM single-particle
three-dimensional reconstruction was determined on three
complexes: purified S. cerevisiae ORC alone; ORC bound to
ARS1 origin-containing 66 bp-long double-stranded DNA
(dsDNA); and ORCbound
S. cerevisiae Cdc6 (see Experimental Procedures) (Speck
et al., 2005). All three samples were maintained in buffer con-
taining 1 mM ATPgS, a slowly hydrolyzable ATP analog.
Although yeast ORC at 414 kDa is small for cryo-EM, by using
a secondary layer of continuous thin-carbon film over the
primary lacy carbon film and by strictly selecting EM grid
regions with the thinnest vitreous ice, we were able to obtain
electron micrographs with adequate particle contrast (Fig-
ure 2A). We found that a small percentage of ORC particles
(<10%) formed dimer-like aggregates in solution and on EM
grids. It was therefore necessary to manually select particles
from micrographs to avoid the occasional putative ORC dimer.
Figure 2B shows five class averages and their corresponding 2D
projections of ORC-DNA (see Figure S1 available online). The
3D EM map of ORC alone is relatively featureless (Figure 2C)
whereas the larger ORC assemblies, ORC-Cdc6-DNA and
ORC-DNA, contain more structural detail (Figures 2D and 3).
This probably indicates that ORC is flexible on its own and
that interaction with its functional partners DNA and Cdc6 stabi-
lizes the ORC structure. Fourier shell correlations (FSCs) of the
to ARS1 originDNA and
Cryo-EM of ORC-Cdc6 Bound to DNA
Structure 20, 534–544, March 7, 2012 ª2012 Elsevier Ltd All rights reserved 535
3D reconstructions indicate that the data self-consistency
ranges from ?1 nm to 2.5 nm (Figure S2). But FSC is not an
accurate measurement of resolution (Grigorieff, 2000). Based
on structural features, the EM map of ORC alone may have
a resolution of 2.5 nm and that of ORC-DNA and ORC-Cdc6-
DNA a resolution of 1.5 nm resolution. The cryo-EM map of
the largest assembly, ORC-DNA-Cdc6, represents a significant
improvement over the previously published negative-stain EM
map (Chen et al., 2008): the previous map reveals only the
shape of the ORC complex but contains no structural features
for the components, whereas in the new cryo-EM map, several
of the ORC subunits are resolved. See below for greater
V 1 2 3 4 5 V 1 2 3 4 5V 1 2 3 4 5
G 6 G 6 G 6 G 6
: GST or
G 6 G 6 G 6 G 6 : GST or
: PCR orc2
1-26541-265111-265 141-2651-230 1-170
G 6 G 6 G 6 G 6 G 6
1-621 (FL) 1-26541-26581-265111-265 141-265171-621 1-2301-170
Figure 1. The Smallest ORC Subunit Orc6
Interacts with Orc2, but Not with Orc3
(A–C) Radiolabeled Orc1/2/3/4/5 (A) or deletion
an in vitro transcription/translation system and
pulled down by GST (G) or GST-Orc6 (6). Input
(5%) and bound materials (30%) were visualized.
FL, full length; V, expression vector.
(D) Comparison of reference-free class averages
of EM images of ORC and the Orc1–5 sub-
complex. Adapted from Chen, Z., Speck, C.,
Wendel, P., Tang, C., Stillman, B., and Li, H.
(2008). The architecture of the DNA replication
origin recognition complex in Saccharomyces
cerevisiae. Proc. Natl. Acad. Sci. USA 105, 10326–
10331. Copyright 2008 National Academy of
(E) A complete assignment of all six subunits
Binding to Origin DNA Induces
a Large Conformational Change
within the Top Lobe of the Bilobed
The cryo-EM 3D map of ORC is similar in
overall architecture to the elongated,
crescent-like, two-lobed shape of the
et al., 2005) (Figure 2C). The approximate
locations oftheORCsubunits aremarked
based on our previous negative-stain EM
mapping results in which the maltose-
binding protein was fused to individual
subunits (Chen et al., 2008). We group
the upper and middle regions into the
top lobe consisting of Orc1, Orc4, and
Orc5. The bottom region forms the
second lobe and is composed of Orc2,
Orc3, and Orc6. The cryo-EM structure
of ORC is slightly twisted out of plane as
compared with the flat shape in the nega-
tive-stain EM map. The flattening was
likely caused by air drying and by the
preferred orientation of ORC in the stain
salt on the carbon film (Speck et al.,
2005). In the EM maps (Figures 2C and
2D, front views), a 3 nm-sized density region at the left side in
the middle region is tentatively assigned to the N-terminal BAH
domain of Orc1 (1-NTD), based on the proximity to the Orc1
main density and the fit to the crystal structure of this domain
The structure of ORC bound to a 66 bp double-stranded
ARS1 origin DNA was determined to better resolution than
ORC alone, as evidenced by increased structural detail (Fig-
ure 2D). When the lower lobes of the ORC and ORC-DNA struc-
tures are aligned, the upper lobe of ORC-DNA appears to be
rotated as a rigid body by ?20?relative to ORC alone (Figures
2C and 2D). Importantly, the upper lobe is composed of Orc1,
Orc4, and Orc5, and thus contains all the potential ATP binding
Cryo-EM of ORC-Cdc6 Bound to DNA
536 Structure 20, 534–544, March 7, 2012 ª2012 Elsevier Ltd All rights reserved
and hydrolysis sites (Bowers et al., 2004; Klemm et al., 1997;
Speck et al., 2005). The large conformational change in the
ATPase-containing top lobe of ORC may explain the long-
standing observation that ORC binding to origin DNA is an
ATP-dependent event (Bell and Stillman, 1992). Due to the
limited resolution (?1.5 nm), the 66 bp DNA could not be demar-
cated in the ORC-DNA structure (Figure 2D).
Cdc6 Binding to the Side of ORC-DNA Switches Orc1
The replication initiator Cdc6 can bind ORC in the presence of
both origin DNA and ATP. In the absence of origin DNA, ATP
hydrolysis disrupts the ORC-Cdc6 complex (Speck et al.,
2005). Thus, a stable ORC-DNA-Cdc6 complex was assembled
in the presence of the slowly hydrolyzing ATPgS. Our previous
negative-stain EM analysis showed that Cdc6 bound to the
side of the crescent-like ORC structure in the absence of origin
Figure 3. Cdc6 Binding onto the ORC-DNA Structure Induces a
Rotation of Orc1
(A and B) Negatively stained EM image averages of ORC-DNA (A) and Cdc6-
ORC-DNA (B). Gain of density in the presence of Cdc6 is marked by filled thick
(C and D) Comparison of the cryo-EM 3D map of ORC-DNA (C) with that of
ORC-DNA-Cdc6 (D).The precise boundary between Orc2 and Orc3 cannot be
Orc1 NTD is painted in cyan. Cdc6 density in (D) is painted in magenta. Re-
arrangement of Orc1 density upon Cdc6 binding is indicated by a pair of blue
arrows. The protruding density in ORC-Cdc6-DNA (D) is painted in red and
tentatively assigned as part of Orc6.
See also Figures S1 and S2 for 3D reconstruction details.
Figure 2. Binding of ORC to Origin DNA Stabilizes the Structure and
Induces a 20?Rigid-Body Rotation at the ORC Top Lobe Composed
of Orc1, Orc4, and Orc5
(A) A raw image of ORC mixed with 66 bp ARS1 dsDNA at a 1:1.2 molar ratio.
Several individual particles are marked in white circles.
(B) Selected 2D reprojections (left) and corresponding reference-based class
averages (right) of the ORC-dsDNA complex.
(C) Cryo-EM 3D map of ORC alone in top (upper) and front side (lower) views.
(D) Cryo-EM 3D map of ORC-dsDNA shown in top (upper) and front side
(lower) views. Individual subunits are labeled.
See also Figures S1 and S2 for 3D reconstruction details.
Cryo-EM of ORC-Cdc6 Bound to DNA
Structure 20, 534–544, March 7, 2012 ª2012 Elsevier Ltd All rights reserved 537
DNA (Speck et al., 2005). In the current studies, we first asked
whether Cdc6 binds to the same location in the presence of
origin DNA. Comparison of the reference-free 2D class averages
of stained ORC-DNA particles with that of ORC-DNA-Cdc6
clearly shows that Cdc6 still binds to the same position, trans-
forming the crescent-shaped structure into a ring-like structure
(Figures 3A and 3B, thick white arrows). However, we found an
important difference in the presence of origin DNA: the orienta-
tion of the density assigned to Orc1 appears to be changed
when Cdc6 binds to ORC-DNA (Figures 3A and 3B, thin white
To visualize the nature of the Orc1 rearrangement, we deter-
mined the cryo-EM structure of ORC-DNA-Cdc6 to ?1.5 nm
resolution (Figure 3). A side-by-side comparison of the ORC-
DNA structure (Figure 3C; the same as Figure 2D but with
additional side and back views) with that of ORC-DNA-Cdc6
(Figure 3D) clearly reveals the density of the bound Cdc6
(highlighted in purple), with the remaining density belonging to
ORC and DNA. The positions of the individual ORC subunits
are again labeled. Orc1 is composed of a highly curved
C-shaped main body (highlighted in light blue) and a separate
NTD (highlighted in cyan). As already hinted in the 2D averages,
upon Cdc6 binding the Orc1 main body rotated, as indicated by
1-914 (FL) 1-400
: GST or
: PCR orc1
301-914 301-400 1-3001-200
1-914 (FL)101-914 201-914 301-914 401-914 501-914 601-914 701-914 801-914 1-914 (FL) 101-914 201-914301-914 401-914 501-914 601-914 701-914 801-914
G 6 G 6 G 6 G 6
1-914 (FL)1-400 301-914301-400 1-300 1-200
Figure 4. Repression of Orc6-Orc1301–400
Binding via the Orc1 N-Terminal Domain
Radiolabeled Orc1 (FL) or its deletion constructs
were expressed in vitro and pulled down by GST-
Orc6 (A; ‘‘6’’ in B) or GST (‘‘G’’ in B). Input (5%) and
bound materials (30%) were visualized.
a pair of blue arrows (Figure 3D; best
viewed in the front and back images).
toward the back of the ORC crescent,
as indicated by a cyan arrow in the top
and back views in Figure 3D. The flip of
Orc1-NTD appears to make room for the
incoming Cdc6, which binds ORC from
the same side. The rotation of the Orc1
main body appears to make better
contact with the incoming Cdc6 (see the
front and the back panels in Figures 3C
and 3D; also see below for docking of
the archaeal Orc1/Cdc6 crystal struc-
ture). Orc1 reorientation also rearranges
Orc4 and pushes Orc4 slightly away
from Orc1 (Figure 3D, black arrow).
Strikingly, upon Cdc6 binding, a small
piece of density crops out toward the
front of the ORC-DNA complex, pro-
truding from the lower lobe where Orc2,
Orc3, and Orc6 reside. The correspond-
ing densities in ORC-DNA and ORC-
DNA-Cdc6 are highlighted in red (Figures
3C and 3D; Movie S1). The identity of this
density is currently unknown. However,
because Orc6 has been determined to
bind on top of Orc2 and is located approximately in this region,
we propose that the protrusion may be part of Orc6. If true, the
central location of Orc6 may enable Orc6 to reach Orc1, which
is located on the opposite end from Orc2 in our model. The
experimental paradox with this idea is that the full-length Orc1
has no significant affinity for GST-Orc6 (Figure 1A). One interpre-
tation is that the flexible Orc1 N-terminal domain, which can be
rotated drastically upon Cdc6 binding to the ORC-DNA complex
(Figures 3C and 3D), may interfere with the binary interaction
between Orc1 and Orc6. To test this possibility, we subjected
a series of deletion constructs of Orc1 to GST-Orc6 pull-down
assays. Deletion of the first 100 or 200 residues (101–914 and
201–914) had no effect on GST-Orc6 binding (Figure 4A). In
contrast, Orc1 lacking the first 300 residues (301–914) showed
interaction with GST-Orc6 (Figure 4A) that was Orc6 dependent
(Figure 4B). Deletion of a further 100 residues (401–914) abol-
ished this binding (Figure 4A), implying that residues 301–400
are important for Orc6 binding. Indeed, a short peptide of Orc1
(301–400) retained affinity for Orc6. Fusion of the N-terminal
300 residues to the short peptide (1–400), however, eliminated
this binding activity (Figure 4B). These results suggest that there
is binary interaction between Orc6 and Orc1301–400that could be
Cryo-EM of ORC-Cdc6 Bound to DNA
538 Structure 20, 534–544, March 7, 2012 ª2012 Elsevier Ltd All rights reserved
has affinity for Orc1, in addition to Orc2 (Figure 1), both of which
are located at opposite ends of ORC in our model. This conclu-
sion lends support to our tentative assignment of Orc6 to the
central protrusion in the ORC-DNA-Cdc6 structure.
Therefore, our cryo-EM study shows that the most prominent
changes in the ORC-DNA structure upon Cdc6 binding are
confined to the rearrangement of Orc1 and probably Orc6.
This observation is in good agreement with previous work
reporting that Cdc6 binding to the ORC-DNA complex increased
the sensitivity of Orc1 and Orc6 to trypsin digestion, whereas
the Orc3, Orc4, and Orc5 subunits were relatively resistant
(Mizushima et al., 2000).
Docking of Archaeal Orc1/Cdc6 Crystal Structures
into the Cryo-EM Map of ORC-DNA-Cdc6
Proteins involved inthe assembly ofthe pre-RC aremostlyAAA+
proteins, many of whose archaeal homolog structures have
been determined by crystallography (Mott and Berger, 2007).
Of particular relevance to the eukaryotic initiators are the
archaeal Orc1/Cdc6 initiator protein that is analogous to the
eukaryotic Orc1 subunit of ORC and the Cdc6 protein, which
are related in amino acid sequence to each other (Dueber
et al., 2007; Gaudier et al., 2007; Liang et al., 1995; Liu et al.,
2000; Singleton et al., 2004). The structure of Pyrobaculum
aerophilum Orc1/Cdc6 in the absence of DNA is in a linear an-
d extended conformation and was proposed to bind DNA
primarily via the C-terminal WHD (Liu et al., 2000) (Figure 5A).
However, the structures of the Aeropyrum pernix and Sulfolobus
solfataricus Orc1/Cdc6-like initiator proteins, either not bound or
bound to DNA, formed a curved, C-shaped conformation with
both the N-terminal ATPase domain and the C-terminal WHD,
interacting with origin DNA when it is present (Figure 5B) (Dueber
et al., 2007; Gaudier et al., 2007; Singleton et al., 2004). In order
to dock the homologous crystal structures, we first segmented
the 3D density map of ORC-DNA-Cdc6 by using the water-
shed-based semiautomatic program Segger (Pintilie et al.,
2010) (Figure 5C). The electron densities of Orc1, Orc4, Orc5,
and Cdc6 are well resolved and separated from their respective
neighbors, and the program Segger was able to demarcate the
boundaries of these four proteins automatically without ambi-
guity. The archaeal structure can be fitted as single rigid body
into the segmented subunit density (Figure S3). However, the
remaining three proteins (Orc2, Orc3, and Orc6) at the lower
lobe are densely packed with little separation, and consequently
Seggerwas unableto automaticallydemarcate their boundaries.
Therefore, the boundaries depicting Orc2, Orc3, and Orc6 are
speculative (Figure 5C). We were unable to trace the 66 bp
DNA. This may be a result of the possibility that DNA density
Archaeal Cdc6Archaeal Orc1
DNA binding regions
Figure 5. Interpretation of the 3D Cryo-EM
Structure of the ORC-DNA-Cdc6 Superas-
(A) Crystal structure of archaeal Orc1/Cdc6 in the
absence of DNA in a cartoon view showing the
three linearly arranged domains (Liu et al., 2000).
(B) Archaeal Orc1 in DNA-binding conformation in
a cartoon view showing the highly curved C-sha-
ped arrangement of the three domains. The DNA
molecule is not shown for clarity. Two pink ovals
mark the approximate DNA-binding regions in the
N-terminal a/b-folded ATPase domain and the
C-terminal WHD (Dueber et al., 2007).
(C) Segmentation of the 3D map. Each subunit is
shown in a different color. Segger (Pintilie et al.,
2010), as implemented in Chimera (Pettersen
et al., 2004), was used to segment the density.
Cdc6 at the side, Orc1 and Orc4 at the top, and
Orc5 in the middle are more separated; their
boundary definition is objective. Orc2, Orc3, and
Orc6 are tightly packed at the bottom lobe, and as
such their boundaries are speculative.
(D) Docking with the highly curved archaeal Orc1/
Cdc6 crystal structure (PDB ID code 2qby) as an
individual rigid body for the segmented Orc1–5
densities (Figure S3). Archaeal Orc1/Cdc6 crystal
structure in linear form (PDB ID code 1fnn) was
docked as a rigid body into the segmented yeast
structure was found, and its density was left un-
docked. The black double arrow in the front view
indicates the distance between the putative
Orc4 arginine finger and the Orc1 nucleotide
See also Figure S3 for how the rigid-body docked
homolog crystal structure fits the segmented
individual subunits Orc1, Orc4, Orc5, and Cdc6.
Cryo-EM of ORC-Cdc6 Bound to DNA
Structure 20, 534–544, March 7, 2012 ª2012 Elsevier Ltd All rights reserved 539
intermingles with protein densities in the EM map at modest
resolution and that bound DNA may be partially flexible and
thus is partially averaged out in the 3D reconstruction.
From the above-described EM density segmentation, it
appears that the large crescent-like ORC structure is actually
formed by a chain of smaller C-shaped protein subunits, Orc1
through Orc5, except for the Orc6 at the front of the lower lobe
(Figure 5C). In the absence of a crystal structure determination
for any of the yeast replication initiators, we used the highly
curved Orc1/Cdc6 structure (Protein Data Bank [PDB] ID code
2qby; Figure 5B) to model Orc1–5 (Figure 5D; Figure S3). We
confirmed with the online protein homology recognition program
Phyre (Kelley and Sternberg, 2009) that S. cerevisiae ORC
subunits 1–5 are indeed orthologs of the archaeal Orc1 struc-
tures (Clarey et al., 2006; Dueber et al., 2007; Gaudier et al.,
Orc1, Orc4, and Orc5 are located but less so in the lower lobe
because of our inability to clearly separate the Orc2 and Orc3
densities, and probably because of the fact that Orc2 and
Orc3 contain extra domains unrelated to the AAA+ and WHD
domains (Figure 5D). Yeast Cdc6, located at the side of ORC,
takes on a nearly linear configuration and bridges the gap
between the two ends of ORC (Figure 5C). Hence, the extended
Orc1/Cdc6 structure (PDB ID code 1fnn; Figure 5A) was used to
model the yeast Cdc6 protein (Liu et al., 2000) (Figure 5D; Fig-
ure S3). The archaeal protein fits well with the segmented
Cdc6 density, with its large ATPase domain contacting Orc1 at
the top and its WHD pointing toward the bottom near Orc3
In summary, five copies of the C-shaped archaeal Orc1 struc-
ture (PDB ID code 2qby) (Dueber et al., 2007) were docked as
individual rigid bodies to the densities assigned to Orc1–Orc5,
with the ATPase domain facing toward the front, the WHD facing
toward the back, and the middle helical domain at the ridge
pointing outward (Figure 5D). We refrained from adjusting the
domains of the archaeal Orc1 structure to improve fitting to the
EM map because the sequence identity between the archaeal
Orc1/Cdc6 and the yeast Orc1–5 subunits is not high, ranging
from 8% for Orc2 to 21% for Orc1. The crystal structure of yeast
Orc1-NTD is known and fits with the assigned density (PDB ID
code 2m4z; Figure 5D; Figure S3; Movie S1) (Zhang et al.,
2002). We note that the backside location of the Orc1-NTD
BAH domain away from the ORC main body is consistent with
the knowledge that deletion of this domain does not affect
in DNA binding but is involved in nucleosome binding and origin
preference within chromatin (Bell et al., 1995; Chen et al., 2008;
Mu ¨ller et al., 2010; Onishi et al., 2007; Zhang et al., 2002).
the arginine finger of the Orc4 ATPase domain faces the putative
nucleotide binding site of Orc1. However, these two sites are too
far apart to enable their cooperative ATP hydrolysis (Figure 5D,
of the crystal structure of the DNA-bound archaeal Orc1/Cdc6
heterodimer, in which one subunit is too distant to activate the
ATPase of the other (Dueber et al., 2007). Thus, our structure
suggests that the Orc1 ATPase is inhibited in the ORC-DNA-
Cdc6 complex and that an additional conformational change in
Orc1 would be required to bring the Orc1 closer to Orc4 in order
for Orc1 ATP hydrolysis to occur. We propose that the required
conformational change of Orc1 may be triggered by ATP hydro-
lysis of Cdc6, which is known to occur before ORC ATPase
activity (Randell et al., 2006). Therefore, the ORC-DNA-Cdc6
complex structure may define the order of ATP hydrolysis,
Cdc6 being the first, followed by Orc1.
An Origin DNA Binding Model as Suggested by the
Putative DNA-Binding Domains of the ORC Subunits
From segmentation and docking of the archaeal homolog
structures into the cryo-EM map, the two ends of each of the
five C-shaped large ORC subunits, namely their WHD and
ATPase domains, all point toward the inside of the crescent-
like ORC structure, with the middle helical domains at the ridge
of the large crescent facing outside (Figure 5D). Based on this
model, the segmented subunit densities were pulled apart to
better visualize their shapes and interconnectivity (Figure 6A).
Both the WHD and ATPase domains in each subunit are pre-
dicted to bind DNA (Dueber et al., 2007; Gaudier et al., 2007).
This structural insight, together with the biochemical data indi-
cating that Orc1–Orc5 all directly bind to origin DNA (Lee and
Bell, 1997), suggests that origin DNA tracks along the interior
found that ARS1 origin DNA is indeed negatively supercoiled
when bound to ORC (S. Mitelheiser and B.S., unpublished
data), similar to DNA wrapping induced by S. pombe and
Drosophila ORCs (Houchens et al., 2008; Remus et al., 2004).
Interestingly, a 72 bp DNA can be modeled along the proposed
interior surface of ORC (Figure 6B; Movie S1). The length of the
model DNA is comparable to the 80 bp DNase I footprint of the
origin DNA in the presence of ORC and Cdc6 (Bell and Stillman,
1992; Speck et al., 2005). Perhaps the most satisfying feature of
our origin DNA binding model is that the DNA enters and exits
ORC at the same side of ORC where Cdc6 binds. This observa-
tion explains the puzzle that although only one Cdc6 binds to
ORC, Cdc6 extends the DNase I footprint of ORC at both ends
of the origin DNA (Figure 6A) (Speck et al., 2005). An earlier
metal-shadowing EM observation of ORC bound to a long
ARS1-containing dsDNA showed that some of the ORC-bound
DNA molecules were indeed highly bent at the ORC binding
site (Chastain et al., 2004).
In this report, we present cryo-EM analyses of eukaryotic ORC
and its complexes with origin DNA and the other replication
initiator, Cdc6. With improved resolution, several of the protein
subunits take on distinctive shapes as compared with the
featureless blobs seen in previously reported negative-stain EM
structures (Clarey et al., 2006; Speck et al., 2005). The improved
resolution allows docking of the single-subunit archaeal ortholog
Orc1/Cdc6 proteins and suggests an emerging view of origin
DNA binding to the highly curved interior surface, but not to the
exterior surface, of the eukaryotic six-subunit ORC.
We have identified the locations and approximate boundaries
of most protein components in the cryo-EM 3D map of the
ORC-DNA-Cdc6 assembly. Identification of these subunits is
based mainly on the improved structural features, a previous
Cryo-EM of ORC-Cdc6 Bound to DNA
540 Structure 20, 534–544, March 7, 2012 ª2012 Elsevier Ltd All rights reserved
subunit-mapping study (Chen et al., 2008), and our in vitro pull-
down data. We also used biochemical knowledge to resolve
ambiguities in distinguishing between Orc4 and Orc5 in the
upper lobe of the ORC structure, because density features and
mapping pattern alone were not sufficient to determine whether
Orc4 or Orc5 follows the largest Orc1 subunit in the top lobe.
Based on the fact that Orc1 ATPase is activated by an arginine
finger in Orc4 (Bowers et al., 2004; Speck et al., 2005), Orc4
was assigned to the density next to Orc1.
The assigned spatial arrangement of these subunits is in good
agreement with DNA crosslinking data showing that Orc1 and
Orc4 bind near the essential A element of ARS1 origin DNA
and that Orc2 and Orc3 bind closer to the B elements (Lee and
Bell, 1997). Importantly, binding of Cdc6 on the side of the cres-
cent adjacent to Orc1 at the top and Orc3 at the bottom of ORC
could explain the peculiar DNase I footprint projection pattern
that extends from both ends of the ORC binding site on ARS1
origin DNA upon Cdc6 binding (Speck et al., 2005; Speck and
Stillman, 2007). We note that in the current ORC-DNA-Cdc6
architecture, Orc1 and Orc3 are located at the two ends of the
tightly packed structure, with Orc6 potentially at the front projec-
ting away from the main structure.
tent with what is known about human ORC (Dhar et al., 2001;
Siddiqui and Stillman, 2007; Vashee et al., 2001), raising the
possibility that all eukaryotic ORCs share a similar architecture.
Indeed, the ScORC and DmORC structures have the same
dimensions, closely resemble each other, and share the essen-
tial features of the half-ring structure and midbody location of
Orc5 (Figure S4). However, the DNA binding mode we propose
for ScORC differs from that proposed for Drosophila ORC
(Clarey et al., 2008). This is not surprising, because the DNA
binding mode of DmORC is known to be different from ScORC:
whereas ScORC recognizes specific origin DNA sequences,
albeit with low binding specificity, DmORC binds DNA with little
sequence specificity, and DmORC was proposed to recognize
the negatively supercoiled DNA topology rather than the DNA
sequence (Remus et al., 2004; Remus and Diffley, 2009). We
also note that the functions of human Orc2 and Orc3 seem to
be swapped compared to ScOrc2 and ScOrc3 in terms of
Orc6 binding (Figure 1) (Siddiqui and Stillman, 2007). As the
molecular masses of HsOrc2 and HsOrc3 are also swapped,
that is, HsOrc2 is smaller than HsOrc3, certain functional switch-
ing between Orc2 and Orc3 might have occurred during
The superhelical arrangement of the bacterial DnaA oligomer
as revealed by crystallography functions to unwind DNA and
can, with the help of DnaC, load the bacterial DnaB helicase
(Erzberger et al., 2006). Upon binding to DNA, the bacterial initi-
ator DnaA locally unwinds the DNA helix to allow assembly of the
helicase. In contrast, ORC loads multiple MCM2-7 double hex-
amers onto the origin, and there is no evidence for duplex DNA
unwinding in the pre-RC. Yeast ORC forms a slightly twisted
and highly curved crescent-like structure that is partially flexible
ence of ATP, particularly at the Orc1 subunit when it binds origin
DNA (Figure 2), which in S. cerevisiae has very weak sequence
specificity and even less specificity in other eukaryotes. Such
a conformational change might increase the affinity of ORC for
the origin DNA or allow efficient Cdc6 binding, where ORC
undergoes even further conformational changes. The Cdc6-
induced ring structure sitting on top of the Orc2-Orc3 stem is
the main feature of the ORC-DNA-Cdc6 superassembly. The
Orc1 N-terminal BAH domain is at the back of the ring. Orc1
BAH is known to interact with either the silencing factor Sir1 at
the silent mating-type loci (Bell et al., 1995; Hsu et al., 2005) or
histones (Onishi et al., 2007). Our observation that the BAH
domain localizes to the back of the ORC-Cdc6-DNA structure
Figure 6. A Model for Replication Origin DNA Recognition by ORC
(A) Individual subunits are pulled apart to reveal the C-shaped structures of
Orc1–Orc5. Cdc6 density is extended. Assuming the yeast ORC subunits bind
replication DNA in a similar manner, that is, each subunit binds DNA with two
of the large crescent-shaped structure of ORC. The DNA path is illustrated by
the dashed orange band.
(B) An atomic model built with 72 bp dsDNA nested at the inner surface of the
crescent-like ORC structure. Such a DNA recognition model satisfies the
general DNA interaction mode, and predicts a highly bent origin DNA onto
which the MCM2-7 helicase will be loaded in the following steps.
See Movie S1. See also Figure S4 for the architectural similarity between
ScORC and DmORC.
Cryo-EM of ORC-Cdc6 Bound to DNA
Structure 20, 534–544, March 7, 2012 ª2012 Elsevier Ltd All rights reserved 541
suggests that it might interact with histones in nucleosomes
without interference with origin DNA binding activity. ORC is
known to interact with and organize nucleosome positioning at
origins of DNA replication (Eaton et al., 2010).
The large conformational changes in ORC induced by Cdc6
binding are probably the physical basis for the molecular switch
that transforms ORC from a recognizer of origin DNA sequences
Orc6 to the front of the ORC-Cdc6 ring to engage Cdt1 that is
bound to the MCM2-7 hexameric helicase (Chen et al., 2007).
The heterohexameric yeast MCM2-7 is a near-symmetric ring
with a similar dimension as the ring formed in the ORC-Cdc6
structure (Speck et al., 2005). At the front surface of ORC-
Cdc6, the initial interaction between Orc6 and Cdt1 would bring
the helicase in close contact with the ORC-DNA-Cdc6 assembly
(Chen et al., 2007), thus inducing subsequent interaction
that results in the eventual loading of the helicase onto the origin
DNA (Evrin et al., 2009; Remus et al., 2009; Tsakraklides and
Bell,2010).Thepathof theDNAwepredict hasbothendsexiting
the ORC-Cdc6 structure adjacent to the Orc1 and Cdc6 ATPase
subunits and on one side of the ring. This is consistent with the
large bend observed in ORC-DNA complexes (Chastain et al.,
Orc6 bound near the B elements in DNA crosslinking experi-
ments (Lee and Bell, 1997), and it has two predicted repeat
domains that are similar in structure to the C-terminal repeats
of the transcription factor TFIIB (Chesnokov et al., 2003). The
C-terminal domain of TFIIB binds promoter DNA in cooperation
with the TATA-binding protein TBP (Tsai and Sigler, 2000), and
TFIIB was recently shown to regulate the closed-to-open
promoter transition when bound to RNA polymerase (Liu et al.,
2010). Because the protrusion in the ORC-Cdc6-DNA structure
is likely to consist of Orc6 (Figure 3C), we suggest that follow-
ing the interaction between Cdt1 and Orc6, the ORC-Cdc6
complex undergoes additional conformational changes to load
the MCM2-7 hexamer and bring the B elements of the origin
nearer to Orc6. In recent studies it is suggested that the two
ment of Cdt1-MCM2-7 to ORC (Labib, 2011; Takara and Bell,
The improved cryo-EM structure of ORC-Cdc6 on origin DNA
is a step forward in determining how this protein machine loads
another AAA+ protein machine onto DNA to mark the location
for initiation of DNA replication in S phase of the cell cycle. As
discussed, the structure suggests an origin DNA binding model,
and provides a framework for understanding pre-RC assembly.
GST Pull-Down Assays
Pull-down assays were performed as previously described (Chen et al., 2008;
Siddiqui and Stillman, 2007) except as follows. GST-Orc6 was cloned and
overexpressed using the pET system (Novagen). Template DNAs for in vitro
transcription/translation of ORC subunits were derived from pCITE-2a(+)
(Novagen) carrying individual full-length ORC genes (Chen et al., 2008);
templates for deletion constructs were prepared using the QuikChange
Site-Directed Mutagenesis Kit (Stratagene) or PCR. The TNT T7 Quick system
for PCR DNA (Promega) was used for transcription/translation when the
PCR-amplified DNAs were templates.
ScORC and Cdc6 were expressed and purified as described (Speck et al.,
2005). The ARS1-containing 66 bp double-stranded DNA was prepared by
PCR.WeincubatedORC at1.9 mg/ml concentrationwith DNAata1:1.2 molar
ratio in a buffer containing 0.5 mM ATPgS for 30 min. The mixture was diluted
to 0.19 mg/ml ORC with a buffer containing 50 mM HEPES/KOH (pH 7.5),
1 mM EDTA, 1 mM EGTA, 1 mM DTT, and 100 mM KGlu. Purified Cdc6 was
added to the diluted ORC-DNA mixture with a molar ratio of 1:1.3 for
ORC:Cdc6 and incubated on ice for an additional 10 min. The solution was
further diluted by 5-fold just before flash-freezing.
The lacy carbon grids (EM Science) were first coated with a thin layer of
continuous carbon film. The grids were glow discharged for 30 s in a vacuum
evaporator (Edwards). A Vitrobot plunge freezer (FEI) was used with the
temperatureset to11?Cand relative humidity set to70%.A 3mldiluted sample
was pipetted onto the EM grids, let sit for 30 s, and blotted, and then the grids
were plunged into liquid ethane to obtain a vitreous sample. Cryo-EM was
carried out with a JEM 2010F TEM transmission electron microscope (JEOL
USA) equipped with a Gatan 626 cryo-specimen holder and a Gatan 4K 3
4K UltraScan CCD camera. Electron micrographs were recorded in low-
dose mode on Kodak SO-163 negative film at a magnification of 60,0003.
To produce adequate contrast for these small particles, we selected regions
of EM grids with very thin ice and used relatively large underfocus values of
3–6 mm. The negative film was developed in Kodak D-19 solution and digitized
with a Nikon Supercool Scanner 8000ED at a step size of 6.35 mm (4,000 dots
per in), corresponding to a pixel size of 1.06 A˚at the sample level.
Image Processing and 3D Reconstruction
All images from ORC, ORC-DNA, and ORC-DNA-Cdc6 samples were compu-
tationally processed in a similar manner. We wrote several python scripts
invoking a series of EMAN commands to facilitate processing a large number
of images (Ludtke et al., 1999). We first produced high-contrast images for
manual selection of particles with a script that processed all scanned raw
images automatically. For each image, the image format was changed from
TIF to MRC, and the image size was shrunk by a factor of 4. Then, low-
pass-filtered (25 A˚) images were calculated. To pick particles based on the
low-pass-filtered images, we used the semiautomatic mode in ‘‘boxer’’ and
manually removed the ‘‘bad’’ particles. The boxer size is 128 3 128 pixels.
For contrast transfer function (CTF) correction, we first made a structure factor
file by ‘‘ctfit’’ using several particle sets at different defoci, and then found
the CTF parameters with ‘‘fitctf’’ and flipped the phase of the images with
‘‘applyctf.’’ Particles from individual images were eventually combined into
one file, contrast inverted, and high-pass filtered.
Although the particles were carefully selected initially, many bad particles
werestill able toenterthedata set. Wesubsequently used reference-freeclas-
sification on the low-pass-filtered and shrunk particles to carry out a second
round of particle selection; particles that did not produce good class averages
were rejected at this stage. For each data set containing more than 100,000
particles, we performed a classification with the class number set to 500.
Raw particles producing well-defined class averages were pooled, and those
that did not were removed. The final number of particles used for projection-
based 3D refinement was 40,000 for ORC, 36,000 for ORC-DNA, and 54,000
for ORC-DNA-Cdc6. Thus, slightly over half of the initially selected particles
Microscopy Data Bank accession number 1156) as the starting model. All
three cryo-EM data sets used this same starting model. This model was deter-
mined previously by the random conical tilting method from negatively stained
EM images (Speck et al., 2005). We first refined the model with the data set at
a reduced sample level (4.24 A˚/pixel) on a Linux workstation with four dual-
core processors. The resulting 3D map was further refined with a finer-
sampled data set (2.12 A˚/pixel). The ‘‘amask’’ option of EMAN’s ‘‘refine’’
command was used to make a mask from the envelope of the model, and
the mask was subsequently applied to the new volume after each refinement
cycle. The Fourier shell correlations of the EM maps were calculated by
‘‘eotest’’ in EMAN. Three-dimensional EM map visualization and docking of
the crystal structures were carried out in UCSF Chimera (Pettersen et al.,
2004). Density segmentation used the semiautomated program Segger
(Pintilie et al., 2010), a program now incorporated into UCSF Chimera. To
maintain objectivity and reproducibility, only program default values were
Cryo-EM of ORC-Cdc6 Bound to DNA
542 Structure 20, 534–544, March 7, 2012 ª2012 Elsevier Ltd All rights reserved
used during segmentation. Subdomains were grouped into putative subunits
based on molecular shape and subunit localization information.
The cryo-EM 3D density map of ScORC-Cdc6-DNA has been deposited in the
Electron Microscopy Data Bank under accession number 5381.
Supplemental Information includes four figures and one movie and can be
found with this article online at doi:10.1016/j.str.2012.01.011.
Chunyan Tang participated in the initial stage of the cryo-EM work. We thank
Sylvain Mitelheiser for suggestions on the in vitro translation of ORC subunits
and Patty Wendel for technical assistance in ORC preparation. This work was
supported by National Institutes of Health grant nos. GM45436 (to B.S.) and
GM74985 (to H.L.) and the United Kingdom Medical Research Council (to
C.S.). H.K. was supported by Postdoctoral Fellowships for Research Abroad
from the Japan Society for the Promotion of Science and the Uehara Memorial
Received: November 1, 2011
Revised: January 16, 2012
Accepted: January 17, 2012
Published: March 6, 2012
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