Concerted loading of Mcm2-7 double hexamers around DNA during DNA replication origin licensing.
ABSTRACT The licensing of eukaryotic DNA replication origins, which ensures once-per-cell-cycle replication, involves the loading of six related minichromosome maintenance proteins (Mcm2-7) into prereplicative complexes (pre-RCs). Mcm2-7 forms the core of the replicative DNA helicase, which is inactive in the pre-RC. The loading of Mcm2-7 onto DNA requires the origin recognition complex (ORC), Cdc6, and Cdt1, and depends on ATP. We have reconstituted Mcm2-7 loading with purified budding yeast proteins. Using biochemical approaches and electron microscopy, we show that single heptamers of Cdt1*Mcm2-7 are loaded cooperatively and result in association of stable, head-to-head Mcm2-7 double hexamers connected via their N-terminal rings. DNA runs through a central channel in the double hexamer, and, once loaded, Mcm2-7 can slide passively along double-stranded DNA. Our work has significant implications for understanding how eukaryotic DNA replication origins are chosen and licensed, how replisomes assemble during initiation, and how unwinding occurs during DNA replication.
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ABSTRACT: Eukaryotic DNA replication initiates from multiple replication origins. To ensure each origin fires just once per cell cycle, initiation is divided into two biochemically discrete steps: the Mcm2-7 helicase is first loaded into prereplicative complexes (pre-RCs) as an inactive double hexamer by the origin recognition complex (ORC), Cdt1 and Cdc6; the helicase is then activated by a set of "firing factors." Here, we show that plasmids containing pre-RCs assembled with purified proteins support complete and semi-conservative replication in extracts from budding yeast cells overexpressing firing factors. Replication requires cyclin-dependent kinase (CDK) and Dbf4-dependent kinase (DDK). DDK phosphorylation of Mcm2-7 does not by itself promote separation of the double hexamer, but is required for the recruitment of firing factors and replisome components in the extract. Plasmid replication does not require a functional replication origin; however, in the presence of competitor DNA and limiting ORC concentrations, replication becomes origin-dependent in this system. These experiments indicate that Mcm2-7 double hexamers can be precursors of replication and provide insight into the nature of eukaryotic DNA replication origins.The EMBO Journal 02/2014; · 9.82 Impact Factor
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ABSTRACT: Graphical Abstract Figure optionsView in workspaceDownload full-size imageDownload high-quality image (160 K)Download as PowerPoint slide Highlights ► Replicative helicase CMG can bypass a lagging strand but not a leading strand roadblock ► This suggests that native CMG translocates along ssDNA in the 3′ to 5′ direction ► MCM2-7 reconfigures from a dsDNA to a ssDNA binding mode during replication initiationCell. 09/2011; 146(6):931–941.
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ABSTRACT: The temporal organization of DNA replication has puzzled cell biologists since before the mechanism of replication was understood. The realization that replication timing correlates with important features, such as transcription, chromatin structure and genome evolution, and is misregulated in cancer and aging has only deepened the fascination. Many ideas about replication timing have been proposed, but most have been short on mechanistic detail. However, recent work has begun to elucidate basic principles of replication timing. In particular, mathematical modeling of replication kinetics in several systems has shown that the reproducible replication timing patterns seen in population studies can be explained by stochastic origin firing at the single-cell level. This work suggests that replication timing need not be controlled by a hierarchical mechanism that imposes replication timing from a central regulator, but instead results from simple rules that affect individual origins.Trends in Genetics 04/2012; 28(8):374-81. · 9.77 Impact Factor
Concerted Loading of Mcm2–7
Double Hexamers around DNA during
DNA Replication Origin Licensing
Dirk Remus,1Fabienne Beuron,2Go ¨khan Tolun,3Jack D. Griffith,3Edward P. Morris,2and John F.X. Diffley1,*
1Clare Hall Laboratories, Cancer Research UK London Research Institute, South Mimms EN6 3LD, UK
2Section of Structural Biology, The Institute of Cancer Research, London SW3 6JB, UK
3Lineberger Comprehensive Cancer Center, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA
The licensing of eukaryotic DNA replication origins,
involves the loading of six related minichromosome
maintenance proteins (Mcm2–7) into prereplicative
complexes (pre-RCs). Mcm2–7 forms the core of the
replicative DNA helicase, which is inactive in the pre-
RC. The loading of Mcm2–7 onto DNA requires the
origin recognition complex (ORC), Cdc6, and Cdt1,
and depends on ATP. We have reconstituted Mcm2–
7 loading with purified budding yeast proteins. Using
biochemical approaches and electron microscopy,
we show that single heptamers of Cdt1?Mcm2–7 are
loaded cooperatively and result in association of
stable, head-to-head Mcm2–7 double hexamers con-
nected via their N-terminal rings. DNA runs through
a central channel in the double hexamer, and, once
loaded, Mcm2–7 can slide passively along double-
stranded DNA. Our work has significant implications
for understanding how eukaryotic DNA replication
origins are chosen and licensed, how replisomes
during DNA replication.
In eukaryotic cells, DNA replication initiates from multiple repli-
cation origins distributed along multiple chromosomes. This
allows cells to replicate large genomes in relatively short periods
of time. However, origin usage must be carefully coordinated to
ensure the genome is completely replicated in each cell cycle,
but no region of the genome is replicated more than once.
A two-step mechanism underpins once-per-cell-cycle replica-
tion in eukaryotes (Bell and Dutta, 2002; Blow and Dutta, 2005;
Diffley, 2004). In the first step, known as licensing, the six-
subunit origin recognition complex (ORC), together with Cdc6
and Cdt1, loads Mcm2–7 onto DNA in a reaction requiring ATP
hydrolysis. Mcm2–7, comprising six related polypeptides, is
believed to be the engine of the replicative helicase but is inac-
tive in this prereplicative complex (pre-RC). The loading of
kinase (CDK) activity is low and the anaphase promoting
complex/cyclosome (APC/C) is active. In budding yeast, CDKs
prevent Mcm2–7 loading by directly phosphorylating and inhib-
iting each pre-RC component. Pre-RCs are activated in S phase
by the combined action of two protein kinases, CDK and the
Dbf4-dependent protein kinase (DDK) comprising a heterodimer
of Dbf4 and Cdc7. CDKs work by phosphorylating Sld2 and
Sld3, which generates binding sites for tandem pairs of BRCT
repeats in Dpb11 (Tanaka et al., 2007; Zegerman and Diffley,
2007), while DDK phosphorylates Mcm2–7 directly (Sheu and
Stillman, 2006). These events together lead to the loading of
Cdc45 and the GINS complex into a preinitiation complex (pre-
IC), which is required for the activation of the Mcm2–7 helicase.
Although we know the order in which initiation factors are
recruited to origins, relatively little is known about the biochem-
ical mechanism of pre-RC and pre-IC assembly and how this
leads to origin unwinding and replisome assembly. To under-
stand these mechanisms in detail, it will be necessary to recon-
stitute these reactions with purified proteins.
Purification of Pre-RC Proteins
We set out to purify pre-RC components for reconstitution
fied from budding yeast cells arrested in G1 phase, a period of
competence for pre-RC assembly in vivo (Piatti et al., 1996)
and in vitro (Seki and Diffley, 2000). ORC was purified from G1
phase cell extracts from a yeast strain that overexpressed all
available online). Because Cdc6 protein is highly unstable during
G1 phase in budding yeast (Drury et al., 2000), it was expressed
in insect cells from a baculovirus vector and purified as an
apparent monomer (Figures 1B and S1C). The purified Cdc6
migrated inSDS-PAGE asaphosphatase-sensitivedoublet (Fig-
ure 1C, lanes 1 and 2), indicating that it is phosphorylated. We
therefore also expressed and purified a mutant Cdc6 lacking
all eight CDK phosphorylation sites (Figure 1C, lanes 3 and 4).
This protein migrated as a fast-migrating single band in the pres-
ence and absence of lambda phosphatase.
Cell 139, 719–730, November 13, 2009 ª2009 Elsevier Inc. 719
terminus with a 3XFLAG epitope and purified from the soluble
(non-chromatin-bound) fraction of G1 phase yeast extracts by
a-FLAG immunoaffinity chromatography followed by Superdex
200 gel-filtration chromatography. Approximately half of the
Mcm4 eluted from the Superdex 200 column in a high-molec-
ular-weight complex that cofractionated with a series of other
polypeptides (Figure 1D). Immunoblot (Figure 1E) and mass-
spectrometric analysis (data not shown) identified the coeluting
polypeptides in the complex as Mcm2, Mcm3, Mcm5, Mcm6,
Mcm7, and Cdt1. The Cdt1?Mcm2–7 complex eluted in the
same fraction as thyroglobulin (670 kDa) (Figure 1D), consistent
(676 kDa). The existence of a stoichiometric complex between
Cdt1 and Mcm2–7 is consistent with earlier biochemical and
Transmission electron microscopy of negatively stained, puri-
fied Cdt1?Mcm2–7 revealed a relatively homogeneous distribu-
tion of globular particles, approximately 15 nm in diameter
(Figure 1F). Reference-free classification of ?10,000 particles
yielded views that we interpret broadly as top/bottom and side
views (Figure 1F). The round top/bottom views feature a central
hole or cavity. Presumptive side views feature two distinct,
parallel layers of protein density (Figure 1F, white arrows in
bottom average panel) separated by a gap and connected by
thin protein bridges. These are similar to views of the homohexa-
thermautotrophicus, which assembles from a single MCM sub-
unit (MthMCM) (Pape et al., 2003). Extra protein density located
(arrowheads, Figure 1F) may be due to the non-MCM Cdt1
subunit. We are currently analyzing these images to determine
void670 kDa158 kDa
12345678910 11 12 13 14
Figure 1. Purification of Pre-RC Proteins
(A and B) Purified ORC (A) and Cdc6 (B) analyzed by SDS-PAGE and Coomassie staining of the gel. Molecular mass markers (kDa) are indicated on the left, and
positions of ORC and Cdc6 are indicated on the right.
(C) Purified Cdc6-wt (lanes 1 and 2) and Cdc6-8A (lanes 3 and 4) were analyzed by SDS-PAGE and Coomassie staining before (lanes 1 and 3) and after (lanes
2 and 4) l-phosphatase treatment.
(D) Gel-filtration analysis of Mcm4-FLAG immunoaffinity purified from whole-cell extract. Fractions were analyzed by SDS-PAGE and silver staining. Elution posi-
tions of molecular size markers (thyroglobulin, 670 kDa; bovine g-globulin, 158 kDa) and the void position are indicated on the top. Gel positions of identified
proteins are indicated on the right.
(E) Western blot analysis of purified Cdt1?Mcm2–7 complex using specific antibodies indicated on the top of each lane.
(F) Transmission electron microscopy of negatively stained purified Cdt1?Mcm2–7.A representative micrograph is shown on the left.Examplesof side views and
top views are encircled in black and white, respectively. Panels on the right show representative reference-free class averages of top/bottom (upper two panels)
or side views (lower two panels). The class averages, resulting from four rounds of SPIDER alignment and IMAGIC classification, correspond to 65, 47, 58, and
55 aligned images (top to bottom). Arrowheads indicate an asymmetric protein appendage. Arrows indicate the two stacked protein layers in the side view.
720 Cell 139, 719–730, November 13, 2009 ª2009 Elsevier Inc.
the 3D structure of Cdt1?Mcm2–7. We conclude from electron-
microscopic (EM) images and gel filtration results that Cdt1 and
Mcm2–7 form a heteroheptameric complex. Significantly, for
the work described herein, double heptamers were never seen.
Reconstitution of Mcm2–7 Loading
To begin to examine pre-RC assembly in vitro, we analyzed the
recruitment of these purified proteins to linear, origin-containing
DNA fragments (1 kb) coupled to paramagnetic beads (DNA
beads). Figure 2A (lanes 1–3) shows that, after incubation with
purified proteins in reactions containing ATP, these DNA beads
(but not beads lacking DNA—data not shown) bind ORC,
Cdc6, Cdt1, and Mcm2–7 subunits. Mcm2–7 loading in vivo
and in extracts has previously been defined by the generation
of Mcm2–7 complexes that remain bound to DNA even after
high-salt treatment (Bowers et al., 2004; Donovan et al., 1997).
of DNA beads from reactions containing ATP quantitatively
removed ORC but not the Mcm2–7 subunits. Mcm2–7 loading
is distinguished from simple recruitment because loading
25 50 100 25 50 100 25 50 100 25 50 100
ORC + Cdc6
1 2 3 45 6 7 89 10 11 12
- + + +
1 2 3 4
17 15 25 4017 15 25 40
5 10 15 20 25 30 35 40
Mcm2, -5, -7 bound
10 20 50
Figure 2. Reconstitution of Mcm2–7 Loading In Vitro
(A) Western blot analysis of DNA-bead-bound fractions of loading reactions performed at a constant concentration of ORC (25 nM) and Cdc6 (50 nM) and indi-
cated concentrations of Cdt1?Mcm2–7. Reactions were performed in presence of 5 mM ATP (lanes 1–6) or 5 mM ATPgS (lanes 7–12). Beads were washed twice
with low-salt buffer (0.3 M K-glutamate; lanes 1–3 and 7–9) or once with low-salt and once with high-salt (0.5 M NaCl) buffer (lanes 4–6 and 10–12). Proteins
analyzed are indicated on the right.
(B) DNA bead association of all six MCM subunits (Mcm2–7) and indicated proteins after loading reactions in the presence of ATP (lanes 1 and 2) or ATPgS (lanes
3 and 4) without (lanes 1 and 3) and with (lanes 2 and 4) high-salt extraction.
(D) Time course analysis of Mcm2–7 loading. ORC and Mcm2–7 binding to DNA beads were monitored at the indicated time points after low-salt (lanes 1–5) or
high-salt (lanes 6–10) wash. Quantified western blot signals for Mcm2, Mcm7, and Mcm5 at the indicated time points after low-salt (closed squares) or high-salt
(open triangles) wash are plotted in the bottom graph relative to loading at 40 min after low-salt wash.
(E) Silver-stained SDS-PAGE gel analysis of loading reactions performed in the presence of 50 nM Cdc6, 50 nM Cdt1?Mcm2–7, and increasing amounts of ORC
as indicated. Visible bands are annotated on the right.
Cell 139, 719–730, November 13, 2009 ª2009 Elsevier Inc. 721
requires ATP hydrolysis by ORC and Cdc6 (Bowers et al., 2004;
Klemm and Bell, 2001; Perkins and Diffley, 1998; Randell et al.,
2006; Seki and Diffley, 2000; Weinreich et al., 1999). When
ATPgS (Figure 2A, lanes 7–12), ORC, Cdc6, Cdt1, and Mcm2–7
subunits were all bound to DNA beads after a low-salt wash;
however, the Mcm2–7 subunits along with ORC and Cdt1 were
all quantitatively removed from DNA beads by high-salt extrac-
tion. Figure 2B shows that all six Mcm2–7 subunits are loaded
onto DNA in a salt-resistant manner in the presence of ATP,
but in the presence of ATPgS, all six Mcm2–7subunits are
removed by high-salt extraction, suggesting that Mcm2–7
subunits remain associated in one complex during recruitment
and after loading onto DNA. From these observations, we
conclude that a salt-resistant, DNA-bound Mcm2–7 complex is
generated upon coincubation of DNA with purified ORC, Cdc6,
and Cdt1?Mcm2–7 in a reaction that requires ATP hydrolysis.
Together, these results show that ORC, Cdc6, and Cdt1 are
sufficient to perform Mcm2–7 loading in vitro, consistent with
previous work (Gillespie et al., 2001; Kawasaki et al., 2006).
Cdt1, along with Mcm2–7, remained associated with the beads
(Figures 2A and 2B). To determine whether these proteins are
actually bound to DNA, we treated the salt-washed beads with
EcoR1, which cleaves the DNA at a site near the biotin-streptavi-
din linkage. Figure 2C shows that Cdc6 and Cdt1 remained
bound to beads after removal of >95% of the DNA with EcoR1.
Mcm2–7, however, were quantitatively removed from beads
with the DNA. Thus, retention of Cdc6 and Cdt1 on DNA beads
is due to nonspecific interactions with the beads after Mcm2–7
loading. By contrast, Mcm2–7 proteins appear to be loaded
directly on DNA. Maximum levels of Mcm2–7 loaded onto DNA
were reached between 25 and 40 min of incubation (Figure 2D),
similar to reported results in extracts (Bowers et al., 2004).
Tofurtherexamine Mcm2–7 loading, wevisualizedDNAbead-
bound proteins by silver staining after SDS-PAGE. Figure 2E
shows that very similar amounts of Mcm2–7 and ORC were
loaded onto DNA during the course of the reaction (lanes 2–4).
In separate experiments, up to 20% of the input Mcm2–7 was
converted into salt-resistant DNA-bound complexes (data not
shown). We conclude that the Mcm2–7 loading reaction with
purified proteins is relatively efficient, unlike the loading of
Mcm2–7 in cell extracts (Seki and Diffley, 2000).
ORC and Cdc6 Act Together to Recruit
and Load Mcm2–7
13), indicating that ORC alone cannot efficiently recruit
Cdt1?Mcm2–7. Similarly, Mcm2–7 subunits were neither re-
cruited nor loaded in the absence of ORC (Figure 3B, lanes 1, 5,
9, and 13). In both Figures 3A and 3B, ATP-dependent Mcm2–7
loading was rescued by titration of the missing component back
into these reactions. These experiments show that ORC and
Cdc6 are both required for recruitment of Cdt1?Mcm2–7 to DNA.
As shown in Figure 1C, the Cdc6 protein used here is at least
partly phosphorylated, presumably by endogenous insect cell
CDKs. Figure 3C shows, however, that loading efficiencies of
Cdc6-wt were indistinguishable from those of Cdc6-8A across
a range of concentrations. Therefore, CDK phosphorylation of
Cdc6 does not directly inhibit Mcm2–7 loading in this system,
consistent with previous work showing that CDK inhibition of
Cdc6 function requires additional protein cofactors, including
Role of DNA Sequence in Mcm2–7 Loading
The initiation of DNA replication in budding yeast, unlike meta-
as specific binding sites for ORC and are also the sites at which
pre-RCs assemble. The A element of the well-characterized
ARS1 contains the essential ARS consensus sequence (ACS),
which, together with the nonessential B1 element, defines the
primary ORC binding site (Bell and Stillman, 1992; Marahrens
and Stillman, 1992; Rao and Stillman, 1995; Rowley et al.,
1995). The B2 element contains a 9/11 match to the ACS and
can bind ORC in the absence of the ACS (Bell and Stillman,
1992). We therefore compared ORC binding and Mcm2–7
loading on wild-type ARS1 and a A?B2?double mutant, which
removes all known ORC binding sites. Surprisingly, Figure 3D
shows that, in the absence of competitor DNA, ORC is recruited
equally well, even at substoichiometric concentrations, to the
wild-type ARS1 and the A?B2?double mutant (Figure 3D, lanes
1, 2, 5, and 6). Figure 3D also shows that Mcm2–7 loading effi-
ciency was indistinguishable between wild-type and the A?B2?
mutant ARS1 in the absence of competitor DNA (lanes 1–8).
Addition of nonspecific competitor DNA strongly suppressed
the binding of ORC as well as Mcm2–7 to both wild-type and
A?B2?mutant DNA (data not shown), and, under these condi-
tions, ORC specifically associated with the wild-type ARS1
DNA (Figure 3D, compare lanes 9 and 10 and lanes13 and 14).
Mcm2–7 loading was also preferentially observed on wild-type
origin DNA in these reactions (Figure 3D, lanes 9–16). These
data suggest that purified ORC, Cdc6, and Cdt1?Mcm2–7 can
mediate sequence-specific Mcm2–7 loading in vitro in the pres-
ence of competitor DNA, yet specific sequences are not mecha-
nistically required for either ORC binding or Mcm2–7 loading.
Mcm2–7 Are Loaded as Double Hexamers
As shown in Figure 2C (lane 4), the loaded Mcm2–7 complexes
can be purified after high-salt wash by removal of the DNA from
ing, the majority of the Mcm2–7 particles present in this fraction
were roughly rectangular and homogeneous in size (150 A˚3
tion of individual particles showed them to be composed of four
parallel layers of protein density (Figure 4C, upper panel), with
height of this four layer Mcm2–7 complex is roughly twice that of
appears to be a double hexamer of Mcm2–7.
To testwhetherthedouble hexamer wastheresult ofenforced
hydrophobic interactions induced by exposure to high salt, we
omitted the high-salt wash and analyzed the entire loading reac-
tion in solution rather than on beads. Reactions were incubated
722 Cell 139, 719–730, November 13, 2009 ª2009 Elsevier Inc.
with free 1 kb DNA fragments and spotted directly onto carbon-
coated copper grids for EM analysis. As shown in the overview
image of Figure 4B, the same rectangular double-hexameric
particles were readily seen against a background of smaller,
more heterogeneous complexes that likely correspond to free
Cdt1?Mcm2–7 complexes and ORC. Image analysis confirmed
that these abundant, larger particles were structurally indistin-
guishable from the Mcm2–7 particles purified after high-salt
wash (Figure 4C, lower panel). Double hexamers were not
detected in reactions that lacked DNA, ORC, or Cdc6. They
were also not detected in complete reactions that were per-
formed in the presence of ATPgS (Figure S2).
These double hexamers are similar to 2D class averages of
electron-microscopic images of double-hexameric assemblies
of the archaeal MthMCM (Costa et al., 2006). In the case of
MthMCM, molecular modeling indicated that the thicker outer
tiers of the double hexamer contain the AAA+ domains, whereas
the two inner tiers correspond to the double-hexameric ring of
the N-terminal domain that mediates hexamer-hexamer associ-
ation. The Mcm2–7 double hexamer appears to have a similar
organization. To obtain independent evidence of the orientation
of the individual hexamers, we took advantage of the FLAG
epitope tag on the Mcm4 C terminus. We performed loading
as in Figure 4A except reactions contained purified a-FLAG
antibody. Figure 4D shows that the extra density resulting from
the antibody labeling of the Mcm4 C terminus was specifically
associated with the outer tiers of the structure, and the sites of
antibody attachment on either end of the double-hexamers
appeared to be spatially related by rotational symmetry. Termi-
nally bound IgG was also observed in cases where only one
IgG was bound to an Mcm2–7 double hexamer or where one
(Figure S3). Thus, Mcm2–7 are loaded as head-to-head double
hexamers with the C-terminal AAA+ domain located in the
thicker tiers on the outside of the double hexamer.
A Three-Dimensional Model of the Mcm2–7
We calculated a 3D reconstruction of the Mcm2–7 double hex-
amer to ?30 A˚resolution from 2292 particles out of a 3700
- 10 20 50
- 10 20 50
- 10 20 50
- 10 20 50
1 2 3 4 5 6 78 9 10 11 12 13 14 15 16
- 10 20 50 - 10 20 50 - 10 20 50 - 10 20 50 [nM]
Cdc6 + Cdt1.MCM2-7
1 2 3 4 56 7 8 9 10 11 12 13 14 15 16
Cdc6 + Cdt1.MCM2-7
25 50 25 50 25 50 25 50 25 50 25 50 25 50
1 2 3 45 6 7 89 10 11 12 13 14 15 16
9 10 116 12
Figure 3. Protein and DNA Sequence Dependencies of Mcm2–7 Loading In Vitro
(A) Loading reactions performed in the absence (lanes 1, 5, 9, and 13) or presence (lanes 2–4, 6–8, 10–12, 14–16) of the indicated amounts of Cdc6. All reactions
contained ORC (25 nM) and Cdt1?Mcm2–7 (50 nM) and either 5 mM ATP (lanes 1–8) or 5 mM ATPgS (lanes 9–16).
(B) Loading reactions carried out in the absence or presence of indicated amounts of ORC. All reactions contained Cdc6 (50 nM) and Cdt1?Mcm2–7 (50 nM) and
either 5 mM ATP (lanes 1–8) or 5 mM ATPgS (lanes 9–16).
(C) Comparison of the loading efficiencies of wild-type Cdc6 (lanes 1–6) and Cdc6-8A (lanes 7–12). Reactions were performed in the presence of 5 mM ATP,
25 nM ORC, and 50 nM Cdt1?Mcm2–7, and Cdc6 protein as indicated.
(D) DNA sequence specificity of Mcm2–7 loading in vitro. Reactions contained 50 nM Cdc6, 50 nM Cdt1?Mcm2–7, 5 mM ATP, and either 25 nM or 50 nM ORC as
indicated. Binding to wild-type ARS1 DNA (lanes 1–4 and 9–12) or A?B2?mutant ARS1 (lanes 5–8 and 13–16) was monitored either in the absence (lanes 1–8) or
presence (lanes 9–16) of 0.8 mg/ml poly(dIdC)?poly(dIdC) and 10 mg/ml BSA.
Cell 139, 719–730, November 13, 2009 ª2009 Elsevier Inc. 723
particles data set (Figure S4). Surface representations of a series
of angular rotations around the long axis of the particles and an
end-on view are shown in Figures 5A–5E. The overall barrel-like
structure (150 A˚3 230 A˚) exhibits four ring-like tiers of protein
density consistent with the density described for the 2D class
agree well with the 2D class averages, supporting the validity of
the 3D map (Figure S4B). The two hexamers are connected by
multiple thin proteinbridgesattheinner andouter circumference
of the N-terminal rings (Figures 5 and S4F). This differs slightly
from the X-ray structure of the double hexameric MthMCM
N-terminal ring, which is connected primarily by interactions
near the inner circumference of the N-terminal rings (Fletcher
et al., 2003). Several eukaryotic Mcm2–7 subunits (Mcm2,
in Archaea, which may contribute to these interhexamer interac-
tions. Similar to the MthMCM hexamer, side openings are found
between the N-terminal and AAA+ rings in each hexamer
i iiiii iv
i iiiii iv
Figure 4. Transmission Electron Micros-
copy of Negatively Stained Mcm2–7 Double
(A) Representative micrograph of DNA-bound
Mcm2–7 complexes eluted from bDNA beads
after high-salt-wash fraction by EcoR1 cleavage.
Rectangular, four-tiered Mcm2–7 double hexam-
ers are encircled.
(B) Micrograph showing proteins of complete
loading reaction performed in solution. Mcm2–7
double hexamers (encircled) are distinguishable
among other particles corresponding to free
Cdt1?Mcm2–7, ORC and Cdc6.
(C) Representative class averages (i–iv) of Mcm2–
7 double hexamers containing ?100 particles
each. Averages in the top row were obtained
from purified particles as in (A), averages in
the bottom row were obtained from particles
zontal arrows mark the four tiers of the Mcm2–7
double hexamer side view; thicker arrows mark
the AAA+ containing outer tiers, thinner arrows
mark the inner N-terminal tiers. Vertical arrows
indicate the path of the central channel.
(D) Class average of ?100 particles of Mcm2–7
double hexamers bound to a-FLAG IgG directed
against the Mcm4 C terminus. Brackets mark
extra density due to bound IgG.
Figure 5. Three-Dimensional Reconstruc-
tion of the Mcm2–7 Double Hexamer
(A–E) Surface representations of a 3D reconstruc-
tion of the Mcm2–7 double hexamer filtered to
30 A˚at the predicted molecular weight for the
Mcm2–7 double hexamer.
(F) Cut-open side view.
724 Cell 139, 719–730, November 13, 2009 ª2009 Elsevier Inc.
(Figures 5A–5D). The end-on view (Figure 5E), the cut-away view
(Figure 5F), and the density sections (Figure S4C) show that
a central channel runs through the entire length of the double
hexamer. Both the cut-away view of the surface-rendered 3D
model and serial density sections through the double-hexameric
structure reveal that the channel bends at the interface between
the two hexameric halves because of off-register stacking of the
hexamers (Figures 5F and S4C).
Mobility of Mcm2–7 Double Hexamers on DNA
The presence of the central channel suggests a path for DNA
through the Mcm2–7 double hexamer, although DNA density
was not directly recovered in the 3D reconstruction. We used
direct mounting and tungsten rotary shadowing of purified,
loaded Mcm2–7 complexes to visualize the DNA associated
with Mcm2–7 double hexamers. Mcm2–7 particles bound to
linear 1 kb DNA fragments (Figure 6A) exhibited similar shape
and dimension (166 ± 15 A˚3 249 ± 13 A˚[n = 75]) to the
Mcm2–7 double hexamer described above. The small increase
sition of tungsten. Very few DNA molecules contained more than
one double hexamer. In specimens where we could unambigu-
to enter and exit from opposite ends of the Mcm2–7 double hex-
amer (Figure 6A). In addition, free DNA molecules exhibited
mean DNA contour lengths that were not significantly different
from those bound by Mcm2–7 double hexamers (318 nm ± 16
nm [n = 30] versus 321 nm ± 31 nm [n = 43], respectively). These
observations are consistent with DNA passing through the
the possibility that Mcm2–7 may also engage DNA by other
mechanisms (Costa et al., 2008).
IfDNApassed throughthiscentral channel,Mcm2–7would be
topologically linked to the DNA. To test this, we asked whether
circularization of the bound DNA prevented dissociation of
Mcm2–7. Figure 6B shows that Mcm2–7 exhibited a half-life on
DNA of only approximately 10 min on 1 kb linear DNA in high-
salt (0.5 M NaCl) buffer (Figure 6B). This could reflect the overall
instability of the complex, or it could reflect the loss of Mcm2–7
from the ends of these short, linear molecules. To distinguish
between these possibilities, we compared residence times of
Mcm2–7 double hexamers on a 1 kb linear DNA template to
a circularized version of the same 1 kb DNA fragment. Strikingly,
Figure 6C shows that Mcm2–7 double hexamers exhibited
a half-life on the circular DNA that greatly exceeded 60 min,
compared to 10 min on linear DNA (Figure 6C, lanes 6–10, and
graph below). Figure 6C also shows that the loss of Mcm2–7
from the linear DNA occurred at the same rate in the presence
or absence of ATP. These data strongly suggest that Mcm2–7
double hexamers can slide off the ends of a linear DNA molecule
2 10 20 40 602 10 20 40 60
0.1 M NaCl
1 2 3 4 5 6 7 8 9 10
10 2030 40 5060
1 2 3 4 5 6 7 8 9 10
2 10 20 40 60 2 10 20 40 60 [min]
10 2030 405060
bound / free
2 10 20 40 60 2 10 20 40 60 [min]
0.5 M NaCl
1020 304050 60
12345678 9 10
i iiiii ivv
Figure 6. Mcm2–7 Double Hexamers Can
Slide on DNA
(A) Mcm2–7 double hexamers bound to linear 1 kb
ARS305-containing DNA visualized after tungsten
rotary shadow casting.
(B) Time course analysis of Mcm2–7 retention on
linear DNA after loading. After the final high-salt
wash of a loading reaction, DNA beads were
resuspended in 25 mM HEPES-KOH (pH 7.6)/
0.5 M NaCl/5 mM Mg(OAc)2/0.02% NP-40/10%
glycerol/1 mM DTT. The suspension was incu-
bated at 30?C and at the indicated time points an
aliquot was removed from the suspension and
DNA-bead-associated fractions (lanes 1–5) and
supernatant fractions (lanes 6–10) analyzed by
western blotting. Quantified western blot signals
are plotted in the graph below (squares, superna-
tant; triangles, DNA bead associated).
(C) Time-dependent Mcm2–7 double hexamer
retention on linear (lanes 1–5) or circular (lanes
6–10) 1 kb ARS305 DNA. DNA beads after
a loading reaction were resuspended in buffer as
in (B) either in the absence (upper panels) or pres-
ence (lower panels) of 5 mM ATP. MCM2, MCM7,
and MCM5 western blot signals in each time
course experiment were quantified, normalized in
each series to the earliest time point, and plotted
in the graph below [closed triangles, circular
DNA (?) ATP; open triangles, circular DNA (+)
ATP; closed squares, linear DNA (?) ATP; open
squares, linear DNA (+) ATP].
(D) As (C), except that Mcm2–7 retention on DNA
was monitored in buffer containing 0.1 M NaCl
instead of 0.5 M NaCl.
(E) Mcm2–7 double hexamers bound to circular
1 kb ARS305-containing DNA visualized as in (A).
Cell 139, 719–730, November 13, 2009 ª2009 Elsevier Inc. 725
in an ATP-independent fashion and are therefore topologically
linked to the DNA. At a more physiological salt concentration
(0.1 M NaCl), the half-life of Mcm2–7 double hexamers on linear
DNA increased to approximately 35 min (Figure 6D, lanes 1–5,
and graph below). This longer half-life on linear DNA is pre-
sumably due to electrostatic interactions between DNA and
Mcm2–7. However, this is still significantly shorter than the half-
life of Mcm2–7 double hexamers on the circular DNA template,
which was greater than 60 min (Figure 6D, lanes 6–10, and graph
below). Again, the rate of Mcm2–7 loss was not affected by the
presence of ATP. Taken together, these experiments show that
Mcm2–7 double hexamers encircle and bind DNA in the central
channel and that, once bound to DNA, the double hexamers
Because this sliding activity might have led to an underesti-
mate of double hexamer formation by EM, we reinvestigated
the binding to circular DNA by EM using rotary shadowing.
Most DNA molecules contained either zero or one double hex-
amer (examples of one double hexamer in Figure 6E, i, ii, and
iii). Although there were DNA molecules with two or three double
hexamers (Figure 6E, iv and v), these accounted for fewer than
5% of the protein-DNA complexes arguing that loading of
multiple double hexamers is unlikely to be processive. The
appearance of relaxed circular DNAbound to Mcm2–7isconsis-
sive single-stranded DNA.
Our results provide the first evidence that ORC and Cdc6 load
the Mcm2–7 proteins from single Cdt1?Mcm2–7 heptamers
into pre-RCs as head-to-head double hexamers. DNA, probably
double stranded, passes through the long, central channel of
this double hexamer. And, once loaded, the double hexamer
is mobile, capable of passive one-dimensional diffusion or
‘‘sliding’’ along DNA. These features of the pre-RC have implica-
tions for how origins are chosen and how replisomes assemble
ATP, consistent with requirements in vivo. The requirement for
Cdt1 was not tested because it is an integral component of the
Mcm2–7 complex. The interaction of Cdt1 with both Mcm2–7
and Orc6 (Chen et al., 2007) suggests that it may act as a bridge
between ORC and Mcm2–7. However, our results demonstrate
that additional interactions are involved in this recruitment.
Surprisingly, loading of Mcm2–7 in vitro does not require
specific ORC binding sites. Our results may contribute to
resolving the long-standing issue of how orthologs of ORC can
act on specific DNA sequences in yeast, but show little or no
sequence preference in metazoans (Gilbert, 2004). Our results
indicate that even yeast ORC has no inherent mechanistic
requirement for specific DNA sequences in the loading of
Mcm2–7. The sequence specific DNA binding of the budding
yeast ORC may be an evolutionary adaptation designed to
ensure sufficient origin activity in a genome containing very little
intergenic DNA (Brewer, 1994). Sequence specificity appears to
be an integral part of the S. cerevisiae core ORC while sequence
specificity of Schizosaccharomyces pombe ORC is conferred by
an extended AT hook domain on the Orc4 subunit (Chuang et al.,
2002; Kong and DePamphilis, 2001; Lee et al., 2001). Recruit-
ment of ORC in metazoans may also involve interactions with
additional sequence specific DNA binding proteins like TRF2
(Atanasiu et al., 2006; Deng et al., 2007; Tatsumi et al., 2008).
Consistent with this idea, recruitment of ORC to a GAL4 DNA
binding site array via fusion of ORC subunits or Cdc6 to the
GAL4 DNA binding domain is sufficient to create a functional
replication origin in human cells (Takeda et al., 2005).
In E. coli, the ATP-bound DnaA initator binds to 9-mer
sequences in oriC causing localized DNA unwinding in adjacent
13-mersequences (Bramhill and Kornberg,1988). ThetwoDnaB
helicase hexamers are then recruited to load around the
unwound 13-mer strands by slightly different mechanisms (Mott
et al., 2008). Unlike DnaA binding, ORC binding does not induce
any detectable DNA unwinding. Thus, the eukaryotic initiator
to encircle. Moreover, previous analysis of yeast origins in vivo
failed to detect any KMnO4-reactive DNA in G1-arrested cells
(Geraghty et al.  and data not shown), indicating that initial
but instead occurs when the helicase is activated during S
phase.Consistentwith this,theability ofloaded Mcm2–7to slide
freely on double-stranded DNA in an ATP-independent manner
(Figures 6B–6D) indicates that Mcm2–7 loaded in vitro encircles
double-stranded, not single-stranded, DNA. Delay of the gener-
ation of single-stranded DNA within the Mcm2–7 double hex-
amer until S phase may protect origin DNA from damage during
The loading of the double hexamer appears to be highly coop-
erative. Double Cdt1?Mcm2–7 heptamers were never seen prior
to loading, and single Mcm2–7 hexamers were never detected
on DNA after loading. These results indicate that the two hexam-
ers are loaded together in a concerted reaction. The stoichiom-
etry of ORC and Cdc6 in this loading reaction is presently
unknown. Although yeast origins generally contain a single
high-affinity ORC binding site, a second potential ORC binding
site resides within the B2 element of ARS1 (Wilmes and Bell,
2002). Additionally, archaeal replication origins, which may
initiate replication by mechanisms similar to eukaryotes, gener-
ally have ORC binding sites arranged symmetrically around an
A/T rich region (Wigley, 2009). The lack of stringent sequence
requirement suggests that a second ORC binding site need not
be a specific sequence element. We note that this concerted
loading reaction requires the coordinated breaking and reform-
ing of four separate rings, two in each hexamer. Further work
will be required to elucidate the mechanism of this complex
loading reaction and to determine the functionality of the loaded
Mcm2–7 double hexamer.
The binding of Mcm2–7 around double-stranded DNA has
implications for how DNA unwinding is ultimately catalyzed by
the Cdc45/Mcm2–7/GINS (CMG) complex (Moyer et al., 2006).
SF3 initiator/helicases including the SV40 large T antigen (TAg)
and the papillomavirus E1 protein. The TAg double hexamer
can bind to double-stranded DNA, and this binding can induce
726 Cell 139, 719–730, November 13, 2009 ª2009 Elsevier Inc.
within one of the two hexamers (Borowiec and Hurwitz, 1988).
Although TAg and E1 can assemble as double hexamers around
double-stranded DNA (Dean et al., 1992; Fouts et al., 1999; Liu
et al., 1998), current models indicate that they act during
unwinding as classical helicases by encircling single-stranded
DNA (Enemark and Joshua-Tor, 2008; Schuck and Stenlund,
2005). If Mcm2–7 act analogously to these proteins, then CDK-
and DDK-dependent events must promote remodeling of the
Mcm2–7 complex to encircle single-stranded DNA during origin
Alternatively, Mcm2–7 may act during replication as a double-
strand DNA translocase (Laskey and Madine, 2003). In this
model, Cdc45 and/or GINS would play a direct, structural role
in strand separation, perhaps acting as a ‘‘plough’’ or ‘‘pin’’
into which DNA is pumped by Mcm2–7 (Takahashi et al.,
2005). This is analogous to the bacterial RuvAB Holliday junction
branch migrating enzyme in which two RuvB hexamers pump
double-stranded DNAthrougha tetramer of RuvA, which coordi-
nates the separation and reannealing of strands (West, 2003).
topological reorganization of Mcm2–7 subunits during initiation
and because it provides a potential biochemical function for
Cdc45 and/or GINS during replication. The helicase activity of
archaeal MCM (Chong etal.,2000; Kelman etal., 1999;Shechter
etal., 2000) aswell aseukaryotic Mcm2–7 complexes (Bochman
and Schwacha, 2008; Ishimi, 1997; Moyer et al., 2006) on single-
stranded DNA substrates need not reflect their mode of action
in vivo: even double-stranded DNA translocases like RuvB can
function in standard helicase assays (Tsaneva et al., 1993),
presumably because they can translocate along one strand of
DNA and displace annealed oligonucleotides.
By analogy to the mode of action of both SV40 large T antigen
and RuvB, the orientation of the Mcm2–7 hexamers suggests
that DNA is translocated from the AAA+ domains toward the
N-terminal domains in each hexamer (Li et al., 2003; VanLoock
et al., 2002; Wessel et al., 1992; West, 2003). Consistent with
this, the archaeal MCM hexamer from Sulfolobus solfataricus
(SsoMCM) can be loaded onto the 30single-stranded overhang
of a forked substrate, with its AAA+ domain facing the fork
(McGeoch et al., 2005).
The purified replisome progression complex (RPC), a protein
assemblage containing the CMG complex as well as other repli-
some components, contains just a single copy of Mcm4 (and
presumably the other Mcm2–7 subunits) (Gambus et al., 2006).
Thus, the functional unit of Mcm2–7 in elongation may be the
single hexamer. We note that DDK, required for helicase
activation, acts on N-terminal tails of Mcm2, Mcm4, and Mcm6
(Sheu and Stillman, 2006). Moreover, the mcm5-bob1 allele,
which bypasses the requirement for DDK, is a mutation in the
N-terminal domain of Mcm5 near the interface between hexam-
ers (Fletcher et al., 2003). We speculate that DDK may activate
the helicase by inducing separation of the two hexamers and
the mcm5-bob1 allele may mimic this by destabilizing interac-
tions between hexamers.
The length of an Mcm2–7 double hexamer (230 A˚) corre-
sponds to approximately 68 bp of B form DNA. This is similar
tothesizeofthepre-RCfootprint atseveralorigins invivo(Diffley
et al., 1994; Santocanale and Diffley, 1996). However, based on
data from Donovan et al. (1997), the equivalent of approximately
five double hexamers are loaded per origin in yeast. The pre-RC
footprint cannot account for this number of double hexamers.
Indeed, the loading of multiple Mcm2–7 complexes per origin
appears to be a common feature in eukaryotes (Hyrien et al.,
2003). Once loaded, Mcm2–7 double hexamers may slide
away from origins, allowing repeated loading. Although multiple
Mcm2–7 complexes are loaded per origin, there is currently no
evidence that this loading is processive and our results indicate
that loading in vitro is distributive. Additional activities would
presumably be required to allow the double hexamers to slide
past nucleosomes bordering origins. Since ORC and Cdc6 can
promote sequence-independent loading of Mcm2–7 (Figure 3D),
it isalso possible thatsomeMcm2–7isloadedoutside oforigins,
perhaps at random locations in vivo, which might not have been
detectable in ChIP-chip experiments (Wyrick et al., 2001). These
extra Mcm2–7 complexes may act as ‘‘spare’’ origins to rescue
stalled replication forks (Ge et al., 2007; Woodward et al., 2006).
The ability of the double hexamer to slide on double-stranded
DNA provides a mechanism by which supernumerary Mcm2–7
double hexamers may be pushed ahead of the replisome during
normal elongation. These excess double hexamers could play
a role in restoring an active helicase at stalled forks. This might
provide an important mechanism for replication restart that
does not require the reloading of soluble Mcm2–-7 and, there-
fore, does not compromise mechanisms designed to ensure
once per cell cycle replication.
Detailed expression and purification protocols are included in the Supple-
ORC was purified from alpha factor-arrested budding yeast strain YDR11,
which overexpresses extra copies of all six ORC subunits using the GAL1,10
inducible promoter. The Orc1 subunit was fused to a modified version of the
C-terminal TAP-tag, which is described in the Supplemental Experimental
Procedures. ORC was purified by calmodulin affinity chromatography, fol-
lowed by proteolytic removal of the tag and conventional column chromatog-
raphy with Superdex 200 gel filtration and successive MonoQ and MonoS
Cdt1?Mcm2–7 complex was purified from alpha factor-arrested budding
yeast strain YDR17 via a C-terminal 3xFLAG affinity tag fused to endogenous
Mcm4. Affinity-purified Mcm4-FLAG and associated proteins were further
fractionated by gel filtration through a Superdex 200 column.
Recombinant wild-type and mutant Cdc6 proteins were purified via
an N-terminal 6xHis tag with a baculovirus overexpression system. After
Ni2+-chelate column chromatography, the proteins were further purified by
Superdex 200 gel filtration.
Linear DNAs containing ARS1 or ARS305 were generated by PCR as
described in the Supplemental Experimental Procedures. Circular ARS305-
containing DNA was prepared as in Pacek et al. (2006).
Loading reactions were performed in 40 ml of 25 mM HEPES-KOH (pH 7.6)/
0.1 M K-glutamate/0.02% NP-40/10 mM Mg(OAc)2/5% glycerol/1 mM DTT
and 5 mM ATP or ATPgS. DNA beads were included at 1 pmol (25 nM) of
DNA molecules. Reactions were mixed on ice and incubated at 30?C. Beads
were washed once with 0.4 ml low-salt wash buffer (25 mM HEPES-
KOH [pH 7.6]/0.3 M K-glutamate/0.02% NP-40/5 mM Mg(OAc)2/1 mM
Cell 139, 719–730, November 13, 2009 ª2009 Elsevier Inc. 727
EDTA/1 mM EGTA/10% glycerol/1 mM DTT) and once with 0.4 ml high-salt
wash buffer (as low-salt buffer, but 0.5 M NaCl instead of 0.3 M K-glutamate).
Mcm2–7-DNA complexes were purified from a standard loading reaction,
followed by cleavage of the DNA in 40 ml of 25 mM HEPES-KOH (pH 7.6)/
0.1 M K-glutamate/0.02% NP-40/5 mM Mg(OAc)2/5% glycerol with EcoR1
for 25 min at 30?C. IgG-labeled Mcm2–7 double hexamers were purified
similartounlabelled complexes,except that1mga-FLAGM2antibody (Sigma)
was added to a loading reaction 10 min before the wash.
Sample preparation and image processing are summarized as follows with
details given in the Supplemental Experimental Procedures. Molecular images
of negatively stained Cdt1?Mcm2–7 and Mcm2–7 double hexamers were
selected from digitized micrographs. Sets of two-dimensional averages
were produced by iterative reference free alignment and classification with
EMAN (Ludtke et al., 1999), Imagic (van Heel et al., 2000), and Spider (Frank
et al., 1996) procedures. A number of the Mcm2–7 double hexamer refer-
ence-free class averages were found to possess either 2-fold rotational
symmetry or mirror symmetry, characteristic of orthogonal projections of
a 3D complex with C2 point group symmetry. An initial 3D model of the
Mcm2–7 double hexamer was calculated by back projection of these orthog-
onal views. The 3D structure was used as an initial reference for 3D refinement
using C2 symmetry.
For rotary shadowing, Mcm2–7 complexes bound to linear DNA were eluted
from beads with EcoR1 in 25 mM HEPES-NaOH (pH 7.6)/50 mM NaCl/5 mM
Mg(OAc)2/5% glycerol. Mcm2–7 complexes bound to circularized DNA
were purified from loading reactions in solution by gel filtration on Superose
6 in EcoR1 digestion buffer. The sample was prepared as described (Griffith
and Christiansen, 1978) with modifications (Supplemental Experimental
Supplemental Data include Supplemental Experimental Procedures and
four figures and can be found with this article online at http://www.cell.com/
This work was funded by Cancer Research UK (D.R., J.F.X.D., F.B., and
E.P.M.), the Institute of Cancer Research (F.B. and E.P.M.), and National Insti-
tutes of Health grants to J.D.G. (GM31819 and ES13773). D.R. was supported
by European Molecular Biology Organization long-term fellowship. We are
grateful to Dale Wigley for help and advice in the early stages of this project
and to Paula da Fonseca for advice on image processing. We thank Mark Ske-
hel and colleagues for mass spectrometry and Karim Labib for antibodies. We
are also grateful to members of the Diffley laboratory for helpful discussions.
Received: June 11, 2009
Revised: August 5, 2009
Accepted: September 24, 2009
Published online: November 5, 2009
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