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Isolation of the Cdc45兾Mcm2–7兾GINS (CMG) complex,
a candidate for the eukaryotic DNA replication
fork helicase
Stephen E. Moyer, Peter W. Lewis, and Michael R. Botchan*
Department of Molecular and Cell Biology, Division of Biochemistry and Molecular Biology, University of California, Berkeley, CA 94720
Edited by Bruce W. Stillman, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, and approved May 22, 2006 (received for review March 23, 2006)
The protein Cdc45 plays a critical but poorly understood role in the
initiation and elongation stages of eukaryotic DNA replication. To
study Cdc45’s function in DNA replication, we purified Cdc45
protein from Drosophila embryo extracts by a combination of
traditional and immunoaffinity chromatography steps and found
that the protein exists in a stable, high-molecular-weight complex
with the Mcm2–7 hexamer and the GINS tetramer. The purified
Cdc45兾Mcm2–7兾GINS complex is associated with an active ATPdependent DNA helicase function. RNA interference knock-down
experiments targeting the GINS and Cdc45 components establish
that the proteins are required for the S phase transition in Drosophila cells. The data suggest that this complex forms the core
helicase machinery for eukaryotic DNA replication.
Drosophila 兩 ATPase
A
lthough in recent years there has been significant progress
in our understanding of the molecular mechanisms of
eukaryotic DNA replication, the identity of the primary activity
that unravels duplex DNA at the growing fork has remained
unclear. Based on a range of indirect data, there is widespread
support for the idea that the Mcm2–7 hexamer constitutes part
of the replicative helicase (1). Each of the genes is essential, and
five of six Mcm2–7 proteins have been shown to be required for
both DNA replication initiation and elongation in vivo in Saccharomyces cerevisiae (2, 3). Assays with Xenopus cell-free extracts have shown that all Mcm2–7 proteins are required for
chromosomal DNA unwinding and that Mcm2, Mcm5, and
Mcm7 localize to sites of DNA unwinding on a plasmid (4–6).
The primary structure of the proteins places each in the helicase
supergroup of the AAA⫹ family, and archaeal forms do show
robust helicase function (7–9). Curiously, the purified Mcm2–7
complex has not displayed helicase activity in vitro; in contrast,
the subcomplex of Mcm4, Mcm6, and Mcm7 does (10–12).
Moreover, the activity of this subcomplex is inhibited by Mcm2,
Mcm3, or Mcm5 (10, 11, 13, 14). Intricate models involving
continuous assembly and rearrangements of Mcm proteins at the
replication fork could somehow accommodate these findings. A
simpler hypothesis posits that other proteins are required to
activate the Mcm 2–7 complex for helicase function.
The protein Cdc45 has emerged as a pivotal factor in the G1
to S phase transition and as a possible helicase cofactor (4, 6, 15,
16). CDC45 is an essential gene product in yeast (17) and
engages proteins assembled in the prereplication complex at
origin sites on the DNA during S phase as origins are activated
(16, 18). Further, in Xenopus egg extracts, Cdc45 associates with
sites of DNA unwinding on a plasmid (6), and interfering
antibodies directed at Cdc45 abolish chromosomal unwinding
(4). There is a conserved genetic interaction between Cdc45 and
the Mcms; specific mutations in these genes can either suppress
or show synthetic lethality with the other (19–21). By coimmunoprecipitation兾immunoblot experiments, Cdc45 has been
shown to associate with a variety of other DNA replication
proteins, including DNA polymerase ␣ (22, 23), various Mcm2–7
proteins (16, 23, 24), Mcm10 (25), Orc2 (26), Sld3 (27), and Sld5
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(28). Many of these interactions may be important but transient
or indirect and may obscure a core function or complex within
which Cdc45 works.
Results
Purification and Identification of the Cdc45兾Mcm2–7兾GINS (CMG)
Complex. A protocol to purify a high-molecular-weight complex
containing the Cdc45 protein from Drosophila embryo extracts
is outlined in Fig. 1A (see Materials and Methods and see also
Table 1 and Supporting Text, which are published as supporting
information on the PNAS web site, for additional details). Final
purification used immunoprecipitation (IP) of Cdc45 protein
from the peak Cdc45-containing fractions of the Mono Q
column and elution from the antibody resin with buffer (pH 2.5).
Before elution, the material in the IP was extensively washed
with buffer containing 1 M KCl and treated with ethidium
bromide and MNase to remove any contaminating DNA. As
shown in Fig. 1B and Fig. 6, which is published as supporting
information on the PNAS web site, Cdc45 coimmunoprecipitates
with 10 other major proteins. The identity of the copurifying
proteins was determined by two methods: (i) mass spectroscopy
of individual protein bands cut out from the gel (see List 1, which
is published as supporting information on the PNAS web site)
and (ii) immunoblotting of the eluate with protein-specific
antibodies.
The 10 binding partners of Cdc45 were found to be the six
proteins of the Mcm2–7 complex and CG14549, CG9187,
CG18013, and CG2222, which BLAST searches revealed are the
Drosophila homologs of the four members of the GINS complex
(Sld5, Psf1, Psf2, and Psf3, respectively) (Fig. 1B). We also
performed mass spectroscopy analysis on the total protein pool
obtained after an anti-Cdc45 IP from the Cdc45-containing
DEAE fractions from the protocol described in Fig. 1 A (see
Table 2, which is published as supporting information on the
PNAS web site). The only DNA replication proteins identified
by this method were also Cdc45, Mcm2–7, and GINS.
Sld5, the founding member of the GINS complex, was first
uncovered in a S. cerevisiae synthetic lethal screen with Dpb11
(29). Dpb11 was itself genetically isolated in a suppressor screen
for a mutant in a subunit of DNA polymerase ␧ (30). The Psf
genes are each partners of Sld5 and are required for DNA
synthesis in yeast and in Xenopus cell-free DNA replication
extracts (28, 29). The GINS proteins form a tetramer, and it was
recently shown that Sld5 localizes to sites of DNA unwinding on
Conflict of interest statement: No conflicts declared.
This paper was submitted directly (Track II) to the PNAS office.
Freely available online through the PNAS open access option.
Abbreviations: CMG, Cdc45兾Mcm2–7兾GINS; IP, immunoprecipitation; RNAi, RNA interference.
*To whom correspondence should be addressed at: Department of Molecular and Cell
Biology, University of California, 16 Barker Hall, Berkeley, CA 94720. E-mail: mbotchan@
berkeley.edu.
© 2006 by The National Academy of Sciences of the USA
www.pnas.org兾cgi兾doi兾10.1073兾pnas.0602400103
a plasmid in Xenopus egg extracts (6), suggesting that all four
GINS proteins may be present in a DNA helicase complex.
Examination of the Superdex-200 column fractions from Fig.
1 A shows that the majority of the Cdc45 protein in these extracts
exists as a free, low-molecular-weight pool (see Fig. 1C and Fig.
7, which is published as supporting information on the PNAS
web site). However, only discrete high-molecular-weight fractions contain a complex that shows an interaction among Cdc45,
the GINS members, and the Mcm proteins. Fig. 1C shows an
immunoblot of the material eluted from an anti-Cdc45 IP from
each Superdex-200 chromatography fraction. The size of the
complex as estimated from the elution position on the column
closely matches the sum of the individual components (see
Discussion). We were not able to reform the high-molecularweight complex by mixing of lower molecular-weight fractions,
which contain an excess of the components (see Fig. 7). We
estimate that ⬇5% of the total Cdc45 and GINS proteins and
⬇1% of the total Mcm proteins in our extracts are in this
complex. These results suggest a role for specific protein modifications that would allow for such associations and would
support a previous report that the Cdc45 protein does not
interact with individual Mcm2–7 proteins (31).
Moyer et al.
To further explore the notion that a discrete 11-member CMG
complex exists, we embarked on independent purification methods. We prepared 0- to 12-h embryo extracts from Drosophila
expressing a functional, FLAG-tagged version of Mcm6 (32). A
single-step affinity purification of FLAG-Mcm6 from these flies
captures mainly FLAG-Mcm6 in complex with Mcm2, Mcm4,
and Mcm7 (Fig. 2A Left). This result was anticipated because
subcomplexes of the Mcm proteins are in vast (⬎100-fold) excess
to the high-molecular-weight complex we had uncovered. IP of
Cdc45 from the purified FLAG-Mcm6 material yielded the
complete CMG complex (Fig. 2 A Right). In another set of
experiments, we fractionated embryo extract, following the
high-molecular-weight Cdc45 through the Mono Q step as in Fig.
1 A, and with antibodies directed against Mcm5, Sld5, Psf2, and
Psf3, asked whether the individual reagents would in turn
precipitate the proteins identified by previous methods. Fig. 2B
shows a Deep Purple stain of the material eluted from the
anti-Psf2 IP. Cdc45 and the complete Mcm2–7 complex coimmunoprecipitated with the GINS complex, and immunoblots of
the material eluted from each IP (see Fig. 8, which is published
as supporting information on the PNAS web site) confirmed the
tight association of each of the 11 members of this complex.
Based on these multiple lines of evidence, we conclude that
Cdc45, Mcm2–7, and GINS form a stable, high-molecular-weight
biochemical unit, which we refer to as the CMG complex. An
immediate question was whether the complex contained an
active form of the hypothetical Mcm2–7 helicase.
Purification of the CMG Complex by Peptide Elution from an Affinity
Resin. We attempted to purify the CMG complex to homogeneity
by conventional chromatography but found that the only bioPNAS 兩 July 5, 2006 兩 vol. 103 兩 no. 27 兩 10237
BIOCHEMISTRY
Fig. 1. Purification of the CMG complex. (A) Purification schematic. See the
supporting information for additional details. (B) Material eluted from the
anti-Cdc45 IP after the Mono Q step was separated by SDS兾PAGE and visualized by Deep Purple stain. For improved resolution of individual bands, the
image is composed of the eluate separated by two different acrylamide gels:
9% (top of gel to IgG) and 12% (IgG to bottom of gel). The complete lane of
each gel used for this image is shown in Fig. 6. All subsequent gels in this study
are 10% acrylamide and contain all proteins in one complete lane. (C) At the
Superdex-200 step shown in A, each fraction was subjected to an anti-Cdc45
IP, and the individual eluates were separated on SDS兾PAGE and immunoblotted with antibodies against the indicated proteins. The antibodies have
variable staining intensity; the ratio of all CMG proteins to each other in the
high-molecular-weight fractions is the same (see Figs. 1B, 2, and 6).
Fig. 2. IPs with antibodies against different CMG complex members. In this
figure, the proteins were identified by immunoblot and Rf value. (A) (Left)
FLAG-Mcm6 material purified from embryo extracts by anti-FLAG chromatography. (Right) From the material first purified by the anti-FLAG chromatography, an
anti-Cdc45 IP and a control (rabbit IgG) IP were performed. The anti-Cdc45
immunoprecipitated material shown here is enriched for Cdc45 ⬇70-fold over the
purified FLAG-Mcm6 material (i.e., ⬇700 ␮l of the material in Left was used to
obtain 10 ␮l of the material in Right). Both gels were stained with Deep Purple.
(B) Peak Cdc45-containing fractions from the Mono Q step were precipitated with
anti-Psf2 antibodies and control antibodies (anti-FLAG), and the eluates were
stained with Deep Purple.
Fig. 3. The CMG complex is a helicase. (A) Schematic of the purification protocol used for the helicase assay. (B) Illustration of helicase substrate (not to scale).
(C) (Top) PhosphorImager picture of helicase assays with anti-Cdc45 peptide B-purified material from each Mono Q fraction. (Middle) Quantitation of the
percentage of primers displaced from duplex DNA in each helicase reaction. (Bottom) Immunoblot with antibodies against indicated proteins of the anti-Cdc45
peptide B-purified material from each Mono Q fraction. The Cdc45 protein peak in fractions 12–15 is monomeric Cdc45 that has copurified with the CMG complex
until this chromatography step. (Left) Deep Purple stain of anti-Cdc45 peptide B-purified material from fraction 20. (D) (Left) Immunoblots of material that has
been immunodepleted with anti-Psf2, anti-Mcm5, or control antibodies. (Right) PhosphorImager picture of helicase assays with protein purified by anti-Cdc45
peptide B IPs from material previously depleted with anti-Psf2, anti-Mcm5, or control antibodies. (E) (Left) Helicase assays with material mock-eluted or Cdc45
peptide B-eluted from anti-Cdc45 peptide B IPs from purified FLAG-Mcm6 material. (Center) Helicase assays with and without ATP with CMG complex isolated
from purified FLAG-Mcm6 material. (Right) Helicase assays with CMG complex purified as in A and with varying concentrations of ATP.
chemical step that accomplished this purification was an antiCdc45 affinity resin. Thus, to assay the CMG complex for
helicase activity, we sought to release the CMG complex from
antibodies with a gentle elution method that did not abolish any
intrinsic enzymatic function. We searched for peptide-specific
anti-Cdc45 antibodies and explored releasing the intact CMG
complex from the anti-Cdc45 antibody beads by peptide elution
(see Fig. 9A, which is published as supporting information on the
PNAS web site). We selected two peptides from a region of
Cdc45 that is hydrophilic and predicted to have no secondary
structure (see Fig. 9B). Purified antibodies specific to peptide B
can immunoprecipitate Cdc45 protein and release Cdc45 when
challenged with excess free peptide B in a neutral buffer (see Fig.
9C). Importantly, the anti-Cdc45 peptide B antibodies can also
bind and release Cdc45 protein that is in the CMG complex (Fig.
3C), indicating that this region is on the surface of the Cdc45
protein and that it is exposed on both free Cdc45 protein and
Cdc45 protein in the CMG complex.
Identification of CMG-Associated Helicase Activity. We used the
anti-Cdc45 peptide B-specific antibodies to purify the CMG
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complex according to the procedure outlined in Fig. 3A and
asked whether the purified material had helicase activity. The
substrate used contains a 40-bp duplex region with a short tail
annealed to a single-stranded circle (Fig. 3B). Fig. 3C shows that
helicase activity peaks with the protein peak of the purified
CMG complex. Fig. 3C Left shows a Deep Purple stain of the
material purified by this protocol from Mono Q fraction 20,
indicating the presence of the complete CMG complex.
Immunodepletion of column fractions with specific antisera
raised to recombinant proteins showed that the activity we had
assayed above was directly associated with the CMG complex.
We pooled material from Mono Q fractions 18–21, divided it
into three portions, and then immunodepleted it with specific
antibodies. The antibody beads were all prepared in the same
buffer to minimize the possibility of a nonspecific inhibitor being
present with any of the antibody resins. Fig. 3D Left examines the
extent of depletion in the material treated with control, antiPsf2, or anti-Mcm5 antibodies. We were able to immunodeplete
Psf2 protein to levels below detection and to at least 50% of the
starting level for Mcm5 protein. From the immunodepleted
Moyer et al.
material, we attempted to purify the CMG complex with antiCdc45 peptide B beads, and the respective eluted proteins were
tested for helicase activity. Fig. 3D Right shows that the Psf2depleted material contains no Cdc45-associated helicase activity, and the Mcm5-depleted material has reduced Cdc45associated helicase activity, consistent with the reduction, but
not complete removal, of Mcm5 protein from the starting
protein pool. The material depleted with control antisera
showed robust Cdc45-associated activity. These results demonstrate that the helicase activity observed in Fig. 3C is associated
with the CMG complex and that Psf2 and Mcm5 are components
of the active helicase complex.
Helicase activity was also identified in CMG complex isolated
from purified FLAG-Mcm6 material (Fig. 3E Left and Center).
Anti-Cdc45 peptide B resin was mixed with purified FLAGMcm6-containing complexes, the resin was washed extensively,
and bound CMG complexes were eluted with a neutral buffer
containing Cdc45 peptide B. To ensure that any observed
helicase activity in these preparations was not from residual
Mcm4,6,7 complex that may have not been completely washed
from the resin after binding CMG to the anti-Cdc45 peptide B
beads, we mock-eluted with buffer containing FLAG peptide
before eluting with buffer containing Cdc45 peptide B. Fig. 3E
Left shows helicase assays with the material eluted from the
anti-Cdc45 peptide B resin by the mock elution or the Cdc45
Moyer et al.
peptide B elution and indicates that the detected helicase in
these assays is associated with the CMG complex. We have found
in our various preparations of the CMG complex that the
helicase activity is ATP-dependent (Fig. 3E Center) and saturates with 1 mM nucleotide (Fig. 3E Right).
Two substrates (Fig. 4A Left) were used to test the directionality of the CMG complex tracking movement on DNA. As
indicated by the displacement of the 5⬘ labeled substrate in Fig.
4A Right, the CMG complex translocates on DNA in a 3⬘–5⬘
direction. We have also determined that the CMG complex will
displace a substrate identical to that in Fig. 3B, except with a 3⬘
30T tail instead of a 5⬘ tail (data not shown). Thus, the CMG
complex can load onto helicase substrates with a gapped single
strand (Fig. 4A) or 5⬘ or 3⬘ overhangs, like Mcm4,6,7 (10, 33).
To assay the processivity of the CMG helicase, we annealed
the primer used in Fig. 3B to m13mp18 ssDNA plasmid and
extended the primer with T7 DNA polymerase to generate a
population of duplex primer–plasmid regions of various and
heterogeneous lengths. Fig. 4B shows the results of these helicase
assays with titrations of the CMG complex, and we conclude that
the complex can work processively for many hundreds of base
pairs. From analysis of side-by-side comparisons with the Drosophila Mcm4,6,7 subcomplex, we conclude that the CMG
complex is as processive as the subcomplex and perhaps more
active at lower levels of protein.
In Vivo Requirement of Cdc45 and GINS. The four proteins of the
GINS complex are required for cell cycle progression in yeast
(29), but parallel in vivo or cell-based data have not been
reported for most GINS members in metazoans. We depleted
Cdc45 or the GINS complex members from Drosophila Kc tissue
culture cells by RNA interference (RNAi) and examined the cell
cycle distribution of the treated cells. Loss of Cdc45 or any one
of the GINS complex members results in a significant impairment of cell cycle progression and an accumulation of cells in G1
and S phase (Fig. 5A). These results support a recent report that
Psf1 is required for embryo development and cell proliferation
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BIOCHEMISTRY
Fig. 4. CMG directionality and processivity. (A) (Left) Cartoon of procedure
for preparing substrates for directionality assays. A 48-mer primer was annealed to m13mp18 ssDNA plasmid and then labeled with 32P at the 5⬘ end
with T4 polynucleotide kinase and [␥-32P]ATP or at the 3⬘ end with T7 DNA
polymerase and [␣-32P]dGTP. The duplex region was digested with SmaI,
creating a linear plasmid with ssDNA in the middle and duplex DNA at both
ends (the 5⬘ labeled primer is 20 bases, and the 3⬘ labeled primer is 28 bases).
The radiolabeled primer can only be approached on ssDNA from one direction, thereby allowing determination of the directionality of helicase movement on DNA. (Right) PhosphorImager picture of CMG complex tested with
both directionality substrates. (B) The primer in Fig. 3B was extended by the
DNA polymerase of T7 (Sequenase), and the heterogeneous products created
substrates for helicase assays. A PhosphorImager picture of DNA substrates
after incubation in helicase assays with increasing amounts of CMG or
Mcm4,6,7 complexes is shown. For measuring the lengths of the duplex DNA
in the substrates, a dsDNA ladder was labeled with 32P and separated on the
gel in either native or boiled form.
Fig. 5. Cdc45 and GINS proteins are required for normal S phase progression.
(A) (Left) Drosophila Kc tissue culture cells were treated with ⬇500 bp of
dsRNA directed against Cdc45, Sld5, Psf1, Psf2, Psf3, or nonspecific (NS) RNA.
After 8 days of RNAi treatment, cells were harvested; FACS profiles are shown.
The x axis is arbitrary fluorescence units, and the y axis is the number of cells
(0 –128). (Right) Cell cycle distribution of RNAi-treated cells. (B) A model of the
CMG complex. Green, Mcm2–7; blue, GINS; yellow, Cdc45.
in mice (34) and indicate an in vivo requirement for GINS during
S phase in metazoans.
Discussion
We have provided evidence that Cdc45 exists in a stable
biochemical unit with the Mcm2–7 and GINS complexes and
that this large complex has associated with it an ATP-dependent
helicase activity. The evidence that the Mcm2–7 complex is
responsible for this activity (as opposed to another helicase or a
subset of the Mcm proteins) is not definitive, but it is an
attractive hypothesis. Reconstitution of the complex from recombinant proteins will be the next step in testing this notion.
The identification of the CMG complex and its associated
helicase activity supports previous reports that implicate the
Mcms, Cdc45, and GINS in chromosome unwinding (4–6), and
it begins to provide a molecular model for the mechanism of
DNA unwinding at the eukaryotic DNA replication fork.
Formation of the CMG Complex. Cdc45 and GINS first associate
with replication origins at the G1 to S phase transition after the
activation of the cyclin-dependent kinase (Cdk) and Cdc7 protein kinases (16, 35), and it is possible that phosphorylation of
one or more of the CMG complex members by Cdk and兾or Cdc7
may promote Cdc45 and GINS association with Mcm2–7. Mcm
proteins have been shown to be phosphorylated at the G1 to S
phase transition (36, 37), and Cdc45 and GINS have been shown
to preferentially associate with Mcms during S phase (15, 16, 38).
Furthermore, the bob-1 mutant, an allele of Mcm5 in S. cerevisiae, suppresses the requirement for the Cdc7 kinase (39), and it
is tempting to speculate that this mutation bypasses a modification that is essential for CMG complex assembly.
The exclusive assembly of Cdc45 and GINS with Mcm2–7 at
a prereplication complex just before and during DNA synthesis
would also clarify why only a small percentage of the total Cdc45,
GINS, and Mcm2–7 proteins in our starting nuclear extract are
found in the complex (see Fig. 7). A free pool of Cdc45 and GINS
proteins may be required for activation of replication origins
throughout S phase. Only a small percentage of Mcm proteins in
the nucleus are in the CMG complex; therefore, most Mcm
proteins may not be located at sites of DNA unwinding. The vast
excess of Mcm2–7 in the extracts that is not in the CMG complex
is consistent with reports that most Mcm proteins do not localize
to sites of DNA replication in metazoans (40, 41).
Taking further the notion that the helicase activity of the
CMG complex is provided by Mcm2–7, what role might Cdc45
and GINS play in this function? It is possible that the CMG
complex forms only at preinitiation sites and that, concomitant
with this assembly, a specific set of posttranslational modifications of the initiation factors activates the helicase activity.
Further dissection of the modification patterns of the proteins of
the CMG complex and an understanding of how the complex is
assembled will answer these questions. Apart from specific
modifications, a simple notion would be that Cdc45 and兾or
GINS induces or stabilizes a conformational change in the
Mcm2–7 hexamer, serving as a molecular switch that converts an
inactive helicase to an active form. Cdc45 and GINS may thus be
a part of the actual helicase machinery, with, for example, Cdc45
possibly serving as a wedge in the recently proposed ‘‘plowshare’’
model for helicase activity (1). A second, nonexclusive possibility
is that Cdc45 and GINS associate with the Mcm2–7 helicase
complex for purposes of coordination with DNA repair factors.
Cdc45 has been shown to associate with the checkpoint proteins
Mrc1 and Tof1 during S phase (42, 43), and we stress that our
stringent purification methods might dislodge the Drosophila
homolog of Claspin兾Mrc1 from the CMG complex. The cell’s
central replicative helicase may only be activated when the
factors that can coordinate helicase function with checkpoint
proteins are fully engaged.
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CMG as a Helicase. The data presented in this study indicate that
a purified Mcm2–7-containing complex has DNA helicase activity. The question may be raised of whether the Mcm2–7
proteins of the CMG complex are functioning as a helicase or
whether the CMG complex either dissociates to a Mcm4,6,7
subcomplex or copurifies with an unrecognized helicase.
Although we cannot formally disprove either of these possibilities, our data suggest that both of these possibilities are
unlikely. The CMG complex is stable through many chromatography steps and high-salt conditions, and we believe that it is
unlikely that the complex would dissociate during the gentle
conditions of the helicase assays. In addition, if it did dissociate,
then the free Mcm2, Mcm3, and Mcm5 that would result from
the dissociation would be expected to inhibit the Mcm4,6,7
activity. The second possibility, that there is an unrecognized
helicase complex that copurifies with the CMG complex, is also
unlikely. The depletion experiments in Fig. 3D indicate that the
observed helicase activity is tightly associated with the CMG
complex. Examination of the CMG complex purified by antiCdc45, anti-FLAG兾anti-Cdc45, or anti-Psf2 chromatography
(Figs. 1 and 2) suggests that CMG does not have any unidentified
binding partners that could provide helicase activity. Hence, the
best explanation of the data shown here is that the observed
helicase activity is manifest from the complex itself.
Molecular Architecture of the CMG Complex. Electron microscope
images of Mcm2–7 and GINS complexes individually have shown
that both complexes form ring-shaped structures (28, 44). These
findings suggest that the two rings may stack on top of each other
to form a common central channel that could surround ssDNA
or dsDNA. Fig. 5B shows a speculative model of the molecular
architecture of the CMG complex. Double hexamers of the
archaeal Mcm rings have been observed (7, 45), and, by analogy,
one might expect the Drosophila Mcm2–7 to form such double
hexamer structures. However, our present model posits that the
active helicase contains a six-member (Mcm2–7) and fourmember (GINS) ring ensemble. The model of one Mcm2–7
hexamer per CMG complex is based on gel filtration data, which
show that the CMG complex migrates at the same position as
thyrogloblulin (669 kDa) on both Superdex-200 (Fig. 1C) and
Superose 6 (see Fig. 10, which is published as supporting
information on the PNAS web site) columns, suggesting that the
mass of the CMG complex is close to 700 kDa. The calculated
molecular mass of a complex containing one Mcm2–7 hexamer,
one GINS tetramer, and one Cdc45 molecule is 708 kDa.
A recent study (6) showed that Mcm2, Mcm5, Mcm7, Sld5, and
Cdc45 localize to sites of DNA unwinding on a plasmid in
Xenopus extracts, a result that is consistent with the present data.
In the study (6), unwinding was uncoupled from DNA synthesis,
and some fraction of DNA replication fork proteins were
associated with the unwinding activity. These researchers suggested that the entire set of Mcm2–7, Cdc45, and the GINS
complex were part of a molecular machine that they referred to
as the ‘‘unwindosome.’’ The term unwindosome may perhaps
refer to a very large collection of proteins yet to be identified that
comprise and associate with the DNA unwinding machinery. In
fact, another recent study (43) shows that a large number of S.
cerevisiae proteins associate with Sld5 and Mcm4 in a highmolecular-weight complex referred to as the ‘‘replisome progression complex.’’ In this study, we show that Cdc45, Mcm2–7,
and GINS form a biochemically discrete complex, and we
propose that the CMG complex per se forms the core of helicase
activity.
Materials and Methods
CMG Complex Purification. The experimental details of CMG com-
plex purification are described in the supporting information.
Moyer et al.
Antibodies. Antibodies against Drosophila Cdc45, Sld5, Psf1, Psf2,
and Psf3 proteins were raised by cloning the respective full-length
genes into pQE-30 (Qiagen, Valencia, CA) and expressing Histagged versions of each protein in the Escherichia coli strain XA-90.
All proteins were solubilized with 6 M Gu-HCl and purified by
binding to Ni-nitrilotriacetic acid resin (Qiagen), followed by imidazole elution. For each protein, two New Zealand White rabbits
were injected with the respective purified protein mixed with Ribi
adjuvant (Corixa, Seattle). Anti-Cdc45 antibodies were affinitypurified by standard methods by using maltose-binding proteinlinked Cdc45 protein as the target protein. Antibodies against
Mcm2, Mcm4, and Mcm5 were a generous gift from T. T. Su
(University of Colorado, Boulder).
Mass Spectroscopy. 2D analysis of total protein eluate from the
anti-Cdc45 IP was performed by the Proteomics兾Mass Spectrometry Facility (University of California, Berkeley). Individual
protein bands were identified by AmProx (Carlsbad, CA).
IPs. For antibody resins, antibodies were mixed with protein A
Peptides. Cdc45 peptides A and B were prepared by GenScript
Mcm4,6,7 Purification. Purified FLAG-Mcm6 material was
loaded onto a Mono Q column and gradient-eluted, which
separates Mcm4,6,7 complexes from Mcm2,4,6,7 complexes.
The peak of Mcm4,6,7 elution is from 280 to 310 mM KCl, and
the Mcm2,4,6,7 peak is from 325 to 380 mM KCl.
(Piscataway, NJ).
Helicase Substrates and Assays. The experimental details of the
helicase substrates and assays are described in the supporting
information.
RNAi and FACS Analysis. dsRNA synthesis, RNA transfection, and
total cellular RNA purification for Cdc45, Sld5, Psf1, Psf2, and
Psf3 were performed as described in ref. 46. RNAi-treated cells
were stained with propidium iodide for FACS analysis.
FLAG-Mcm6 Material Purification. Extract was prepared from 0- to
12-h embryos of flies expressing FLAG-Mcm6. The extract was
mixed with anti-FLAG M2 resin (Sigma) overnight and washed
extensively with buffer containing 1 M KCl. Material was eluted
from the anti-FLAG resin by competition with ‘‘Cdc45 buffer’’
We dedicate this paper to the memory of our mentor, friend, and
colleague, Nicholas Cozzarelli. We thank Brian Calvi (University of
Pennsylvania, Philadelphia) for the FLAG-Mcm6 strain of Drosophila,
Tin Tin Su for anti-Mcm antibodies, and Sue Cotterill (St. Georges,
University of London, London) for anti-Cdc45 antibodies used in the
initial stages of this work. This work was supported by National Institutes
of Health Grant CA R37-30490.
1. Takahashi, T. S., Wigley, D. B. & Walter, J. C. (2005) Trends Biochem. Sci. 30,
437–444.
2. Labib, K., Tercero, J. A. & Diffley, J. F. (2000) Science 288, 1643–1647.
3. Forsburg, S. L. (2004) Microbiol. Mol. Biol. Rev. 68, 109–131.
4. Pacek, M. & Walter, J. C. (2004) EMBO J. 23, 3667–3676.
5. Shechter, D., Ying, C. Y. & Gautier, J. (2004) J. Biol. Chem. 279, 45586–45593.
6. Pacek, M., Tutter, A. V., Kubota, Y., Takisawa, H. & Walter, J. C. (2006) Mol.
Cell 21, 581–587.
7. Chong, J. P., Hayashi, M. K., Simon, M. N., Xu, R. M. & Stillman, B. (2000)
Proc. Natl. Acad. Sci. USA 97, 1530–1535.
8. Kelman, Z., Lee, J. K. & Hurwitz, J. (1999) Proc. Natl. Acad. Sci. USA 96,
14783–14788.
9. Shechter, D. F., Ying, C. Y. & Gautier, J. (2000) J. Biol. Chem. 275,
15049–15059.
10. Ishimi, Y. (1997) J. Biol. Chem. 272, 24508–24513.
11. Lee, J. K. & Hurwitz, J. (2000) J. Biol. Chem. 275, 18871–18878.
12. Kaplan, D. L., Davey, M. J. & O’Donnell, M. (2003) J. Biol. Chem. 278,
49171–49182.
13. Ishimi, Y., Komamura, Y., You, Z. & Kimura, H. (1998) J. Biol. Chem. 273,
8369–8375.
14. You, Z., Komamura, Y. & Ishimi, Y. (1999) Mol. Cell. Biol. 19, 8003–8015.
15. Masuda, T., Mimura, S. & Takisawa, H. (2003) Genes Cells 8, 145–161.
16. Zou, L. & Stillman, B. (2000) Mol. Cell. Biol. 20, 3086–3096.
17. Moir, D., Stewart, S. E., Osmond, B. C. & Botstein, D. (1982) Genetics 100,
547–563.
18. Aparicio, O. M., Stout, A. M. & Bell, S. P. (1999) Proc. Natl. Acad. Sci. USA
96, 9130–9135.
19. Hennessy, K. M., Lee, A., Chen, E. & Botstein, D. (1991) Genes Dev. 5,
958–969.
20. Zou, L., Mitchell, J. & Stillman, B. (1997) Mol. Cell. Biol. 17, 553–563.
21. Miyake, S. & Yamashita, S. (1998) Genes Cells 3, 157–166.
22. Mimura, S., Masuda, T., Matsui, T. & Takisawa, H. (2000) Genes Cells 5,
439–452.
23. Mimura, S. & Takisawa, H. (1998) EMBO J. 17, 5699–5707.
24. Hopwood, B. & Dalton, S. (1996) Proc. Natl. Acad. Sci. USA 93, 12309–12314.
25. Christensen, T. W. & Tye, B. K. (2003) Mol. Biol. Cell 14, 2206–2215.
26. Saha, P., Thome, K. C., Yamaguchi, R., Hou, Z., Weremowicz, S. & Dutta, A.
(1998) J. Biol. Chem. 273, 18205–18209.
27. Kamimura, Y., Tak, Y. S., Sugino, A. & Araki, H. (2001) EMBO J. 20,
2097–2107.
28. Kubota, Y., Takase, Y., Komori, Y., Hashimoto, Y., Arata, T., Kamimura, Y.,
Araki, H. & Takisawa, H. (2003) Genes Dev. 17, 1141–1152.
29. Takayama, Y., Kamimura, Y., Okawa, M., Muramatsu, S., Sugino, A. & Araki,
H. (2003) Genes Dev. 17, 1153–1165.
30. Araki, H., Leem, S. H., Phongdara, A. & Sugino, A. (1995) Proc. Natl. Acad.
Sci. USA 92, 11791–11795.
31. Ramachandran, N., Hainsworth, E., Bhullar, B., Eisenstein, S., Rosen, B., Lau,
A. Y., Walter, J. C. & LaBaer, J. (2004) Science 305, 86–90.
32. Schwed, G., May, N., Pechersky, Y. & Calvi, B. R. (2002) Mol. Biol. Cell 13,
607–620.
33. You, Z., Ishimi, Y., Mizuno, T., Sugasawa, K., Hanaoka, F. & Masai, H. (2003)
EMBO J. 22, 6148–6160.
34. Ueno, M., Itoh, M., Kong, L., Sugihara, K., Asano, M. & Takakura, N. (2005)
Mol. Cell. Biol. 25, 10528–10532.
35. Jares, P. & Blow, J. J. (2000) Genes Dev. 14, 1528–1540.
36. Lei, M., Kawasaki, Y., Young, M. R., Kihara, M., Sugino, A. & Tye, B. K. (1997)
Genes Dev. 11, 3365–3374.
37. Takahashi, T. S. & Walter, J. C. (2005) Genes Dev. 19, 2295–2300.
38. Yamada, Y., Nakagawa, T. & Masukata, H. (2004) Mol. Biol. Cell 15,
3740–3750.
39. Hardy, C. F., Dryga, O., Seematter, S., Pahl, P. M. & Sclafani, R. A. (1997)
Proc. Natl. Acad. Sci. USA 94, 3151–3155.
40. Dimitrova, D. S., Todorov, I. T., Melendy, T. & Gilbert, D. M. (1999) J. Cell
Biol. 146, 709–722.
41. Madine, M. A., Khoo, C. Y., Mills, A. D., Musahl, C. & Laskey, R. A. (1995)
Curr. Biol. 5, 1270–1279.
42. Katou, Y., Kanoh, Y., Bando, M., Noguchi, H., Tanaka, H., Ashikari, T.,
Sugimoto, K. & Shirahige, K. (2003) Nature 424, 1078–1083.
43. Gambus, A., Jones, R. C., Sanchez-Diaz, A., Kanemaki, M., van Deursen, F.,
Edmondson, R. D. & Labib, K. (2006) Nat. Cell Biol. 8, 358–366.
44. Adachi, Y., Usukura, J. & Yanagida, M. (1997) Genes Cells 2, 467–479.
45. Gomez-Llorente, Y., Fletcher, R. J., Chen, X. S., Carazo, J. M. & San Martin,
C. (2005) J. Biol. Chem. 280, 40909–40915.
46. Lewis, P. W., Beall, E. L., Fleischer, T. C., Georlette, D., Link, A. J. & Botchan,
M. R. (2004) Genes Dev. 18, 2929–2940.
Moyer et al.
PNAS 兩 July 5, 2006 兩 vol. 103 兩 no. 27 兩 10241
BIOCHEMISTRY
Sepharose CL-4B beads (Amersham Pharmacia) and crosslinked by standard methods. IPs were performed by mixing
antibody兾protein A resins with protein samples overnight, followed by extensive washing of the resin. Except where noted in
the text, bound proteins were eluted from antibody兾protein A
beads by elution with glycine buffer (pH 2.5) and immediately
neutralized with 1 M Tris䡠Cl (pH 8.0). For Cdc45 peptide B
elutions, the anti-Cdc45 peptide B antibody resins were incubated for 10 min with a buffer containing 50 mM Hepes (pH
7.58), 10 mM magnesium acetate, 50 mM sodium acetate, 10%
glycerol, 0.25 mg兾ml insulin (Sigma), and 200 ␮g兾ml Cdc45
peptide B.
(see the CMG complex purification protocol in the supporting
information) containing 100 ␮g兾ml FLAG peptide.