Cdt1p, through its interaction with Mcm6p, is required
for the formation, nuclear accumulation and chromatin
loading of the MCM complex
Rentian Wu1,2, Jiafeng Wang1and Chun Liang1,2,*
1Division of Life Science and Center for Cancer Research and2Bioengineering graduate program, Hong Kong University of Science and Technology,
Clear Water Bay, Kowloon, Hong Kong, China
*Author for correspondence (email@example.com)
Accepted 22 August 2011
Journal of Cell Science 125, 209–219
? 2012. Published by The Company of Biologists Ltd
Regulation of DNA replication initiation is essential for the faithful inheritance of genetic information. Replication initiation is a
multi-step process involving many factors including ORC, Cdt1p, Mcm2-7p and other proteins that bind to replication origins to form
a pre-replicative complex (pre-RC). As a prerequisite for pre-RC assembly, Cdt1p and the Mcm2-7p heterohexameric complex
accumulate in the nucleus in G1 phase in an interdependent manner in budding yeast. However, the nature of this interdependence is
not clear, nor is it known whether Cdt1p is required for the assembly of the MCM complex. In this study, we provide the first evidence
that Cdt1p, through its interaction with Mcm6p with the C-terminal regions of the two proteins, is crucial for the formation of the
MCM complex in both the cytoplasm and nucleoplasm. We demonstrate that disruption of the interaction between Cdt1p and Mcm6p
prevents the formation of the MCM complex, excludes Mcm2-7p from the nucleus, and inhibits pre-RC assembly and DNA
replication. Our findings suggest a function for Cdt1p in promoting the assembly of the MCM complex and maintaining its integrity
by interacting with Mcm6p.
Key words: DNA replication, Pre-replicative complex, Cdt1p–Mcm6p interaction, MCM complex formation, Saccharomyces cerevisiae
DNA replication is one of the most fundamental cellular
processes. To maintain the integrity of the genetic information
as it is passed from one generation to the next, the initiation
of DNA replication must be stringently controlled to ensure
that genome duplication occurs precisely once per cell
cycle. Replication initiation is a multi-step process, including
replication origin licensing and activation. During origin
licensing at the M-to-G1 transition, pre-RCs are formed by the
stepwise assembly of Cdc6p, Cdt1p and the minichromsome
maintenance (MCM) complex onto the platform formed by the
origin recognition complex (ORC) and other proteins at
replication origins (Bell and Dutta, 2002; Me ´ndez and Stillman,
2003; Labib, 2010). Subsequently, pre-initiation complexes (pre-
ICs) are formed by the loading of other replication-initiation
proteins onto replication origins. Activation of replication
initiation is then achieved by the actions of cyclin-dependent
kinases (CDKs) and the Dbf4p-dependent Cdc7p kinase (DDK),
which phosphorylate several pre-RC and pre-IC components.
Cdt1p was first identified as a licensing factor in Xenopus
(Maiorano et al., 2000). Along with Cdc6p and other licensing
factors, Cdt1p is required for pre-RC formation but not DNA
replication elongation (Devault et al., 2002; Tanaka and Diffley,
2002). Unlike other proteins involved in DNA replication, the
primary sequences of Cdt1p homologues have a low degree of
conservation among diverse species; for example, there is only
11% identity between yeast and human CDT1 proteins. In
addition, the regulation of Cdt1 varies significantly among
eukaryotes. It is regulated by ubiquitin-mediated proteolysis in
fission yeast (Nishitani et al., 2004) and other model organisms
(Higa et al., 2003; Arias and Walter, 2005); by binding of the
inhibitor Geminin in Xenopus (Wohlschlegel et al., 2000) and
mammalian cells (Yanagi et al., 2002); and by nuclear export in
budding yeast (Tanaka and Diffley, 2002).
In budding yeast, Mcm2-7p and Cdt1p are imported into the
nucleus during the M-to-G1 transition when CDK activity is low,
whereas their export to the cytoplasm in late G1 and S phases is
promoted by high CDK activity (Labib et al., 1999; Liku et al.,
2005; Nguyen et al., 2000). Cdt1p, which does not possess a
nuclear localization signal (NLS), requires Mcm2-7p to enter the
nucleus (Tanaka and Diffley, 2002). Furthermore, nuclear
accumulation of Mcm2-7p requires the formation of a complex
containing all six MCM subunits and Cdt1p (Pasion and
Forsburg, 1999; Labib et al., 2001; Tanaka and Diffley, 2002).
However, the reason why the nuclear localization of the Mcm2-
7p complex, in which Mcm2p and Mcm3p have NLSs, depends
on Cdt1p is still unknown.
In this study, we discovered that Cdt1p is essential for the
formation of the MCM complex in both the cytoplasm and
nucleoplasm before pre-RC formation. We found that disruption
of the interaction between Cdt1p and Mcm6p prevents the
assembly of the MCM complex, abolishes nuclear retention of
Mcm2-7p, and inhibits pre-RC formation and DNA replication.
These findings suggest a function for Cdt1p in facilitating the
assembly and maintaining the integrity of the hexameric MCM
complex by interacting with Mcm6p.
Research Article 209
Journal of Cell Science
Depletion of either Mcm6p or Cdt1p in M phase prevents
MCM complex formation
Cdt1p and the MCM proteins are exported separately from the
nucleus: Cdt1p is exported to the cytoplasm before replication
initiation (Tanaka and Diffley, 2002), whereas Mcm2-7p is
exported, possibly as individual subunits, during DNA replication
(Nguyen et al., 2000) (see the Discussion). Cdt1p and Mcm2-7p
associate to form an MCM–Cdt1p complex in the cytoplasm in M
phase (Tanaka and Diffley, 2002). Previous studies showed that
depletion of Cdt1p or any one of the MCM subunits abolishes the
nuclear import of the MCM complex during the M-to-G1
transition (Labib et al., 2001; Tanaka and Diffley, 2002).
localization signal and hence Mcm2-7p needs to be imported
into the nucleus as a heterohexameric complex, we speculated
that the failure of Mcm2-7p nuclear importation when Cdt1p or
an MCM subunit is depleted might result from the failure of the
MCM subunits to form a complex. To test this hypothesis, we
between different MCM subunits in yeast extracts prepared
from mcm6-td (td, temperature-inducible degron) and cdt1-td
cells depleted of the Mcm6-td and Cdt1-td protein, respectively.
synchronized in M phase with the microtubule-depolymerising
drug nocodazole. The Mcm6-td or Cdt1-td protein was then
depleted at the restrictive temperature of 37˚C upon GAL-UBR1
induction, which facilitates td protein degradation. Cells
maintained at the permissive temperature of 25˚C without GAL-
UBR1 induction were used as a control. As shown in Fig. 1A,
Mcm3p and Cdt1p were co-immunoprecipitated with Mcm2p in
the anti-Mcm2 immunoprecipitation using the extracts prepared
from MCM6 wild-type cells at both 25˚C and 37˚C and from
mcm6-td cells at 25˚C (lanes 1–9). By contrast, neither Cdt1p nor
Mcm3p was co-immunoprecipitated with Mcm2p using the
extract from mcm6-td cells at 37˚C when the Mcm6-td protein
was degraded (Fig. 1A, lanes 10–12). As controls for the
specificity of the co-immunoprecipitation, Mcm2p, Mcm3p or
Cdt1p was not precipitated by the control mouse IgG (Fig. 1A).
These results indicate that Mcm2p, Mcm3p and Cdt1p can
no longer be in a complex without Mcm6p. Similarly, co-
immunoprecipitation experiments using cdt1-td cells showed that
Mcm3p, Mcm4–GFP, Mcm6–HA and Cdt1p or Cdt1-td were co-
immunoprecipitated with Mcm2p using the extracts from CDT1
wild-type cells at both 25˚C and 37˚C and from cdt1-td cells at
25˚C when Cdt1p or Cdt1-td was present (Fig. 1B, lanes 1–9),
but not from cdt1-td cells at 37˚C when the Cdt1-td protein was
depleted (Fig. 1B, lanes 10–12). These results suggest that
depletion of Cdt1p prevents Mcm2-7p complex formation.
To corroborate the anti-Mcm2 co-immunoprecipitation results,
we performed anti-Mcm3, anti-Mcm4–GFP and anti-Mcm6-HA
co-immunoprecipitation experiments, separately, under similar
conditions as those for Fig. 1B. The results showed that Mcm2p,
Mcm3p, Mcm4–GFP, Mcm6-HA and Cdt1p or Cdt1-td were all
co-immunoprecipitated by each of the relevant antibodies using
extracts from CDT1 wild-type cells at both 25˚C and 37˚C and
from cdt1-td cells at 25˚C, but not from cdt1-td cells at 37˚C
(supplementary material Fig. S1A–C). These results support
the conclusion from the anti-Mcm2 co-immunoprecipitation
experiments that Cdt1p is required for the formation of the
toexamine the interactions
To confirm that Mcm2-7p cannot assemble into a complex
after Cdt1-td depletion, we examined the co-sedimentation of
different MCM subunits by sucrose gradient centrifugation.
Extracts from wild-type and Cdt1-td-depleted M phase cells were
separated on a 20–50% sucrose gradient, and fractions were
immunoblotted for the different MCM subunits (Fig. 1C; see
Coomassie-stained gels in supplementary material Fig. S1D).
Mcm2p, Mcm3p, Mcm4–GFP and Mcm6–HA from wild-type
cells co-migrated as both monomeric (,140 kDa) and hexameric
(,540 kDa) forms, whereas they only appeared in the fractions
of the monomers in Cdt1-td-depleted extract. Together with the
co-immunoprecipitation data, these results demonstrate that
Cdt1p is required for the formation of the Mcm2-7p complex
in M phase before the MCM complex enters the nucleus,
providing an explanation for the failure of the MCM complex to
enter the nucleus in the absence of Cdt1p.
Depletion of Cdt1p in G1 phase abolishes the formation
and nuclear retention of non-chromatin-bound Mcm2-7p
To investigate the possible role of Cdt1p in maintaining the
integrity of the MCM complex in G1 phase when most of MCM
proteins are in the complex and normally locate in the nucleus,
we examined the localization of Mcm4–GFP in G1 phase
cdt1-td cells depleted of the Cdt1-td protein. When the cells
synchronized in G1 phase by the yeast mating pheromone a-
factor at 25˚C were shifted to 37˚C to deplete the Cdt1-td protein,
most Mcm4–GFP was excluded from the nucleus, whereas
most Mcm4–GFP remained in the nucleus in wild-type cells
(supplementary material Fig. S2A,B). However, there remained a
low level of GFP signal in the nucleus even after 4 hours of
Cdt1-td depletion (supplementary material Fig. S2A). We
speculated that this might result from the stabilization of the
chromatin-bound MCM complex as a component of the pre-RC.
To test this possibility, we examined the chromatin association of
Mcm2p in cells depleted of Cdt1p as above. The chromatin-
bound Mcm2p in cdt1-td cells at 37˚C, although decreased to
some extent compared with levels in wild-type cells, appeared
constantduring the time course
(supplementary material Fig. S2C). These results suggest that
depletion of Cdt1p results in nuclear export of most Mcm2-7p,
but does not remove the chromatin-associated MCM proteins
once pre-RCs have formed. Consistent with this finding, flow
cytometry confirmed that depletion of Cdt1p in G1 phase did not
result in any observable defect in cell cycle progression after the
cells were released into fresh medium (supplementary material
Fig. S2D), indicating that the MCM proteins stably bound on
chromatin after Cdt1-td depletion were functional for DNA
To more clearly demonstrate the role of Cdt1p in the nuclear
retention of the non-chromatin-bound MCM proteins in G1 cells,
we needed to prevent MCM proteins from binding to chromatin
in G1 phase before Cdt1-td protein depletion. To do this, we first
synchronized cells in G1 phase and then released them from the
G1 block while shutting off CDC6 expression under the control
of the MET3 promoter to prevent pre-RC formation in the next
G1 phase (Labib et al., 1999; Nguyen et al., 2000). These G1
cells were then shifted to 37˚C to deplete the Cdt1-td protein (see
diagram in Fig. 2A). Fluorescence microscopy showed that
without Cdt1p, the nuclear Mcm4–GFP signal decreased
dramatically compared with that in the CDT1 wild-type control
cells and was reduced to a non-detectable level after 120 minutes
Journal of Cell Science 125 (1)210
Journal of Cell Science
of Cdt1-td depletion (Fig. 2B,C). Quantification of the Mcm4–
GFP nuclear localization indicated that wild-type and cdt1-td
cells at 25˚C showed a similar level of the nuclear accumulated
Mcm4–GFP (Fig. 2C). By contrast, few cdt1-td cells contained
nuclear Mcm4–GFP after 2 hours of Cdt1-td depletion at 25˚C,
whereas wild-type cells accumulated nuclear Mcm4–GFP
normally (Fig. 2C). Flow cytometry confirmed that the cells
remained arrested in G1 phase throughout the experiment
(supplementary material Fig. S4). The chromatin-bound and
soluble proteins from the cells at each time point were also
immunoblotted to confirm that there was no pre-RC (i.e. absence
of Mcm4–GFP on chromatin) without Cdc6p, and that the
decrease of the nuclear Mcm4–GFP signal during the time course
of Cdt1-td depletion was not due to any notable change in the
total Mcm4–GFP protein level (Fig. 2D). Together, these results
reveal that depletion of Cdt1p abolishes the nuclear retention of
non-chromatin-bound MCM proteins.
Because depletion of Cdt1p prevents the formation of the
MCM complex in M phase cells (Fig. 1), we reasoned that the
loss of nuclear retention of non-chromatin-bound MCM proteins
in Cdt1-td-deleted G1 cells probably also resulted from
disassembly of the MCM complex. To validate this hypothesis,
we performed co-immunoprecipitation with extracts from G1
cells depleted of the Cdt1-td protein and without pre-RC
formation as described above. The results showed that neither
Mcm3p nor Mcm4–GFP could be co-immunoprecipitated with
Mcm2p in G1-phase cell extracts without Cdt1p (Fig. 2E, lanes
10–12), but they were co-immunoprecipitated with Mcm2p in the
control G1 extracts in the presence of Cdt1p or Cdt1-td (Fig. 2E,
lanes 1–9). Together, these data indicate that Cdt1p is required
for the nuclear retention of non-chromatin-bound Mcm2-7p in G1
cells by maintaining the integrity of the MCM complex. These
findings might have implications in the nuclear export of MCM
proteins in S phase (see the Discussion).
Cdt1p and Mcm6p interact through their
As a component of pre-RC, Cdt1p is known to interact with
Mcm6p physically in metazoans (Teer and Dutta, 2008; Wei
et al., 2010; Zhang et al., 2010). To further investigate the role of
Cdt1p in maintaining the MCM complex integrity in budding
yeast, we focused on the interaction between Cdt1p and Mcm6p.
Based on the mapping of interaction domains of human CDT1
and MCM6 (Zhang et al., 2010), we truncated Cdt1p and Mcm6p,
each into a large N-terminal and a small C-terminal fragment
designated Cdt1N (amino acids 1–421), Cdt1C (422–604),
Mcm6N (1–914) and Mcm6C (915–1017), respectively (see
diagram in Fig. 3A). We performed yeast two-hybrid assays and
found that Cdt1p and Cdt1C interacted with both Mcm6p
and Mcm6C, whereas Cdt1N and Mcm6N had no interaction
either with each other, or with Mcm6p or Cdt1p (Fig. 3B).
Immunoblotting showed that the hybrid proteins used in this
study were expressed at similar levels in the host cells
(supplementary material Fig. S3). These data suggest that
Cdt1p and Mcm6p interact through the small C-terminal
domain present in each of the two proteins.
To confirm the interactions detected by the yeast two-hybrid
analysis, we performed co-immunoprecipitation to examine
Fig. 1. Depletion of either Mcm6p or Cdt1p in
M phase cells causes dissolution of the MCM
complex. (A) YL136 (MCM6) and YL135
(mcm6-td) cells arrested in M phase with
nocodazole were kept at 25˚C or 37˚C for
1.5 hours before harvest. Whole cell extracts
(WCEs) were immunoprecipitated with anti-
Mcm2p antibody (a-Mcm2) or with the control
mouse IgG (Ctrl. IgG), and WCEs and the
immunoprecipitates were immunoblotted with
anti-HA antibody to detect the Mcm6-td protein.
Mcm3p and Mcm2p were also immunoblotted. A
dash between two lane numbers in A and B
indicates a blank lane. (B) YL1217 (cdt1-td) and
YL1218 (CDT1) cells arrested in M phase were
kept at 25˚C or 37˚C for 1.5 hours before harvest.
WCEs were immunoprecipitated with anti-Mcm2
antibody, and WCEs and the immunoprecipitates
were immunoblotted for Cdt1-td, Cdt1p, Mcm6–
HA, Mcm4–GFP, Mcm3p and Mcm2p.
(C) WCEs from strains YL1217 (cdt1-td) and
YL1218 (CDT1) were separated on a 20–50%
sucrose gradient. Alkaline phosphatase
(140 kDa) and b-galactosidase (540 kDa) were
applied as molecular size makers. The numbers
above each lane represent the fraction numbers.
Cdt1p and MCM complex formation 211
Journal of Cell Science
interactions of the FLAG-tagged Cdt1p, Cdt1N and Cdt1C with
Mcm6–HA and the interactions of FLAG-tagged Mcm6p,
Mcm6N and Mcm6C with Cdt1-HA in yeast extracts. The
results showed that Mcm6–HA was co-immunoprecipitated with
Cdt1–FLAG (Fig. 3C, lane 7) and Cdt1C–FLAG (Fig. 3C, lane
11), but not with Cdt1N–FLAG (Fig. 3C, lane 9). Likewise,
Cdt1–HA was co-immunoprecipitated with Mcm6–FLAG and
Mcm6C–FLAG,but not with
Together, the results from the yeast two-hybrid and co-
immunoprecipitation assays strongly suggest that Cdt1p and
Mcm6p interact with each other through their C-terminal
Our yeast two-hybrid analysis (Fig. 3B) also confirmed the
previously reported results using the same assay (Asano et al.,
2007) and the GST-pull down assay (Chen et al., 2007) that
Cdt1p interacts with Orc6p, and that Mcm6p interacts with
Mcm2p, as reported for the homologous proteins in mammalian
cells (Kneissl et al., 2003; Yu et al., 2004). Our results further
showed that the N-terminal domain of Cdt1p interacts with
Orc6p, and that the N-terminal domain of Mcm6p interacted with
Mcm2p (Fig. 3B).
To identify the interacting amino acid residues in the C-
terminal domains of Cdt1p and Mcm6p, we introduced point
mutations in the respective C-terminal domain for yeast two-
hybrid analysis (Fig. 3B). The Cdt1p-binding, C-terminal domain
of human Mcm6p contains several acidic residues. Our previous
study showed that mutations at these and other residues in the C-
terminal domain of human MCM6 (Glu757Ala, Glu763Ala and
Leu766Ala) significantly reduced the affinity between Cdt1p and
Mcm6p (Wei et al., 2010). Sequence alignment showed that three
acidic amino acids, Glu945, Asp947 and Glu953, in the C-
terminal domain of Mcm6p are conserved from yeast to human
(supplementary material Fig. S5A). In addition, Leu951 and
Tyr954, corresponding to the Glu763 and Leu766 in human
MCM6, are conserved in most species (supplementary material
Fig. S5A). Because these residues are on the interacting interface
of the human Cdt1p and Mcm6 (Wei et al., 2010), we reasoned
that these five residues are likely to be important for the
interaction between Cdt1p and Mcm6p. We substituted these
residues with alanine in Mcm6p and Mcm6C to construct the
Mcm6-5A (full length) and Mcm6C-5A (C-terminal domain)
mutants, respectively. Yeast two-hybrid analysis showed that
Fig. 2. Cdt1p is essential for the nuclear retention and integrity of non-chromatin-bound MCM complex. (A) Outline of the experiments to examine the
localization and chromatin association of Mcm4–GFP and the integrity of the MCM complex. YL1211 (cdt1-td MET-CDC6) and YL1213 (CDT1 MET-CDC6)
cells were cultured in medium lacking methionine, and a-factor was added to synchronize cells in G1 phase at 25˚C (–Met +a-F, 25˚C). Cells were then released
into fresh methionine-containing medium (+Met –a-F, 25˚C) grown until buds emerged, and a-factor was then added to block cells in the next G1 phase
(+Met +a-F, 25˚C). Cells were then shifted to 37˚C for 2 hours (+Met +a-F, 37˚C) and harvested for analysis. (B) Fluorescence microscopic analysis was
performed with living cells in the final G1 phase at 25˚C and at 37˚C with 30 minute intervals. Scale bar: 10 mm. (C) The proportion of cells with nuclear Mcm4–
GFP shown in Fig. 2B was quantified (n5300). (D) WCEs and chromatin fraction (Chr.) prepared from the cells were immunoblotted for Mcm2p, Orc3p, Cdc6p,
Cdt1-td, Cdt1p and Mcm4–GFP. (E) WCEs were immunoprecipitated with anti-Mcm2 antibody or the control mouse IgG (Ctrl IgG), and WCEs and the
immunoprecipitates were immunoblotted for Cdt1-td, Cdt1p, Mcm4–GFP, Mcm3p and Mcm2p. A dash between two lane numbers indicates a blank lane.
Journal of Cell Science 125 (1) 212
Journal of Cell Science
Mcm6-5A and Mcm6C-5A could no longer interact with either
Cdt1p or Cdt1C, whereas Mcm6-5A still reserved the interaction
with Mcm2p (Fig. 3B). We also determined that Mcm6p fused to
the activation domain (AD–Mcm6p), but not AD–Mcm6-5A,
expressed from the yeast two-hybrid plasmids that were used for
the interaction study in Fig. 3B supported the growth of the GAL-
MCM6 cells in which the expression of the endogenous MCM6
was repressed by glucose (supplementary material Fig. S5C),
biologically functional. These results suggest that some, if not
all of the mutated residues in Mcm6p are crucial for the Cdt1
p–Mcm6p interaction, and that the C-terminal domain of Mcm6p
is essential for cell proliferation through its interaction with
Given that the Cdt1p-interacting surface of Mcm6p contains
acidic amino acids, we speculated the residues on the Mcm6p-
interacting surface of Cdt1p might contain basic amino acids. We
aligned the C-terminal sequences of Cdt1p from different species
and identified five highly conserved basic amino acids, Arg486,
Arg488, Arg490, Arg501 and Lys512 (supplementary material
Fig. S5B). We changed all of the five amino acids to alanine to
construct the Cdt1-5A (full length) and Cdt1C-5A (C-terminal
Fig. 3. Cdt1p and Mcm6p interact through the C-terminal domain of the two proteins. (A) Diagrams of the coding sequences of Cdt1p and Mcm6p and their
N- and C-terminal fragments used in the experiments. The numbers above the diagrams of the proteins or fragments refer to the amino acid numbers in each
protein or fragment. (B) Testing the interactions between the full-length protein and fragments of the wild-type and mutant of Cdt1p and Mcm6p. Interactions
between pairs of fusion proteins were examined by yeast two-hybrid analysis on SCM –Trp –Leu (synthetic complete medium, lacking tryptophan and leucine;
selective for the two vectors but non-selective for the reporter gene) and SCM –Trp –Leu –His –Ade (lacking tryptophan, leucine, histidine and adenine; selective
for the reporter gene) plates. As negative controls, the host yeast cells were co-transformed with the empty BD or AD vector together with the individual fusion
proteins as indicated. (C) WCEs from YL1225 (MCM6-6HA) cells containing pESC (FLAG vector), pESC-Cdt1, pESC-Cdt1N or pESC-Cdt1C were
immunoprecipitated with anti–FLAG antibody (a-FLAG) or control mouse IgG (Ctrl. IgG). WCEs and immunoprecipitates (IP) were immunoblotted with anti-
HA antibody and anti-FLAG antibody. (D) WCEs from YL1226 (CDT1-3HA) cells containing pESC, pESC-Mcm6, pESC-Mcm6N or pESC-Mcm6C were
immunoprecipitated with anti-FLAG antibody (a-FLAG) or control mouse IgG (Ctrl. IgG). WCEs and immunoprecipitates were immunoblotted with anti-HA
antibody and anti-FLAG antibody. (E) A model for the interactions of Cdt1p and Mcm6p with other pre-RC components. A small C-terminal region of Cdt1p
interacts with a small C-terminal region of Mcm6p. Cdt1p interacts with ORC through its N-terminal domain, whereas Mcm6p interacts with other MCM proteins
to form the MCM complex through its large N-terminal domain.
Cdt1p and MCM complex formation 213
Journal of Cell Science
domain) mutants. As predicted, Cdt1-5A and Cdt1C-5A no
longer interacted with Mcm6p or Mcm6C, but Cdt1-5A still
interacted with Orc6p in the two-hybrid assay (Fig. 3B).
Furthermore, BD–Cdt1p (Cdt1p fused to the DNA binding
domain), but not BD–Cdt1-5A, supported the growth of the GAL-
CDT1 cells when the expression of the endogenous CDT1 was
repressed (supplementary material Fig. S5C), indicating that BD–
Cdt1-5A is no longer functional, unlike BD-Cdt1p. These results
suggest that the conserved basic residues in the C-terminal
domain of Cdt1p are crucial for the Cdt1p–Mcm6p interaction
and essential for Cdt1p function. Taken together, our two-hybrid
and co-immunoprecipitation results strongly suggest that the
interaction between the small C-terminal domains of Cdt1p and
Mcm6p, at least partly through charge–charge interactions, is a
part of the bridge that brings ORC and the MCM complex
together to form the pre-RC (see diagram in Fig. 3E).
Overexpression of the Mcm6p-interacting domain of Cdt1p
impairs cell growth, MCM nuclear localization and
initiation of DNA replication
Because the interaction between the C-terminal domains of
Mcm6p and Cdt1p is crucial for MCM complex assembly,
overexpression of these domains might exert negative dominant
effects oncell proliferation.
(supplementary material Fig. S5C), therefore we overexpressed
different BD-fusion proteins in wild-type yeast cells to examine
cell growth. The growth of the cells expressing BD–Cdt1C, but
not BD–Cdt1p or BD–Cdt1N, was severely defective (Fig. 4A).
However, overexpression of the BD–Cdt1C-5A mutant, which
does not interact with Mcm6p as described above, did not show
any effect on cell growth (Fig. 4A). These results suggest that the
overexpressed BD–Cdt1C hindered cell growth through its
interaction with the endogenous Mcm6p.
If overexpression of BD–Cdt1C interferes with the interaction
between the endogenous Cdt1p and Mcm6p, and Cdt1p is
essential for the formation as well as chromatin loading of the
MCM complex, overexpression of BD–Cdt1C should result in
mislocalization of the MCM complex. To investigate this
possibility, we examined the localization of Mcm4–GFP in
cells expressing BD–Cdt1C in G1 phase. By comparing the
average Mcm4–GFP signal per unit area in the nucleus and in the
whole cell (see the Materials and Methods), we found that cells
overexpressing BD–Cdt1C, but not BD–Cdt1 or BD–Cdt1C-5A,
showed in a reduced relative intensity in the nuclear Mcm4–GFP
BD-Cdt1p is functional
Fig. 4. Overexpression of the C-terminal region of Cdt1p impairs MCM nuclear accumulation, initiation of DNA replication and cell growth. (A) Tenfold
serial dilutions of YL1216 (Mcm4–GFP) cells containing the empty BD vector or the plasmid expressing BD–Cdt1, BD–Cdt1N, BD–Cdt1C or BD–Cdt1C-5A
were plated on a SCM –Trp plate (selective for the BD vector) and incubated for 3 days at 23˚C. (B) Microscopic images of G1-phase-synchronized YL1216
(Mcm4–GFP) cells containing the empty BD vector or the plasmid expressing BD–Cdt1, BD–Cdt1C or BD–Cdt1C-5A. Scale bar: 10 mm. Insets represent the
single cells in the white boxes. (C) Quantification of the average fluorescence signal intensity of the nucleus normalized to the average fluorescence signal
intensity of the whole cell (n530). The error bars represent s.d. *P,0.001. (D) Quantitative plasmid loss rates were measured for the cells containing the empty
BD vector or the plasmid expressing BD–Cdt1C or BD–Cdt1C-5A plus either p1ARS or p8ARSs grown in SCM-Trp medium at 25˚C for 10–11 generations.
Plasmid loss rates are represented as the means of percentage loss per generation ± s.d. of three separate experiments.
Journal of Cell Science 125 (1)214
Journal of Cell Science
signal (Fig. 4B,C). These results suggest that disruption of
nuclear localization of Mcm2-7p is part of the mechanism by
which overexpression of BD–Cdt1C impairs cell growth.
To determine whether overexpression of BD–Cdt1C impairs
replication initiation, the loss rates of a pair of tester plasmids,
p1ARS and p8ARSs (Zhang et al., 2002), in the cells were
measured. p1ARS contains one replication origin ARS1, and
p8ARSs carries seven additional copies of H4-ARS inserted into
p1ARS. It has been well documented that all known replication
initiation mutants have a higher rate of p1ARS loss compared
with p8ARSs (Hogan and Koshland, 1992; Loo et al., 1995;
Zhang et al., 2002; Cheng et al., 2010; Ma et al., 2010; Wang
et al., 2010; Zhai et al., 2010). In control cells containing the
empty BD vector, the loss rates of p1ARS and p8ARSs were
1.2% and 0.5% per generation, respectively (Fig. 4D), as
expected for wild-type cells. In the cells expressing BD–Cdt1C,
the loss rate of p1ARS was as high as 15.3% per generation,
whereas the loss rate of p8ARSs was significantly reduced to 3.0%
(Fig. 4D), indicating that the high p1ARS loss rate was due to
defective replication initiation in BD–Cdt1C- expressing cells.
Plasmid loss rates ofthe cells expressingthe mutant BD–Cdt1C-5A
weresimilartothoseof thevectorcontrolcells(Fig. 4D).Together,
data in Fig. 4 suggest that the overexpressed BD–Cdt1C impaired
between the endogenous Cdt1p and Mcm6p and impairing nuclear
accumulation of the MCM complex and replication initiation.
Disruption of the Cdt1p–Mcm6p interaction by mutation of
Cdt1-5A or Mcm6-5A impairs MCM complex formation and
nuclear localization, pre-RC assembly and DNA replication
To further probe the physiological significance of the Cdt1p–
Mcm6p interaction, we examined the consequences of disrupting
the interaction using Cdt1-5A or Mcm6-5A mutants. Because
BD–Cdt1p and AD–Mcm6p are functional (supplementary
material Fig. S5C), we expressed BD–Cdt1p and BD–Cdt1-5A
in cdt1-td cells, and AD–Mcm6p and AD–Mcm6-5A in mcm6-td
cells to investigate the effects of the mutations in Cdt1-5A and
Mcm6-5A on MCM complex formation and nuclear localization,
pre-RC formation and DNA replication.
We first performed co-immunoprecipitation between Mcm2p
and other MCM subunits using yeast extracts to determine
whether the Cdt1-5A or Mcm6-5A mutant could support MCM
complex formation when the corresponding Cdt1-td or Mcm6-td
protein is degraded. cdt1-td cells containing the empty BD vector
or the plasmid expressing BD–Cdt1p or BD–Cdt1-5A were
synchronized in M phase using nocodazole and then shifted to
37˚C to degrade the Cdt1-td protein. Mcm2p, Mcm3p and
Mcm4–GFP were co-immunoprecipitated with extracts from
cells expressing wild-type BD–Cdt1p but not BD–Cdt1-5A
mutant or the cells containing the empty vector (Fig. 5A).
These results suggest that the Cdt1-5A mutant cannot support the
formation of the Mcm2-7p complex. Similarly, using mcm6-td
cells containing the empty AD vector or the plasmid expressing
AD–Mcm6p or AD–Mcm6-5A, we found that the AD–Mcm6-5A
mutant was unable to support MCM complex formation
(Fig. 5B). These data strongly suggest that the Cdt1p–Mcm6p
interaction through the C-terminal regions of the two proteins is
required for the assembly and integrity of the Mcm2-7p complex.
We reasoned that when the BD–Cdt1-5A or AD–Mcm6-5A
mutant failed to support MCM complex formation, MCM nuclear
accumulation should also be abolished. To verify this, cells from
the same strains used in Fig. 5A,B were blocked in M phase
using nocodazole, shifted to 37˚C to degrade the Cdt1-td or
Mcm6-td protein, respectively, and then released into G1 phase
in fresh medium containing a-factor at 37˚C to examine
localization of Mcm4–GFP. G1-phase cells at 25˚C and M-
phase cells at 37˚C were used as the positive and negative
controls, respectively, for MCM nuclear localization. The results
showed that the BD–Cdt1-5A mutant did not support Mcm4–
GFP nuclear accumulation in G1 phase at 37˚C when the Cdt1-td
protein was depleted. However, Mcm4–GFP localized normally
in cells expressing wild-type BD–Cdt1p at both 25˚C and 37˚C
and those expressing BD–Cdt1-5A at 25˚C (Fig. 5C). Similarly,
the Mcm6-5A mutant failed to allow nuclear accumulation of
Mcm4–GFP when the Mcm6-td protein was depleted (Fig. 5C).
We further investigated the effects of the mutations in Cdt1-5A
on pre-RC formation during the M-to-G1 transition. cdt1-td cells
expressing BD–Cdt1p or BD–Cdt1-5A were synchronized in M
phase, shifted to 37˚C, and then released into G1 phase in
medium containing a-factor at 37˚C. Chromatin binding assays
were performed to examine Mcm2p and other proteins on
chromatin to evaluate pre-RC formation. The results showed that
the BD–Cdt1-5A mutant did not support pre-RC formation
because Mcm2p was absent from chromatin when cells passed
through M phase into G1 phase, whereas in cells expressing
the wild-type BD–Cdt1p, Mcm2p was loaded onto chromatin
30 minutes after release from the M-phase block (Fig. 5D,E).
These results indicate that the interaction between Cdt1p and
Mcm6p is essential for pre-RC formation.
To investigate the effects of the Cdt1-5A mutations on DNA
replication, we studied the cell cycle profile of cdt1-td cells
expressing BD–Cdt1p or BD–Cdt1-5A. Cells were induced to
degrade the Cdt1-td protein in M phase and then released into a-
factor-containing medium at 37˚C. The synchronized G1 cells
were then released into fresh medium at 37˚C, and the DNA
content of the cells in different time points was analyzed by flow
cytometry. As Fig. 5F shows, when the Cdt1-td protein was
depleted, the cells expressing the wild-type BD–Cdt1p replicated
normally, whereas the cells expressing the BD–Cdt1-5A mutant
could not duplicate their DNA, even though cell budding
occurred normally. These results indicate that BD–Cdt1-5A
does not support DNA replication. Together, data shown in
Figs 4 and 5 indicate that disruption of the Cdt1p–Mcm6p
interaction by the mutations in Cdt1-5A impairs MCM complex
formation and nuclear localization, pre-RC assembly and cell
In our effort to better understand the physiological roles of Cdt1p
in pre-RC formation and the assembly mechanism of the MCM
complex, we provide the first evidence that Cdt1p, through
its interaction with Mcm6p with a small C-terminal region of
each of the two proteins, is crucial for both the assembly and
integrity of the non-chromatin-bound MCM complex. Once
this interaction is disrupted, by depleting Cdt1p or Mcm6p,
expressing the Mcm6p-interacting region of Cdt1p, or mutating
the interacting interface between Cdt1p and Mcm6p, the MCM
complex disassembles and loses its ability to accumulate in the
nucleus and to form the pre-RC. Furthermore, we provide a
molecular explanation as to why Cdt1p is required for the nuclear
import of MCM proteins that contain NLSs.
Cdt1p and MCM complex formation215
Journal of Cell Science
The Cdt1p–Mcm6p interaction is crucial for MCM complex
formation in vivo
Several models have been proposed for the assembly of the MCM
complex. Studies from E. coli to metazoans suggest that the
architecture of replicative helicases, including Mcm2-7p, is a ring-
shaped hexamer. In addition, depletion of any subunit of Mcm2-7p
results in the mislocalization of the MCM complex (Pasion and
Forsburg,1999;Labib et al., 2001).These and other results suggest
that Mcm2-7p function together as a heterohexamer (Davey et al.,
2003; Evrin et al., 2009; Remus et al., 2009). However, it is not
clear how Mcm2-7p assembles to form a hexameric complex, or
whether Cdt1p plays a role in this process.
Fig. 5. Mutations in Mcm6-5A and Cdt1-5A abolish Mcm2-7p nuclear accumulation, MCM complex integrity, pre-RC formation and DNA replication.
(A) The Cdt1-td protein was depleted in M-phase-synchronized YL1215 (cdt1-td) cells containing the empty BD vector or the plasmid expressing BD-Cdt1 or
BD-Cdt1-5A. WCEs were immunoprecipitated with anti-Mcm2p antibody or the control mouse IgG (Ctrl. IgG), and WCEs and immunoprecipitates were
immunoblotted with anti-Myc antibody to detect the BD–Cdt1p and BD–Cdt1-5A fusion proteins. Mcm2p, Mcm3p and Mcm4–GFP were also immunoblotted.
(B) Mcm6-td was depleted in M-phase-synchronized YL1224 (mcm6-td) cells containing the empty AD vector or plasmid expressing AD–Mcm6 or AD–Mcm6-
5A. WCEs were immunoprecipitated with anti-Mcm2p antibody or the control mouse IgG (Ctrl. IgG), and WCEs and immunoprecipitates were immunoblotted
with anti-HA antibody to detect the AD–Mcm6p and AD–Mcm6-5A fusion proteins. Mcm2p, Mcm3p and Mcm4–GFP were also immunoblotted. (C) YL1224
(mcm6-td) and YL1215 (cdt1-td) cells containing the empty vector or the plasmid expressing Mcm6p, Cdt1p, Mcm6-5A or Cdt1-5A, were synchronized by a-
factor (a-F.) before being released into fresh medium containing nocodazole (Noc.). Cells blocked in M phase were shifted to 37˚C to degrade the Mcm6-td or
Cdt1-td protein, respectively, and then released to fresh medium at 37˚C containing a-factor to synchronize cells in G1 phase. The cells in G1 phase at 25˚C (a-F.,
25˚C) and those synchronized in M (Noc., 37˚C) or G1 (a-F., 37˚C) phase at 37˚C were taken for analysis. The proportion of the cells with nuclear Mcm4–GFP
was quantified (n5300). (D) YL1208 (cdt1-td) cells containing the plasmid expressing Cdt1p or Cdt1-5A were synchronized in M phase and were then shifted to
37˚C for 1.5 hours before being released into fresh medium containing a-factor at 37˚C for two hours. Samples were taken at 30 minute intervals. The chromatin
fraction (Chr.) and (WCEs) of the cell samples were immunoblotted for Mcm2p, Orc3p, Cdt1p and Cdt1-5A. (E) Flow cytometry analysis performed for the cells
from D. % Bud., percentage of budded cells. (F) YL1208 (cdt1-td) cells containing the plasmid expressing Cdt1p or Cdt1-5A were synchronized in M phase
before being shifted to 37˚C for 1.5 hours to deplete the Cdt1-td protein. Afterwards, cells were released into fresh medium containing a-factor at 37˚C for 2 hours
and then released into fresh medium at 37˚C. Flow cytometry was performed for the cell samples taken at the indicated time points.
Journal of Cell Science 125 (1)216
Journal of Cell Science
It was reported that approximately equimolar Cdt1p was
readily co-purified with the Mcm2-7p complex from yeast cell
extracts (Kawasaki et al., 2006; Remus et al., 2009; Tsakraklides
and Bell, 2010). It was also shown that, without co-
overexpression of Cdt1p, most of the overexpressed MCM
subunits in yeast cell extracts formed sub-complexes other than
the Mcm2-7p hexamer (Tsakraklides and Bell, 2010). These
observations are consistent with our results that non-chromatin-
bound MCM proteins cannot form a heterohexameric complex
after Cdt1p depletion in vivo. However, a fraction of the
overexpressed Mcm2-7p subunits could be purified as hexamers
from yeast extracts without Cdt1p co-overexpression in G1 phase
cells (Evrin et al., 2009; Tsakraklides and Bell, 2010). The MCM
complex purified this way might originate from the chromatin-
bound MCM proteins, because we found that depletion of Cdt1p
does not affect the integrity of the chromatin-bound MCM
complex in G1 phase. In an in vitro system, six separately
purified recombinant MCM subunits at 17.6 mM each could form
a hexamer without Cdt1p (Davey et al., 2003). However, it is
noteworthy that the concentrations of most MCM subunits in the
cell are around 1 mM or lower (Ghaemmaghami et al., 2003). It is
possible that the in vitro environment with relatively high
concentrations of MCM proteins, low ion strength and lack of
competition from other proteins facilitates the intermolecular
interactions and thus MCM complex formation. In some other
reports, the MCM complex was purified from insect cells co-
overexpressing Mcm2-7p (Schwacha and Bell, 2001; Bochman
and Schwacha, 2007). However, these reports also showed a
significant increase of the molecular weight of the purified MCM
complex (Schwacha and Bell, 2001), suggesting that the MCM
complex is associated with other proteins, possibly including
Cdt1p from insect cells.
By contrast, it is reported that Mcm4p–Mcm6p–Mcm7p and
Mcm3p–Mcm5p form separated sub-complexes in vitro (You
et al., 1999). Our results from yeast two-hybrid assay support
these observations in budding yeast, because MCM subunits that
are not neighbors in the hexamer also had interactions (data not
shown), which is consistent with the data in human and murine
MCM (Kneissl et al., 2003; Yu et al., 2004). However, the
results from the co-immunoprecipitation and sucrose gradient
centrifugation assay in this study did not show the clear existence
of sub-complexes. There at least two possible reasons for the
absence of sub-complexes in our experiments. First, the salt
concentration of our lysis buffer is relatively high (200 mM K-
Glutamate) which might break the sub-complexes. Second, the
resolution of gradient ultra-centrifugation might not be high
enough to differentiate the monomers and sub-complexes.
Because it has been shown that the functional MCM complex
is the heterohexamer (Gambus et al., 2006; Moyer et al., 2006),
we did not further investigate the role of Cdt1p in sub-complex
In this study, we used three different methods, namely cdt1-td
protein depletion, overexpression of the Mcm6p-interacting
domain of Cdt1p, and mutation of the interacting surface of
Mcm6p and Cdt1p, to disrupt the Cdt1p–Mcm6p interaction, and
observed similar phenotypes: disassembly and failed nuclear
accumulation of the MCM complex, defective pre-RC formation
and DNA replication. A recent study showed in budding yeast
that Cdt1p C-terminal mutations, which include some of the
mutation sites in Cdt1-5A cause a lethal phenotype (Jee et al.,
2010), consistent with our results that the Mcm6-5A and Cdt1-5A
mutations disrupt the Cdt1p–Mcm6p interaction. These results
demonstrate that the interaction between Cdt1p and Mcm6p is
crucial for the assembly and integrity of the Mcm2-7p complex in
The C-terminal region of Cdt1p is conserved in eukaryotes
Despite the fact that Cdt1p is not highly conserved in eukaryotes
with only 10–12% protein sequence identity among the
homologues, we noted high sequence similarity in the C-
terminal region of Cdt1p from different species (supplementary
material Fig. S5B). Consistently with the possibility that this
conserved domain might perform some essential functions, we
demonstrate that, like its homologues in other eukaryotes, the C-
terminal domain of yeast Cdt1p interacts with Mcm6p. Our
findings also suggest that Cdt1p, as a stabilizer to maintain the
integrity of the MCM complex, can be another safeguard to
prevent re-replication by ensuring the timely disassembly and
nuclear export of the excess MCM complex, which is not loaded
onto pre-RCs. Our study also raised a question as to whether the
function of Cdt1p in maintaining the Mcm2-7p integrity is
conserved in other eukaryotes.
A model for the role of Cdt1p in ensuring timely MCM
The timing of MCM complex nuclear export has been
extensively studied. Mcm2-7p that is not chromatin bound is
excluded from the nucleus at the G1-to-S transition (Labib et al.,
1999; Nguyen et al., 2000) (see model in supplementary material
Fig. S7A). However, the MCM complexes on the replication
forks are exported to the cytoplasm during DNA synthesis (Labib
et al., 1999; Nguyen et al., 2000) (see model in supplementary
material Fig. S7A). Similarly to the nuclear export of the excess
Mcm2-7p in late G1 phase, nuclear exclusion of Cdt1p occurs
before activation of Cdc7p–Dbf4p (DDK) (Labib et al., 1999;
Nguyen et al., 2000; Tanaka and Diffley, 2002). The coincidence
of the timing of nuclear export of Cdt1p and Mcm2-7p at the G1-
to-S transition suggests that they are exported together as an
MCM–Cdt1p complex during this time window.
It was suggested that, of the six MCM subunits, only Mcm3p
contains an NES as a part of the mechanism that regulates the
localization of the MCM complex (Liku et al., 2005). However,
by carefully analyzing the protein sequences of the six MCM
subunits using the eukaryotic linear motif server (Puntervoll et al.,
2003), we found that all MCM subunits contained putative NES
(supplementary material Fig. S6). Interestingly, most of these
NESs, except that in Mcm3p, are located at or near the ATPase
motifs or regions that are homologous to the so called ‘A-
domain’ of the Sulfolobus solfataricus MCM protein (Brewster
et al., 2008) (supplementary material Fig. S6). Because MCM
subunits form the MCM complex by interacting with one another
through their ATPase motifs (Davey et al., 2003), and the
A-domains might also localize to the interface of the MCM
subunits, which is suggested by the crystal structure of the
Sulfolobus solfataricus MCM protein (Brewster et al., 2008), the
NESs in the budding yeast MCM proteins, except that in Mcm3p,
might be blocked in the MCM complex.
Based on our findings, we propose a model for the regulation
of Cdt1p and MCM complex localization (supplementary
material Fig. S7). The NESs on the MCM subunits, except that
in Mcm3p, are protected from being recognized by binding with
other MCM subunits (supplementary material Fig. S7B). At the
Cdt1p and MCM complex formation217
Journal of Cell Science
G1-to-S transition, CDK activates the NES on Mcm3p to
export the non-chromatin-bound MCM–Cdt1p complex to the
cytoplasm (supplementary material Fig. S7A). The MCM
complex on chromatin disassociates from replication forks
during S-phase progression, and disintegrates because there is
no Cdt1p in the nucleus to maintain its integrity (supplementary
material Fig. S7A). Dissolution of the Mcm2-7p complex causes
the exposure of the NESs and nuclear exportation of all MCM
subunits (supplementary material Fig. S7A). In the cytoplasm,
the six MCM subunits and Cdt1p assemble to form a Cdt1–MCM
complex, which is still excluded from the nucleus by the NES on
Mcm3p until late mitosis when the CDK activity is low
(supplementary material Fig. S7A). This model, which is
mostly supported by data presented here and from others,
explains why depletion of any one subunit of the Cdt1p–Mcm2-
7p complex abolishes the nuclear importation and retention of
other subunits, and why the nuclear localization of the Mcm2-7p
complex, in which Mcm2p and Mcm3p have NLSs, depends on
Cdt1p, thus providing new insight into the regulation of the
Cdt1p–MCM complex in DNA replication.
Materials and Methods
Strains, plasmids and antibodies
The strains and plasmids used in this study are listed in supplementary material
Tables S1 and S2, respectively. Anti-Orc3p, anti-Mcm2p, anti-Mcm3p and anti-
Cdt1p antibody were kind gifts from Bruce Stillman (Cold Spring Harbor
Laboratory, NY, US) and John Diffley, (London Research Institute, London, UK).
Anti-HA, anti–FLAG, anti-Mcm4p and anti–GFP antibodies were purchased from
Roche, Sigma-Aldrich and Santa Cruz Biotechnology, respectively.
Yeast two-hybrid assay
Matchmaker system III from Clontech was used for the yeast two-hybrid assay,
conducted as previously described (Kan et al., 2008; Lai et al., 2011; Wang et al.,
Cell cycle synchronization and flow cytometry
Cell cycle block and release with a-factor or nocodazole was carried out as
described previously for the cdt1-td cells (Tanaka and Diffley, 2002) or mcm6-td
cells (Labib et al., 2001). Doxycycline (20 mg/ml) was added to cdt1-td cells
before shifting the cells to 37˚C. Flow cytometry was performed as previously
described (Zhang et al., 2002). 16107cells were collected and fixed with 70%
ethanol at 4˚C for at least 1 hour. Cells were resuspended with 1 ml of 50 mM
sodium citrate containing 0.25 mg/ml RNaseA. After incubation at 50˚C for
1 hour, cells were further treated with 1 mg/ml Proteinase K for 1 hour at 50˚C.
Finally, cells were stained with 2 mg/ml propidium iodide. Fluorescence of each
sample was measured on FACSort (Becton Dickinson).
Chromatin binding assay
The chromatin binding assay to examine the cell cycle patterns of chromatin-
associated proteins was performed as previously described (Liang and Stillman,
1997; Zhang et al., 2002) with minor modifications. The spheroplasts were
prepared by digesting the cell wall with lyticase. The spheroplasts were lysed in
extraction buffer containing 0.1% Triton X-100. The lysates were centrifuged at
500 g for 10 minutes to eliminate most of unlysed cells. The clarified lysates were
underlaid with a 30% sucrose cushion and centrifuged at 21,500 g for 10 minutes
to separate the soluble and crude chromatin fractions.
Cells expressing Mcm4–GFP were washed once with ice-cold PBS before
observation. Images were captured using Nikon TE2000 E microscope with SPOT
RT1200 camera. Quantification of fluorescence intensity was carried out by using
the Metamorph 6.2 software to measure the average signal intensity in the nucleus
compared with the whole cell region. Thirty cells from each sample were studied.
Quantification of the percentage of cells with nuclear accumulated Mcm4–GFP
was conducted by counting more than 300 cells for each time point.
Quantitative plasmid loss assay
The quantitative plasmid loss assay was performed as previously described (Ma
et al., 2010; Zhang et al., 2002) with minor modifications. Cells containing p1ARS
or p8ARSs were grown to early log phase (26106cells/ml) in SCM –Trp –Leu
medium at 25˚C, and plated on SCM –Trp or SCM –Trp –Leu for the initial time
point. Cells were then diluted to 26105cells/ml in SCM –Trp and cultured for 10–
11 generations at 25˚C before they were plated for the final time point. Loss rates
(percentage per generation) were calculated using the equation [1–(F/I)1/N]
6100%, where I is the initial percentage of plasmid-containing cells and F is the
percentage of plasmid-containing cells after N generations. All loss rates were the
average of three separate experiments.
100 ml of yeast culture at OD60052 (,26107cells/ml) were harvested for one co-
immunoprecipitation. Yeast whole cell extracts were prepared by bead beating in
Buffer K-200 (25 mM HEPES, pH 7.5, 5 mM magnesium acetate, 1 mM EDTA,
1 mM EGTA, 200 mM K-glutamate, 0.5% Triton X-100, 10% glycerol, 16
protease inhibitor). Extracts were diluted to 10 mg/ml, and pre-cleared by
centrifugation at 21,500 g for 30 minutes and then immunoprecipitated with
immunoprecipitates were then collected with Protein-G beads for 2 hours at 4˚C.
The immunoprecipitates were washed five times with pre-chilled Buffer K-200
(Evrin et al., 2009) before elution with SDS sample buffer at 95˚C.
mouseIgGovernight at4˚C. The
Sucrose gradient centrifugation
50 ml of yeast culture at OD52 (,26107cells/ml) were harvested for each
sample. Yeast whole cell extracts were prepared by bead beating in Buffer GF
(50 mM HEPES, pH 7.5, 150 mM NaCl, 16 protease inhibitor). Extracts
containing 1 mg of total protein together with molecular size makers (0.5 U
alkaline phosphatase and 0.5 U b-galactosidase) were applied on the top of a 2ml
20–50% sucrose gradient in Buffer GF with protease inhibitors. The sucrose
gradient was centrifuged at 55,000 r.p.m. for 14 hours at 4˚C in a TSL-55 rotor.
Twenty-six fractions were collected from the top of the gradient after
centrifugation. The fractions containing alkaline phosphatase and b-galactosidase
were detected by activity assay (Chen et al., 2010).
We gratefully acknowledge Bruce Stillman, John Diffley and
Etienne Schwob for kindly providing yeast strains and antibodies,
Chuchu Zhang and Siu Wong Chung for technical assistance, Bik-
Kwoon Tye and John Diffley for helpful discussion and suggestions,
and Bik-Kwoon Tye for critical reading of the manuscript.
Supported by the Hong Kong Research Grants Council [grant
numbers HKUST6140/03M, HKUST6114/04M and HKUST6430/
06M] to C.L.
Supplementary material available online at
Arias, E. E. and Walter, J. C. (2005). Replication-dependent destruction of Cdt1 limits
DNA replication to a single round per cell cycle in Xenopus egg extracts. Genes Dev.
Asano, T., Makise, M., Takehara, M. and Mizushima, T. (2007). Interaction between
ORC and Cdt1p of Saccharomyces cerevisiae. FEMS Yeast Res. 7, 1256-1262.
Bell, S. P. and Dutta, A. (2002). DNA replication in eukaryotic cells. Annu. Rev.
Biochem. 71, 333-374.
Bochman, M. L. and Schwacha, A. (2007). Differences in the single-stranded DNA
binding activities of MCM2-7 and MCM467. J. Biol. Chem. 282, 33795-33804.
Brewster, A. S., Wang, G., Yu, X., Greenleaf, W. B., Carazo, J. M., Tjajadi, M.,
Klein, M. G. and Chen, X. S. (2008). Crystal structure of a near-full-length archaeal
MCM: functional insights for an AAA+ hexameric helicase. Proc. Natl. Acad. Sci.
USA 105, 20191-20196.
Chen, S., de Vries, M. A. and Bell, S. P. (2007). Orc6 is required for dynamic
recruitment of Cdt1 during repeated Mcm2-7 loading. Genes Dev. 21, 2897-2907.
Chen, V. P., Xie, H. Q., Chan, W. K., Leung, K. W., Chan, G. K., Choi, R. C., Bon,
S., Massoulie ´, J. and Tsim, K. W. (2010). The PRiMA-linked cholinesterase
tetramers are assembled from homodimers: hybrid molecules composed of
acetylcholinesterase and butyrylcholinesterase dimers are up-regulated during
development of chicken brain. J. Biol. Chem. 285, 27265-27278.
Cheng, X., Xu, Z., Wang, J., Zhai, Y., Lu, Y. and Liang, C. (2010). ATP-dependent
pre-replicative complex assembly is facilitated by Adk1p in budding yeast. J. Biol.
Chem. 285, 29974-29980.
Davey, M. J., Indiani, C. and O’Donnell, M. (2003). Reconstitution of the Mcm2-7p
heterohexamer, subunit arrangement, and ATP site architecture. J. Biol. Chem. 278,
Devault, A., Vallen, E. A., Yuan, T., Green, S., Bensimon, A. and Schwob, E. (2002).
Identification of Tah11/Sid2 as the ortholog of the replication licensing factor Cdt1 in
Saccharomyces cerevisiae. Curr. Biol. 12, 689-694.
Journal of Cell Science 125 (1)218
Journal of Cell Science
Evrin, C., Clarke, P., Zech, J., Lurz, R., Sun, J., Uhle, S., Li, H., Stillman, B. and
Speck, C. (2009). A double-hexameric MCM2-7 complex is loaded onto origin DNA
during licensing of eukaryotic DNA replication. Proc. Natl. Acad. Sci. USA 106,
Gambus, A., Jones, R. C., Sanchez-Diaz, A., Kanemaki, M., van Deursen, F.,
Edmondson, R. D. and Labib, K. (2006). GINS maintains association of Cdc45 with
MCM in replisome progression complexes at eukaryotic DNA replication forks. Nat.
Cell Biol. 8, 358-366.
Ghaemmaghami, S., Huh, W. K., Bower, K., Howson, R. W., Belle, A., Dephoure,
N., O9Shea, E. K. and Weissman, J. S. (2003). Global analysis of protein expression
in yeast. Nature 425, 737-741.
Higa, L. A., Mihaylov, I. S., Banks, D. P., Zheng, J. and Zhang, H. (2003). Radiation-
mediated proteolysis of CDT1 by CUL4-ROC1 and CSN complexes constitutes a new
checkpoint. Nat. Cell Biol. 5, 1008-1015.
Hogan, E. and Koshland, D. (1992). Addition of extra origins of replication to a
minichromosome suppresses its mitotic loss in cdc6 and cdc14 mutants of
Saccharomyces cerevisiae. Proc. Natl. Acad. Sci. USA 89, 3098-3102.
Jee, J., Mizuno, T., Kamada, K., Tochio, H., Chiba, Y., Yanagi, K., Yasuda, G.,
Hiroaki, H., Hanaoka, F. and Shirakawa, M. (2010). Structure and mutagenesis
studies of the C-terminal region of licensing factor Cdt1 enable the identification of
key residues for binding to replicative helicase Mcm proteins. J. Biol. Chem. 285,
Kan, J., Zou, L., Zhang, J., Wu, R., Wang, Z. and Liang, C. (2008). Origin
recognition complex (ORC) mediates histone 3 lysine 4 methylation through
cooperation with Spp1 in Saccharomyces cerevisiae. J. Biol. Chem. 283, 33803-
Kawasaki, Y., Kim, H. D., Kojima, A., Seki, T. and Sugino, A. (2006). Reconstitution
of Saccharomyces cerevisiae prereplicative complex assembly in vitro. Genes Cells
Kneissl, M., Pu ¨tter, V., Szalay, A. A. and Grummt, F. (2003). Interaction and
assembly of murine pre-replicative complex proteins in yeast and mouse cells. J. Mol.
Biol. 327, 111-128.
Labib, K. (2010). How do Cdc7 and cyclin-dependent kinases trigger the initiation of
chromosome replication in eukaryotic cells? Genes Dev. 24, 1208-1219.
Labib, K., Diffley, J. F. and Kearsey, S. E. (1999). G1-phase and B-type cyclins
exclude the DNA-replication factor Mcm4 from the nucleus. Nat. Cell Biol. 1, 415-
Labib, K., Kearsey, S. E. and Diffley, J. F. (2001). MCM2-7 proteins are essential
components of prereplicative complexes that accumulate cooperatively in the nucleus
during G1-phase and are required to establish, but not maintain, the S-phase
checkpoint. Mol. Biol. Cell 12, 3658-3667.
Lai, F., Wu, R., Wang, J., Li, C., Zou, L., Lu, Y. and Liang, C. (2011). Far3p domains
involved in the interactions of Far proteins and pheromone-induced cell cycle arrest in
budding yeast. FEMS Yeast Res. 11, 72-79.
Liang, C. and Stillman, B. (1997). Persistent initiation of DNA replication and
chromatin bound MCM proteins during the cell cycle in cdc6 mutants. Genes Dev. 11,
Liku, M. E., Nguyen, V. Q., Rosales, A. W., Irie, K. and Li, J. J. (2005). CDK
phosphorylation of a novel NLS-NES module distributed between two subunits of the
Mcm2-7 complex prevents chromosomal rereplication. Mol. Biol. Cell 16, 5026-5039.
Loo, S., Fox, C. A., Rine, J., Kobayashi, R., Stillman, B. and Bell, S. (1995). The
origin recognition complex in silencing, cell cycle progression, and DNA replication.
Mol. Biol. Cell 6, 741-756.
Ma, L., Zhai, Y., Feng, D., Chan, T., Lu, Y., Fu, X., Wang, J., Chen, Y., Li, J., Xu,
K. and et al. (2010). Identification of novel factors involved in or regulating initiation
of DNA replication by a genome-wide phenotypic screen in Saccharomyces
cerevisiae. Cell Cycle 9, 4399-4410.
Maiorano, D., Moreau, J. and Mechali, M. (2000). XCDT1 is required for the
assembly of pre-replicative complexes in Xenopus laevis. Nature 404, 622-625.
Me ´ndez, J. and Stillman, B. (2003). Perpetuating the double helix: molecular machines
at eukaryotic DNA replication origins. BioEssays 25, 1158-1167.
Moyer, S. E., Lewis, P. W. and Botchan, M. R. (2006). Isolation of the Cdc45/Mcm2-
7/GINS (CMG) complex, a candidate for the eukaryotic DNA replication fork
helicase. Proc. Natl. Acad. Sci. USA 103, 10236-10241.
Nguyen, V. Q., Co, C., Irie, K. and Li, J. J. (2000). Clb/Cdc28 kinases promote nuclear
export of the replication initiator proteins Mcm2-7. Curr. Biol. 10, 195-205.
Nishitani, H., Lygerou, Z. and Nishimoto, T. (2004). Proteolysis of DNA replication
licensing factor Cdt1 in S-phase is performed independently of geminin through its N-
terminal region. J. Biol. Chem. 279, 30807-30816.
Pasion, S. G. and Forsburg, S. L. (1999). Nuclear localization of Schizo-
saccharomycespombe Mcm2/Cdc19p requires MCM complex assembly. Mol. Biol.Cell
Puntervoll, P., Linding, R., Gemu ¨nd, C., Chabanis-Davidson, S., Mattingsdal, M.,
Cameron, S., Martin, D. M. A., Ausiello, G., Brannetti, B., Costantini, A. et al.
(2003). ELM server: a new resource for investigating short functional sites in modular
eukaryotic proteins. Nucleic Acids Res. 31, 3625-3630.
Remus, D., Beuron, F., Tolun, G., Griffith, J. D., Morris, E. P. and Diffley, J. F.
(2009). Concerted loading of Mcm2-7 double hexamers around DNA during DNA
replication origin licensing. Cell 139, 719-730.
Schwacha, A. and Bell, S. P. (2001). Interactions between two catalytically distinct
MCM subgroups are essential for coordinated ATP hydrolysis and DNA replication.
Mol. Cell 8, 1093-1104.
Tanaka, S. and Diffley, J. F. (2002). Interdependent nuclear accumulation of budding
yeast Cdt1 and Mcm2-7 during G1 phase. Nat. Cell Biol. 4, 198-207.
Teer, J. K. and Dutta, A. (2008). Human Cdt1 lacking the evolutionarily conserved
region that interacts with MCM2-7 is capable of inducing re-replication. J. Biol.
Chem. 283, 6817-6825.
Tsakraklides, V. and Bell, S. P. (2010). Dynamics of pre-replicative complex assembly.
J. Biol. Chem. 285, 9437-9443.
Wang, J., Wu, R., Lu, Y. and Liang, C. (2010). Ctf4p facilitates Mcm10p to promote
DNA replication in budding yeast. Biochem. Biophys. Res. Commun. 395, 336-341.
Wei, Z., Liu, C., Wu, X., Xu, N., Zhou, B., Liang, C. and Zhu, G. (2010).
Characterization and structure determination of the Cdt1 binding domain of human
Minichromosome Maintenance (Mcm) 6. J. Biol. Chem. 285, 12469-12473.
Wohlschlegel, J. A., Dwyer, B. T., Dhar, S. K., Cvetic, C., Walter, J. C. and Dutta, A.
(2000). Inhibition of eukaryotic DNA replication by geminin binding to Cdt1. Science
Yanagi, K., Mizuno, T., You, Z. and Hanaoka, F. (2002). Mouse geminin inhibits not
only Cdt1-MCM6 interactions but also a novel intrinsic Cdt1 DNA binding activity. J.
Biol. Chem. 277, 40871-40880.
You, Z., Komamura, Y. and Lshimi, Y. (1999). Biochemical analysis of the intrinsic
Mcm4-Mcm6-mcm7 DNA helicase activity. Mol. Cell. Biol. 19, 8003-8015.
Yu, Z., Feng, D. and Liang, C. (2004). Pairwise interactions of the six human MCM
protein subunits. J. Mol. Biol. 340, 1197-1206.
Zhai, Y., Yung, P. Y. K., Huo, L. and Liang, C. (2010). Cdc14p resets the competency
of replication licensing by dephosphorylating multiple initiation proteins during
mitotic exit in budding yeast. J. Cell Sci. 123, 3933-3943.
Zhang, J., Yu, L., Wu, X., Zou, L., Sou, K. K. L., Wei, Z., Cheng, X., Zhu, G. and
Liang, C. (2010). The interacting domains of hCdt1 and hMcm6 involved in the
chromatin loading of the MCM complex in human cells. Cell Cycle 9, 4848-4857.
Zhang, Y., Yu, Z., Fu, X. and Liang, C. (2002). Noc3p, a bHLH protein, plays an
integral role in the initiation of DNA replication in budding yeast. Cell 109, 849-860.
Cdt1p and MCM complex formation219
Journal of Cell Science