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 (firstname.lastname@example.org)
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
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