© 2015. Published by The Company of Biologists Ltd | Journal of Cell Science (2015) 128, 2482-2496 doi:10.1242/jcs.166553
RESEARCH ARTICLE
Control of Cdc6 accumulation by Cdk1 and MAPK is essential for
completion of oocyte meiotic divisions in Xenopus
ABSTRACT
Vertebrate oocytes proceed through the first and the second meiotic
division without an intervening S-phase to become haploid. Although
DNA replication does not take place, unfertilized oocytes acquire the
competence to replicate DNA one hour after the first meiotic division
by accumulating an essential factor of the replicative machinery,
Cdc6. Here, we show that the turnover of Cdc6 is precisely regulated
in oocytes to avoid inhibition of Cdk1. At meiosis resumption, Cdc6 is
expressed but cannot accumulate owing to a degradation mechanism
that is activated through Cdk1. During transition from the first to the
second meiotic division, Cdc6 is under the antagonistic regulation of
B-type cyclins (which interact with and stabilize Cdc6) and the Mos–
MAPK pathway (which negatively controls Cdc6 accumulation).
Because overexpressing Cdc6 inhibits Cdk1 reactivation and drives
oocytes into a replicative interphasic state, the fine-tuning of Cdc6
accumulation is essential to ensure two meiotic waves of Cdk1
activation and to avoid unscheduled DNA replication during meiotic
maturation.
KEY WORDS: Meiotic division, Cdc6, Cdk1, Xenopus oocyte
INTRODUCTION
In proliferative cells, Cdc6 is an important protein for the initiation
of DNA replication by allowing the assembly of pre-replicative
complexes ( pre-RCs) during G1 on replicative origins (Diffley,
2004). Independently of this licensing activity, Cdc6 is required for
spindle formation during mitosis and meiosis (Anger et al., 2005;
Illenye and Heintz, 2004; Narasimhachar et al., 2012; Yim and
Erikson, 2010). Cdc6 further regulates M-phase progression by
activating checkpoint mechanisms at the G2/M transition and by
inhibiting Cdk1 activity during mitosis exit (Borlado and Mendez,
2008; Yim and Erikson, 2010, 2011). Intriguingly, little is known
regarding the role and the regulation of Cdc6 during the meiotic cell
cycle besides its requirement for the acquisition of competence to
replicate DNA in female germ cells.
Xenopus laevis oocytes are naturally arrested in prophase of the
first meiotic division (meiosis I) and resume meiosis at the time of
ovulation following progesterone stimulation. Oocytes progress
through the first and then second meiotic division (meiosis II),
where they halt at the metaphase (M)II stage, awaiting fertilization.
Importantly, DNA replication is repressed between the two meiotic
divisions in order to generate haploid gametes. Prophase oocytes
have developed a similar strategy to that of quiescent cells arrested
in G0 to avoid unscheduled DNA replication during their
1
UPMC Univ Paris 06, UMR7622, Biologie du Dé veloppement, Institut de Biologie
2
Paris-Seine (IBPS), 75005 Paris, France. CNRS, UMR7622, Biologie du
Dé veloppement, Institut de Biologie Paris-Seine (IBPS), 75005 Paris, France.
*Author for correspondence ([email protected])
Received 2 December 2014; Accepted 19 May 2015
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long-lasting arrest. In both cases, the inability to replicate is due
to the absence of Cdc6. As cells re-enter the cell cycle, either in G1
for somatic cells or in meiosis I for oocytes, the ability to replicate is
restored through Cdc6 accumulation (Cook et al., 2002; Duursma
and Agami, 2005; Lemaître et al., 2002, 2004; Mailand and Diffley,
2005; Stoeber et al., 1998; Whitmire et al., 2002). In Xenopus oocytes,
Cdc6 is translated from maternal mRNAs and starts to accumulate 45–
60 min after germinal vesicle breakdown (GVBD, which marks entry
into the first meiotic division) (Lemaître et al., 2002; Whitmire et al.,
2002). Once Cdc6 is present, the replicative machinery becomes
immediately functional, but DNA replication is inhibited (Lemaître
et al., 2002; Whitmire et al., 2002). Because Cdc6 accumulation
coincides with entry into meiosis II, the timing of Cdc6 accumulation
should be essential to prevent unscheduled DNA replication between
the two meiotic divisions. The mechanisms regulating the timing of
Cdc6 accumulation and the functional consequences of its
misregulation on meiotic divisions remain unknown.
Two master kinases orchestrating meiotic divisions, the Cdk1–
Cyclin-B complex and mitogen-activated protein kinase (MAPK;
only one MAPK protein is expressed in Xenopus oocytes), are good
candidates for controlling the timing of Cdc6 accumulation. The
first one, called M-phase promoting factor (MPF), is a complex
between Cdk1 and Cyclin B, and a potent inhibitor of DNA
replication in somatic cells (Diffley, 2004; Gautier et al., 1990,
1988). In Xenopus prophase oocytes, Cdk1–Cyclin-B complexes
are inactive owing to inhibitory phosphorylation at residues T14 and
Y15 on Cdk1 (Karaiskou et al., 2001). Upon progesterone
stimulation, Cdk1 is dephosphorylated and activated, leading to
GVBD and entry into meiosis I (Gautier et al., 1989; Karaiskou
et al., 2001). Once oocytes reach MI, the degradation of Cyclin B
inactivates Cdk1 within 1 h of GVBD to promote anaphase I and
exit from meiosis I. At this stage, although degradation of Cyclin B
is not yet complete, newly synthesized Cyclin B reaccumulates and
reactivates Cdk1 to initiate entry into meiosis II. In Xenopus oocytes,
inhibiting Cdk1 reactivation abolishes entry into meiosis II and
triggers the formation of interphasic replicative nuclei (Furuno et al.,
1994; Huchon et al., 1993; Iwabuchi et al., 2000; Ohsumi et al., 1994).
Therefore, the MI–MII transition is a sensitive period during which
the activity of Cdk1, the main replicative inhibitor, decreases, but
Cdc6 synthesis has already started. Importantly, DNA must not
replicate during the transition to reduce oocyte ploidy.
The second kinase network that might regulate DNA replication
is the Mos–MAPK pathway. Mos is a germ-line specific kinase that
is synthesized in response to progesterone from maternal mRNA
that is indirectly responsible for the activation of MAPK at GVBD
(Dupré et al., 2011). The Mos–MAPK pathway remains active from
GVBD to MII, where it plays an essential role in maintaining MII
arrest until fertilization (Bhatt and Ferrell, 1999; Dupré et al., 2011,
2002; Gross et al., 2000; Haccard et al., 1993; Sagata et al., 1989).
As for Cdk1, inhibiting this pathway during the MI–MII transition
promotes DNA replication in oocytes (Dupré et al., 2002; Furuno
Journal of Cell Science
Enrico M. Daldello1,2, Tran Le1,2, Robert Poulhe1,2, Catherine Jessus1,2, Olivier Haccard1,2 and Aude Dupré 1,2, *
et al., 1994; Gross et al., 2000), clearly demonstrating that both
Cdk1 and the Mos–MAPK pathway negatively control DNA
replication in oocytes.
Our results provide new insights into the mechanisms regulating
Cdc6 accumulation during the meiotic divisions. We show here that
Cdc6 becomes unstable very early upon entry into meiosis I, being
degraded by a mechanism that is dependent on the proteasome and
a Skp1–Cullins–F-box complex in which Cdc4 is the F-box
protein (SCFCdc4; Cdc4 is also known as Fbxw7). Thereafter, both
the Cdk1–Cyclin-B complex and the Mos–MAPK pathway control
the turnover of Cdc6 in opposing ways, leading to the gradual
accumulation of Cdc6 during entry into meiosis II. Tight regulation
of Cdc6 accumulation is essential for meiotic maturation because
the precocious expression of Cdc6 in prophase or in MI impairs
meiosis resumption or drives the oocyte into a pseudo-interphasic
replicative state by inhibiting Cdk1. Therefore, the accumulation of
Cdc6 needs to be strictly synchronized with Cdk1 reactivation after
meiosis I to ensure the success of the meiotic cell cycle progression.
RESULTS
Ectopic Cdc6 is degraded at meiosis I entry in a Cdk1- and
SCFCdc4-dependent manner
To analyze Cdc6 turnover during meiotic maturation, prophase
oocytes were injected with mRNA encoding Xenopus histidinetagged wild-type Cdc6 (His–WT-Cdc6) and then stimulated with
progesterone to induce resumption of meiosis (Fig. 1A). Cdk1 and
MAPK activation was examined by immunoblotting for Cyclin B2
(also known as Ccnb2), of which the phosphorylation and up-shift
by SDS-PAGE depends on Cdk1 activation, and MAPK
phosphorylation (Fig. 1A). Cdc6 protein was visualized using an
antibody against histidine (Fig. 1A). As expected, both Cdk1 and
MAPK were activated at GVBD and in MII (Fig. 1A). His–WTCdc6 was efficiently translated in prophase but became
undetectable 2 h after progesterone addition (Fig. 1A). Cdc6 was
detected 30 min later at GVBD and was still present in MII
(Fig. 1A). To confirm that loss of His–WT-Cdc6 before GVBD was
due to proteolytic degradation, prophase oocytes were injected with
recombinant GST-tagged Xenopus wild-type Cdc6 protein (GST–
WT-Cdc6) and Cdc6 stability was verified using an antibody against
GST. GST–WT-Cdc6 remained stable for at least 5 h following its
injection in prophase (supplementary material Fig. S1A). Oocytes
were further induced to mature by using progesterone (Fig. 1B).
GST–WT-Cdc6 was stable up to 180 min following hormonal
stimulation but became undetectable in oocytes, at the time when
50% of oocytes reached GVBD (GVBD50), independently of GVBD
status (Fig. 1B). Therefore, progesterone induces degradation of
Cdc6 just before GVBD. Because the proteasome inhibitor MG132
or the prior injection of the Cdk inhibitor p21Cip1 (also known as
Cdkn1a) abolished degradation of GST–WT-Cdc6 at GVBD
(Fig. 1C), the underlying mechanism relies on the proteasome and
on the initial activation of Cdk1.
In various cell lines, Cdc6 turnover depends on multiple domains,
including one TPxxS binding site for the F-box protein Cdc4, which
mediates SCFCdc4-dependent degradation (Clijsters and Wolthuis,
2014; Perkins et al., 2001; Petersen et al., 2000). We mutated the
F-box-binding motif TPxxS of Xenopus Cdc6 into APxxS, and
the corresponding mRNA or recombinant GST–APxxS-Cdc6 were
injected into prophase oocytes (Fig. 1A,C). Following progesterone
addition, overexpressed His–APxxS-Cdc6 and recombinant GST–
APxxS-Cdc6 proteins were no longer degraded during meiosis
resumption (Fig. 1A,C). Therefore, ectopic Cdc6 becomes
unstable and is degraded shortly before GVBD by a Cdk1- and
Journal of Cell Science (2015) 128, 2482-2496 doi:10.1242/jcs.166553
proteasome-dependent mechanism involving its F-box-binding
domain and presumably SCFCdc4.
We then investigated whether the absence of endogenous Cdc6 is
due to its degradation or the inhibition of its translation in prophase
or during meiosis resumption. Prophase oocytes were selected at
various steps of oogenesis from stage I to full-grown stage VI
(Dumont, 1972), and lysates were immunoblotted for Cdc6. The
protein was absent from stage-II oocytes onwards (Fig. 1D).
Moreover, inhibiting either Cdk1 activation with p21Cip1 or the
proteasome with MG132 was not sufficient to promote Cdc6
stabilization and detection in prophase oocytes, or in response to
progesterone (Fig. 1E; supplementary material Fig. S1B),
suggesting that the synthesis of endogenous Cdc6 is repressed
during prophase arrest of growing oocytes and is re-initiated around
GVBD following Cdk1 activation.
The stabilization of ectopic Cdc6 during the MI–MII transition
depends on Cyclin B
Cdc6 stability was further analyzed during the MI–MII transition by
injecting GST–WT-Cdc6 or GST–APxxS-Cdc6 at GVBD (Fig. 2).
Following progesterone stimulation, Cyclin B2 was phosphorylated
at GVBD and degraded during the MI–MII transition, and then reaccumulated in MII (Fig. 2). Cdk1 activity was estimated by
monitoring the phosphorylation at residue T320 of a peptide derived
from the protein phosphatase 1 subunit PPP1CC (GST–PP1C-S), as
described in Lewis et al. (2013) (Fig. 2). In agreement with the
Cyclin B2 pattern, Cdk1 underwent two successive waves of
activation, corresponding respectively to the GVBD–MI period and
entry into MII (Fig. 2). MAPK was phosphorylated at GVBD and
remained active until MII (Fig. 2). When injected at GVBD, both
GST–WT-Cdc6 and GST–APxxS-Cdc6 remained detectable up to
4 h after GVBD, and oocytes proceeded normally through the MI–
MII transition (Fig. 2). Therefore, a mechanism that stabilizes Cdc6
becomes effective during the MI–MII transition following Cdk1
activation in meiosis I. The same experiment was performed in the
presence of an inhibitor of protein synthesis, cycloheximide, added
at GVBD. As expected, cycloheximide prevented the accumulation
of Cyclin B2 and Wee1, a kinase that accumulates during entry into
MII (Fig. 2). Under these conditions, GST–WT-Cdc6 and GST–
APxxS-Cdc6 protein levels decreased substantially 180 min after
GVBD (Fig. 2), revealing that Cdc6 is unstable in meiosis I and is
degraded by a SCFCdc4-independent mechanism. Taken together,
these results demonstrate that once Cdk1 is activated, Cdc6 is
stabilized in a protein-synthesis-dependent manner during the MI–
MII transition. Because the addition of cycloheximide at GVBD
prevents Cyclin B2 reaccumulation, and consequently leads to
Cdk1 and MAPK inactivation, this opens up the possibility that one
of these activities, or both of them, control Cdc6 stability after
GVBD.
In yeast and somatic cells, Cdc6 can be stabilized by Cdkdependent phosphorylation at serine or threonine residues [(S/T)-P
sites], or by direct interaction with mitotic cyclins (Borlado and
Mendez, 2008; Mimura et al., 2004). We first generated a mutant of
Cdc6 (GST–7A-Cdc6) in which the (S/T)-P sites were mutated into
alanine residues. Although GST–WT-Cdc6 and GST–APxxS-Cdc6
were efficiently thiophosphorylated in vitro by Cdk1 and MAPK
(supplementary material Fig. S2A,B), the 7A mutant was not
(supplementary material Fig. S2C,D). Furthermore, GST–7A-Cdc6
remained stable when injected at GVBD and was degraded after
cycloheximide addition (Fig. 2B). Therefore, Cdc6 phosphorylation
at (S/T)-P sites is not required to protect Cdc6 from degradation
after MI.
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Journal of Cell Science (2015) 128, 2482-2496 doi:10.1242/jcs.166553
Fig. 1. Recombinant Cdc6 is degraded in response to progesterone. (A) Prophase-arrested oocytes (Pro) were injected with mRNA encoding histidinetagged WT-Cdc6 (His–WT-Cdc6) or histidine-tagged APxxS-Cdc6 (His–APxxS-Cdc6) and then incubated with progesterone (Pg). Oocytes were immunoblotted
at the indicated times for His–Cdc6, Cyclin B2, phosphorylated MAPK ( pMAPK) and total MAPK. Time corresponding to 50% of oocytes exhibiting GVBD
(GVBD50)=150 min. (B) Prophase-arrested oocytes were injected with GST–WT-Cdc6 (0.05 µM) then incubated with progesterone. Oocytes were immunoblotted
at the indicated times for GST–Cdc6, Cyclin B2 (the up-shifted band represents the phosphorylated form), phosphorylated MAPK and total MAPK. No GVBD,
oocytes that had not undergone pigment rearrangement at the time of GVBD50. GVBD50: 240 min. (C) Prophase-arrested oocytes were incubated with MG132 or
injected with p21Cip1. Oocytes were further injected with either GST–WT-Cdc6 or GST–APxxS-Cdc6 (0.05 µM) and then incubated with progesterone. Oocytes
were immunoblotted at the indicated times for GST–Cdc6, Cyclin B2, phosphorylated MAPK and total MAPK. No GVBD: oocytes that had not undergone pigment
rearrangement at the time of GVBD50. GVBD50: 240 min for oocytes incubated with MG132, and 300 min for control oocytes corresponding to p21Cip1-injected
oocytes. (D) Expression of endogenous Cdc6 during oogenesis and meiotic maturation. Equal amounts (25 µg) of proteins from stage-I to stage-VI oocytes,
collected in prophase, at GVBD or in MII were immunoblotted for endogenous Cdc6, Cyclin B2, total MAPK and Cdk1. (E) Prophase-arrested oocytes were
injected or not with p21Cip1 then stimulated or not with progesterone. Oocytes were immunoblotted in prophase (−Pg) or at the time of GVBD (+Pg) for Greatwall
(Gwl; note, the up-shifted band represents the phosphorylated form), Cdc6, phosphorylated MAPK and Cdk1 phosphorylated at residue Y15 ( pY15-Cdk1).
To address the role of B-type cyclins in Cdc6 stabilization, low levels
of a non-degradable form of a truncated mutant termed Δ90-Cyclin-B
were injected together with GST–WT-Cdc6 at GVBD, and
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cycloheximide was concomitantly added (Fig. 2A). Although the
amount of injected Δ90-Cyclin-B was too low to reactivate Cdk1 and
MAPK in the presence of cycloheximide, GST–WT-Cdc6 was no
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Fig. 2. Recombinant Cdc6 is stabilized in meiosis I by Cyclin B. Oocytes were immunoblotted at the indicated times for GST–Cdc6, Wee1, Cyclin B2,
phosphorylated MAPK ( pMAPK) and total MAPK. Cdk1 activity was assayed by monitoring the phosphorylation status at residue T320 of GST–PP1C-S ( pT320PP1C). The total amount of GST–PP1C-S was ascertained with a GST antibody (GST–PP1C-S). (A) Prophase-arrested oocytes (Pro) were incubated with
progesterone and then injected at GVBD with GST–WT-Cdc6 (0.05 µM) together with non-degradable Cyclin B. Oocytes were further incubated or not
with cycloheximide (CHX). (B) Prophase-arrested oocytes were incubated with progesterone and then injected at GVBD with either GST–APxxS-Cdc6 or
GST–7A-Cdc6 (0.05 µM), in the presence or absence of cycloheximide.
longer degraded following GVBD (Fig. 2A). Non-degradable Cyclin B
is thus sufficient to stabilize Cdc6 independently of Cdk1 activity. We
next asked whether GST–WT-Cdc6 interacts with Cdk1–Cyclin-B
complexes. GST–WT-Cdc6 was added to prophase or MII extracts,
and GST was pulled down. Both Cyclin B2 and Cdk1 were recovered
with GST–WT-Cdc6 in MII extracts, as seen by immunoblotting
(Fig. 3A). Control MII-arrested oocytes or oocytes injected at GVBD
with GST–WT-Cdc6 were then fractionated using gel filtration
chromatography on Superose-12 columns (Fig. 3B,C). Cdk1 and
Cyclin B2 eluted in distinct fractions; fractions 7–9 correspond to highmolecular-mass complexes, and fractions 24–26 correspond to the
dimeric Cdk1–Cyclin-B complex, as previously described in De
Smedt et al. (2002). Interestingly, endogenous Cdc6 was recovered
together with Cdk1 and Cyclin B2 in the high-molecular-mass
complexes (Fig. 3B,C). In Cdc6-injected oocytes, the recombinant
protein was also recovered in the fraction containing high-molecularmass complexes (Fig. 3C), suggesting that GST–WT-Cdc6 interacts
with Cdk1–Cyclin-B complexes during the MI–MII transition.
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To determine whether this interaction exists in ovo, Cdk1–Cyclin-B
complexes were affinity-purified from prophase or MII-arrested
oocytes using p13suc1 beads (beads coated with the product of
the yeast gene suc1). Importantly, endogenous Cdc6 was detected by
immunoblotting on p13suc1 beads from MII oocytes (Fig. 4A).
To further confirm the interaction between endogenous Cdc6 and
Cdk1–Cyclin-B, MII extracts were incubated with an antibody against
Cdc6 and then fractionated on a Superose-6 column (Fig. 4B). As
previously observed, endogenous Cdc6 eluted with Cdk1 and
Cyclin B2 in control oocytes (Fig. 4B). In the presence of an
antibody against Cdc6, Cdc6 still co-eluted with Cdk1–Cyclin-B in
fraction 3 (Fig. 4B). IgGs were then recovered from fraction 3 by adding
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protein-G-coupled beads and Cyclin B2, Cdk1 and Cdc6 were detected
on the beads (Fig. 4C). Therefore, Cdk1–Cyclin-B and Cdc6 form
a trimeric complex in meiosis-II oocytes. Altogether, these data
strongly suggest that the interaction between Cdc6 and Cdk1–Cyclin-B
complexes contributes to its stabilization after GVBD.
Cyclin B reaccumulation and MAPK activity regulate Cdc6
accumulation in opposing ways after GVBD
To investigate the roles of B-type cyclins and the Mos–MAPK
pathway on Cdc6 stabilization, the accumulation of endogenous
Cdc6 was monitored in oocytes that had been injected with antisense
oligonucleotides targeting B-type cyclins or Mos mRNAs in order to
Fig. 3. GST–WT-Cdc6 interacts with Cdk1–Cyclin-B during the MI–MII transition. (A) GST beads coupled to GST–WT-Cdc6 or not (Mock) were added
to extracts from prophase- and MII-arrested oocytes (Pro and MII, respectively). The interaction between Cdc6 and Cdk1–Cyclin-B complexes was analyzed
by immunoblotting the beads for GST–Cdc6, Cyclin B2 and Cdk1. The electrophoretic shift of Cdk1 in prophase is due to its phosphorylation at residues T14 and
Y15. (B) Prophase-arrested oocytes were incubated with progesterone and then injected with buffer at GVBD. At 4 h after GVBD, oocytes were collected, and
protein extracts were fractionated on a Superose-12 column. Each fraction was immunoblotted for GST–Cdc6, endogenous Cdc6, Cyclin B2, phosphorylated
MAPK ( pMAPK) and Cdk1. (C) The same experiment as that shown in B, except that oocytes were injected at GVBD with GST–WT-Cdc6 (0.5 µM). Fraction
numbers highlighted in red correspond to high-molecular-mass complexes containing Cdk1–Cyclin-B complexes and Cdc6.
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abolish Cdk1 reactivation and MAPK activation, respectively
(Fig. 5). In control oocytes, Cdk1 activity followed the usual twowave pattern, whereas MAPK was activated at GVBD and remained
active until MII (Fig. 5A). As already reported by Lemaître et al.
(2002) and Whitmire et al. (2002), Cdc6 accumulated progressively,
starting 45–60 min after GVBD until MII (Fig. 5A). According to
Haccard and Jessus (2006) and Hochegger et al. (2001), injecting
antisense oligonucleotides against B-type cyclins did not block the
first wave of activation of Cdk1 and GVBD, but did abolish Cdk1
reactivation and entry into meiosis II (Fig. 5B). As a consequence of
Cdk1 inactivation, MAPK was inactivated 180 min after GVBD
(Fig. 5B). Under these conditions, Cdc6 accumulation was strongly
delayed compared with that in controls – it was only detectable
150 min after GVBD (Fig. 5B) – suggesting that B-type cyclins
positively regulate the accumulation of endogenous Cdc6, in
agreement with our previous hypothesis. It is noteworthy that Cdc6
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accumulation started when MAPK was inactivated in oocytes
that had been injected with antisense oligonucleotides against
B-type cyclins (Fig. 5B), suggesting that MAPK could negatively
regulate Cdc6 accumulation. We then impeded the synthesis of
Mos by injecting morpholino antisense oligonucleotides and, as a
consequence, MAPK was neither phosphorylated nor activated
following hormonal stimulation (Fig. 5C). Interestingly, the
accumulation of endogenous Cdc6 occurred earlier than in control
oocytes, almost at the same time as GVBD (Fig. 5C). The
accumulation of endogenous Cdc6 was also advanced in
progesterone-treated oocytes that had been incubated with the
MEK inhibitor U0126, which prevents MAPK activation during
meiotic maturation (supplementary material Fig. S3). Altogether, our
results demonstrate the positive role of the accumulation of B-type
cyclins on endogenous Cdc6 accumulation, counterbalanced by a
negative regulation that is exerted by the Mos–MAPK pathway.
Fig. 4. Endogenous Cdc6 interacts with Cdk1–Cyclin-B complexes in meiosis II. (A) Sepharose beads coupled to p13suc1 or not (Mock) were incubated in extracts
from prophase- and MII-arrested oocytes (Pro and MII, respectively). The association between endogenous Cdc6 and Cdk1–Cyclin-B complexes was analyzed by
immunoblotting the beads for Cdc6, Cyclin B2 and Cdk1. (B) Extracts from MII-arrested oocytes were incubated or not (Control) with an antibody against Cdc6 and then
fractionated on a Superose-6 column. Each fraction was immunoblotted for endogenous Cdc6, Cyclin B2 and Cdk1. (C) Protein-G-coupled beads were added to
fraction 3 (shown in B, highlighted in red) and immunoblotted for Cdk1, Cyclin B2 and endogenous Cdc6. *Non-specific band associated with beads.
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Ectopic expression of stable Cdc6 inhibits meiosis
resumption
Interestingly, Cdc6 can inhibit Cdk1 during the G2/M transition
(Archambault et al., 2003; Calzada et al., 2001; Clay-Farrace
et al., 2003; El Dika et al., 2014; Elsasser et al., 1996; Murakami
et al., 2002; Oehlmann et al., 2004). If such effect were to operate
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in oocytes, deregulation of Cdc6 accumulation is expected to
disturb meiotic divisions. As seen in Fig. 1, injection of 0.05 µM
GST–WT-Cdc6 or GST–APxxS-Cdc6 did not prevent meiosis
resumption. To determine whether Cdk1 inhibition by Cdc6 is
dose-dependent, increasing amounts of GST–WT-Cdc6 were
added to extracts from MII-arrested oocytes, and Cdk1 activity
Fig. 5. Cdc6 turnover is regulated by Cdk1–Cyclin-B and the Mos–MAPK pathway after meiosis I. Prophase-arrested oocytes (Pro) were injected or not
with antisense oligonucleotides against mRNA encoding either Cyclin B or Mos and then incubated with progesterone. Oocytes were immunoblotted at the
indicated times after GVBD for endogenous Cdc6, Cyclin B2, phosphorylated MAPK ( pMAPK) and total MAPK. Cdk1 activity was assayed by monitoring the
phosphorylation level of GST–PP1C-S at residue T320 ( pT320-PP1C). The total amount of GST–PP1C-S was monitored with an antibody against GST
(GST–PP1C-S). The signals corresponding to T320-phosphorylated GST–PP1C-S (Cdk1 activity, blue line), phosphorylated MAPK ( pMAPK, red line) and Cdc6
level (Cdc6 quantity) were further quantified using ImageJ software (a.u., arbitrary unit). The time of Cdc6 appearance is indicated in blue for control oocytes and in
red for antisense-oligonucleotides-injected oocytes. (A) Representative experiment for control oocytes. The MI–MII transition and the MII arrest are indicated with
red lines. (B) A representative experiment for oocytes that had been injected with antisense oligonucleotides against B-type cyclins. This experiment was
performed five times. (C) Representative experiment of oocytes injected with morpholinos against Mos. This experiment was performed six times.
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was assayed in vitro using GST–PP1C-S (supplementary
material Fig. S4A). Cdk1 was fully active in MII extracts and was
inhibited in a dose-dependent manner by GST–WT-Cdc6
(supplementary material Fig. S4A). Cdk1 activity was inhibited
by 70% with 0.5 µM of GST–WT-Cdc6 in MII extracts
(supplementary material Fig. S4A). This concentration is ten
times higher than that used in Fig. 1, but corresponds to the
physiological concentration of Cdc6 in MII-arrested oocytes
(supplementary material Fig. S4B).
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Next, the amount of GST–WT-Cdc6 or GST–APxxS-Cdc6 that
was injected into prophase oocytes was increased to 0.5 µM (Fig. 6).
Following the addition of progesterone, GVBD induction was
slightly delayed in the presence of GST–WT-Cdc6 and strongly
impaired with GST–APxxS-Cdc6 (Fig. 6A). Moreover, the levels of
Cdk1 activity at GVBD were reduced in GST–WT-Cdc6-injected
oocytes compared with those of control oocytes, as determined by
using a Cdk1 activity assay and phosphorylation of Cyclin B2 and
Greatwall (also known as Mastl) (Fig. 6B). Following injection of
Fig. 6. Overexpression of a stable form of Cdc6 impairs meiosis resumption. Prophase-arrested oocytes (Pro) were injected with GST–WT-Cdc6 or
GST–APxxS-Cdc6 (0.5 µM) and then incubated with progesterone (Pg). (A) Time courses of GVBD induction in response to progesterone. (B) Oocytes from the
experiment described in A were collected either in prophase, at the time of GVBD (+ and − denote whether oocytes exhibited GVBD or not) or 4 h after GVBD.
Cdk1 activity was assayed in oocytes by monitoring the T320 phosphorylation level of GST–PP1C-S (pT320-PP1C). The total amount of GST–PP1C-S was
ascertained with an antibody against GST (GST–PP1C-S) and quantified with ImageJ software (a.u., arbitrary units). Oocytes were immunoblotted for Greatwall
(Gwl), GST–Cdc6, Cyclin B2, phosphorylated MAPK ( pMAPK) and total MAPK. (C) External morphology of prophase oocytes (Pro; view from animal poles) that had
been injected with GST–WT-Cdc6 or GST–APxxS-Cdc6 (0.5 µM) and then collected either at GVBD or 4 h after GVBD. Percentages, oocytes that underwent GVBD.
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RESEARCH ARTICLE
GST–APxxS-Cdc6, Cdk1 activity was strongly reduced in oocytes
that reached GVBD and was not detectable in oocytes that failed to
mature (Fig. 6B). Therefore, the presence of a stable form of Cdc6 in
prophase impairs Cdk1 activation in response to progesterone.
Oocytes that performed GVBD after injection of GST–WT-Cdc6 or
GST–APxxS-Cdc6 were further analyzed 4 h later. Cdk1 activity
was strongly reduced, Cyclin B2 and Greatwall were
dephosphorylated, and MAPK was inactivated (Fig. 6B),
suggesting that the oocyte did not enter meiosis II. The external
morphology of these oocytes was altered (Fig. 6C), with pigment rearrangements that were similar to those at egg activation, as well as
Journal of Cell Science (2015) 128, 2482-2496 doi:10.1242/jcs.166553
at exit from meiosis and entry into interphase. Therefore, depending
on the amount of Cdc6 present in prophase, Cdc6 has the potential
to inhibit Cdk1 activation before GVBD but also after MI.
Ectopic expression of Cdc6 at GVBD forces oocytes into an
interphasic state
We then examined whether deregulating Cdc6 expression levels
after the first meiotic division prevents entry into meiosis II.
Oocytes were injected at GVBD with 0.5 µM of GST–WT-Cdc6,
and Cdk1 activity was monitored at the indicated times (Fig. 7A). In
control oocytes, Cdk1 activity increased at GVBD, then decreased
Fig. 7. Overexpression of WT–Cdc6 in MI prevents Cdk1 reactivation and entry into meiosis II. Prophase-arrested oocytes (Pro) were incubated or not
with caffeine and then induced to mature with progesterone (Pg). At GVBD, oocytes were injected or not with GST–WT-Cdc6 (0.5 µM), or with buffer and collected
at the indicated times after GVBD. (A) Cdk1 activity was assayed by monitoring the T320-phosphorylation level of GST–PP1C-S ( pT320-PP1C). The total
amount of GST–PP1C-S was ascertained by using an antibody against GST (GST–PP1C-S), and the signal was further quantified using ImageJ software (a.u.,
arbitrary units). (Inset) Immunoblot analysis of the phosphorylation of Chk1 ( pChk1) in MII-arrested oocytes that had been injected or not with double-strand-break
DNA (DSB) in the presence or not of caffeine. *non-specific band. (B) Oocytes shown in A were also immunoblotted for Greatwall (Gwl), Wee1, GST–Cdc6, Cyclin
B2, phosphorylated MAPK ( pMAPK) and total Cdk1.
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RESEARCH ARTICLE
RESEARCH ARTICLE
1 h later and rose again when oocytes entered MII (Fig. 7A). In
oocytes that had been injected with 0.5 µM of GST–WT-Cdc6 at
GVBD, Cdk1 activity decreased after GVBD, as in control oocytes,
but did not increase thereafter (Fig. 7A). Furthermore, these oocytes
did not enter the second meiotic division, as determined
by immunoblotting for several molecular markers of meiosis II
entry – phosphorylation of Greatwall, accumulation of Wee1 and
Cyclin B2, as well as activation of MAPK (Fig. 7B). These results
indicate that oocytes injected at GVBD with high amounts of GST–
WT-Cdc6 exit from meiosis I but do not proceed through meiosis II.
Instead, they are driven into a pseudo-interphasic state owing to
inhibition of Cdk1 reactivation.
In somatic cells and in Xenopus egg extracts, overexpression of
Cdc6 prevents Cdk1 activation by activating checkpoints that are
dependent on ATM and/or ATR, or Chk1 (Borlado and Mendez,
2008). To determine whether this pathway mediates the negative
effect of Cdc6 towards Cdk1, prophase oocytes were incubated with
an inhibitor of ATM and ATR, caffeine, and then stimulated with
progesterone to resume meiosis (Fig. 7). Oocytes were further
injected at GVBD with 0.5 µM of GST–WT-Cdc6 (Fig. 7). As
expected, caffeine prevented Chk1 phosphorylation in MII-arrested
oocytes that had been injected with a DNA substrate that contained
double-strand breaks (Fig. 7A, inset), but neither affected meiosis
resumption nor Cdk1 activation in progesterone-stimulated oocytes
(Fig. 7). Moreover, caffeine was unable to restore Cdk1 reactivation
when GST–WT-Cdc6 was injected at GVBD, as seen by using
GST–PP1C-S in assays (Fig. 7A) and by immunoblotting for
Greatwall, Wee1, Cyclin B2 and phosphorylated MAPK (Fig. 7B).
Therefore, the inhibitory effect of Cdc6 on Cdk1 activation is
unlikely to be mediated by a DNA-damage-related checkpoint in
Xenopus oocytes. Because 0.5 µM of GST–WT-Cdc6 interacts with
Cdk1–Cyclin-B complexes without promoting their dissociation
during the MI–MII transition (Fig. 3C), this direct interaction
probably accounts for the inhibition of Cdk1 through Cdc6 after
meiosis I.
Journal of Cell Science (2015) 128, 2482-2496 doi:10.1242/jcs.166553
concentration of GST–WT-Cdc6 (0.05 µM) at GVBD did not affect
entry into meiosis II – Cdk1 was efficiently reactivated, and no
DNA replication was observed (Fig. 8C). By contrast, the injection
of 0.5 µM GST–WT-Cdc6 at GVBD abolished Cdk1 reactivation,
and DNA replication was promoted after meiosis I (Fig. 8C).
We then tested whether 0.05 µM of GST–WT-Cdc6, which does not
prevent entry into meiosis II as shown above, is sufficient to support
DNA replication. For this purpose, the synthesis of endogenous Cdc6
was prevented by using antisense oligonucleotides before injection
of biotin–dUTP (Fig. 8B). Oocytes were then stimulated with
progesterone, injected at GVBD with 0.05 µM of GST–WT-Cdc6
and, 45 min after GVBD, were further incubated with cycloheximide
to monitor DNA replication (Fig. 8B). The inhibition of endogenous
Cdc6 synthesis abolished the competence to replicate DNA, but
injection of 0.05 µM of GST–WT-Cdc6 at GVBD rescued DNA
replication in oocytes (Fig. 8C). Therefore, GST–WT-Cdc6 injected at
a concentration that is too low to inhibit Cdk1 is sufficient to support
DNA replication after meiosis I.
To ascertain whether DNA replication following GST–WT-Cdc6
injection at GVBD is a consequence of Cdk1 inhibition by Cdc6,
the same experiment was performed in the presence of aphidicolin
(APD), an inhibitor of DNA replication (Fig. 8D). Prophase oocytes
were injected with biotin–dUTP, stimulated with progesterone and
injected at GVBD with 0.5 µM of GST–WT-Cdc6 in the presence or
absence of APD (Fig. 8D). APD efficiently inhibited DNA
replication in progesterone-stimulated oocytes that had been
incubated in cycloheximide 45 min after GVBD (Fig. 8D). As
previously shown, GST–WT-Cdc6 inhibited the activity of Cdk1 by
60% and led to DNA replication (Fig. 8D). As expected, no DNA
replication was observed in the presence of APD, but Cdk1 activity
was still inhibited by GST–WT-Cdc6 under these conditions
(Fig. 8D). This demonstrates that precocious expression of Cdc6 at
the beginning of the first meiotic division inhibits Cdk1
reactivation, which forces the cell into a replicative interphasic state.
Our results prompted us to analyze DNA replication in oocytes that
had been injected at GVBD with GST–WT-Cdc6 by monitoring the
in vivo incorporation of biotin–dUTP into genomic DNA using
horseradish peroxidase (HRP)-coupled streptavidin. Cdc6
accumulation starts 45 min after GVBD, conferring the ability to
replicate DNA (Lemaître et al., 2002; Whitmire et al., 2002).
However, this competence is repressed and DNA is not replicated
between the two meiotic divisions. Adding cycloheximide 45 min
after GVBD, when Cdc6 is already expressed at low levels, induces
DNA replication (Furuno et al., 1994; Lemaître et al., 2002)
(Fig. 8A). Treatment with cycloheximide is thus a convenient way to
test the competence of oocytes to replicate DNA. As seen in Fig. 8A,
no incorporation of biotin–dUTP was detected in control oocytes,
whether they were treated or not with cycloheximide at GVBD;
however, 45 min later, adding cycloheximide promoted DNA
replication (Fig. 8A). To study the effect of Cdc6 on DNA
replication, prophase oocytes were first injected with biotin–dUTP,
stimulated with progesterone and then injected at GVBD with
0.05 µM or 0.5 µM of GST–WT-Cdc6 (Fig. 8B). Oocytes were
collected 4 h 45 min after GVBD to monitor Cdk1 activity and
DNA replication, as well as levels of GST–Cdc6 and endogenous
Cdc6 (Fig. 8C). In control oocytes, Cdk1 was active, and biotin–
dUTP was not incorporated (Fig. 8C). As expected, adding
cycloheximide 45 min after GVBD prevented Cdk1 reactivation,
and oocytes underwent DNA replication (Fig. 8C). Injecting a low
In this paper we show that the regulation of Cdc6 turnover during
meiotic maturation is essential to coordinate the levels of Cdc6
accumulation with entry into the second meiotic division, avoiding
Cdk1 inhibition and unscheduled DNA replication.
Our data revealed that recombinant Cdc6 is stable in prophase and
becomes unstable in response to progesterone, being degraded by the
proteasome shortly before entry into meiosis I. This degradation
mechanism relies on a SCFCdc4-dependent proteolytic system,
controlled by initial activation of Cdk1, either directly or indirectly.
In prophase, transcription is dormant, and Cdc6 expression is shut
down during the long period of oocyte growth. Inhibiting the
proteasome with MG132 does not permit precocious expression of
endogenous Cdc6 following progesterone addition. Although we
cannot exclude that our antibody is not sensitive enough to detect very
small amounts of Cdc6, this result supports the idea that Cdc6
translation is repressed during prophase arrest. Moreover, because
endogenous Cdc6 does not accumulate in p21Cip1-injected oocytes
following progesterone stimulation, this further indicates that the
translation of endogenous Cdc6 is specifically activated downstream
of the first Cdk1 activation, possibly by the polyadenylation of its
mRNA, as proposed by Lemaître et al. (2002). Therefore, Cdk1
activation concomitantly initiates Cdc6 synthesis and activates
SCFCdc4. Hence, endogenous Cdc6 protein is absent until GVBD.
The protein starts to be translated at GVBD depending on Cdk1
activation but cannot accumulate at that time owing to SCFCdc4dependent degradation. This intricate regulation of the stability of
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DISCUSSION
High levels of Cdc6 induce DNA replication by inhibiting Cdk1
Journal of Cell Science (2015) 128, 2482-2496 doi:10.1242/jcs.166553
Fig. 8. Ectopic expression of Cdc6 promotes DNA replication after meiosis I in a Cdk1-dependent manner. (A) Prophase-arrested oocytes (Pro) were
injected with biotin–dUTP and then incubated with progesterone (Pg). Oocytes were treated with cycloheximide (CHX) either at GVBD or 45 min after GVBD
(45′ post-GVBD), in the presence or in the absence of APD. Oocytes were collected 4 h 45 min after GVBD and the incorporation of biotin–dUTP was visualized
by southern blot analyses using HRP-coupled streptavidin (streptavidin-HRP). (B) Representative scheme of the experiment shown in C. Prophase oocytes
were injected or not with antisense oligonucleotides against Cdc6 (Cdc6 AS) and with biotin–dUTP. Following progesterone stimulation, oocytes were further
injected at GVBD with GST–WT-Cdc6 (0.05 µM or 0.5 µM) or buffers (buffer 1 and buffer 2, see Materials and Methods). Some Cdc6-AS-injected oocytes were
further injected or not with GST–WT-Cdc6 (0.05 µM) and then treated with cycloheximide 45 min after GVBD to induce DNA replication. As a positive control for
DNA replication, progesterone-stimulated oocytes were incubated 45 min after GVBD in the presence of cycloheximide to promote DNA replication. Oocytes were
collected 4 h 45 min after GVBD. (C) Cdk1 activity was assayed 4 h 45 min after GVBD by monitoring the T320-phosphorylation status of GST–PP1C-S ( pT320PP1C). The total amount of GST–PP1C-S was ascertained by using an antibody against GST (GST–PP1C-S), and the signal was quantified with ImageJ
software (a.u., arbitrary units). The incorporation of biotin–dUTP into DNA was monitored 4 h 45 min after GVBD by southern blotting using streptavidin–HRP.
GST–Cdc6 and endogenous Cdc6 were detected by immunoblotting. (D) The same experiment as those shown in B and C except that oocytes were injected at
GVBD with GST–WT-Cdc6 (0.5 µM) in the presence or APD.
Cdc6 in response to progesterone is important because the presence of
high Cdc6 protein levels before GVBD can inhibit the first activation
of Cdk1 in a dose-dependent manner. Although overexpressing Cdc6
through injection of mRNA does not delay meiosis resumption in
Xenopus (our results) and mouse oocytes (Anger et al., 2005), we
show here that injection of recombinant Cdc6 protein, at
concentrations corresponding to endogenous protein levels in MII
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(0.5 µM), decreases Cdk1 activation following hormonal stimulation.
This inhibitory effect of Cdc6 over Cdk1 is stronger when a nondegradable mutant of Cdc6 is injected, because meiosis resumption is
abolished in the majority of oocytes. Hence, the SCFCdc4-dependent
proteolytic system that is responsible for degradation of Cdc6 acts as a
safety mechanism to prevent any precocious accumulation of Cdc6
before GVBD, which would compromise meiosis resumption.
Journal of Cell Science
RESEARCH ARTICLE
Starting from GVBD, the expression levels of Cdc6 and thus its
accumulation, result from two mechanisms regulating its turnover,
one being responsible for its stabilization and the other for its
degradation. The stabilizing mechanism is set up when Cdk1
activity is established in MI and does not rely on the Cdk1dependent phosphorylation of Cdc6, as demonstrated here with the
7A mutant of Cdc6 that remains stable after GVBD. Interestingly,
the binding of mitotic cyclins stabilizes Cdc6 in yeast (Drury et al.,
2000; Elsasser et al., 1999; Mimura et al., 2004; Perkins et al., 2001;
Weinreich et al., 2001). This mechanism is probably conserved in
Xenopus oocytes. When oocytes are treated with cycloheximide at
GVBD, the degradation of ectopic Cdc6 is blocked by the presence
of a non-degradable Cyclin B when it is injected at a dose too low to
reactivate Cdk1. Moreover, the accumulation of endogenous Cdc6
is strongly delayed when synthesis of B-type cyclins is prevented.
Because recombinant and endogenous Cdc6 both interact with
Cdk1–Cyclin-B complexes, this mechanism certainly accounts for
Cdc6 stabilization in MI and during entry into meiosis II. However,
Cdc6 does not accumulate during the first hour following GVBD
owing to partial degradation of B-type cyclins and a second effector,
the Mos–MAPK pathway, which control Cdc6 turnover. The
suppression of the Mos–MAPK pathway promotes precocious
accumulation of endogenous Cdc6. Furthermore, endogenous Cdc6
appears unexpectedly in oocytes that have been injected with
antisense oligonucleotides against B-type cyclins when the Mos–
MAPK pathway is inactivated. This is the first demonstration that
the Mos–MAPK pathway acts independently of B-type cyclin
turnover to negatively regulate Cdc6 accumulation, certainly by
inducing its degradation. Interestingly, DNA is decondensed and a
nucleus is reformed after meiosis I, when the Mos–MAPK module
is inactive (Furuno et al., 1994). Whether these nuclei or the state of
DNA compaction are involved in controlling the turnover of
endogenous Cdc6 warrants further analysis.
This tight regulation of Cdc6 turnover through B-type cyclins and
the Mos–MAPK pathway after meiosis I synchronizes the timing of
Cdc6 accumulation, which is essential for proper succession of the
two meiotic divisions without DNA replication. The precocious
expression of 0.5 µM Cdc6 in MI impairs Cdk1 reactivation. As a
consequence, oocytes enter a replicative interphase-like stage. This
process involves the direct interaction between Cdc6 and Cdk1–
Cyclin-B, a mechanism already known to contribute to exit from
M-phase in Xenopus extracts, yeast and human cells (Archambault
et al., 2003; Calzada et al., 2001; El Dika et al., 2014; Elsasser et al.,
1996; Perkins et al., 2001; Weinreich et al., 2001). Interestingly,
Cdc6 specifically associates with Cyclin B2 and Cdk1 in highmolecular-mass complexes, which are distinct from the canonical
dimeric Cdk1–Cyclin-B complexes (De Smedt et al., 2002).
Whether these high-molecular-mass complexes play a pivotal role
in the activation of Cdk1 is unknown. However, these results
highlight the existence of a feedback loop between Cdc6 and Cdk1–
Cyclin-B complexes that operates during the MI–MII transition –
Cdc6 inhibits Cdk1 activity, whereas accumulation of B-type
cyclins stabilizes Cdc6. Because ectopic Cdc6 is stabilized at
GVBD, this explains why high levels of injected Cdc6 inhibit Cdk1
reactivation. Hence, the MI–MII transition is a crucial period for
oocytes where Cdc6 starts to be synthesized and stabilized, with the
potential to prevent Cdk1 reactivation and entry into meiosis II.
Why does endogenous Cdc6 not perturb meiotic divisions under
physiological conditions? At the time of entry into meiosis I,
although stabilized by binding to B-type cyclins, Cdc6 is present at
very low levels because, at the start of its synthesis, the levels are at
zero. During the MI–MII transition, B-type cyclins are degraded
Journal of Cell Science (2015) 128, 2482-2496 doi:10.1242/jcs.166553
and, as a consequence, no longer protect Cdc6 from degradation. At
the same time, the Mos–MAPK pathway, the activity of which is
maintained at a high level, negatively regulates Cdc6 accumulation,
probably by destabilizing the protein. For this reason, Cdc6 levels
at the end of meiosis I are insufficient to inhibit Cdk1 reactivation
and entry into meiosis II, although they are sufficient to support
DNA replication. The Mos–MAPK pathway is therefore key to
synchronize the MI–MII transition by coordinating Cdc6 expression
levels with Cdk1 reactivation. Indeed, this pathway stimulates the
reaccumulation of a positive regulator of Cdk1, namely B-type
cyclins, and postpones the accumulation of a Cdk1 inhibitor, Cdc6,
to enable entry into meiosis II. Furthermore, it prevents unscheduled
DNA replication by ensuring that no pre-RCs are assembled when
Cdk1 activity is low. As the oocyte proceeds through the second
meiotic division and then arrests at MII, the mechanism stabilizing
Cdc6 through its interaction with newly synthesized B-type cyclins
bypasses the negative effect that is exerted by the Mos–MAPK
pathway. Cdc6 is therefore stabilized and can accumulate. The
balance between the Mos–MAPK pathway, which remains
constantly active from GVBD to MII, and Cdk1–Cyclin-B
complexes, the quantity and activity of which decrease and then
rise again during the same period, results in a progressive and
gradual accumulation of Cdc6. Importantly, this dual regulation of
Cdc6 turnover by B-type cyclins and the Mos–MAPK pathway
tightly controls the expression levels of endogenous Cdc6, which
thus does not disturb normal progression of the meiotic divisions.
Hence, the competence to replicate the DNA that is necessary for
the embryonic cell cycles following fertilization is established but is
repressed by the high and stable activity of Cdk1 in MII. Cdc6 is
therefore a focal point that can control the correct order of events
during meiotic maturation by coordinating the competence to
replicate and the succession of the two meiotic divisions.
MATERIAL AND METHODS
Materials
Xenopus laevis adult females [Centre de Ressources Biologiques Xenopes,
UMS3387, Centre National de la Recherche Scientifique (CNRS), France]
were bred and maintained under laboratory conditions (Animal Facility
Agreement no. B75-05-13; Charles Darwin ethics committee, no. 5).
Reagents, unless otherwise specified, were from Sigma.
Preparation and handling of Xenopus oocytes
Xenopus prophase oocytes were obtained as described in Haccard et al.
(2012). Oocytes of different sizes (stage I to full-grown stage VI) were
sorted on binocular using a micrometer and according to Dumont (1972).
The microinjected volume per oocyte was 50 nl. Meiosis resumption was
triggered with 2 µM progesterone. Cycloheximide, MG132, aphidicholin
(APD) and caffeine were used at concentrations of 355 µM, 100 µM, 60 µM
and 100 µM, respectively, 1 h before progesterone addition. Prophase
oocytes were injected with antisense oligonucleotides directed against
mRNAs encoding all B-type cyclins (Cyc8/B2-5 cocktail; Hochegger et al.,
2001), Mos (Dupré et al., 2002) or Cdc6 (Lemaître et al., 2002). The nondegradable sea urchin Δ90-Cyclin was purified as described in FrankVaillant et al. (1999). Oocytes referred to as GVBD were collected at
the time of the first pigment rearrangement at the animal pole.
Oocytes were homogenized in ten volumes of extraction buffer (80 mM
β-glycerophosphate, pH 7.3; 20 mM EGTA; 15 mM MgCl2), centrifuged at
10,000 g for 15 min, and the supernatants were used for further analysis.
Antibodies and western blots
An equivalent of half or one oocyte was loaded onto 10% or 12%
SDS-polyacrylamide gels (Laemmli, 1970) and immunoblotted as described
in Dupré et al. (2002). Antibodies against the following proteins were used –
phosphorylated MAPK (1:1000, Cell Signaling Technology, 9106); total
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Journal of Cell Science (2015) 128, 2482-2496 doi:10.1242/jcs.166553
MAPK (1:1000, Abcam, SC-154); Cyclin B2 (1:1000, Abcam, ab18250);
total Cdk1 (1:1000, Abcam 3E1, ab20151); Wee1 (1:1000, Zymed, 511700); PP1C phosphorylated at T320 (1:30,000, Abcam, ab62334); alkylated
thiophosphates (1:30,000, Abcam, ab92570); Cdk1 phosphorylated at Y15
(1:1000, Cell Signaling Technology, 9111) and Chk1 phosphorylated at
S345 (1:1000, Cell Signaling Technology, 2348). HRP-conjugated
antibodies directed against either GST (1:5000, Sigma, A-7340) or
Histidine (1:10,000, Sigma) were also used. The antibody against
Greatwall has been described in Dupré et al. (2013). The antibody directed
against Xenopus Cdc6 was raised by immunizing rabbits with recombinant
GST–WT-Cdc6; it was then purified by retro-elution as described in Burke
et al. (1982). Appropriate HRP-labeled secondary antibodies (Jackson
ImmunoResearch) were used in chemiluminescent analysis (Pierce).
Production of mRNA encoding His–WT-Cdc6 and His–APxxSCdc6
Full-length wild-type Xenopus Cdc6 cDNA was purchased from Open
Biosystem (clone ID, 6634013; accession no., BC070554). DNA encoding
histidine-tagged WT-Cdc6 was generated by PCR amplification of
full-length Cdc6 cDNA and pRN3 vector (containing a T3 promoter,
kindly provided by Dr J. Moreau, CNRS, Paris) using the following primers:
5′-CATCACCACCACCACCACGGTATGCCAAGCACCAGGTCTCGGTC-3′ (encoding six histidine residues) and 5′-CACCCTCACAACGCTAAATCCCTGAATTGAGAACATTCC-3′ for Xenopus Cdc6; 5′-CACCCTCACAACGCTAAATCCCTGAATTGAGAACATTCC-3′ and 5′-GTGGTGGTGGTGGTGATGCATGAATTCAGATCTGCCAAAGTTGA-3′ for pRN3.
Xenopus histidine-tagged Cdc6 was cloned into pRN3 vector using the
ligation-independent cloning technique (Aslanidis and de Jong, 1990) and
was then transformed into JM109 bacteria, the resulting construct was
sequenced. cDNA encoding histidine-tagged APxxS-Cdc6 was generated
by PCR amplification of full-length Cdc6 in pRN3 using the following
primers: APxxS Fw, 5′-AAAGGGGCAAGAGGCCCCACCCAGCTC-3′;
APxxS Rev, 5′-GAGCTGGGTGGGGCCTCTTGCCCCTTT-3′.
mRNAs encoding histidine-tagged WT- and APxxS-Cdc6 were produced
using the AmpliCap-Max T3 high-yield message maker kit (Epicentre).
2005) and purified as previously described (Frank-Vaillant et al., 1999;
Lewis et al., 2013).
In vitro thiophosphorylation assays of GST–Cdc6
Inactive or active Cdk1 was affinity purified from four prophase- or MIIarrested oocytes using p13suc1 beads and then incubated with 0.6 µg of GST–
Cdc6 for 30 min at 30°C in kinase buffer (20 mM Hepes, pH 7.4; 2 mM
EGTA; 10 mM β-mercaptoethanol; 0.1 mM ATPγS; 10 mM MgCl2). Active
MAPK (100 ng; Sigma, E7407) was incubated with 0.6 µg of GST–Cdc6 for
30 min at 30°C in kinase buffer (25 mM MOPS, pH 7.2; 12 mM glycerol
2-phosphate; 25 mM MgCl2; 5 mM EGTA; 2 mM EDTA; 0.1 mM ATPγS
and 0.25 mM DTT). Each reaction was stopped by adding 20 mM EDTA and
then incubated for 1 h with 12.5 mM p-nitrobenzyl mesylate (Abcam,
Ab138910) for alkylation. Incorporated thiophosphates were immunoblotted
using an antibody directed against alkylated thiophosphates.
Cdk1 kinase assay using the GST–PP1C-S peptide
An extract equivalent to 0.1 oocyte was incubated with 0.2 µg of GST–PP1C-S
peptide for 30 min at 30°C in kinase buffer (20 mM Hepes, pH 7.4; 2 mM
EGTA; 10 mM β-mercaptoethanol; 0.1 mM ATP; 10 mM MgCl2; 10 µM
okadaic acid). The reaction was stopped with Laemmli buffer (Laemmli, 1970)
and then immunoblotted for GST and PP1C that had been phosphorylated at
residue T320. The signals were quantified using ImageJ software.
GST and p13suc1 pull-down experiments
GST pull-down experiments were performed with 1 µg of GST–WT-Cdc6
that had been pre-coupled to 30 µl of glutathione–agarose beads (Sigma,
G4520), which was then incubated for 1 h at 4°C with 100 µl of extracts of
prophase or MII oocytes. Beads were then recovered, and the resultant
proteins were immunoblotted.
For p13suc1 pull down, 20 µl of control Sepharose or p13suc1-coupled
beads were incubated for 2 h with 50 µl of lysate from prophase or MII
oocytes. Beads were then recovered by centrifugation, and the resultant
proteins were immunoblotted.
Gel filtration
cDNAs encoding histidine-tagged WT- or APxxS-Cdc6 were subcloned
into pGex4T1 (GE Healthcare) within EcoR1 and Not1 sites. GST–7ACdc6 was generated by mutating residues S45, S54, S74, S88, S108,
S120 and S411 into alanine residues within GST–WT-Cdc6 using the
primers described in Pelizon et al. (2000). The expression of
recombinant GST–Cdc6 proteins was induced in BL21 pLys bacteria
(Promega) by autoinduction for 3 days at 17°C (Studier, 2005). Bacteria
were lysed in lysis buffer (20 mM Hepes, pH 7.8; 0.5 M NaCl; 1 mM
EDTA; 1 mM EGTA) supplemented with 0.1% lysozyme, 0.1% Triton
X-100 and 0.1% Igepal. After sonication, lysates were centrifuged and
incubated for 1 h 30 min at 4°C with glutathione–agarose beads (Sigma,
G4510) that had been previously equilibrated overnight at 4°C in lysis
buffer. Glutathione beads were washed in washing buffer (20 mM
Hepes, pH 7.8; 0.5 M NaCl; 0.1 mM EDTA; 0.1 mM EGTA; 5 mM
DTT), and Cdc6 was eluted with 8 ml of washing buffer supplemented
with 5 mM glutathione. The purification of Cdc6 proteins in each
fraction was examined by using Coomassie-stained SDS-polyacrylamide
gels. Fractions containing a high amount of Cdc6 were pooled and
concentrated against polyethyleneglycol in the presence of 0.5 M NaCl
to prevent precipitation. The final concentration of GST–Cdc6 was
10 µM. In experiments using 0.5 µM GST–Cdc6, control oocytes were
injected with the corresponding purification buffer containing 0.5 M
NaCl (buffer 1). In experiments using 0.05 µM GST–Cdc6, GST–Cdc6
was diluted ten times with the same buffer without salt to reach 0.05 M
NaCl (buffer 2).
Expression and purification of GST–p21
Cip1
and GST–PP1C-S
Recombinant GST–PP1C-S peptide was expressed from a pGEX plasmid
(a kind gift from R. Golsteyn, University of Lethbridge, Canada). GST–
p21Cip1 and GST–PP1C-S were produced by using autoinduction (Studier,
2494
Extracts from MII oocytes were prepared as described in De Smedt et al.
(2002), and were incubated or not with an antibody against Cdc6 for 1 h.
The extracts were then fractionated on either a Superose-12 or a Superose-6
gel filtration column (Amersham Biosciences). Fractions of 0.25 ml or
0.5 ml, respectively, were collected and subjected to immunoblotting. In the
experiment using Superose-6 columns, 400 µl of fraction 3 was further
incubated with 25 µl of protein-G-coupled beads for 2 h at 4°C;
immunoblotting was then performed.
DNA replication assay
Prophase oocytes were injected with 50 µM of biotin-11–dUTP (Jena
Bioscience). DNA was extracted from ten oocytes as described in Dupré
et al. (2002), separated on 0.8% agarose gel and transferred overnight onto a
nitrocellulose membrane by southern blotting, as described in Southern
(1975). The membrane was saturated in 5% milk for 90 min and incubated
overnight with streptavidin–HRP (1:10,000, Thermo Scientific) to detect
incorporation of biotin–dUTP into genomic DNA.
Injection of MII-arrested oocytes with HindIII-λ-digested DNA
MII-arrested oocytes were incubated for 1 h in buffer M [10 mM HEPES,
pH 7.6; 88 mM NaCl; 1 mM KCl; 0.33 mM Ca(NO3)2; 0.41 mM CaCl2;
0.82 mM MgSO4] that had been supplemented with 0.1 M EGTA to prevent
oocyte activation. Oocytes were then injected with HindIII-λ-digested DNA
(Fermentas, SM0101), which mimics DNA double-strand breaks, and
collected 1 h later for further analysis.
Acknowledgements
We thank all members of our laboratory for helpful discussions and Dr K. Wassmann
for the critical reading of the manuscript.
Competing interests
The authors declare no competing or financial interests.
Journal of Cell Science
Cloning, expression and purification of GST-tagged Cdc6
proteins
Author contributions
C.J., O.H. and A.D. conceived the original idea. E.M.D., C.J., O.H. and A.D.
designed and planned the experiments, analyzed the data and wrote the paper.
E.M.D., T.L., R.P. and A.D. performed experiments.
Funding
This work was supported by CNRS; Université Pierre et Marie Curie, UPMC [grant
EME1022 to A.D.]; Agence Nationale de la Recherche [grant ANR-13-BSV2-000801 to C.J.]; and Association pour la Recherche Contre le Cancer (ARC) to E.M.D.
Supplementary material
Supplementary material available online at
http://jcs.biologists.org/lookup/suppl/doi:10.1242/jcs.166553/-/DC1
References
Anger, M., Stein, P. and Schultz, R. M. (2005). CDC6 requirement for spindle
formation during maturation of mouse oocytes. Biol. Reprod. 72, 188-194.
Archambault, V., Li, C. X., Tackett, A. J., Wasch, R., Chait, B. T., Rout, M. P. and
Cross, F. R. (2003). Genetic and biochemical evaluation of the importance of
Cdc6 in regulating mitotic exit. Mol. Biol. Cell 14, 4592-4604.
Aslanidis, C. and de Jong, P. J. (1990). Ligation-independent cloning of PCR
products (LIC-PCR). Nucleic Acids Res. 18, 6069-6074.
Bhatt, R. R. and Ferrell, J. E., Jr. (1999). The protein kinase p90 rsk as an essential
mediator of cytostatic factor activity. Science 286, 1362-1365.
Borlado, L. R. and Mendez, J. (2008). CDC6: from DNA replication to cell cycle
checkpoints and oncogenesis. Carcinogenesis 29, 237-243.
Burke, B., Griffiths, G., Reggio, H., Louvard, D. and Warren, G. (1982). A
monoclonal antibody against a 135-K Golgi membrane protein. EMBO J. 1,
1621-1628.
Calzada, A., Sacristá n, M., Sá nchez, E. and Bueno, A. (2001). Cdc6 cooperates
with Sic1 and Hct1 to inactivate mitotic cyclin-dependent kinases. Nature 412,
355-358.
Clay-Farrace, L., Pelizon, C., Santamaria, D., Pines, J. and Laskey, R. A. (2003).
Human replication protein Cdc6 prevents mitosis through a checkpoint
mechanism that implicates Chk1. EMBO J. 22, 704-712.
Clijsters, L. and Wolthuis, R. (2014). PIP-box-mediated degradation prohibits reaccumulation of Cdc6 during S phase. J. Cell Sci. 127, 1336-1345.
Cook, J. G., Park, C.-H., Burke, T. W., Leone, G., DeGregori, J., Engel, A. and
Nevins, J. R. (2002). Analysis of Cdc6 function in the assembly of mammalian
prereplication complexes. Proc. Natl. Acad. Sci. USA 99, 1347-1352.
De Smedt, V., Poulhe, R., Cayla, X., Dessauge, F., Karaiskou, A., Jessus, C. and
Ozon, R. (2002). Thr-161 phosphorylation of monomeric Cdc2. Regulation by
protein phosphatase 2C in Xenopus oocytes. J. Biol. Chem. 277, 28592-28600.
Diffley, J. F. X. (2004). Regulation of early events in chromosome replication. Curr.
Biol. 14, R778-R786.
Drury, L. S., Perkins, G. and Diffley, J. F. X. (2000). The cyclin-dependent kinase
Cdc28p regulates distinct modes of Cdc6p proteolysis during the budding yeast
cell cycle. Curr. Biol. 10, 231-240.
Dumont, J. N. (1972). Oogenesis in Xenopus laevis (Daudin). I. Stages of oocyte
development in laboratory maintained animals. J. Morphol. 136, 153-179.
Dupré , A., Jessus, C., Ozon, R. and Haccard, O. (2002). Mos is not required for the
initiation of meiotic maturation in Xenopus oocytes. EMBO J. 21, 4026-4036.
Dupré , A., Haccard, O. and Jessus, C. (2011). Mos in the oocyte: how to use
MAPK independently of growth factors and transcription to control meiotic
divisions. J. Signal Transduct. 2011, 350412.
Dupré , A., Buffin, E., Roustan, C., Nairn, A. C., Jessus, C. and Haccard, O.
(2013). The phosphorylation of ARPP19 by Greatwall renders the autoamplification of MPF independently of PKA in Xenopus oocytes. J. Cell Sci.
126, 3916-3926.
Duursma, A. M. and Agami, R. (2005). CDK-dependent stabilization of Cdc6:
linking growth and stress signals to activation of DNA replication. Cell Cycle 4,
1725-1728.
El Dika, M., Laskowska-Kaszub, K., Koryto, M., Dudka, D., Prigent, C., Tassan,
J.-P., Kloc, M., Polanski, Z., Borsuk, E. and Kubiak, J. Z. (2014). CDC6 controls
dynamics of the first embryonic M-phase entry and progression via CDK1
inhibition. Dev. Biol. 396, 67-80.
Elsasser, S., Lou, F., Wang, B., Campbell, J. L. and Jong, A. (1996). Interaction
between yeast Cdc6 protein and B-type cyclin/Cdc28 kinases. Mol. Biol. Cell 7,
1723-1735.
Elsasser, S., Chi, Y., Yang, P. and Campbell, J. L. (1999). Phosphorylation
controls timing of Cdc6p destruction: a biochemical analysis. Mol. Biol. Cell 10,
3263-3277.
Frank-Vaillant, M., Jessus, C., Ozon, R., Maller, J. L. and Haccard, O. (1999).
Two distinct mechanisms control the accumulation of cyclin B1 and Mos in
Xenopus oocytes in response to progesterone. Mol. Biol. Cell 10, 3279-3288.
Furuno, N., Nishizawa, M., Okazaki, K., Tanaka, H., Iwashita, J., Nakajo, N.,
Ogawa, Y. and Sagata, N. (1994). Suppression of DNA replication via Mos
function during meiotic divisions in Xenopus oocytes. EMBO J. 13, 2399-2410.
Journal of Cell Science (2015) 128, 2482-2496 doi:10.1242/jcs.166553
Gautier, J., Norbury, C., Lohka, M., Nurse, P. and Maller, J. (1988). Purified
maturation-promoting factor contains the product of a Xenopus homolog of the
fission yeast cell cycle control gene cdc2+. Cell 54, 433-439.
Gautier, J., Matsukawa, T., Nurse, P. and Maller, J. (1989). Dephosphorylation
and activation of Xenopus p34cdc2 protein kinase during the cell cycle. Nature
339, 626-629.
Gautier, J., Minshull, J., Lohka, M., Glotzer, M., Hunt, T. and Maller, J. L. (1990).
Cyclin is a component of maturation-promoting factor from Xenopus. Cell 60,
487-494.
Gross, S. D., Schwab, M. S., Taieb, F. E., Lewellyn, A. L., Qian, Y.-W. and Maller,
J. L. (2000). The critical role of the MAP kinase pathway in meiosis II in Xenopus
oocytes is mediated by p90(Rsk). Curr. Biol. 10, 430-438.
Haccard, O. and Jessus, C. (2006). Redundant pathways for Cdc2 activation in
Xenopus oocyte: either cyclin B or Mos synthesis. EMBO Rep. 7, 321-325.
Haccard, O., Sarcevic, B., Lewellyn, A., Hartley, R., Roy, L., Izumi, T., Erikson, E.
and Maller, J. L. (1993). Induction of metaphase arrest in cleaving Xenopus
embryos by MAP kinase. Science 262, 1262-1265.
Haccard, O., Dupré , A., Liere, P., Pianos, A., Eychenne, B., Jessus, C. and
Ozon, R. (2012). Naturally occurring steroids in Xenopus oocyte during meiotic
maturation. Unexpected presence and role of steroid sulfates. Mol. Cell.
Endocrinol. 362, 110-119.
Hochegger, H., Klotzbucher, A., Kirk, J., Howell, M., le Guellec, K., Fletcher, K.,
Duncan, T., Sohail, M. and Hunt, T. (2001). New B-type cyclin synthesis is
required between meiosis I and II during Xenopus oocyte maturation.
Development 128, 3795-3807.
Huchon, D., Rime, H., Jessus, C. and Ozon, R. (1993). Control of metaphase I
formation in Xenopus oocyte: effects of an indestructible cyclin B and of protein
synthesis. Biol. Cell 77, 133-141.
Illenye, S. and Heintz, N. H. (2004). Functional analysis of bacterial artificial
chromosomes in mammalian cells: mouse Cdc6 is associated with the mitotic
spindle apparatus. Genomics 83, 66-75.
Iwabuchi, M., Ohsumi, K., Yamamoto, T. M., Sawada, W. and Kishimoto, T.
(2000). Residual Cdc2 activity remaining at meiosis I exit is essential for meiotic
M-M transition in Xenopus oocyte extracts. EMBO J. 19, 4513-4523.
Karaiskou, A., Dupré , A., Haccard, O. and Jessus, C. (2001). From progesterone
to active Cdc2 in Xenopus oocytes: a puzzling signalling pathway. Biol. Cell 93,
35-46.
Laemmli, U. K. (1970). Cleavage of structural proteins during the assembly of the
head of bacteriophage T4. Nature 227, 680-685.
Lemaître, J.-M., Bocquet, S. and Mé chali, M. (2002). Competence to replicate in
the unfertilized egg is conferred by Cdc6 during meiotic maturation. Nature 419,
718-722.
Lemaître, J.-M., Bocquet, S., Terret, M.-E., Namdar, M., Ait-Ahmed, O., Kearsey, S.,
Verlhac, M.-H. and Mechali, M. (2004). The regulation of competence to replicate
in meiosis by Cdc6 is conserved during evolution. Mol. Reprod. Dev. 69, 94-100.
Lewis, C. W., Taylor, R. G., Kubara, P. M., Marshall, K., Meijer, L. and Golsteyn,
R. M. (2013). A western blot assay to measure cyclin dependent kinase activity in
cells or in vitro without the use of radioisotopes. FEBS Lett. 587, 3089-3095.
Mailand, N. and Diffley, J. F. X. (2005). CDKs promote DNA replication origin
licensing in human cells by protecting Cdc6 from APC/C-dependent proteolysis.
Cell 122, 915-926.
Mimura, S., Seki, T., Tanaka, S. and Diffley, J. F. X. (2004). Phosphorylationdependent binding of mitotic cyclins to Cdc6 contributes to DNA replication
control. Nature 431, 1118-1123.
Murakami, H., Yanow, S. K., Griffiths, D., Nakanishi, M. and Nurse, P. (2002).
Maintenance of replication forks and the S-phase checkpoint by Cdc18p and
Orp1p. Nat. Cell Biol. 4, 384-388.
Narasimhachar, Y., Webster, D. R., Gard, D. L. and Coué , M. (2012). Cdc6 is
required for meiotic spindle assembly in Xenopus oocytes. Cell Cycle 11,
524-531.
Oehlmann, M., Score, A. J. and Blow, J. J. (2004). The role of Cdc6 in ensuring
complete genome licensing and S phase checkpoint activation. J. Cell Biol. 165,
181-190.
Ohsumi, K., Sawada, W. and Kishimoto, T. (1994). Meiosis-specific cell cycle
regulation in maturing Xenopus oocytes. J. Cell Sci. 107, 3005-3013.
Pelizon, C., Madine, M. A., Romanowski, P. and Laskey, R. A. (2000).
Unphosphorylatable mutants of Cdc6 disrupt its nuclear export but still support
DNA replication once per cell cycle. Genes Dev. 14, 2526-2533.
Perkins, G., Drury, L. S. and Diffley, J. F. X. (2001). Separate SCF(CDC4)
recognition elements target Cdc6 for proteolysis in S phase and mitosis. EMBO J.
20, 4836-4845.
Petersen, B. O., Wagener, C., Marinoni, F., Kramer, E. R., Melixetian, M.,
Lazzerini Denchi, E., Gieffers, C., Matteucci, C., Peters, J.-M. and Helin, K.
(2000). Cell cycle- and cell growth-regulated proteolysis of mammalian CDC6 is
dependent on APC-CDH1. Genes Dev. 14, 2330-2343.
Sagata, N., Watanabe, N., Vande Woude, G. F. and Ikawa, Y. (1989). The c-mos
proto-oncogene product is a cytostatic factor responsible for meiotic arrest in
vertebrate eggs. Nature 342, 512-518.
Southern, E. M. (1975). Detection of specific sequences among DNA fragments
separated by gel electrophoresis. J. Mol. Biol. 98, 503-517.
2495
Journal of Cell Science
RESEARCH ARTICLE
RESEARCH ARTICLE
Whitmire, E., Khan, B. and Coué , M. (2002). Cdc6 synthesis regulates replication
competence in Xenopus oocytes. Nature 419, 722-725.
Yim, H. and Erikson, R. L. (2010). Cell division cycle 6, a mitotic substrate of pololike kinase 1, regulates chromosomal segregation mediated by cyclin-dependent
kinase 1 and separase. Proc. Natl. Acad. Sci. USA 107, 19742-19747.
Yim, H. and Erikson, R. L. (2011). Regulation of the final stage of mitosis by
components of the pre-replicative complex and a polo kinase. Cell Cycle 10,
1374-1377.
Journal of Cell Science
Stoeber, K., Mills, A. D., Kubota, Y., Krude, T., Romanowski, P., Marheineke, K.,
Laskey, R. A. and Williams, G. H. (1998). Cdc6 protein causes premature entry
into S phase in a mammalian cell-free system. EMBO J. 17, 7219-7229.
Studier, F. W. (2005). Protein production by auto-induction in high-density shaking
cultures. Protein Expr. Purif. 41, 207-234.
Weinreich, M., Liang, C., Chen, H.-H. and Stillman, B. (2001). Binding of cyclindependent kinases to ORC and Cdc6p regulates the chromosome replication
cycle. Proc. Natl. Acad. Sci. USA 98, 11211-11217.
Journal of Cell Science (2015) 128, 2482-2496 doi:10.1242/jcs.166553
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