MOLECULAR AND CELLULAR BIOLOGY, Sept. 2008, p. 5328–5336
Copyright © 2008, American Society for Microbiology. All Rights Reserved.
Vol. 28, No. 17
Cyclin-Specific Control of Ribosomal DNA Segregation?
Matt Sullivan, Liam Holt, and David O. Morgan*
Departments of Physiology and Biochemistry & Biophysics, University of California, San Francisco, California
Received 12 February 2008/Returned for modification 28 March 2008/Accepted 24 June 2008
Following chromosome duplication in S phase of the cell cycle, the sister chromatids are linked by cohesin.
At the onset of anaphase, separase cleaves cohesin and thereby initiates sister chromatid separation. Separase
activation results from the destruction of its inhibitor, securin, which is triggered by a ubiquitin ligase called
the anaphase-promoting complex (APC). Here, we show in budding yeast that securin destruction and, thus,
separase activation are not sufficient for the efficient segregation of the repetitive ribosomal DNA (rDNA). We
find that rDNA segregation also requires the APC-mediated destruction of the S-phase cyclin Clb5, an activator
of the protein kinase Cdk1. Mutations that prevent Clb5 destruction are lethal and cause defects in rDNA
segregation and DNA synthesis. These defects are distinct from the mitotic-exit defects caused by stabilization
of the mitotic cyclin Clb2, emphasizing the importance of cyclin specificity in the regulation of late-mitotic
events. Efficient rDNA segregation, both in mitosis and meiosis, also requires APC-dependent destruction of
Dbf4, an activator of the protein kinase Cdc7. We speculate that the dephosphorylation of Clb5-specific Cdk1
substrates and Dbf4-Cdc7 substrates drives the resolution of rDNA in early anaphase. The coincident destruc-
tion of securin, Clb5, and Dbf4 coordinates bulk chromosome segregation with segregation of rDNA.
Cyclin-dependent kinases (Cdks) determine the order of cell
cycle events by phosphorylating different substrates at different
points in the cell cycle. The temporal control of substrate
phosphorylation is made possible by the periodic synthesis and
destruction of multiple cyclin cofactors that bind and activate
the Cdk (17). Different cyclins have different functional capac-
ities, in part because they target the associated Cdk to specific
substrates (2, 4). In the budding yeast Saccharomyces cerevisiae,
for example, a complex of the S-phase cyclin Clb5 and Cdk1
displays high specificity for a small subset of Cdk1 substrates,
due to a docking site on Clb5 that greatly increases its affinity
for those substrates (16). Many Clb5-specific substrates are
involved in DNA synthesis, helping to explain why Clb5-Cdk1
drives early cell cycle events.
The final events of M phase require dephosphorylation of
Cdk1 targets, which results from a combination of cyclin de-
struction, induction of Cdk1 inhibitors, and activation of phos-
phatases, such as Cdc14 of budding yeast, that remove phos-
phates from Cdk1 sites (30). Clb5 and the major mitotic cyclin
Clb2 are both targeted for destruction by the anaphase-pro-
moting complex (APC), a ubiquitin ligase that is activated in
metaphase by association with the cofactor Cdc20 (APCCdc20)
(22, 32). APCCdc20triggers the complete destruction of Clb5
before anaphase but promotes only partial destruction of Clb2,
which is completely destroyed later in mitosis when the APC is
activated by Cdh1 (24, 39). These differences in the timing of
cyclin destruction might provide a mechanism to allow the
dephosphorylation of Clb5-specific substrates in early ana-
phase, while the general substrates of Clb2-Cdk1 remain phos-
phorylated until later (30). The Clb5-specific substrates Fin1
and Ase1, for example, are dephosphorylated in early ana-
phase, allowing them to help initiate important changes in
mitotic spindle dynamics (12, 40).
If the order of cyclin destruction determines the timing of
late-mitotic events, then the stabilization of different cyclins
should have distinct effects on mitotic processes. Indeed, the sta-
bilization of metazoan cyclin A (by mutation of sequences re-
quired for APC recognition) causes phenotypes very different
than those caused by stabilized cyclin B (21, 25). In budding yeast,
stabilization or overexpression of Clb2 causes defects in spindle
breakdown and mitotic exit, whereas partially stabilized Clb5 has
little apparent effect on late-mitotic events (39). Overexpression
excess Clb5-Cdk1 activity are not clear.
The initiating event of anaphase is sister chromatid separa-
tion, which results primarily from the proteolytic cleavage of
cohesin by separase (19). In budding yeast, however, separase-
mediated cohesin destruction is not sufficient for resolution of
the highly repetitive ribosomal DNA (rDNA) locus on chro-
mosome XII. Segregation of the rDNA also requires separase-
dependent activation of the phosphatase Cdc14 (5, 28), sug-
gesting that dephosphorylation of Cdk1 substrates is required.
It remains unclear if destruction of APCCdc20substrates other
than securin is required for resolution of the rDNA in anaphase.
two additional APCCdc20substrates, Clb5 and Dbf4, contributes
to rDNA segregation. Our results also provide evidence for cyclin
specificity in late-mitotic control, as rDNA resolution depends on
loss of Clb5 activity but not on loss of activity of the major mitotic
cyclin Clb2. We also show that degradation of Clb5 is critical for
origin relicensing and, hence, DNA replication. Finally, we dem-
cohesion in mitosis are similar to those in anaphase I of the
MATERIALS AND METHODS
All mitotic experiments were performed in strain W303, and all meiotic ex-
periments were performed in strain SK1. Epitope tagging of endogenous genes
* Corresponding author. Mailing address: UCSF Mailcode 2200,
Genentech Hall Room N312B, 600 16th Street, San Francisco, CA
94158-2517. Phone: (415) 476-6695. Fax: (415) 476-5233. E-mail:
?Published ahead of print on 30 June 2008.
at UNIV OF CALIF-SAN FRANCISCO on June 8, 2010
and gene deletions was performed by gene targeting using PCR products. The
CLB5 gene, along with three C-terminal hemagglutinin epitopes and 451 bp of
the CLB5 promoter, was amplified from budding yeast and cloned to allow
integration of an extra copy of CLB5-HA3 at the URA3 locus. This plasmid was
modified to generate the N-terminal deletion, CLB5-?N, by removal of the
coding sequence for amino acids 2 to 95. For expression in meiosis, the coding
sequence of CLB5-?N-TAP was placed in front of 341 bp of the DMC1 promoter
to allow integration of pDMC1-CLB5-?N-TAP at the HIS3 locus. To express high
levels of cyclins in mitosis, CLB5-?N-TAP or CLB2-?N-HA3 (encoding Clb2
lacking amino acids 2 to 176) was cloned in front of the GAL1-10 promoter. To
make stabilized Dbf4, a plasmid that contained 523 bp of the DBF4 promoter
and a portion of the DBF4 gene with the coding sequence for amino acids 2 to 65
to switch the endogenous gene to DBF4-?N and was confirmed by PCR.
Conditions for growth and release of synchronous cultures from arrest in G1
by ?-factor were performed as described previously (16). In the GAL-CDC20
shutoff experiment (Fig. 1), cells were grown in raffinose/galactose media for
?-factor arrest and released into glucose media. In the GAL-CLB experiment
(Fig. 2), cells were grown in raffinose media for ?-factor arrest and released into
raffinose/galactose media. In experiments involving GAL-SIC1, cells were grown
in raffinose/galactose media for ?-factor arrest (Fig. 3, 4, and 5). Once arrested,
glucose was added for 30 min, and cells were released from the arrest into fresh
glucose media. Synchronous meiotic experiments were performed as described
previously (29). Western blotting, budding counts, and immunofluorescence
analysis were performed as described previously (28). The antibodies used were
?-Nop1 clone MCA28F2 (Encor), ?-tubulin YOL1/34 (Serotec), ?-hemaggluti-
nin clone 16B12 (Babco), and ?-myc clone 9E10 (Babco).
Resolution of the rDNA requires APCCdc20-mediated degra-
dation of Clb5 and securin. To test whether the destruction of
APCCdc20targets other than securin governs rDNA resolution,
we examined chromosome segregation in budding-yeast cells
cdc20? pds1? strains, expressing Cdc20 from the repressible
GAL1-10 promoter, were arrested in G1by ?-factor treatment
FIG. 1. APCCdc20-dependent degradation of securin and Clb5 allows rDNA segregation. Wild-type (WT), cdc20?, cdc20? pds1?, and cdc20?
pds1? clb5? yeast expressing GAL-CDC20 were arrested in G1with ?-factor and then released into fresh dextrose media to repress CDC20
transcription. Samples were collected every 20 min for analysis of budding, binucleate formation (bulk chromosome segregation, assessed by DAPI
[4?,6-diamidino-2-phenylindole] staining), and Nop1 segregation (a marker of nucleolar rDNA segregation) (top panel). The bottom panel shows
representative images of cdc20? pds1? and cdc20? pds1? clb5? yeast 140 min after release from G1.
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and then released into dextrose media to repress transcription of
CDC20. Budding and binucleate formation were scored. To ex-
amine rDNA segregation, we used immunofluorescence analysis
of Nop1, a nucleolar protein that is involved in rDNA processing.
Nop1 associates with the rDNA throughout the budding-yeast
cell cycle, and its division into two distinct foci at anaphase is a
useful marker of rDNA segregation. Wild-type cells segregated
bulk DNA and rDNA with roughly equivalent timing, while
cdc20? cells failed to segregate either (Fig. 1). In contrast, bulk
chromosome segregation in cdc20? pds1? cells occurred with
normal timing, but rDNA segregation was delayed. Destruction
of APCCdc20targets other than securin might therefore be nec-
essary for efficient rDNA segregation.
Deletion of CLB5 is known to restore viability to cdc20?
pds1? mutants (24). As Clb5 is an APCCdc20substrate, this
suggests that the failure of cdc20? pds1? yeast to efficiently
segregate their rDNA might be due to the continued presence
of Clb5. Consistent with this possibility, we found that deletion
of CLB5 almost completely abolished the rDNA segregation
delay in the cdc20? pds1? strain (Fig. 1). Thus, APCCdc20
promotes rDNA segregation at anaphase onset by targeting
both securin and Clb5 for destruction.
rDNA cohesion is controlled specifically by Clb5-Cdk1 ac-
tivity. The rDNA segregation defect caused by Clb5 could be
due to either a specific role for Clb5-Cdk1 or a general in-
crease in Cdk1 activity. To distinguish between these possibil-
ities, we compared the effects of different cyclins on rDNA
segregation. Cells were released from G1in the presence of
galactose-inducible Clb5 or Clb2 protein (each stabilized by
removal of amino-terminal APC-targeting domains, as dis-
cussed below). Cells overexpressing either cyclin arrested as
large-budded cells with segregated DNA masses (Fig. 2A), and
examination of chromosome III segregation revealed that bulk
chromatin segregated accurately (data not shown). Strikingly,
however, rDNA segregation was almost completely blocked in
cells overexpressing Clb5 but was unperturbed in cells overex-
pressing Clb2. This result indicates that rDNA cohesion is
mediated specifically by Clb5-Cdk1 activity.
Stabilized Clb5 mutants cause rDNA segregation defects. If
efficient rDNA resolution requires Clb5 destruction, then seg-
FIG. 2. Clb5 inhibits rDNA segregation. (A) Cells carrying either GAL-CLB5-?N or GAL-CLB2-?N were arrested in G1with ?-factor and then
released into fresh galactose media to induce cyclin expression. Samples were collected every 15 min for analysis of binucleate formation and Nop1
segregation. Representative images are shown of GAL-CLB5-?N yeast and GAL-CLB2-?N yeast 120 min after release from G1. (B) Asynchronous
CLB5 (WT) and CLB5-?db cells were collected and binucleates analyzed for the number of segregated and unsegregated Nop1 masses.
Representative images are shown (left).
5330 SULLIVAN ET AL.MOL. CELL. BIOL.
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regation defects should result when stabilized mutant forms of
Clb5 are expressed at physiological levels. APCCdc20recog-
nizes its substrates through interactions with specific sequence
motifs such as the D-box (22). The amino-terminal region of
Clb5 contains a D-box, the removal of which partly stabilizes it
in anaphase (the Clb5-?db mutant) (39). Clb5-?db is not lethal
and has no clear effect on mitotic progression. We examined
rDNA segregation in asynchronous populations of wild-type
and CLB5-?db cells. 92% of wild-type cells that had segre-
gated their chromosomes had also segregated their rDNA,
whereas this was the case for only 65% of CLB5-?db cells (Fig.
2B). Thus, partly stabilized Clb5 slows rDNA segregation.
Many APC substrates contain multiple destruction determi-
nants in addition to the D-box. Full stabilization of cyclins, for
example, often requires the deletion of their entire N-terminal
regions. To produce fully stabilized Clb5, we removed its 95
amino-terminal residues, leaving its Cdk1-binding domain in-
tact (the Clb5-?N mutant). Transformation of wild-type cells
with a plasmid carrying CLB5-?N under the control of its own
promoter was lethal. This lethality was suppressed by overex-
pression of SIC1 from the GAL1-10 promoter (Fig. 3A). When
expression of GAL-SIC1 was repressed by plating on glucose
media, cells carrying the CLB5-?N formed inviable microcolo-
nies. To confirm that Clb5 was stabilized by the removal of its
N terminus, cells containing an extra copy of either CLB5 or
CLB5-?N were released from G1arrest in the absence of SIC1
expression. Levels of wild-type Clb5 fell at anaphase onset,
while the amount of Clb5-?N was unchanged (Fig. 3B). Thus,
removal of the N terminus of Clb5 stabilizes it in anaphase and
causes cell lethality.
We analyzed anaphase progression in CLB5 and CLB5-?N
cells, again shutting off SIC1 expression after release from G1.
FIG. 3. CLB5-?N is lethal and causes an rDNA segregation delay. (A) Wild-type or rDNA? cells, carrying GAL-SIC1 and an extra copy of the
CLB5 or CLB5-?N gene (expressed from the CLB5 promoter), were plated on either galactose or dextrose media. (B) GAL-SIC1 cells carrying
an extra copy of CLB5 or CLB5-?N were arrested in G1by ?-factor treatment and released into fresh dextrose media to repress SIC1 transcription.
Samples were taken every 20 min, and the levels of Clb5 and Clb2 were analyzed by Western blotting. (C) Procedure was as in panel B, but ?-factor
was added 90 min after release to arrest cells in the following G1. Samples were collected every 15 min for analysis of budding, binucleate
formation, and Nop1 segregation. Images on the right show representative cells 140 min after release from G1.
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?-Factor was added back to the cultures after the initiation of
budding. In CLB5-?N cells, budding and chromosome segre-
gation occurred with normal timing (Fig. 3C). However, there
was a significant delay in rDNA segregation in these cells,
demonstrating that Clb5 degradation is necessary for timely
resolution of the rDNA. Unlike wild-type cells, CLB5-?N cells
failed to arrest in the following G1, suggesting that Clb5-?N
prevented the G1arrest mediated by mating pheromone.
We considered the possibility that Clb5-?N prevents rDNA
segregation by blocking activation of the phosphatase Cdc14,
which is required for rDNA segregation (5, 28). However, the
phenotypes of the CLB5-?N mutant are clearly distinct from
those of temperature-sensitive cdc14 mutants. cdc14-1 cells, in
addition to delaying rDNA segregation, fail to break down the
anaphase spindle, fail to undergo cytokinesis and do not enter
the next cell cycle (26). In contrast, CLB5-?N cells, like CLB5-
?db cells (39), showed no significant defects in mitotic exit, as
judged by spindle disassembly, cytokinesis, or new cycle entry,
although Clb2 destruction was slightly delayed (Fig. 3B). Fur-
thermore, previous studies of cdc20? pds1? cells showed that
Cdc14 is released efficiently from the nucleolus at anaphase
onset even though Clb5 levels remain high (24). Together,
these lines of evidence suggest that the delayed rDNA segre-
gation of CLB5-?N cells is not due to problems with Cdc14
activation but instead that Cdk1-Clb5 promotes rDNA cohe-
sion through another mechanism.
One possibility is that Clb5 opposes the effects of Cdc14 on
the phosphorylation state of Cdk1 targets involved in rDNA
cohesion. Consistent with this possibility, we found that dele-
tion of CLB5 partly suppressed rDNA segregation defects in
cdc14-1 cells. When arrested for 2.5 h at 37°C, rDNA segre-
gation occurred in 44% of cdc14-1 cells and 68% of cdc14-1
?clb5 cells in three separate experiments.
We next tested if the lethality of the CLB5-?N mutation
results from a failure to efficiently segregate chromosome XII,
which contains the rDNA. We used a yeast strain from which
the entire rDNA locus is deleted and viability is supported by
multiple copies of a plasmid carrying an rDNA repeat (36). A
GAL-SIC1 plasmid was introduced into this strain, along with
a plasmid carrying either CLB5 or CLB5-?N. Deletion of the
rDNA locus did not restore the viability of CLB5-?N cells
when SIC1 expression was repressed on dextrose media (Fig.
3A). We conclude that rDNA segregation defects are not the
sole cause of CLB5-?N lethality.
DNA replication fails in CLB5-?N cells. Clb5-Cdk1 is a
critical regulator of DNA replication. To investigate whether
DNA replication is aberrant in cells expressing stabilized Clb5,
we measured the DNA content of cells expressing CLB5 or
CLB5-?N after release from G1in the absence of Sic1. Both
strains completed the first round of DNA replication and pro-
gressed into mitosis with similar timing, revealing no significant
defects in the first cycle of DNA replication (Fig. 4A). The
CLB5 strain then entered the next cell cycle and completed
another S phase and progressed through the second and sub-
sequent cell cycles. In contrast, CLB5-?N cells failed to un-
dergo a second round of DNA replication and arrested as
large-budded cells with a 1n DNA content (Fig. 4A). Thus, the
continued presence of Clb5 after anaphase prevents DNA rep-
lication and thereby causes cell death.
We suspected that Clb5-?N blocked DNA replication by
inhibiting the formation of prereplicative complexes (pre-RCs)
at replication origins. From early S to late M phases, Cdk1
blocks pre-RC assembly by phosphorylating and thereby inhib-
iting several pre-RC subunits; Cdk1 inactivation in late mitosis
then triggers pre-RC assembly (1, 20). A key pre-RC compo-
nent is the Mcm complex, the phosphorylation of which pre-
vents its nuclear import (14, 15). We examined the localization
of a GFP-tagged Mcm complex in cells released from a G1
arrest. In cells expressing wild-type CLB5, the Mcm complex
moved from the nucleus to the cytoplasm during S phase and
reentered the nucleus in late mitosis (Fig. 4B). In contrast,
late-mitotic nuclear reentry of the Mcm complex did not occur
in CLB5-?N cells, and it remained cytoplasmic. Thus, the
continued presence of Clb5 after anaphase blocks the nuclear
import of the Mcm complex, presumably preventing efficient
pre-RC formation. It seems likely that this problem leads to
defects in the initiation of DNA replication, and the resulting
replication stress triggers a DNA damage response that blocks
cell cycle progression.
APCCdc20-mediated destruction of Dbf4 contributes to
timely rDNA disjunction. We noted earlier that cdc20? pds1?
cells exhibited a 30-min delay in rDNA segregation (Fig. 1).
However, expression of Clb5-?N in otherwise wild-type cells
caused only a 15-min delay in rDNA segregation (Fig. 3C).
One explanation for this difference is that Clb5-?N is not fully
stabilized, but we could find no evidence for that (Fig. 3B).
An alternate possibility is that degradation of an additional
APCCdc20substrate contributes to rDNA segregation. One ap-
pealing candidate was Dbf4, the activating subunit for the
protein kinase Cdc7. Dbf4 is known to be an APC substrate
FIG. 4. CLB5-?N causes DNA replication defects. (A) CLB5 and
CLB5-?N cells were released from G1arrest in the absence of SIC1 as
in Fig. 3B, and flow cytometry was used to analyze cellular DNA
content. (B) Procedure was as in panel A but with MCM4-GFP cells,
which were analyzed for budding, binucleates, and the localization of
the Mcm4-GFP protein.
5332SULLIVAN ET AL.MOL. CELL. BIOL.
at UNIV OF CALIF-SAN FRANCISCO on June 8, 2010
whose levels drop in mitosis, although the precise timing of
its destruction and the APC cofactor involved are not known
(7). We found that APCCdc20ubiquitinates Dbf4 in vitro and
that Dbf4 is destroyed at anaphase onset, independently of
CDH1 (Fig. 5A and data not shown), suggesting that Dbf4 is
an APCCdc20substrate. Removal of its amino-terminal de-
struction sequences stabilized Dbf4 in mitosis and G1(Fig.
5A and B).
To assess the contribution of Dbf4 to rDNA segregation, we
constructed a strain in which the endogenous DBF4 gene was
FIG. 5. Stabilization of Dbf4 contributes to the rDNA segregation delay. (A) Representative images of Dbf4-myc in metaphase or anaphase
from wild-type or DBF4-?N cells. (B) DBF4 and DBF4-?N cells were arrested in M phase by nocodazole treatment or G1by ?-factor treatment,
and the levels of Dbf4 were analyzed. The asterisk indicates a nonspecific background band recognized by the anti-myc antibody. (C) DBF4 and
DBF4-?N cells, carrying an extra copy of either CLB5 or CLB5-?N, were released from a G1arrest as in Fig. 3C, and ?-factor was re-added 80
min after release to arrest cells in the following G1. Samples were collected every 20 min for analysis of budding, binucleate formation, and Nop1
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replaced with DBF4-?N. This strain displayed no apparent
defects in cell viability, mitotic progression, or rDNA segrega-
tion (Fig. 5C). We then introduced either CLB5 or CLB5-?N
in combination with GAL-SIC1 and examined cell cycle pro-
gression. Notably, cells carrying both CLB5-?N and DBF4-?N
exhibited an rDNA segregation defect that was more severe
than that seen in CLB5-?N cells. The average delay in rDNA
segregation relative to bulk chromatin in the CLB5-?N
DBF4-?N cells was similar to that in cdc20? pds1? cells, and
many cells failed to separate their nucleolus before cytokinesis.
Thus, Clb5 and Dbf4 are APCCdc20targets that must both be
destroyed for efficient rDNA segregation.
Meiotic rDNA segregation fails in the continued presence of
Clb5 and Dbf4. In anaphase I of meiosis, homologs are resolved
by loss of cohesin on the chromosome arms. However, rDNA
cohesion must also be resolved to allow segregation of the chro-
mosome XII homologs. We investigated whether meiotic rDNA
segregation involves the same mechanisms as those we identified
in mitosis. Immunofluorescence analysis revealed that both Clb5
and Dbf4 were present at high levels in metaphase I and absent in
anaphase I, consistent with degradation via APCCdc20(data not
shown). We then expressed stabilized Clb5 and/or Dbf4 in the
SK1 budding-yeast strain, which allows analysis of synchronous
meiosis. Replacement of endogenous DBF4 with DBF4-?N had
no apparent impact on meiosis (Fig. 6A and B). Expression of
CLB5-?N from the meiosis-specific DMC1 promoter caused a
delay in meiotic progression and a small decrease in spore viabil-
ity (Fig. 6A and B) but no apparent defect in rDNA segregation
in anaphase I. However, cells expressing both Clb5-?N and
in spore viability. Strikingly, rDNA segregation failed completely
I and II cells, where only a single, unsegregated nucleolar mass
was visible (Fig. 6C and D). Thus, APCCdc20-mediated destruc-
tion of both Clb5 and Dbf4 contributes to rDNA segregation in
meiosis, and a failure to destroy both in anaphase I completely
prevents rDNA disjunction.
Sister chromatid cohesion depends primarily on cohesin
linkages (19). In budding yeast, an additional mechanism op-
FIG. 6. Meiotic rDNA segregation fails in the presence of stabilized Dbf4 and Clb5. Diploid DBF4 and DBF4-?N SK1 cells, with or without
pDMC1-CLB5-?N, were followed through synchronous meiosis. Chromosome divisions were scored (A), as were the survival rates of resulting
spores (B) and the segregation patterns of Nop1 through both anaphase I (C) and anaphase II (D). Histograms are labeled WT (wild type), d
(DBF4-?N), c (pDMC1-CLB5-?N), and dc (DBF4-?N with pDMC1-CLB5-?N).
5334SULLIVAN ET AL.MOL. CELL. BIOL.
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erates specifically at the rDNA locus of chromosome XII.
Removal of both cohesive mechanisms requires APCCdc20-
dependent degradation of securin and the liberation of sepa-
rase, which cleaves a subunit of cohesin and also promotes
dissolution of rDNA cohesion by activating the phosphatase
Cdc14 (5, 27, 28, 31). Although it is well established that the
only critical role of APCCdc20in cohesin removal is the pro-
motion of securin degradation, it was not known if the destruc-
tion of additional targets is necessary for the resolution of
rDNA. Our results demonstrate that efficient rDNA resolution
also depends on APCCdc20-dependent destruction of Clb5 and
Although delayed, rDNA segregation does eventually occur
in cells expressing stabilized forms of Clb5 and Dbf4, indicat-
ing that the destruction of these proteins is not essential for
rDNA segregation in mitosis. In meiosis, however, rDNA seg-
regation fails completely in the presence of stabilized Clb5 and
Dbf4, revealing a critical function for the inactivation of these
kinases in meiotic anaphase.
Previous evidence that the phosphatase Cdc14 promotes
rDNA segregation (5, 28) suggested that rDNA cohesion de-
pends on the phosphorylation of Cdk1 substrates. Our results
are consistent with this possibility and also suggest that rDNA
resolution depends specifically on the dephosphorylation of
Clb5-Cdk1 targets, as rDNA segregation was not significantly
affected by Clb2 overexpression. Given the known timing of
Clb5 and Clb2 destruction, this cyclin specificity supports a
model (Fig. 7) in which Clb5 destruction and Cdc14 activation
trigger the dephosphorylation in early anaphase of specific
Cdk1 targets involved in rDNA segregation. The significant
levels of Clb2 that remain in the anaphase cell are apparently
insufficient to maintain the phosphorylation of these targets in
the face of separase-dependent Cdc14 activation. However, we
speculate that Clb2-Cdk1 activity in anaphase does maintain
the phosphorylation state of other Cdk1 targets involved in
spindle disassembly and mitotic exit; the dephosphorylation of
these targets occurs after anaphase as a result of Clb2 destruc-
tion, perhaps coupled with the increased Cdc14 activity that
results from activation of the mitotic-exit network (30). Stabi-
lization of Clb2 therefore blocks mitotic exit but not rDNA
segregation or other anaphase processes.
What are the key targets of Clb5-Cdk1 in rDNA cohesion?
The molecular basis of rDNA cohesion is not well understood
but seems to depend on complex mechanisms involving topo-
isomerase II, sumoylation, condensin, and RNA polymerase I
(5, 10, 18, 28, 33, 34, 38). Clb5-Cdk1 may act through one or
multiple substrates to govern these mechanisms. We identified
several Clb5-specific Cdk1 substrates in our previous work
(16), but none of these appear to be a clear candidate for the
control of rDNA cohesion. A significant future challenge will
be the systematic identification of all Clb5-specific Cdk1 sub-
strates, which should allow discovery of the critical Clb5 sub-
strates involved in rDNA resolution and other anaphase pro-
We also identified the Cdc7-binding partner Dbf4 as a
potential regulator of rDNA cohesion in budding yeast. Dbf4-
Cdc7 is known primarily as a regulator of the initiation of DNA
synthesis, and no mitotic roles have been described for this
kinase. How might Dbf4-Cdc7 regulate rDNA cohesion? The
fact that the DBF4-?N mutant has no apparent cell cycle de-
fect alone but enhances the segregation delay from the
CLB5-?N mutant suggests that one function of Dbf4-Cdc7
might be to enhance Clb5-Cdk1 function, either by increasing
its intrinsic kinase activity or by enhancing its activity toward
some Clb5-Cdk1 target. Alternatively, Dbf4-Cdc7 could pro-
mote cohesion by directly phosphorylating some component of
the rDNA machinery. Notably, recent work suggests that Dbf4-
Cdc7 helps promote DNA recombination in meiotic prophase
and monopolar chromosome attachment in meiosis I (23, 35,
37). Dbf4-Cdc7 might therefore be involved in the control of
multiple processes other than DNA replication.
In fission yeast and higher eukaryotes, it is not clear if there
are cohesin-independent chromosome linkages like those op-
erating at budding-yeast rDNA. The fission yeast ortholog of
Cdc14, Clp1, is not required for rDNA segregation (3), but it
remains possible that other phosphatases contribute to the
FIG. 7. Model of regulatory mechanisms governing chromosome segregation in budding yeast. Bulk chromosome separation occurs when
cohesin is cleaved by separase, the activation of which results from the APCCdc20-dependent destruction of securin. Efficient removal of rDNA
cohesion also requires the dephosphorylation of Clb5-specific Cdk1 targets, which results from the activation of Cdc14 by separase and the
destruction of Clb5 via APCCdc20. By uncertain mechanisms, APCCdc20-dependent Dbf4 destruction also contributes. Destruction of Clb2 allows
dephosphorylation of its targets after anaphase, leading to spindle disassembly and mitotic exit.
VOL. 28, 2008 CYCLIN-SPECIFIC CONTROL OF rDNA SEGREGATION 5335
at UNIV OF CALIF-SAN FRANCISCO on June 8, 2010
dephosphorylation of Cdk1 substrates involved in rDNA co-
hesion. In mammalian cells, noncohesin rDNA linkages, if they
exist, may be lost early in mitosis as a result of the “prophase”
pathway that drives chromosome arm resolution and separa-
tion before metaphase (8, 13). Alternatively, rDNA resolution
in animal cells might depend on the degradation of cyclin A in
prometaphase. In some cell types, expression of APC-resistant
cyclin A leads to a metaphase-like arrest with unseparated
sister chromatids (6, 9, 21, 25), and cohesin-independent link-
ages may contribute to this separation defect.
Future models of mitosis should take into account the qual-
itative contributions of individual cyclin-Cdk1 complexes, in
terms of both phosphorylation events early in the cell cycle and
dephosphorylation events as cyclins are sequentially destroyed
in late mitosis. Early destruction of cyclins that display strong
substrate specificity, such as Clb5 and cyclin A, may be re-
quired for the correct timing of anaphase events. The substrate
specificity of phosphatases such as Cdc14 provides another
layer of complexity in the control of Cdk substrate dephos-
phorylation. These mechanisms collaborate to ensure that key
Cdk substrates involved in processes such as chromosome seg-
regation and spindle elongation are dephosphorylated at an-
aphase onset, while dephosphorylation of other Cdk substrates
is delayed until later in mitosis.
We are grateful to A. Amon, F. Cross, J. Diffley, K. Nasmyth, M.
Nomura, and F. Uhlmann for reagents and to members of the Morgan
laboratory for discussions and comments on the manuscript.
This work was supported by funding from the National Institute of
General Medical Sciences.
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