Cdc7p-Dbf4p Regulates Mitotic Exit by Inhibiting Polo
Charles T. Miller1,2, Carrie Gabrielse2, Ying-Chou Chen2,3, Michael Weinreich2*
1Graduate Program in Cell and Molecular Biology, Michigan State University, East Lansing, Michigan, United States of America, 2Laboratory of Chromosome Replication,
Van Andel Research Institute, Grand Rapids, Michigan, United States of America, 3Graduate Program in Genetics, Michigan State University, East Lansing, Michigan, United
States of America
Cdc7p-Dbf4p is a conserved protein kinase required for the initiation of DNA replication. The Dbf4p regulatory subunit
binds Cdc7p and is essential for Cdc7p kinase activation, however, the N-terminal third of Dbf4p is dispensable for its
essential replication activities. Here, we define a short N-terminal Dbf4p region that targets Cdc7p-Dbf4p kinase to Cdc5p,
the single Polo kinase in budding yeast that regulates mitotic progression and cytokinesis. Dbf4p mediates an interaction
with the Polo substrate-binding domain to inhibit its essential role during mitosis. Although Dbf4p does not inhibit Polo
kinase activity, it nonetheless inhibits Polo-mediated activation of the mitotic exit network (MEN), presumably by altering
Polo substrate targeting. In addition, although dbf4 mutants defective for interaction with Polo transit S-phase normally,
they aberrantly segregate chromosomes following nuclear misorientation. Therefore, Cdc7p-Dbf4p prevents inappropriate
exit from mitosis by inhibiting Polo kinase and functions in the spindle position checkpoint.
Citation: Miller CT, Gabrielse C, Chen Y-C, Weinreich M (2009) Cdc7p-Dbf4p Regulates Mitotic Exit by Inhibiting Polo Kinase. PLoS Genet 5(5): e1000498.
Editor: Orna Cohen-Fix, National Institute of Diabetes and Digestive and Kidney Diseases, United States of America
Received December 4, 2008; Accepted April 29, 2009; Published May 29, 2009
Copyright: ? 2009 Miller et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This work was supported by the Van Andel Research Institute and American Cancer Society grant RSG0506301GMC to MW. The funders had no role in
study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
* E-mail: email@example.com
Accurate ordering of cell cycle events is an important
requirement for the viability of all eukaryotic organisms. Once
cells commit to duplicate their genome they must restrain mitosis
until replication is complete and then accurately coordinate
mitosis with cytokinesis to ensure the faithful transmission of
chromosomes to daughter cells . Importantly, errors in cell
cycle checkpoints that enforce this ordering can be deleterious for
accurate chromosome transmission. For instance, DNA damage or
replication fork arrest during S-phase elicits a reversible block to
mitotic progression by the budding yeast Mec1p (HsATR) and
Rad53p (HsChk2) checkpoint kinases [2,3]. In the absence of
Mec1p or Rad53p, replication fork arrest during S-phase is not
sensed leading to premature mitotic events and cell death
(reviewed by ). Additionally, since daughter cell growth is
highly polarized in the budding yeast, exit from mitosis is
prevented until sister chromatids segregate through the bud neck
and into the daughter cell [5–7]. This ensures that spindle
disassembly and mitotic exit are not initiated until accurate
chromosome partitioning between mother and daughter cells has
occurred. Failure to block mitotic exit when nuclear division takes
place within the mother cell results in polyploid and anucleate
progeny [8,9]. It is not surprising therefore, that both entry into
and exit from mitosis are delayed by cellular checkpoints that
respond to replication stress, chromosome damage, or spindle
disruption . Errors in these mitotic checkpoints are catastrophic
and result in ploidy defects and genetic alterations, which are
frequently observed in human cancers (reviewed by ).
The Cdc7p-Dbf4p kinase is required to catalyze the initiation of
DNA synthesis at the beginning of S-phase (reviewed by ).
Cdc7p kinase activity is tightly regulated during the cell cycle by
binding the Dbf4p regulatory subunit, which is cyclically expressed.
Dbf4p accumulates in late G1, is present throughout S-phase and
then is destroyed during mitosis and early G1 by anaphase
promoting complex (APC)-dependent degradation [12–17]. There-
fore, Cdc7p-Dbf4p kinase activity is low following exit from mitosis
and entry into G1-phase until it is needed to initiate a new round of
DNA synthesis in late G1-phase of the following cell cycle. Multiple
lines of evidence suggest that Cdc7p-Dbf4p activates the MCM
DNA helicase [18–20] that is assembled at origins of replication in
early G1 in an inactive form (reviewed in [21,22]).
In addition to its essential role in replication initiation, several
studies suggest that the Cdc7p-Dbf4p kinase responds to DNA
damage or replication fork stalling but its precise role in these
activities is unknown [17,23–25]. Dbf4p encodes a dispensable
BRCT-like domain in the N-terminus that might target the kinase
to stalled replication forks [26,27]. In fission yeast, the Cdc7p-
Dbf4p ortholog Hsk1p-Dfp1p interacts with Swi1p (budding yeast
Tof1p), a component of replication forks required for fork stability
and also promotes centromeric cohesion in early mitosis [28,29].
Rad53p also phosphorylates Dbf4p in response to replication stress
and this regulation requires N-terminal Dbf4p sequences through
which Rad53p physically interacts [17,25,30]. Interestingly, the
absence of the BRCT-like domain results in a defect in late origin
activation suggesting that this domain might alter Cdc7p-Dbf4p
binding at early versus late replication origins . Together,
these data suggest that the Dbf4p N-terminus encodes non-
PLoS Genetics | www.plosgenetics.org1 May 2009 | Volume 5 | Issue 5 | e1000498
essential regulatory functions that target the kinase to particular
To identify proteins that interact with the Dbf4p N-terminus,
we performed a yeast two-hybrid screen with an N-terminal region
of Dbf4p and identified an interaction with the Cdc5p kinase, the
only Polo ortholog in yeast. Budding yeast Polo, like Drosophila
Polo and human Polo-like kinase 1 (Plk1), functions as a master
regulator of mitotic progression and is also required for cytokinesis
(reviewed by [31,32]). Polo activity is regulated by several
independent cellular mechanisms. Polo protein levels are con-
trolled by APC-dependent degradation in mitosis/G1-phase and
activation of Polo catalytic activity requires phosphorylation by
Cdk1 kinase early in G2 [33–35]. In addition, Polo function is
inhibited by cell cycle checkpoints that are induced following DNA
or spindle damage [36–38]. A genetic and physical interaction
between Dbf4 and Polo was described previously [39,40], however
the biological significance of this interaction was not known.
Polo controls multiple mitotic events to ensure accurate
chromosome segregation. After anaphase initiation, Polo is
required to activate the FEAR (Cdc14 early anaphase release)
and MEN (mitotic exit network) pathways that promote nucleolar
release of Cdc14p phosphatase [37,41–43]. Limited Cdc14p
release by the FEAR pathway promotes accurate rDNA and
telomere segregation [44–46]. Subsequent full nucleolar release of
Cdc14p by the MEN reverses Cdk substrate phosphorylation that
leads to APC-Cdh1p activation, cyclin destruction and mitotic
spindle disassembly (reviewed by ). Activation of the MEN is
promoted by Tem1p-GTP and antagonized by Bfa1p-Bub2p, a
two-component GTPase activating protein (GAP) [48–50]. To
promote mitotic exit, Polo phosphorylates Bfa1p-Bub2p to inhibit
its GAP activity and is also required for activation of Dbf2p kinase
activity, independently of Bfa1p-Bub2p [37,49,51,52]. The Polo
requirement for Dbf2p kinase activation may reflect that Polo also
promotes Cdc14p release in the FEAR pathway, which primes the
MEN . Therefore, Polo promotes accumulation of Tem1p-
GTP and activation of the downstream MEN kinases Cdc15p and
Dbf2p, which ultimately cause full release of Cdc14p from the
nucleolus. In response to replication fork arrest, Rad53 inhibits
MEN activation, which may or may not impact Polo activity since
the molecular basis of this regulation is not understood [53,54].
Spindle position defects also counteract Polo activity by targeting
Kin4p kinase to the spindle poles where it inhibits Polo-dependent
Bfa1p phosphorylation [8,9,55]. Failure to execute the spindle
position checkpoint (SPOC) results in premature exit from mitosis
and nuclear partitioning defects.
Here we define an N-terminal Dbf4p polo-box interaction
region (that we refer to as the ‘‘PIR’’) that binds directly to Polo
and show that Dbf4p inhibits Polo and Dbf2p activity. Deletion of
the PIR allows Cdc14p nucleolar release in a cdc5-1 mutant at the
non-permissive temperature. In response to nuclear misposition-
ing, a dbf4 mutant lacking the PIR fails to arrest in mitosis and
prematurely exits the cell cycle. Thus, Dbf4 protein is required for
proper functioning of the spindle position checkpoint most likely
by antagonizing the ability of Polo to promote Cdc14p release in
either the FEAR or MEN pathways. Our work therefore reveals a
previously unrecognized function for Dbf4p in the regulation of
mitotic progression through a direct interaction with Polo.
The Cdc5p Polo-Box Domain Interacts with the Dbf4p N-
We conducted a yeast two-hybrid screen to identify proteins
that interact with the Dbf4p N-terminus (residues 67–227) and
recovered multiple clones encoding the polo-box domain (PBD) of
Cdc5p. Polo kinase has two conserved domains; an N-terminal
kinase domain and a C-terminal region called the polo-box
domain (PBD) (reviewed by ), which is a phospho-Ser/Thr
binding module that targets the kinase to its mitotic substrates
[57,58]. The crystallographic structure of the Plk1 PBD bound to
a phospho-threonine peptide has been solved . Since the
Dbf4p BRCT-like region alone (residues 110–227) failed to
interact with the Polo PBD (Figure 1A), this suggested that the
PBD interaction was occurring through Dbf4p N-terminal
sequences from 67–109. Residues 67–109 were similarly required
for the Polo interaction within the context of full length Dbf4p
(Figure 1B) and were sufficient to interact with the Polo PBD
(Figure 1A). Dbf4p residues 67–109 with all serines/threonines
changed to alanine still interacted with the PBD (Figure 1A)
suggesting that the PBD can bind to this Dbf4p region
independently of phosphorylation. Further deletion and point
mutant analysis (Y-C.C. and M.W., unpublished data) revealed
that residues 82–88 are essential for the Dbf4p-Polo interaction
(Figure 1A, B).
The PBD is composed of three conserved regions called the
Polo-cap (Pc), Polo-box 1 (PB1) and Polo-box 2 (PB2) that fold
together to form a functional phosphopeptide-binding domain
. We deleted conserved residues within the PBD to test their
requirement for interaction with Dbf4p (Figure 1C). Deletion of
residues preceding the PBD (GAD-Polo454–705) had little effect on
the Dbf4p-Polo interaction. However, elimination of the Pc (GAD-
Polo510–705) completely disrupted the interaction with Dbf4p.
These data suggest that the structural integrity of the Polo PBD is
required for Dbf4p binding.
Dbf4p Directly Interacts with Polo
Dbf4p binds and activates the Cdc7p kinase subunit in yeast
and has no known role apart from its interaction with Cdc7p
[14,60]. To determine whether the interaction between Dbf4p and
Polo occurred in the context of the full-length Cdc7p-Dbf4p
kinase, Sf9 cells were co-infected with baculoviruses expressing
Polo, wild type HA-Cdc7p-Dbf4p or wild type HA-Cdc7p with
various Dbf4p deletion derivatives. HA-Cdc7p-Dbf4p kinase was
immunoprecipitated using an antibody against the HA tag and
examined for the presence of Polo. All the Dbf4p deletion
Cdc7p-Dbf4p is a two-subunit enzyme required to copy
the genetic material present on every chromosome in a
process termed DNA replication. Dbf4p is an essential
regulatory subunit of this enzyme that likely directs the
Cdc7p subunit to its targets within the cell. We found that
Dbf4p physically interacts with another protein called Polo
that acts during mitosis, a later step in the cell cycle when
the newly copied chromosomes are equally divided to
mother and daughter cells. Polo is a master regulator of
mitosis and impacts many other proteins required for cell
division. We determined that Cdc7p-Dbf4p is a Polo
inhibitor and, further, that Cdc7p-Dbf4p delayed or
prevented chromosome segregation when errors occurred
during the cell division process. Interestingly, Dbf4p may
bind the Polo substrate-binding domain using a type of
interaction not previously described. Thus, we have
uncovered a new activity for Cdc7p-Dbf4p in the cell
cycle to inhibit chromosome segregation, and these
findings impact multiple fields that investigate how cells
accurately copy and segregate their chromosomes.
Dbf4p Inhibits Polo Kinase
PLoS Genetics | www.plosgenetics.org2May 2009 | Volume 5 | Issue 5 | e1000498
of the Dbf4p PIR does not enhance Cdc14 release when combined
with deletion of FOB1. Dfob1 CDC14-EGFP (M3149) and Dfob1
dbf4-ND109 CDC14-EGFP (M3148) were arrested in G1 with
mating pheromone and released into the cell cycle at 30uC. Alpha-
factor was added back after budding to follow a single cell cycle.
Samples were taken at the indicated time points and scored for
release of Cdc14 from the nucleolus.
Found at: doi:10.1371/journal.pgen.1000498.s004 (0.22 MB TIF)
unperturbed cell cycle. PDS1-HA3 DBF4-Myc18 (M3161) was
arrested in G1 with mating pheromone and released into the cell
cycle at 20uC. Samples were taken at the indicated time points.
Protein extracts were made for Western blotting and cells
processed for DNA content analysis by flow cytometry. Western
blots were probed with 9E10 a-Myc (Dbf4-Myc18) and 12CA5 a-
HA (Pds1-HA) antibodies.
Found at: doi:10.1371/journal.pgen.1000498.s005 (3.24 MB TIF)
Some Dbf4p persists after Cdc20 activation during an
Found at: doi:10.1371/journal.pgen.1000498.s006 (0.08 MB
Yeast strains used in this study.
Plasmids used in this study.
Found at: doi:10.1371/journal.pgen.1000498.s007 (0.05 MB
kinase activity, and SPB localization; nucleolar segregation assay;
and Dbf4 cell cycle abundance.
Found at: doi:10.1371/journal.pgen.1000498.s008 (0.03 MB
Supplementary methods: Cdc5 protein abundance,
We thank A. Amon, E. Craig, L. Hartwell, A. Hoyt, L. Johnston, A.
Murray, K. Nasmyth, E. Schiebel, A. Sugino and B. Stillman for yeast
strains and plasmids; D. Morgan for communicating results prior to
publication; C. Miranti and V. Schulz for technical assistance with
microscopy; H. Scott for construction and purification of Dbf4p SUMO-
derivatives; D. Dornbos for strain construction; C. Fox for comments on
the manuscript; A. Amon, G. Pereira, F. Uhlmann and members of the
Weinreich lab for helpful discussions.
Conceived and designed the experiments: MW. Performed the experi-
ments: CTM CG YCC MW. Analyzed the data: CTM CG YCC MW.
Contributed reagents/materials/analysis tools: CTM CG YCC MW.
Wrote the paper: MW.
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PLoS Genetics | www.plosgenetics.org13May 2009 | Volume 5 | Issue 5 | e1000498