Cdc7p-Dbf4p Regulates Mitotic Exit by Inhibiting Polo Kinase

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.


Introduction
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 [1]. 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 [4]). 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][6][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 [1]. Errors in these mitotic checkpoints are catastrophic and result in ploidy defects and genetic alterations, which are frequently observed in human cancers (reviewed by [10]).
The Cdc7p-Dbf4p kinase is required to catalyze the initiation of DNA synthesis at the beginning of S-phase (reviewed by [11]). 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][13][14][15][16][17]. Therefore, 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][19][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][24][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 [26]. Together, these data suggest that the Dbf4p N-terminus encodes non-essential regulatory functions that target the kinase to particular substrates.
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 controlled by APC-dependent degradation in mitosis/G1-phase and activation of Polo catalytic activity requires phosphorylation by Cdk1 kinase early in G2 [33][34][35]. In addition, Polo function is inhibited by cell cycle checkpoints that are induced following DNA or spindle damage [36][37][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][42][43]. Limited Cdc14p release by the FEAR pathway promotes accurate rDNA and telomere segregation [44][45][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 [47]). Activation of the MEN is promoted by Tem1p-GTP and antagonized by Bfa1p-Bub2p, a two-component GTPase activating protein (GAP) [48][49][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 [43]. 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 mispositioning, 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.

Results
The Cdc5p Polo-Box Domain Interacts with the Dbf4p N-Terminus 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 [56]), 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 [59]. 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 [59]. We deleted conserved residues within the PBD to test their requirement for interaction with Dbf4p ( Figure 1C). Deletion of residues preceding the PBD (GAD-Polo 454-705 ) had little effect on the Dbf4p-Polo interaction. However, elimination of the Pc (GAD-Polo 510-705 ) 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

Author Summary
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.
derivatives we examined interact with Cdc7p and activate normal Cdc7p kinase activity ( [26] and data not shown). Whereas Polo interacted with full-length Cdc7p-Dbf4p and Cdc7p-Dbf4-ND65p, Polo did not interact with Cdc7p-Dbf4p complexes that lacked the Dbf4p N-terminal 109 residues required for the Polo two-hybrid interaction ( Figure 2A). These data indicate that full length Cdc7p-Dbf4p kinase interacts with Polo but that Dbf4p residues 65-109 are required for this interaction. Importantly, HA-Cdc7p-Dbf4p interacts with Cdc5p in yeast when the proteins are expressed at endogenous levels and this interaction also depends on the Dbf4p N-terminus ( Figure 2B).
We next tested whether Polo bound directly to Dbf4p using purified proteins. GST-PBD and Sumo-Dbf4p 67-109 fusion proteins purified from E. coli were mixed, pulled down using glutathione-Sepharose and analyzed by immunoblotting. Although Sumo alone did not interact with GST-PBD, Sumo-Dbf4p 67-109 interacted with GST-PBD but not with GST alone ( Figure 2C). These data indicate that Dbf4p residues 67-109 (that we refer to as the Dbf4p PIR) are sufficient for a direct interaction with the Polo PBD.

Dbf4p Inhibits Polo Activity
Cdc7p-Dbf4p is required to initiate DNA replication, but is present throughout S-phase and during the metaphase to anaphase transition. Dbf4p is subject to APC-Cdc20p dependent degradation [16] but some protein is still present in late mitotic mutants that have activated the Cdc20p but not the Cdh1p form of the APC [17]. We examined Dbf4 protein abundance relative to Pds1 protein in cells moving synchronously through the cell cycle, since Pds1p is degraded at the onset of anaphase by APC-Cdc20 [61]. We found that although the abundance of both proteins declines at the same time, Pds1p is absent during mitosis while some fraction of Dbf4p persists ( Figure S5). In contrast, Dbf4p has very low abundance or is absent in cells arrested in G1phase by mating-pheromone when the APC-Cdh1p is active [12,13,15,17] and Dbf4p is stabilized by inactivation of the APC in G1 or by removal of its N-terminal D-box [13,[15][16][17]. Together these data suggest that Dbf4p degradation can occur via both APC-Cdc20p and APC-Cdh1p mediated ubiquitylation.
Since budding yeast Polo is not required for DNA replication but promotes multiple mitotic activities, we reasoned that Cdc7p-Dbf4p might influence Polo activity during mitosis. The dbf4-ND109 mutant progresses normally through the cell cycle and does not exhibit any obvious growth defects or temperature sensitivity ( Figure 3A, B) [26]. We therefore tested for genetic interactions between dbf4-ND109 and the cdc5-1 temperature sensitive (ts) mutant. At the restrictive temperature cdc5-1 cells arrest in late telophase with divided nuclei, elongated spindles and high Cdk1p-Clb2p levels indicating a failure to exit mitosis [34,35,62]. The cdc5-1 mutant is ts at 30uC on rich media, but we found that dbf4-ND109 suppressed the cdc5-1 temperature sensitivity up to 35uC indicating a strong suppression of its growth defect ( Figure 3A). The dbf4-D82-88 mutant defective for interaction with Polo also suppressed the cdc5-1 ts ( Figure 3A). Since the cdc5-1 ts suppression was reduced in heterozygous dbf4-ND109/DBF4 diploids compared to dbf4-ND109/dbf4-ND109 diploids, i.e. dbf4-ND109 was haploinsufficient ( Figure 3C), our data strongly suggests that Dbf4p is a Polo inhibitor and that loss of the Dbf4-Polo interaction leads to increased cdc5-1 activity.
We confirmed that Dbf4p inhibited Polo activity using several independent genetic tests. An extra plasmid copy of wild type DBF4 but not dbf4-ND109 inhibited the growth of cdc5-1 cells ( Figure 3D). A dbf4-ND65 mutant that disrupts the D-box (residues 62-70) resulting in elevated protein levels was synthetically sick or lethal in combination with cdc5-1 ( Figure 3E). Since the dbf4-ND65 mutant exhibits a wild type growth rate and normal S-phase entry and progression (data not shown; [26]) but binds to Polo, this suggests that elevated Dbf4p levels are deleterious to cdc5-1 activity. Finally, elevated expression of the Dbf4p N-terminus from the GAL1 promoter completely inhibited the growth of cdc5-1 cells but had no effect on the growth of wild type (not shown) or a mcm2-1 ts mutant. Mcm2 is a component of the MCM helicase, which is thought to be the physiological target of Cdc7p-Dbf4p during the initiation of DNA replication [11]. The inhibition of cdc5-1 growth depended on the Dbf4p-Polo interaction, since deletion of the PIR (residues 66-109) or Dbf4p residues 82-88 abrogated the growth inhibition ( Figure 4A, B). Since the Dbf4p N-terminus interacts with the PBD, this data suggest that overexpression of Dbf4 N-terminal peptides interferes with essential Polo-substrate interactions by competitive inhibition. Together, these data indicate that the Dbf4p N-terminus inhibits Polo activity and that this inhibition requires residues 66-109, which are also required for the Dbf4p-Polo physical interaction.
We wanted to determine whether the Cdc7p kinase subunit is required to inhibit Polo in the FEAR or MEN pathways. This is not straightforward since Cdc7p is an essential protein kinase. Importantly, inhibiting Cdc7p activity would not only inhibit replication origin firing but would also likely induce the replication checkpoint that inhibits the metaphase to anaphase transition and MEN activation [4]. Inhibiting Cdc7p activity would thus interfere with the mitotic pathways we would like to measure. Therefore, we addressed this question indirectly by taking advantage of our observation that high copy dbf4-ND109 suppressed the cdc5-1 ts phenotype ( Figure 4C). Since Dbf4p residues required for interaction with Cdc7p map between residues 312-704 (C.G. and M.W. unpublished data), Dbf4-ND109 protein (expressed in high copy) will compete with full length Dbf4p (in single copy) for Cdc7p binding. Therefore, our finding suggested that high copy expression of Dbf4-ND109p reduced the cellular concentration of wild type Cdc7p-Dbf4p, the likely Cdc5p inhibitor, which suppressed the cdc5-1 ts allele (Fig 4C). High copy expression of Dbf4-D82-88p that does not interact with Polo also suppressed the cdc5-1 ts (Fig 4C). Importantly, deleting Dbf4p C-terminal residues required for interaction with Cdc7p (CD312-704) eliminated the ts suppression by high copy dbf4-ND109 and dbf4-D82-88. These data are consistent with full-length Cdc7p-Dbf4p kinase acting as the physiological Polo inhibitor.

Cdc7p-Dbf4p Phosphorylates the PBD
Wild type Cdc7p-Dbf4p might inhibit Polo abundance or kinase activity during the cell cycle and thus explain our genetic data. However, we saw little difference in Polo protein levels, cell cycle expression or Polo kinase activity comparing wild type yeast with the dbf4-ND109 mutant ( Figure S2). This suggests that Dbf4p inhibits Polo independently of altering its expression or kinase activity. This is consistent with our genetic data since loss the Dbf4p PIR suppresses the cdc5-1 allele yet the Cdc5-1 protein retains considerable protein abundance and kinase activity at the non-permissive temperature [63]. The mitotic exit defect associated with cdc5-1 is due to a single P511L amino acid substitution preceding polo-box 1 of the PBD [64], strongly suggesting that the cdc5-1 growth defect is caused by a defect in substrate recognition. Since genetically DBF4 is a negative CDC5 regulator we hypothesized that Cdc7p-Dbf4p phosphorylates Polo to prevent its access to key substrates in the MEN. Consistent with this possibility, we found that purified Cdc7p-Dbf4p phosphorylated recombinant GST-PBD but not GST alone ( Figure 2D).

Dbf4p Inhibits Cdc14p Nucleolar Release
The Cdc14p phosphatase is sequestered within the nucleolus during the cell cycle prior to FEAR and MEN pathway activation [50,65]. Activation of the FEAR pathway allows limited Cdc14p nucleolar release, which promotes rDNA and telomere segregation during early anaphase [42,[44][45][46]. Cdc14p is then fully released by MEN activation and antagonizes Cdk activity to trigger exit from mitosis [66]. Since the cdc5-1 mutant fails to release Cdc14p at the restrictive temperature [66], suppression of the cdc5-1 ts by deletion of the Dbf4p PIR ( Figure 3A) suggested that Cdc14p release is likely restored in these cells at higher temperatures. We quantitated the nucleolar release of Cdc14-EGFP in wild type, dbf4-ND109, cdc5-1 and cdc5-1 dbf4-ND109 cells at a restrictive temperature for cdc5-1. Cells were arrested in G1-phase at the permissive temperature and then released into the cell cycle at 34uC. The dbf4-ND109 cells progressed through the cell cycle and released Cdc14p similarly to wild type cells ( Figure 5A). Consistent with previous reports, the cdc5-1 mutant failed to release Cdc14p from the nucleolus but a significant amount of Cdc14p was released from the nucleolus in the cdc5-1 dbf4-ND109 mutant ( Figure 5A). We noticed a delay in mitotic progression at 34uC in the cdc5-1 dbf4-ND109 cells evidenced by a somewhat longer duration of Cdc14p release compared to the wild type and delayed cytokinesis (indicated by the delayed appearance of unbudded (G1) cells). This data indicates that Dbf4p inhibits Polo activity to prevent Cdc14p release, which might be significant during a slowed S-phase or during periods of replication stress.

Dbf4p Also Inhibits the MEN Kinase Dbf2p
Inactivation of Bfa1p-Bub2p is required to activate the MEN [37]. This signaling cascade is partially activated as a result of Bfa1p phosphorylation by Polo, which leads to activation of the Cdc15p and Dbf2p-Mob1p kinases (reviewed by [47]). Dbf2p kinase activation requires a Bub2p-Bfa1p independent function of Polo as well [49], indicating that Polo either directly promotes Dbf2p kinase activity or promotes a MEN-independent pathway that activates Dbf2p. We tested whether Dbf4p functions as a negative regulator of MEN activation by examining whether deletion of the Dbf4p PIR could suppress growth defects associated with additional ts mutants in the MEN ( Figure 5B). We examined the growth of double mutants of cdc5-1, cdc15-2, dbf2-1, cdc14-1, or cdc14-3 with dbf4-D109. As with cdc5-1, loss of the DBF4 PIR rescued the growth of dbf2-1 cells at the nonpermissive temperature. In contrast, the dbf4-D109 mutant failed to suppress the ts phenotype of cdc15-2, cdc15-4 (not shown), cdc14-1, or cdc14-3 mutants ( Figure 5B). This suggests that Dbf4p may specifically inhibit Polo activation of Dbf2p and not Polo inactivation of Bub2p-Bfa1p GAP activity. Taken together, our observations suggest that Dbf4p antagonizes Polo activation of some Bfa1p-Bub2p independent step in MEN activation. This interpretation is further supported by the following experiments.
Dbf4p Regulates a Bfa1p-Bub2p Independent Polo Activity during Mitotic Exit Deletion of BUB2 (or BFA1) is sufficient to cause premature mitotic exit when cells are arrested in metaphase with the spindle poison nocodazole [67,68]. This causes large budded cells to exit mitosis and rebud in the absence of chromosome segregation or cytokinesis. Since Dbf4p is a negative regulator of Polo activity, we examined whether deletion of the Dbf4p PIR was sufficient to induce rebudding in the presence of spindle poisons. Cells were arrested in G1, released into media containing nocodazole and quantitated for rebudding ( Figure 6A). In contrast to bub2D, dbf4-ND109 did not allow rebudding in a wild type background nor did this mutation advance rebudding in a bub2D background. This indicated that loss of the Dbf4p Polo interaction is not sufficient to cause mitotic exit during metaphase and suggested that Dbf4p Polo inhibition may act independently of Bub2p-Bfa1p.
Null alleles of CDC5 fail to activate the MEN and arrest in telophase with unphosphorylated Bfa1 [37]. Although the cdc5-1 mutant is also defective in MEN activation it is proficient for Bfa1p phosphorylation and retains substantial Polo kinase activity at the non-permissive temperature (NPT) [37,63]. This suggests that the cdc5-1 mutant is defective in activating a Bfa1p-independent function of the MEN, perhaps in FEAR pathway activation or activating a downstream MEN target. In contrast to cdc5-1, cdc5-2 cells neither phosphorylate Bfa1p nor activate the MEN at the NPT [37]. The cdc5-2 temperature sensitivity is partially rescued by deletion of either BFA1 or BUB2 [37] but not by dbf4-ND109 ( Figure 6B). However, in a bub2D the cdc5-2 temperature sensitivity was further suppressed by deletion of the Dbf4p PIR. So, eliminating the requirement for Bfa1p-Bub2p inactivation in cdc5-2 cells (i.e. cdc5-2 bub2D), allowed dbf4-ND109 to further suppress the cdc5-2 ts and promote mitotic exit at 34uC ( Figure 6B). These data are consistent with the interpretation that Dbf4p primarily inhibits Polo activation of a Bub2p-Bfa1p independent step in MEN activation, e.g. Dbf2p activity, Cdc14p release in the FEAR pathway, or some unknown activity.

DBF4 Prevents Mitotic Exit When the Nucleus Is Mispositioned
In the budding yeast, KAR9 and DYN1 encode cytoplasmic microtubule-associated and motor proteins, respectively, that operate in two redundant pathways essential for the correct positioning of the nucleus during anaphase (reviewed by [7]). Deletion of either gene results in a small percentage of cells with nuclear orientation defects but deletion of both genes is lethal. In response to nuclear misorientation, S. cerevisiae inhibits premature activation of the MEN via the spindle position checkpoint (SPOC). When the SPOC is activated the Kin4p kinase localizes to spindle pole bodies (SPB) to counteract Polo Bfa1p phosphorylation [8,9,55]. Kin4p thus counteracts Polo inactivation of the Bfap1-Bub2p GAP and this inhibits MEN activation. Failure to adequately respond to nuclear mispositioning allows inappropriate nuclear division within the mother cell, leading to anueploidy and loss of viability. Given that Polo Bfa1p phosphorylation is prevented when the SPOC is activated, we hypothesized Dbf4p may also inhibit Polo to prevent mitotic exit in response to nuclear misorientation.
To test whether DBF4 inhibited mitotic exit when nuclei were mispositioned, we examined wild type, dbf4-ND109, kar9D and kar9D dbf4-ND109 strains for evidence of mitotic exit in the presence of mispositioned nuclei. Asynchronous cultures were grown at 25uC and shifted to 30uC for 4 or 24 hours prior to analysis to increase the penetrance of the nuclear mispositioning phenotype. Although wild type and dbf4 mutant cells did not misorient their nuclei ( Figure 7C), both kar9D and kar9D dbf4-ND109 strains had approximately equal number of cells with nuclear positioning defects. Importantly, deletion of the Dbf4p PIR resulted in a 2 to 3-fold increase (to 6%) in binucleate, anucleate and multinucleate cells at 4 hours ( Figure 7A, C, D), which was CDC5-dependent, suggesting that premature mitotic exit occurred. At 24 hours the number of aberrant chromosome segregation events due to loss of the Dbf4p PIR increased six-fold (12%) relative to kar9D alone (2%). Since exit from mitosis causes spindle disassembly, we quantitated the spindle morphology in cells containing correctly segregated nuclei (between the mother and daughter cells) and in those cells where anaphase had initiated solely within the mother cell. Although kar9D and kar9D dbf4-ND109 cells had similar frequencies of intact or disassembled spindles when nuclear division proceeded normally, kar9D dbf4-ND109 cells showed a three-fold increase in spindle disassembly within the mother cell (leading to a bi-nucleate mother cell) compared to kar9D single mutants at 4 hours ( Figure 7D). These data indicate that mitotic exit occurs in these cells in the absence of the Dbf4p PIR.
Similarly, we tested whether deletion of the Dbf4p PIR allowed premature mitotic exit in cells disrupted in the dynein pathway ( Figure 7B, E, F). The spindle-positioning defect associated with deletion of DYN1 is especially prominent at low temperatures. Although after 8 hours at 14uC only ,3% of dyn1D cells had exited mitosis inappropriately, deletion of the Dbf4p PIR resulted in a 3-to 4-fold increase in mitotic exit as evidence by the appearance of multinucleate and anucleate cells ( Figure 7E) and this frequency was increased at 24 hours. For comparison, a dyn1D bub2D strain had a similar but higher frequency of segregation defects ( Figure 7E, F). Therefore, the bypass of the SPOC following loss of the Dbf4p-Polo interaction is comparable to deletion of the MEN inhibitor BUB2. As with the kar9D mutant, we observed that a higher percentage of dyn1D dbf4-ND109 cells compared to dyn1D single mutants that divided nuclei within the mother cell had disassembled their spindles ( Figure 7F). These observations indicate that deletion of the Dbf4p N-terminus (including the PIR) overrides the mitotic arrest normally activated by the SPOC.

Discussion
We found that Dbf4p inhibited Polo kinase during mitosis very likely through a direct interaction with the polo-box domain. This interaction inhibited MEN pathway activation, nucleolar release of the Cdc14p phosphatase and was likely critical to maintain genome integrity during activation of the spindle position checkpoint. These results therefore have important implications for understanding Dbf4p function and the regulation of mitotic progression in eukaryotic cells.

Defining the Interaction between Dbf4p and Polo
The N-terminal third of Dbf4p encodes multiple functions: a destruction box (residues 62-70), two putative nuclear localization signals (residues 55-61, 251-257) and a BRCT-like domain (residues ,117-220). Nonetheless the 265 N-terminal amino acids of Dbf4p are not essential as long as a nuclear localization signal is present [26]. Deletion of the Dbf4p N-terminus through the PIR has no observable effect on growth, viability, or cell cycle progression either under normal growth conditions or in the presence of replication or spindle poisons ( [26] and data not shown). Here, we discovered an interaction between Dbf4p and the Polo PBD that mapped to a short sequence of ,40 amino acids preceding the BRCT-like domain. Although a two-hybrid interaction between Polo and Dbf4p was reported before [39], the significance of this interaction was not determined.
The Polo PBD functions as a module for binding phosphorylated proteins and thereby targets Polo to its cellular substrates [57,59]. The question naturally arises as to whether phosphorylation of the Dbf4p PIR is required for PBD binding. Currently, our observations suggest that phosphorylation is not required. A polo-box binding consensus sequence (S(pS/pT)P/X) is not present within this region of Dbf4p [59] and mutation of all putative serine and threonine residues within the Dbf4p PIR did not significantly diminish the interaction in the two-hybrid assay. These data suggest that phosphorylation of Dbf4p is not crucial for Polo binding. Our observation that the Dbf4p PIR purified from E. coli directly interacted with the Polo PBD also supports this notion. Thus phosphorylation of Dbf4p was not critical for binding to Polo in vitro, but we cannot exclude the possibility that phosphorylation contributes to Dbf4p-Polo binding in vivo when the two proteins are present at physiological concentrations.

When Does Dbf4p Inhibit Polo and Mitotic Exit?
The finding that deletion of the Dbf4p PIR significantly suppressed the ts phenotype of cdc5-1 suggests that Cdc7p-Dbf4p inhibits Polo during the normal cell cycle and perhaps during periods of replication stress, when Cdc7p-Dbf4p is stabilized [17]. Our data clearly demonstrate a role for Dbf4p in inhibiting mitotic exit, since loss of the Dbf4p PIR suppressed both cdc5 and dbf2 ts mutants and allowed sustained Cdc14p phosphatase release and cytokinesis in the cdc5-1 mutant at the NPT. However, during an unperturbed cell cycle the absence of this regulation had little impact. This is likely attributable to the fact that the cell cycle regulation of Polo activity is complex and modulated by multiple cell cycle checkpoints. Since dbf4-ND109 bub2D double mutants were more sensitive to growth on spindle poisons than either mutant alone ( Figure S3), the Dbf4p-Polo and Bfa1p-Bub2p pathways may work together to suppress premature activation of the mitotic exit network.
It was shown very recently that increased expression of a nondestructible form of Dbf4p (Dbf4-ND65p) could delay rDNA segregation when Clb5p was also stabilized [16]. This raises the possibility that Dbf4p inhibits Cdc14p release via the FEAR pathway under some circumstances and is consistent with our data showing that Dbf4p is a Polo inhibitor. However, we found no evidence that the dbf4-ND109 allele promoted premature rDNA segregation (a FEAR pathway event) in the cdc5-1 mutant ( Figure  S4). Similarly, we found no evidence that dbf4-ND109 caused premature Cdc14p release in a strain deleted for FOB1, which is thought help sequester Cdc14p in the nucleolus ( Figure S4). These data suggest that Dbf4p is not specifically inhibiting the FEAR pathway.
The budding yeast SPOC prevents premature exit from mitosis in part by inducing Kin4p phosphorylation of Bfa1p, which antagonizes the Polo-dependent inhibition of the Bfa1p-Bub2p GAP [8,9,55]. Premature exit from mitosis in dbf4-ND109 dyn1 and dbf4-ND109 kar9 double mutants suggests that Dbf4p regulation of Polo is critical for robust cell cycle control in response to nuclear mispositioning. What remains unclear however, is whether this Dbf4p activity is regulated following activation of the SPOC. For instance, Cdc7p-Dbf4p may inhibit Polo to buffer against premature release of Cdc14p during late Sphase or early M-phase whether or not the SPOC is activated. Therefore, in the absence of the Dbf4p-Polo regulation Polo may prematurely activate the MEN before a nuclear orientation defect is sensed by the SPOC. Following APC-Cdc20p and APC-Cdh1p activation during anaphase onset and exit, we suggest that degradation of Dbf4p provides a positive feedback loop for full Polo activation of the MEN and ultimately, cytokinesis.

How Does Dbf4p Inhibit Polo and Mitotic Exit?
Dbf4p did not influence Polo protein levels or overall kinase activity. Dbf4p nonetheless inhibited Polo activity since dbf4 mutants unable to interact with Polo significantly suppressed the cdc5-1 temperature sensitivity. Since the cdc5-1 allele retains significant Polo protein expression, Bfa1p phosphorylation and overall Polo kinase activity at the non-permissive temperature [37,63], the primary cdc5-1 MEN defect is in a Bfa1p-independent requirement for MEN pathway activation, perhaps Dbf2p activation or some other MEN-dependent step. Our observation that the dbf2-1 temperature sensitivity was also suppressed by loss of the Dbf4p PIR suggests that Dbf4p may specifically inhibit Polo activation of Dbf2p kinase independently of Bfa1p-Bub2p phosphorylation. Dbf2p was recently shown to promote cytoplasmic Cdc14p localization following MEN activation [69]. In addition, deletion of both BUB2 and the DBF4 PIR suppressed the cdc5-2 ts better than either single mutant alone. In other words, since dbf4-ND109 further suppressed the ts phenotype of a cdc5-2 bub2D strain, this supports the contention that Dbf4p regulates MEN activity independently of Bfa1p-Bub2p. Deletion of the Dbf4p PIR also did not allow rebudding in the presence of spindle poisons as seen in bub2D mutants, again suggesting that Dbf4p plays a minor or redundant role to inhibit Polo phosphorylation of Bfa1p-Bub2p.
Cdc7p-Dbf4p kinase phosphorylated the Polo PBD in vitro suggesting that Cdc7p-Dbf4p may antagonize Polo substrate binding. This possibility is consistent with the requirement for the PBD for targeting Polo to sites of MEN activity. Polo, Cdc15p, Dbf2p and Bfa1p-Bub2p are localized to SPB prior to activation of the mitotic exit network [47]. Thus, we favor a model whereby Cdc7p-Dbf4p kinase inhibits precocious Polo binding to critical MEN substrate(s) by phosphorylating the Polo PBD. It will be interesting to investigate whether the Cdc7p-Dbf4p inhibition of Polo is regulated by cell cycle checkpoints and to determine the precise activity of Polo that is affected.
For yeast two-hybrid analyses, a Gal4 DNA binding domain (GBD) fusion to Dbf4p 67-227 was constructed by PCR amplification of Dbf4p residues 67-227 (NcoI-PstI) and cloned into pGBKT7 (Clonetech). Deletion of 71 bp within the ADH1 promoter sequence of pGBKT7 (-647 to -717 from the ATG) removed a Rap1p binding site and reduced the strength of GBD-Dbf4p 67-227 expression (which was otherwise lethal) to give pCG60. Point mutations and deletions were generated by sitedirected mutagenesis using the QuikChange system (Stratagene). For all mutations, the entire coding sequence was verified by DNA sequencing. Construction of baculovirus plasmids encoding WT, ND65Dbf4p, ND109Dbf4p, ND221Dbf4p and HA-Cdc7p was previously described [26]. The baculovirus transfer plasmid containing 3Myc-ND65Polo was constructed in pAcSG2 (BD Biosciences). High-titer baculoviruses were generated by transfection of Sf9 cells using the BaculoGold kit (BD Biosciences) followed by plaque purification and virus amplification.
For in vitro interaction assays, DNA encoding Polo amino acids 357-705 were PCR amplified with BamHI-XmaI linkers and cloned into pGEX-KG for expression of GST-Polo PBD in E. coli. The region encoding Dbf4p amino acids 66-109 was PCR amplified from pMW489 with BsaI-BamHI linkers and cloned into pSUMO (LifeSensors Inc.) for expression of Sumo-Dbf4p 67-109 .

Growth Media and Cell Cycle Experiments
Cells were cultured in YPD (1% yeast extract, 2% bacto peptone, 2% glucose). Synchronous G1 cultures were obtained after addition of 5 mg/ml (0.1 mg/ml in bar1D cells) alpha-factor to cells for 3 hours. DNA content was analyzed by flow cytometry as previously described [17]. Drugs were added directly to plates immediately before pouring.

Two-Hybrid Experiments
PJ69-4a cells containing pCG60 were transformed with a S. cerevisiae two-hybrid library. Interacting clones were recovered on medium lacking tryptophan, leucine, and histidine but containing 2 mM 3-aminotriazole at 30uC. Positive interactors were streakpurified and also tested for ADE2 reporter activity. Prey plasmids that activated both HIS3 and ADE2 expression were confirmed by retransformation in PJ69-4a and then sequenced. To quantify twohybrid interactions, co-transformed cells were spotted at ten-fold serial dilutions on selective media and grown for 2-3 days.

Fluorescence Microscopy
For direct fluorescence analysis of Cdc14-EGFP, cells were fixed in 3.7% formaldehyde at room temperature for 1 hour. DNA was stained using DAPI (1 mg/ml) for 10 minutes at room temperature. For the experiments in Figure 5A, the absence of a distinct Cdc14-EGFP fluorescent signal (but accompanied by diffuse nuclear and cytoplasmic fluorescence) was scored as ''released.'' Any cell that had a distinct Cdc14-EGFP nucleolar fluorescence was counted as sequestered. Spindle morphology was detected after spheroplasting cells and incubation in methanol/acetone prior to incubation with antibodies: rat anti-tubulin (YOL1/34 Accurate Chemicals, 1:10) and goat anti-rat FITC (Jackson Immunoresearch, 1:50). Cells were imaged using a 606 objective.  Figure S4 (A) Deletion of the Dbf4p PIR does not enhance nucleolar segregation in cdc5-1 cells. DBF4, dbf4-ND109, cdc5-1 and cdc5-1 dbf4-ND109 cells transformed with a centromeric EGFP-Nop1 plasmid were arrested in G1 with mating pheromone and released into the cell cycle in YPD at 34uC. Alpha-factor was added back after budding to permit a single cell cycle. Samples were taken at the indicated time points and scored for the presence of one or two GFP signals by florescence microscopy. (B) Deletion 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. Alphafactor 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.