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 
. 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 
. 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 
. 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 
. 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 
that is assembled at origins of replication in early G1 in an inactive form (reviewed in 
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 
. Dbf4p encodes a dispensable BRCT-like domain in the N-terminus that might target the kinase to stalled replication forks 
. 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 
. Rad53p also phosphorylates Dbf4p in response to replication stress and this regulation requires N-terminal Dbf4p sequences through which Rad53p physically interacts 
. 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-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 
). 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 
. In addition, Polo function is inhibited by cell cycle checkpoints that are induced following DNA or spindle damage 
. A genetic and physical interaction between Dbf4 and Polo was described previously 
, 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 
. Limited Cdc14p release by the FEAR pathway promotes accurate rDNA and telomere segregation 
. 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) 
. 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 
. 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 
. Spindle position defects also counteract Polo activity by targeting Kin4p kinase to the spindle poles where it inhibits Polo-dependent Bfa1p phosphorylation 
. 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.