Cdc7p-Dbf4p Regulates Mitotic Exit by Inhibiting Polo Kinase

Graduate Program in Cell and Molecular Biology, Michigan State University, East Lansing, MI, USA.
PLoS Genetics (Impact Factor: 7.53). 06/2009; 5(5):e1000498. DOI: 10.1371/journal.pgen.1000498
Source: PubMed


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.

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    • "Interestingly, ScDbf4 (with Cdc7-S1) also rescued the defect in sporulation with HsDDK. Not surprisingly, interaction of ScDDK with Cdc5, required for meiosis, is mediated by ScDbf4 (Chen and Weinreich 2010; Matos et al. 2008; Miller et al. 2009). "
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    ABSTRACT: The DDK kinase complex, composed of Cdc7 and Dbf4, is required for S-phase progression. The two component proteins show different degrees of sequence conservation between human and yeast. Here, we determine that Saccharomyces cerevisiae bearing human CDC7 and DBF4 grows comparably to cells with yeast DDK under standard growth conditions. HsDrf1 (a second human Dbf4-like protein) does not support growth, suggesting that HsDbf4 is the true ortholog of ScDbf4. Both human subunits are required to complement yeast cdc7Δ or dbf4Δ due to the inability of human Cdc7 or Dbf4 to interact with the corresponding yeast protein. Flow cytometry indicates normal cell cycle progression for yeast containing human DDK. However, yeast containing human DDK is sensitive to long-term exposure to hydroxyurea and fails to sporulate, suggesting that human DDK substitutes for some, but not all, of yeast DDK's functions. We mapped the region of Cdc7 required for species-specific function of DDK to the C-terminus of Cdc7 by substituting the yeast C-terminal 55 amino acid residues in place of the equivalent human residues. The resulting hybrid protein supported growth of a cdc7Δ strain only in the presence of ScDBF4. The strain supported by the hybrid CDC7 was not sensitive to HU and formed tetrads. Together, our data indicate that DDK's targeting of its essential substrate is conserved between species, whereas the interactions within DDK are species specific.
    G3-Genes Genomes Genetics 09/2011; 1(4):317-25. DOI:10.1534/g3.111.000430 · 3.20 Impact Factor
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    • "Rsc2 could also bind the polo-box domain (PBD) of Cdc5, which normally binds substrates previously primed by phosphorylation by another kinase (Elia et al., 2003a). Indeed, Rsc2- HA3 bound to a recombinant GST-PBD fusion protein (Miller et al., 2009) but not to GST alone (Fig. 9 "
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    ABSTRACT: Upon prolonged activation of the spindle assembly checkpoint, cells escape from mitosis through a mechanism called adaptation or mitotic slippage, which is thought to underlie the resistance of cancer cells to antimitotic drugs. We show that, in budding yeast, this mechanism depends on known essential and nonessential regulators of mitotic exit, such as the Cdc14 early anaphase release (FEAR) pathway for the release of the Cdc14 phosphatase from the nucleolus in early anaphase. Moreover, the RSC (remodel the structure of chromatin) chromatin-remodeling complex bound to its accessory subunit Rsc2 is involved in this process as a novel component of the FEAR pathway. We show that Rsc2 interacts physically with the polo kinase Cdc5 and is required for timely phosphorylation of the Cdc14 inhibitor Net1, which is important to free Cdc14 in the active form. Our data suggest that fine-tuning regulators of mitotic exit have important functions during mitotic progression in cells treated with microtubule poisons and might be promising targets for cancer treatment.
    The Journal of Cell Biology 11/2010; 191(5):981-97. DOI:10.1083/jcb.201007025 · 9.83 Impact Factor
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    • "In addition to its highly conserved function in origin firing, other, less well understood, functions have been reported for Cdc7 kinase. These include activation of the ATR-Chk1 pathway in response to DNA damage and DNA replication stress (Takeda et al, 1999; Costanzo et al, 2003; Dierov et al, 2004; Tenca et al, 2007; Kim et al, 2008), cohesin loading onto chromatin required for chromosomal segregation in mitosis (Takahashi et al, 2008), regulation of exit from mitosis (Miller et al, 2009) and double-strand break formation during meiotic recombination (Matos et al, 2008). As the two-step-replication model excludes the formation of replication-competent origins once S phase has started, it has been argued on the basis of experimental evidence that a putative cell cycle checkpoint could delay progression from G1 into S phase if replication initiation is perturbed (Blow and Gillespie, 2008). "
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    ABSTRACT: Perturbation of DNA replication initiation arrests human cells in G1, pointing towards an origin activation checkpoint. We used RNAi against Cdc7 kinase to inhibit replication initiation and dissect this checkpoint in fibroblasts. We show that the checkpoint response is dependent on three axes coordinated through the transcription factor FoxO3a. In arrested cells, FoxO3a activates the ARF-∣Hdm2-∣p53 → p21 pathway and mediates p15(INK4B) upregulation; p53 in turn activates expression of the Wnt/β-catenin signalling antagonist Dkk3, leading to Myc and cyclin D1 downregulation. The resulting loss of CDK activity inactivates the Rb-E2F pathway and overrides the G1-S transcriptional programme. Fibroblasts concomitantly depleted of Cdc7/FoxO3a, Cdc7/p15, Cdc7/p53 or Cdc7/Dkk3 can bypass the arrest and proceed into an abortive S phase followed by apoptosis. The lack of redundancy between the checkpoint axes and reliance on several tumour suppressor proteins commonly inactivated in human tumours provides a mechanistic basis for the cancer-cell-specific killing observed with emerging Cdc7 inhibitors.
    The EMBO Journal 10/2010; 29(19):3381-94. DOI:10.1038/emboj.2010.201 · 10.43 Impact Factor
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