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.     

Characterization of the yeast Cdc7p/Dbf4p complex purified from insect cells. Its protein kinase activity is regulated by Rad53p

The yeast Saccharomyces cerevisiae Cdc7p/Dbf4p protein kinase complex was purified to near homogeneity from insect cells. The complex efficiently phosphorylated yeast Mcm2p and less efficiently the remaining Mcm proteins or other replication proteins. Significantly, when pretreated with alkaline phosphatase, Mcm2p became completely inactive as a substrate, suggesting that it must be phosphorylated by other protein kinase(s) to be a substrate for the Cdc7p/Dbf4p complex. Mutant Cdc7p/Dbf4p complexes containing either Cdc7-1p or Dbf4-1 approximately 5p were also partially purified from insect cells and characterized in vitro. Furthermore, the autonomously replicating sequence binding activity of various dbf4 mutants was also analyzed. These studies suggest that the autonomously replicating sequence-binding and Cdc7p protein kinase activation domains of Dbf4p collaborate to form an active Cdc7p/Dbf4p complex and function during S phase in S. cerevisiae. It is shown that Rad53p phosphorylates the Cdc7p/Dbf4p complex in vitro and that this phosphorylation greatly inhibits the kinase activity of Cdc7p/Dbf4p. This result suggests that Rad53p controls the initiation of chromosomal DNA replication by regulating the protein kinase activity associated with the Cdc7p/Dbf4p complex.     

Cdc7-Dbf4 and the human S checkpoint response to UVC

The S checkpoint response to ultraviolet radiation (UVC) that inhibits replicon initiation is dependent on the ATR and Chk1 kinases. Downstream effectors of this response, however, are not well characterized. Data reported here eliminated Cdc25A degradation and inhibition of Cdk2-cyclin E as intrinsic components of the UVC-induced pathway of inhibition of replicon initiation in human cells. A sublethal dose of UVC (1 J/m(2)), which selectively inhibits replicon initiation by 50%, failed to reduce the amount of Cdc25A protein or decrease Cdk2-cyclin E kinase activity. Cdc25A degradation was observed after irradiation with cytotoxic fluences of UVC, suggesting that severe inhibition of DNA chain elongation and activation of the replication checkpoint might be responsible for the UVC-induced degradation of Cdc25A. Another proposed effector of the S checkpoint is the Cdc7-Dbf4 complex. Dbf4 interacted weakly with Chk1 in vivo but was recognized as a substrate for Chk1-dependent phosphorylation in vitro. FLAG-Dbf4 formed complexes with endogenous Cdc7, and this interaction was stable in UVC-irradiated HeLa cells. Overexpression of FLAG- or Myc-tagged Dbf4 abrogated the S checkpoint response to UVC but not ionizing radiation. These findings implicate a Dbf4-dependent kinase as a possible target of the ATR- and Chk1-dependent S checkpoint response to UVC.     

Molecular mechanism of activation of human Cdc7 kinase: bipartite interaction with Dbf4/activator of S phase kinase (ASK) activation subunit stimulates ATP binding and substrate recognition

Cdc7 is a serine/threonine kinase conserved from yeasts to human and is known to play a key role in the regulation of initiation at each replication origin. Its catalytic function is activated via association with the activation subunit Dbf4/activator of S phase kinase (ASK). It is known that two conserved motifs of Dbf4/ASK are involved in binding to Cdc7, and both are required for maximum activation of Cdc7 kinase. Cdc7 kinases possess unique kinase insert sequences (kinase insert I-III) that are inserted at defined locations among the conserved kinase domains. However, precise mechanisms of Cdc7 kinase activation are largely unknown. We have identified two segments on Cdc7, DAM-1 (Dbf4/ASK interacting motif-1; amino acids 448-457 near the N terminus of kinase insert III) and DAM-2 (C-terminal 10-amino acid segment), that interact with motif-M and motif-C of ASK, respectively, and are essential for kinase activation by ASK. The C-terminal 143-amino acid polypeptide (432-574) containing DAM-1 and DAM-2 can interact with Dbf4/ASK. Characterization of the purified ASK-free Cdc7 and Cdc7-ASK complex shows that ATP binding of the Cdc7 catalytic subunit requires Dbf4/ASK. However, the "minimum" Cdc7, lacking the entire kinase insert II and half of kinase insert III, binds to ATP and shows autophosphorylation activity in the absence of ASK. However, ASK is still required for phosphorylation of exogenous substrates by the minimum Cdc7. These results indicate bipartite interaction between Cdc7 and Dbf4/ASK subunits facilitates ATP binding and substrate recognition by the Cdc7 kinase.     

First the CDKs, now the DDKs.

In budding yeast, Dbf4p and Cdc7p control initiation of DNA synthesis. They form a protein kinase - Cdc7p being the catalytic subunit and Dbf4p a cyclin-like molecule that activates the kinase in late G1 phase. Dbf4p also targets Cdc7p to origins of replication, where probable substrates include certain Mcm proteins. Recent studies have identified Dbf4p- and Cdc7p-related proteins in fission yeast and metazoans. These homologues also phosphorylate Mcm proteins and could have a similar function to that of Dbf4p-Cdc7p in budding yeast. Thus, it seems likely that, like the cyclin-dependent kinases (CDKs), the Dbf4p-Cdc7p activity is conserved in all eukaryotes.     

Bipartite binding of a kinase activator activates Cdc7-related kinase essential for S phase

Dfp1/Him1 protein of fission yeast, Schizosaccharomyces pombe, encodes the regulatory subunit for Hsk1 kinase, a homologue of budding yeast Cdc7 kinase essential for initiation and progression of the S phase of the cell cycle. This protein binds and activates Hsk1 kinase, which phosphorylates the MCM2 protein. Comparison of the amino acid sequences of the Cdc7 regulatory subunits from various eukaryotes revealed the presence of three small stretches of conserved amino acid sequences, namely Dbf4 motifs N, M, and C. We report here that the Dbf4 motif M, a unique proline-rich motif, and the Dbf4 motif C, a C(2)H(2)-type zinc finger motif, are essential for mitotic functions of Dfp1/Him1 protein as well as for full-level activation of Hsk1 kinase. In vitro, a small segment containing the Dbf4 motif M or C alone binds to and partially activates Hsk1. Co-expression of these two segments augments the extent of activation. Furthermore, a fused polypeptide containing only Dbf4 motifs M and C without any spacer can activate Hsk1 and is capable of rescuing the growth defect of him1 null cells. Insertion of a long stretch of amino acids between the motif M and motif C can be tolerated for mitotic functions. On the other hand, internal deletion of Dbf4 motif N, which has some similarity with the BRCA C-terminal domain motif, results in a defect in hydroxyurea-induced checkpoint responses and sensitivity to methyl methane sulfonate, yet mitotic functions and kinase activation are intact. In one-hybrid assays with budding yeast Dbf4, motif N mutants exhibit reduced interaction with a replication origin. Our observations suggest the molecular architecture of Cdc7.Dbf4-related kinase complexes at the origins, in which they are tethered to replication machinery through Dbf4 motif N and the catalytic subunits are activated through bipartite binding of Dbf4 motifs M and C of the regulatory subunits.     

Cdc7p-Dbf4p kinase binds to chromatin during S phase and is regulated by both the APC and the RAD53 checkpoint pathway.

Eukaryotic cells coordinate chromosome duplication by assembly of protein complexes at origins of DNA replication and by activation of cyclin-dependent kinase and Cdc7p-Dbf4p kinase. We show in Saccharomyces cerevisiae that although Cdc7p levels are constant during the cell division cycle, Dbf4p and Cdc7p-Dbf4p kinase activity fluctuate. Dbf4p binds to chromatin near the G(1)/S-phase boundary well after binding of the minichromosome maintenance (Mcm) proteins, and it is stabilized at the non-permissive temperature in mutants of the anaphase-promoting complex, suggesting that Dbf4p is targeted for destruction by ubiquitin-mediated proteolysis. Arresting cells with hydroxyurea (HU) or with mutations in genes encoding DNA replication proteins results in a more stable, hyper-phosphorylated form of Dbf4p and an attenuated kinase activity. The Dbf4p phosphorylation in response to HU is RAD53 dependent. This suggests that an S-phase checkpoint function regulates Cdc7p-Dbf4p kinase activity. Cdc7p may also play a role in adapting from the checkpoint response since deletion of CDC7 results in HU hypersensitivity. Recombinant Cdc7p-Dbf4p kinase was purified and both subunits were autophosphorylated. Cdc7p-Dbf4p efficiently phosphorylates several proteins that are required for the initiation of DNA replication, including five of the six Mcm proteins and the p180 subunit of DNA polymerase alpha-primase.     

High levels of Cdc7 and Dbf4 proteins can arrest cell-cycle progression

Cdc7-Dbf4 serine/threonine kinase is essential for initiation of DNA replication. It was previously found that overexpression of certain replication proteins such as Cdc6 and Cdt1 in fission yeast resulted in multiple rounds of DNA replication in the absence of mitosis. Since this phenomenon is dependent upon the presence of wild-type Cdc7/Hsk1, we hypothesized that high levels of Cdc7 and/or Dbf4 could also cause multiple rounds of DNA replication, or could facilitate entry into S phase. To test this hypothesis, we transiently overexpressed hamster Cdc7, Dbf4 or both in CHO cells. Direct observations of individual cells by fluorescence microscopy and flow cytometric analysis on cell populations suggest that overexpression of Cdc7 and/or Dbf4 does not result in multiple rounds of DNA replication or facilitating entry into S phase. In contrast, moderately increased levels of Dbf4, but not Cdc7, cause cell-cycle arrest in G2/M. This G2/M arrest coincides with hyperphosphorylation of Cdc2/Cdk1 at Tyr-15, raising the possibility that high levels of Dbf4 may activate a G2/M cell-cycle checkpoint. Further increase in Cdc7 and/or Dbf4 by 2–4 fold can arrest cells in G1 and significantly slow down S-phase progression for the cells already in S phase.     

Kinase activity of fission yeast Mph1 is required for Mad2 and Mad3 to stably bind the anaphase promoting complex

Defects in chromosome segregation result in aneuploidy, which can lead to disease or cell death [1, 2]. The spindle checkpoint delays anaphase onset until all chromosomes are attached to spindle microtubules in a bipolar fashion [3, 4]. Mad2 is a key checkpoint component that undergoes conformational activation, catalyzed by a Mad1-Mad2 template enriched at unattached kinetochores [5]. Mad2 and Mad3 (BubR1) then bind and inhibit Cdc20 to form the mitotic checkpoint complex (MCC), which binds and inhibits the anaphase promoting complex (APC/C). Checkpoint kinases (Aurora, Bub1, and Mps1) are critical for checkpoint signaling, yet they have poorly defined roles and few substrates have been identified [6-8]. Here we demonstrate that a kinase-dead allele of the fission yeast MPS1 homolog (Mph1) is checkpoint defective and that levels of APC/C-associated Mad2 and Mad3 are dramatically reduced in this mutant. Thus, MCC binding to fission yeast APC/C is dependent on Mph1 kinase activity. We map and mutate several phosphorylation sites in Mad2, producing mutants that display reduced Cdc20-APC/C binding and an inability to maintain checkpoint arrest. We conclude that Mph1 kinase regulates the association of Mad2 with its binding partners and thereby mitotic arrest.     

Characterization of a Drosophila Ortholog of the Cdc7 Kinase: A ROLE FOR Cdc7 IN ENDOREPLICATION INDEPENDENT OF CHIFFON*

Cdc7 is a serine-threonine kinase that phosphorylates components of the pre-replication complex during DNA replication initiation. Cdc7 is highly conserved, and Cdc7 orthologs have been characterized in organisms ranging from yeast to humans. Cdc7 is activated specifically during late G₁/S phase by binding to its regulatory subunit, Dbf4. Drosophila melanogaster contains a Dbf4 ortholog, Chiffon, which is essential for chorion amplification in Drosophila egg chambers. However, no Drosophila ortholog of Cdc7 has yet been characterized. Here, we report the functional and biochemical characterization of a Drosophila ortholog of Cdc7. Co-expression of Drosophila Cdc7 and Chiffon is able to complement a growth defect in yeast containing a temperature-sensitive Cdc7 mutant. Cdc7 and Chiffon physically interact and can be co-purified from insect cells. Cdc7 phosphorylates the known Cdc7 substrates Mcm2 and histone H3 in vitro, and Cdc7 kinase activity is stimulated by Chiffon and inhibited by the Cdc7-specific inhibitor XL413. Drosophila egg chamber follicle cells deficient for Cdc7 have a defect in two types of DNA replication, endoreplication and chorion gene amplification. However, follicle cells deficient for Chiffon have a defect in chorion gene amplification but still undergo endocycling. Our results show that Cdc7 interacts with Chiffon to form a functional Dbf4-dependent kinase complex and that Cdc7 is necessary for DNA replication in Drosophila egg chamber follicle cells. Additionally, we show that Chiffon is a member of an expanding subset of DNA replication initiation factors that are not strictly required for endoreplication in Drosophila.     

Regulation of the mitotic exit protein kinases Cdc15 and Dbf2

In budding yeast, the release of the protein phosphatase Cdc14 from its inhibitor Cfi1/Net1 in the nucleolus during anaphase triggers the inactivation of Clb CDKs that leads to exit from mitosis. The mitotic exit pathway controls the association between Cdc14 and Cfi1/Net1. It is comprised of the RAS-like GTP binding protein Tem1, the exchange factor Lte1, the GTPase activating protein complex Bub2-Bfa1/Byr4, and several protein kinases including Cdc15 and Dbf2. Here we investigate the regulation of the protein kinases Dbf2 and Cdc15. We find that Cdc15 is recruited to both spindle pole bodies (SPBs) during anaphase. This recruitment depends on TEM1 but not DBF2 or CDC14 and is inhibited by BUB2. Dbf2 also localizes to SPBs during anaphase, which coincides with activation of Dbf2 kinase activity. Both events depend on the mitotic exit pathway components TEM1 and CDC15. In cells lacking BUB2, Dbf2 localized to SPBs in cell cycle stages other than anaphase and telophase and Dbf2 kinase was prematurely active during metaphase. Our results suggest an order of function of mitotic exit pathway components with respect to SPB localization of Cdc15 and Dbf2 and activation of Dbf2 kinase. BUB2 negatively regulates all 3 events. Loading of Cdc15 on SPBs depends on TEM1, whereas loading of Dbf2 on SPBs and activation of Dbf2 kinase depend on TEM1 and CDC15.     

A 63-base pair DNA segment containing an Sp1 site but not a canonical E2F site can confer growth-dependent and E2F-mediated transcriptional stimulation of the human ASK gene encoding the regulatory subunit for human Cdc7-related kinase

Cdc7-Dbf4 kinase complexes, conserved widely in eukaryotes, play essential roles in initiation and progression of the S phase. Cdc7 kinase activity fluctuates during cell cycle, and this is mainly the result of oscillation of expression of the Dbf4 subunit. Therefore, it is crucial to understand the mechanisms of regulation of Dbf4 expression. We have isolated and characterized the promoter region of the human ASK gene encoding Dbf4-related regulatory subunit for human Cdc7 kinase. We have identified a 63-base pair ASK promoter segment, which is sufficient for mediating growth stimulation. This minimal promoter segment (MP), containing an Sp1 site but no canonical E2F site, can be activated by ectopic E2F expression as well. Within the 63-base pair region, the Sp1 site as well as other elements are essential for stimulation by growth signals and by E2F, whereas an AT-rich sequence proximal to the coding region may serve as an element required for suppression in quiescence. Gel shift assays in the presence of an antibody demonstrate the presence of E2F1 in the protein-DNA complexes generated on the MP segment. However, the complex formation on MP was not competed by a DHFR promoter fragment, known to bind to E2F, nor by a consensus E2F binding oligonucleotide. Gel shift assays with point mutant MP fragments indicate that a non-canonical E2F site in the middle of this segment is critical for generation of the E2F complex. Our results suggest that E2F regulates the ASK promoter through an atypical mode of recognition of the target site.     

A novel role for Cdc5p in DNA replication.

DNA replication initiates from specific chromosomal sites called origins, and in the budding yeast Saccharomyces cerevisiae these sites are occupied by the origin recognition complex (ORC). Dbf4p is proposed to play a role in targeting the G1/S kinase Cdc7p to initiation complexes late in G1. We report that Dbf4p may also recruit Cdc5p to origin complexes. Cdc5p is a member of the Polo family of kinases that is required for the completion of mitosis. Cdc5p and Cdc7p each interact with a distinct domain of Dbf4p. cdc5-1 mutants have a plasmid maintenance defect that can be suppressed by the addition of multiple origins. cdc5-1 orc2-1 double mutants are synthetically lethal. Levels of Cdc5p were found to be cell cycle regulated and peaked in G2/M. These results suggest a role for Cdc5p and possibly Polo-like kinases at origin complexes.     

Cdc7 kinases (DDKs) and checkpoint responses: lessons from two yeasts

Principally characterized for its requirement in the initiation of DNA replication, compelling evidence from two yeast model organisms now points to a central role for the Dbf4/Cdc7 kinase complex in S-phase checkpoint responses. Among the key findings supporting this view are observations that orthologs Dfp1 (Schizosaccharomyces pombe) and Dbf4 (Saccharomyces cerevisiae) interact with equivalent checkpoint kinases Cds1 and Rad53, respectively, and that mutants for Dbf4 and Cdc7 in these species are sensitive to genotoxic agents. Recently, these findings have been extended through mutational analyses of conserved regions in both Dfp1 and Dbf4, leading to the identification of distinct motifs which mediate cellular responses to DNA damage and replication fork arrest. The present review is a comparative survey of data obtained from studies conducted with S. pombe and S. cerevisae, and a consideration of models for the role played by Dbf4/Cdc7 in checkpoint responses.     

Mutual regulation of cyclin-dependent kinase and the mitotic exit network

The mitotic exit network (MEN) is a spindle pole body (SPB)–associated, GTPase-driven signaling cascade that controls mitotic exit. The inhibitory Bfa1–Bub2 GTPase-activating protein (GAP) only associates with the daughter SPB (dSPB), raising the question as to how the MEN is regulated on the mother SPB (mSPB). Here, we show mutual regulation of cyclin-dependent kinase 1 (Cdk1) and the MEN. In early anaphase Cdk1 becomes recruited to the mSPB depending on the activity of the MEN kinase Cdc15. Conversely, Cdk1 negatively regulates binding of Cdc15 to the mSPB. In addition, Cdk1 phosphorylates the Mob1 protein to inhibit the activity of Dbf2–Mob1 kinase that regulates Cdc14 phosphatase. Our data revise the understanding of the spatial regulation of the MEN. Although MEN activity in the daughter cells is controlled by Bfa1–Bub2, Cdk1 inhibits MEN activity at the mSPB. Consistent with this model, only triple mutants that lack BUB2 and the Cdk1 phosphorylation sites in Mob1 and Cdc15 show mitotic exit defects.     

Hierarchy of S-phase-promoting factors: yeast Dbf4-Cdc7 kinase requires prior S-phase cyclin-dependent kinase activation

In all eukaryotes, the initiation of DNA synthesis requires the formation of prereplicative complexes (pre-RCs) on replication origins, followed by their activation by two S-T protein kinases, an S-phase cyclin-dependent kinase (S-CDK) and a homologue of yeast Dbf4-Cdc7 kinase (Dbf4p-dependent kinase [DDK]). Here, we show that yeast DDK activity is cell cycle regulated, though less tightly than that of the S-CDK Clb5-Cdk1, and peaks during S phase in correlation with Dbf4p levels. Dbf4p is short-lived throughout the cell cycle, but its instability is accentuated during G(1) by the anaphase-promoting complex. Downregulating DDK activity is physiologically important, as joint Cdc7p and Dbf4p overexpression is lethal. Because pre-RC formation is a highly ordered process, we asked whether S-CDK and DDK need also to function in a specific order for the firing of origins. We found that both kinases are activated independently, but we show that DDK can perform its function for DNA replication only after S-CDKs have been activated. Cdc45p, a protein needed for initiation, binds tightly to chromatin only after S-CDK activation (L. Zou and B. Stillman, Science 280:593-596, 1998). We show that Cdc45p is phosphorylated by DDK in vitro, suggesting that it might be one of DDK's critical substrates after S-CDK activation. Linking the origin-bound DDK to the tightly regulated S-CDK in a dependent sequence of events may ensure that DNA replication initiates only at the right time and place.     

Order of function of the budding-yeast mitotic exit-network proteins Tem1, Cdc15, Mob1, Dbf2, and Cdc5

The Dbf2 protein kinase functions as part of the mitotic-exit network (MEN), which controls the inactivation of the Cdc28-Clb2 kinase in late mitosis [1]. The MEN includes the Tem1 GTP binding protein; the kinases Cdc15 and Cdc5; Mob1, a protein of unknown function; and the phosphatase Cdc14 [2]. Here we have used Dbf2 kinase activity to investigate the regulation and order of function of the MEN. We find that Tem1 acts at the top of the pathway, upstream of Cdc15, which in turn functions upstream of Mob1 and Dbf2. The Cdc5 Polo-like kinase impinges at least twice on the MEN since it negatively regulates the network, probably upstream of Tem1, and is also required again for Dbf2 kinase activation. Furthermore, we find that regulation of Dbf2 kinase activity and actin ring formation at the bud neck are causally linked. In metaphase-arrested cells, the MEN inhibitor Bub2 restrains both Dbf2 kinase activity [3] and actin ring formation [4]. We find that the MEN proteins that are required for Dbf2 kinase activity are also required for actin ring formation. Thus, the MEN is crucial for the regulation of cytokinesis, as well as mitotic exit.