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.     

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.     

Hsk1-Dfp1/Him1, the Cdc7-Dbf4 kinase in Schizosaccharomyces pombe, associates with Swi1, a component of the replication fork protection complex

The protein kinase Hsk1 is essential for DNA replication in Schizosaccharomyces pombe. It associates with Dfp1/Him1 to form an active complex equivalent to the Cdc7-Dbf4 protein kinase in Saccharomyces cerevisiae. Swi1 and Swi3 are subunits of the replication fork protection complex in S. pombe that is homologous to the Tof1-Csm3 complex in S. cerevisiae. The fork protection complex helps to preserve the integrity of stalled replication forks and is important for activation of the checkpoint protein kinase Cds1 in response to fork arrest. Here we describe physical and genetic interactions involving Swi1 and Hsk1-Dfp1/Him1. Dfp1/Him1 was identified in a yeast two-hybrid screen with Swi1. Hsk1 and Dfp1/Him1 both co-immunoprecipitate with Swi1. Swi1 is required for growth of a temperature-sensitive hsk1 (hsk1ts) mutant at its semi-permissive temperature. Hsk1ts cells accumulate Rad22 (Rad52 homologue) DNA repair foci at the permissive temperature, as previously observed in swi1 cells, indicating that abnormal single-stranded DNA regions form near the replication fork in hsk1ts cells. hsk1ts cells were also unable to properly delay S-phase progression in the presence of a DNA alkylating agent and were partially defective in mating type switching. These data suggest that Hsk1-Dfp1/Him1 and Swi1-Swi3 complexes have interrelated roles in stabilization of arrested replication forks.     

Novel Fission Yeast Cdc7-Dbf4-Like Kinase Complex Required for the Initiation and Progression of Meiotic Second Division

Cdc7, a conserved serine/threonine protein kinase, controls initiation of DNA replication. A regulatory subunit, Dbf4, stimulates the kinase activity of Cdc7 and recruits it to the replication origins. Schizosaccharomyces pombe has a homologous kinase complex, composed of Hsk1 and Dfp1/Him1. Here, we report a novel protein kinase of S. pombe, Spo4, which shares common structural features with the Cdc7 kinases. In spite of the structural similarities, Spo4 is dispensable for mitotic growth and premeiotic DNA replication. Intriguingly, spo4 null mutants are defective in initiation and progression of the second meiotic division. Spindles for meiosis II are often fragmented. Spo4 kinase activity is markedly enhanced when the enzyme is associated with its regulatory subunit, Spo6, a Dbf4-like protein. Expression of Spo4 is specifically induced during meiosis. Spo4 is preferentially present in nuclei, but this nuclear localization does not require Spo6. These results suggest that Spo4 is a Cdc7 kinase whose primary role is in meiosis, not in DNA replication. This is the first report of an organism which has two Cdc7-related kinase complexes with different biological functions.     

Regulation of initiation of S phase, replication checkpoint signaling, and maintenance of mitotic chromosome structures during S phase by Hsk1 kinase in the fission yeast

Hsk1, Saccharomyces cerevisiae Cdc7-related kinase in Shizosaccharomyces pombe, is required for G1/S transition and its kinase activity is controlled by the regulatory subunit Dfp1/Him1. Analyses of a newly isolated temperature-sensitive mutant, hsk1-89, reveal that Hsk1 plays crucial roles in DNA replication checkpoint signaling and maintenance of proper chromatin structures during mitotic S phase through regulating the functions of Rad3 (ATM)-Cds1 and Rad21 (cohesin), respectively, in addition to expected essential roles for initiation of mitotic DNA replication through phosphorylating Cdc19 (Mcm2). Checkpoint defect in hsk1-89 is indicated by accumulation of cut cells at 30 degrees C. hsk1-89 displays synthetic lethality in combination with rad3 deletion, indicating that survival of hsk1-89 depends on Rad3-dependent checkpoint pathway. Cds1 kinase activation, which normally occurs in response to early S phase arrest by nucleotide deprivation, is largely impaired in hsk1-89. Furthermore, Cds1-dependent hyperphosphorylation of Dfp1 in response to hydroxyurea arrest is eliminated in hsk1-89, suggesting that sufficient activation of Hsk1-Dfp1 kinase is required for S phase entry and replication checkpoint signaling. hsk1-89 displays apparent defect in mitosis at 37 degrees C leading to accumulation of cells with near 2C DNA content and with aberrant nuclear structures. These phenotypes are similar to those of rad21-K1 and are significantly enhanced in a hsk1-89 rad21-K1 double mutant. Consistent with essential roles of Rad21 as a component for the cohesin complex, sister chromatid cohesion is partially impaired in hsk1-89, suggesting a possibility that infrequent origin firing of the mutant may affect the cohesin functions during S phase.     

Cdc7 kinase complex: a key regulator in the initiation of DNA replication

DNA replication results from the action of a staged set of highly regulated processes. Among the stages of DNA replication, initiation is the key point at which all the G1 regulatory signals culminate. Cdc7 kinase is the critical regulator for the ultimate firing of the origins of initiation. Cdc7, originally identified in budding yeast and later in higher eukaryotes, forms a complex with a Dbf4-related regulatory subunit to generate an active kinase. Genetic evidence in mammals demonstrates essential roles for Cdc7 in mammalian DNA replication. Mini-chromosome maintenance protein (MCM) is the major physiological target of Cdc7. Genetic studies in yeasts indicate additional roles of Cdc7 in meiosis, checkpoint responses, maintenance of chromosome structures, and repair. The interplay between Cdc7 and Cdk, another kinase essential for the S phase, is also discussed.     

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.     

G(1)/S CDK is inhibited to restrain mitotic onset when DNA replication is blocked in fission yeast

Cyclin-dependent kinase (CDK) Tyr15 phosphorylation plays a major role in regulating G(2)/M CDKs, but the role of this phosphorylation in regulating G(1)/S CDKs is less clear. We have studied the regulation and function of Cdc2-Tyr15 phosphorylation in the fission yeast Schizosaccharomyces pombe G(1)/S CDK Cig2/Cdc2. This complex is subject to high level Cdc2-Tyr15 phosphorylation inhibiting its kinase activity in hydroxyurea-treated cells blocked in S-phase. We show that this Tyr15 phosphorylation is required to maintain efficient mitotic checkpoint arrest, because Cig2 accumulates during the block and this accumulation can advance mitotic onset. This mitotic induction operates, at least in part, through activation of the normal G(2)/M CDK complex Cdc13/Cdc2. Thus, Tyr15 phosphorylation of G(1)/S CDK complexes is important in the checkpoint control blocking mitotic onset when DNA replication is inhibited.     

Cdc2 Tyrosine Phosphorylation is Not Required for the S-Phase DNA Damage Checkpoint in Fission Yeast

The S-phase DNA damage checkpoint slows replication when damage occurs during S phase. Cdc25, which activates Cdc2 by dephosphorylating tyrosine-15, has been shown to be a downstream target of the checkpoint in metazoans, but its role is not clear in fission yeast. The dephosphorylation of Cdc2 has been assumed not to play a role in S-phase regulation because cells replicate in the absence of Cdc25, demonstrating that tyrosine-15 phosphorylated Cdc2 is sufficient for S phase. However, it has been reported recently that Cdc25 is required for the slowing of S phase in response to damage in fission yeast, suggesting a modulatory role for Cdc2 dephosphorylation in S phase. We have investigated the role of Cdc25 and the tyrosine phosphorylation of Cdc2 in the S-phase damage checkpoint, and our results show that Cdc2 phosphorylation is not a target of the checkpoint. The checkpoint was not compromised in a Cdc25 overexpressing strain, a strain carrying non-phosphorylatable form of Cdc2, or in a strain lacking Cdc25. Our results are consistent with a strictly Cdc2-Y15 phosphorylation-independent mechanism of the fission yeast S-phase DNA damage checkpoint.     

Hsk1 kinase and Cdc45 regulate replication stress-induced checkpoint responses in fission yeast

In fission yeast, replication fork arrest activates the replication checkpoint effector kinase Cds1^Chk2/Rad53 through the Rad3^ATR/Mec1-Mrc1^Claspin pathway. Hsk1, the Cdc7 homolog of fission yeast required for efficient initiation of DNA replication, is also required for Cds1 activation. Hsk1 kinase activity is required for induction and maintenance of Mrc1 hyperphosphorylation, which is induced by replication fork block and mediated by Rad3. Rad3 kinase activity does not change in an hsk1 temperature-sensitive mutant, and Hsk1 kinase activity is not affected by rad3 mutation. Hsk1 kinase vigorously phosphorylates Mrc1 in vitro, predominantly at non-SQ/TQ sites, but this phosphorylation does not seem to affect the Rad3 action on Mrc1. Interestingly, the replication stress-induced activation of Cds1 and hyperphosphorylation of Mrc1 is almost completely abrogated in an initiation-defective mutant of cdc45, but not significantly in an mcm2 or polε mutant. These results suggest that Hsk1-mediated loading of Cdc45 onto replication origins may play important roles in replication stress-induced checkpoint.Key words: Cdc7, Cdc45, checkpoint, DNA replication, Mrc1     

Hsk1 kinase and Cdc45 regulate replication stress-induced checkpoint responses in fission yeast

In fission yeast, replication fork arrest activates the replication checkpoint effector kinase Cds1^Chk2/Rad53 through the Rad3^ATR/Mec1-Mrc1^Claspin pathway. Hsk1, the Cdc7 homologue of fission yeast required for efficient initiation of DNA replication, is also required for Cds1 activation. Hsk1 kinase activity is required for induction and maintenance of Mrc1 hyperphosphorylation, which is induced by replication fork block and mediated by Rad3. Rad3 kinase activity does not change in an hsk1 temperature-sensitive mutant, and Hsk1 kinase activity is not affected by rad3 mutation. Hsk1 kinase vigorously phosphorylates Mrc1 in vitro, predominantly at non-SQ/TQ sites, but this phosphorylation does not seem to affect the Rad3 action on Mrc1. Interestingly, the replication stress-induced activation of Cds1 and hyperphosphorylation of Mrc1 is almost completely abrogated in an initiation-defective mutant of cdc45, but not in an mcm2 or polε mutant. The results suggest that Hsk1-mediated loading of Cdc45 onto replication origins may play important roles in replication stress-induced checkpoint.     

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.     

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.     

DDK phosphorylates checkpoint clamp component Rad9 and promotes its release from damaged chromatin

When inappropriate DNA structures arise, they are sensed by DNA structure-dependent checkpoint pathways and subsequently repaired. Recruitment of checkpoint proteins to such structures precedes recruitment of proteins involved in DNA metabolism. Thus, checkpoints can regulate DNA metabolism. We show that fission yeast Rad9, a 9-1-1 heterotrimeric checkpoint-clamp component, is phosphorylated by Hsk1(Cdc7), the Schizosaccharomyces pombe?Dbf4-dependent kinase (DDK) homolog, in response to replication-induced DNA damage. Phosphorylation of Rad9 disrupts its interaction with replication protein A (RPA) and is dependent on 9-1-1 chromatin loading, the Rad9-associated protein Rad4/Cut5(TopBP1), and prior phosphorylation by Rad3(ATR). rad9 mutants defective in DDK phosphorylation show wild-type checkpoint responses but abnormal DNA repair protein foci and decreased viability after replication stress. We propose that Rad9 phosphorylation by DDK releases Rad9 from DNA damage sites to facilitate DNA repair.     

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.     

Cloning and characterization of Chinese hamster homologue of yeast DBF4 (ChDBF4)

The Dbf4 protein is the regulatory subunit of Cdc7 serine/threonine kinase, which is essential for entry into S phase. We report here the cloning and initial characterization of the Chinese hamster homologue of yeast DBF4. The deduced ChDbf4 protein contains 676 amino acids with a predicted molecular mass of 75.8 kDa, and shares extensive identity overall with those of human (68%) and mouse (73%). The ChDBF4 mRNA level was barely detectable in the cells arrested in the quiescent stage (G(0)) by isoleucine starvation. When cells in G(0) were released into the cell cycle, the ChDBF4 mRNA level did not significantly change until the cells reached the G(1)/S boundary, when the level rapidly increased and reached approximately 70% of the maximum level that was observed in mid to late S phase. Interestingly, gamma-irradiation rapidly and transiently downregulated the level of ChDBF4 mRNA in asynchronous cell populations. Since Dbf4-Cdc7 kinase is involved in the regulation of replication initiation, which can be transiently downregulated by irradiation (Larner et al., 1994. Mol. Cell. Biol. 14, 1901, our data raise the possibility that the downregulation of DBF4 (and, thus, the Cdc7 kinase activity) by irradiation may play a role in the cell-cycle checkpoint that functions at the G(1)/S transition and in S phase (Lee et al., 1997. Proc. Natl. Acad. Sci. USA 94, 526).