Protein kinase A is required for chromosomal DNA replication.

Passage through mitosis resets cells for a new round of chromosomal DNA replication [1]. In late mitosis, the pre-replication complex - which includes the origin recognition complex (ORC), Cdc6 and the minichromosome maintenance (MCM) proteins - binds chromatin as a pre-requisite for DNA replication. S-phase-promoting cyclin-dependent kinases (Cdks) and the kinase Dbf4-Cdc7 then act to initiate replication. Before the onset of replication Cdc6 dissociates from chromatin. S-phase and M-phase Cdks block the formation of a new pre-replication complex, preventing DNA over-replication during the S, G2 and M phases of the cell cycle [1]. The nuclear membrane also contributes to limit genome replication to once per cell cycle [2]. Thus, at the end of M phase, nuclear membrane breakdown and the collapse of Cdk activity reset cells for a new round of chromosomal replication. We showed previously that protein kinase A (PKA) activity oscillates during the cell cycle in Xenopus egg extracts, peaking in late mitosis. The oscillations are induced by the M-phase-promoting Cdk [3] [4]. Here, we found that PKA oscillation was required for the following phase of DNA replication. PKA activity was needed from mitosis exit to the formation of the nuclear envelope. PKA was not required for the assembly of ORC2, Cdc6 and MCM3 onto chromatin. Inhibition of PKA activity, however, blocked the release of Cdc6 from chromatin and subsequent DNA replication. These data suggest that PKA activation in late M phase is required for the following S phase.     

Clb/Cdc28 kinases promote nuclear export of the replication initiator proteins Mcm2-7

BACKGROUND: In the budding yeast Saccharomyces cerevisiae, the cyclin-dependent kinases of the Clb/Cdc28 family restrict the initiation of DNA replication to once per cell cycle by preventing the re-assembly of pre-replicative complexes (pre-RCs) at replication origins that have already initiated replication. This assembly involves the Cdc6-dependent loading of six minichromosome maintenance (Mcm) proteins, Mcm2-7, onto origins. How Clb/Cdc28 kinases prevent pre-RC assembly is not understood. RESULTS: In living cells, the Mcm proteins were found to colocalize in a cell-cycle-regulated manner. Mcm2-4, 6 and 7 were concentrated in the nucleus in G1 phase, gradually exported to the cytoplasm during S phase, and excluded from the nucleus by G2 and M phase. Tagging any single Mcm protein with the SV40 nuclear localization signal made all Mcm proteins constitutively nuclear. In the absence of functional Cdc6, Clb/Cdc28 kinases were necessary and sufficient for efficient net nuclear export of a fusion protein between Mcm7 and the green fluorescent protein (Mcm7-GFP), whereas inactivation of these kinases at the end of mitosis coincided with the net nuclear import of Mcm7-GFP. In contrast, in the presence of functional Cdc6, which loads Mcm proteins onto chromatin, S-phase progression as well as Clb/Cdc28 kinases was required for Mcm-GFP export. CONCLUSIONS: We propose that Clb/Cdc28 kinases prevent pre-RC reassembly in part by promoting the net nuclear export of Mcm proteins. We further propose that Mcm proteins become refractory to this regulation when they load onto chromatin and must be dislodged by DNA replication before they can be exported. Such an arrangement could ensure that Mcm proteins complete their replication function before they are removed from the nucleus.     

Cdc25B and Cdc25C differ markedly in their properties as initiators of mitosis.

We have used time-lapse fluorescence microscopy to study the properties of the Cdc25B and Cdc25C phosphatases that have both been implicated as initiators of mitosis in human cells. To differentiate between the functions of the two proteins, we have microinjected expression constructs encoding Cdc25B or Cdc25C or their GFP-chimeras into synchronized tissue culture cells. This assay allows us to express the proteins at defined points in the cell cycle. We have followed the microinjected cells by time-lapse microscopy, in the presence or absence of DNA synthesis inhibitors, and assayed whether they enter mitosis prematurely or at the correct time. We find that overexpressing Cdc25B alone rapidly causes S phase and G2 phase cells to enter mitosis, whether or not DNA replication is complete, whereas overexpressing Cdc25C does not cause premature mitosis. Overexpressing Cdc25C together with cyclin B1 does shorten the G2 phase and can override the unreplicated DNA checkpoint, but much less efficiently than overexpressing Cdc25B. These results suggest that Cdc25B and Cdc25C do not respond identically to the same cell cycle checkpoints. This difference may be related to the differential localization of the proteins; Cdc25C is nuclear throughout interphase, whereas Cdc25B is nuclear in the G1 phase and cytoplasmic in the S and G2 phases. We have found that the change in subcellular localization of Cdc25B is due to nuclear export and that this is dependent on cyclin B1. Our data suggest that although both Cdc25B and Cdc25C can promote mitosis, they are likely to have distinct roles in the controlling the initiation of mitosis.     

The Cdc4/34/53 pathway targets Cdc6p for proteolysis in budding yeast.

The budding yeast Cdc6 protein (Cdc6p) is essential for formation of pre-replicative complexes (pre-RCs) at origins of DNA replication. Regulation of pre-RC assembly plays a key role in making initiation of DNA synthesis dependent upon passage through mitosis and in limiting DNA replication to once per cell cycle. Cdc6p is normally only present at high levels during the G1 phase of the cell cycle. This is partly because the CDC6 gene is only transcribed during G1. In this article we show that rapid degradation of Cdc6p also contributes to this periodicity. Cdc6p degradation rates are regulated during the cell cycle, reaching a peak during late G1/early S phase. Removal of a 47-amino-acid domain near the N-terminus of Cdc6p prevents degradation of Cdc6p. Likewise, mutations in the Cdc4/34/53 pathway involved in ubiquitin-mediated degradation block proteolysis and genetic evidence is presented indicating that the N-terminus of Cdc6p interacts with the Cdc4/34/53 pathway, probably through Cdc4p. A stable Cdc6p mutant which is no longer degraded by the Cdc4/34/53 pathway is, none the less, fully functional. Constitutive overexpression of either wild-type or stable Cdc6p does not induce re-replication and does not induce assembly of pre-replicative complexes after DNA replication is complete.     

Control of the Cdc6 replication licensing factor in metazoa: The role of nuclear export and the CUL4 ubiquitin ligase

A central requirement to maintain genome stability is that DNA replication must be tightly controlled so that genomic DNA is replicated only once in a single cell cycle. The prevention of DNA re-replication is achieved by restricting the assembly of pre-replicative complexes (pre RCs) to the period prior to S phase, and ensuring that pre-RCs cannot reform during S phase. The regulation of the replication licensing factors Cdt1 and Cdc6 during S phase is critical to prevent the reformation of pre-RCs. In yeast, Cdc6 is degraded during S phase to block DNA re-replication. In mammals, Cdc6 is exported from the nucleus; however, a variable percentage of endogenous Cdc6 remains nuclear throughout S phase. The perdurance of nuclear Cdc6 has led a number of groups to question whether the nuclear export of Cdc6 is relevant in restricting its activity. A recent study in C. elegans shows that the nuclear export of Cdc6 is in fact critical to prevent DNA re-replication. This work also identifies the CUL-4 ubiquitin ligase as a master regulator that controls DNA replication by regulating both Cdt1 and Cdc6 replication licensing factors.     

The functional role of Cdc6 in S-G2/M in mammalian cells

The Cdc6 protein is required for licensing of replication origins before the onset of DNA replication in eukaryotic cells. Here, we examined whether Cdc6 has other roles in mammalian cell-cycle progression from S to G2/M phase. Using RNA interference, we showed that depletion of Cdc6 in synchronous G1 cells blocks G1 to S transition, confirming the essential role of Cdc6 in the initiation of DNA replication. In contrast, depletion of Cdc6 in synchronous S-phase cells slowed DNA replication and led to mitotic lethality. The Cdc6-depleted S-phase cells showed fewer newly fired origins; however, established replication forks remained active, even during chromatin condensation. Despite such DNA replication abnormalities, loss of Cdc6 failed to activate Chk1 kinase. These results show that Cdc6 is not only required for G1 origin licensing, but is also crucial for proper S-phase DNA replication that is essential for DNA segregation during mitosis.     

Human replication protein Cdc6 prevents mitosis through a checkpoint mechanism that implicates Chk1

In yeasts, the replication protein Cdc6/Cdc18 is required for the initiation of DNA replication and also for coupling S phase with the following mitosis. In metazoans a role for Cdc6 has only been shown in S phase entry. Here we provide evidence that human Cdc6 (HuCdc6) also regulates the onset of mitosis, as overexpression of HuCdc6 in G(2) phase cells prevents entry into mitosis. This block is abolished when HuCdc6 is expressed together with a constitutively active Cyclin B/CDK1 complex or with Cdc25B or Cdc25C. An inhibitor of Chk1 kinase activity, UCN-01, overcomes the HuCdc6 mediated G(2) arrest indicating that HuCdc6 blocks cells in G(2) phase via a checkpoint pathway involving Chk1. When HuCdc6 is overexpressed in G(2), we detected phosphorylation of Chk1. Thus, HuCdc6 can trigger a checkpoint response, which could ensure that all DNA is replicated before mitotic entry. We also present evidence that the ability of HuCdc6 to block mitosis may be regulated by its phosphorylation.     

Stable association of mitotic cyclin B/Cdc2 to replication origins prevents endoreduplication

We show that in fission yeast the mitotic B type cyclin Cdc13/Cdc2 kinase associates with replication origins in vivo. This association is dependent on the origin recognition complex (ORC), is established as chromosomes are replicated, and is maintained during G2 and early mitosis. Cells expressing an orp2 (ORC2) allele that reduces binding of Cdc13 to replication origins are acutely prone to chromosomal reduplication. In synchronized endoreduplicating cells, following Cdc13 ablation, replication origins are coordinately licensed prior to each successive round of S phase with the same periodicity as in a normal cell cycle. Thus, ORC bound mitotic Cyclin B/Cdc2 kinase imposes the dependency of S phase on an intervening mitosis but not the temporal licensing of replication origins between each S phase.     

Cell cycle-dependent nuclear export of Cdh1p may contribute to the inactivation of APC/C(Cdh1)

Cdh1p is a substrate-specific subunit of the anaphase-promoting complex (APC/C), which functions as an E3 ubiquitin ligase to degrade the mitotic cyclin Clb2p and other substrates during the G(1) phase of the cell cycle. Cdh1p is phosphorylated and thereby inactivated at the G(1)/S transition predominantly by Cdc28p-Clb5p. Here we show that Cdh1p is nuclear during the G(1) phase of the cell cycle, but redistributes to the cytoplasm between S phase and the end of mitosis. Nuclear export of Cdh1p is regulated by phosphorylation and requires active Cdc28p kinase. Cdh1p binds to the importin Pse1p and the exportin Msn5p, which is necessary and sufficient to promote efficient export of Cdh1p in vivo. Although msn5delta cells are viable, they are sensitive to Cdh1p overexpression. Likewise, a mutant form of Cdh1p, which is constitutively nuclear, prevents accumulation of Clb2p and leads to cell cycle arrest when overexpressed in wild-type cells. Taken together, these results suggest that phosphorylation-dependent nuclear export of Cdh1p by Msn5p contributes to efficient inactivation of APC/C(Cdh1).     

Cell Cycle Control of Cdc7p Kinase Activity through Regulation of Dbf4p Stability

In Saccharomyces cerevisiae, the heteromeric kinase complex Cdc7p-Dbf4p plays a pivotal role at replication origins in triggering the initiation of DNA replication during the S phase. We have assayed the kinase activity of endogenous levels of Cdc7p kinase by using a likely physiological target, Mcm2p, as a substrate. Using this assay, we have confirmed that Cdc7p kinase activity fluctuates during the cell cycle; it is low in the G1 phase, rises as cells enter the S phase, and remains high until cells complete mitosis. These changes in kinase activity cannot be accounted for by changes in the levels of the catalytic subunit Cdc7p, as these levels are constant during the cell cycle. However, the fluctuations in kinase activity do correlate with levels of the regulatory subunit Dbf4p. The regulation of Dbf4p levels can be attributed in part to increased degradation of the protein in G1 cells. This G1-phase instability is cdc16 dependent, suggesting a role of the anaphase-promoting complex in the turnover of Dbf4p. Overexpression of Dbf4p in the G1 phase can partially overcome this elevated turnover and lead to an increase in Cdc7p kinase activity. Thus, the regulation of Dbf4p levels through the control of Dbf4p degradation has an important role in the regulation of Cdc7p kinase activity during the cell cycle.     

A Yeast GSK-3 Kinase Mck1 Promotes Cdc6 Degradation to Inhibit DNA Re-Replication

Cdc6p is an essential component of the pre-replicative complex (pre-RC), which binds to DNA replication origins to promote initiation of DNA replication. Only once per cell cycle does DNA replication take place. After initiation, the pre-RC components are disassembled in order to prevent re-replication. It has been shown that the N-terminal region of Cdc6p is targeted for degradation after phosphorylation by Cyclin Dependent Kinase (CDK). Here we show that Mck1p, a yeast homologue of GSK-3 kinase, is also required for Cdc6 degradation through a distinct mechanism. Cdc6 is an unstable protein and is accumulated in the nucleus only during G1 and early S-phase in wild-type cells. In mck1 deletion cells, CDC6p is stabilized and accumulates in the nucleus even in late S phase and mitosis. Overexpression of Mck1p induces rapid Cdc6p degradation in a manner dependent on Threonine-368, a GSK-3 phosphorylation consensus site, and SCF^CDC4. We show evidence that Mck1p-dependent degradation of Cdc6 is required for prevention of DNA re-replication. Loss of Mck1 activity results in synthetic lethality with other pre-RC mutants previously implicated in re-replication control, and these double mutant strains over-replicate DNA within a single cell cycle. These results suggest that a GSK3 family protein plays an unexpected role in preventing DNA over-replication through Cdc6 degradation in Saccharomyces cerevisiae. We propose that both CDK and Mck1 kinases are required for Cdc6 degradation to ensure a tight control of DNA replication.     

Separate SCF(CDC4) recognition elements target Cdc6 for proteolysis in S phase and mitosis

The Cdc6 DNA replication initiation factor is targeted for ubiquitin-mediated proteolysis by the E3 ubiquitin ligase SCF(CDC4) from the end of G1phase until mitosis in the budding yeast Saccharomyces cerevisiae. Here we describe a dominant-negative CDC6 mutant that, when overexpressed, arrests the cell cycle by inhibiting cyclin-dependent kinases (CDKs) and, thus, prevents passage through mitosis. This mutant protein inhibits CDKs more efficiently than wild-type Cdc6, in part because it is completely refractory to SCF(CDC4)-mediated proteolysis late in the cell cycle and consequently accumulates to high levels. The mutation responsible for this phenotype destroys a putative CDK phosphorylation site near the middle of the Cdc6 primary amino acid sequence. We show that this site lies within a novel Cdc4-interacting domain distinct from a Cdc4-interacting site identified previously near the N-terminus of the protein. We show that both sites can target Cdc6 for proteolysis in late G1/early S phase whilst only the newly identified site can target Cdc6 for proteolysis during mitosis.     

Stockpiling of Cdc25 during a DNA replication checkpoint arrest in Schizosaccharomyces pombe.

The DNA replication checkpoint couples the onset of mitosis with the completion of S phase. It is clear that in the fission yeast Schizosaccharomyces pombe, operation of this checkpoint requires maintenance of the inhibitory tyrosyl phosphorylation of Cdc2. Cdc25 phosphatase induces mitosis by dephosphorylating tyrosine 15 of Cdc2. In this report, Cdc25 is shown to accumulate to a very high level in cells arrested in S. This shows that mechanisms which modulate the abundance of Cdc25 are unconnected to the DNA replication checkpoint. Using a Cdc2/cyclin B activation assay, we found that Cdc25 activity increased approximately 10-fold during transit through M phase. Cdc25 was activated by phosphorylations that were dependent on Cdc2 activity in vivo. Cdc25 activation was suppressed in cells arrested in G1 and S. However, Cdc25 was more highly modified and appeared to be somewhat more active in S than in G1. This finding might be connected to the fact that progression from G1 to S increases the likelihood that constitutive Cdc25 overproduction will cause inappropriate mitosis.     

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.     

Mitotic effects of a constitutively active mutant of the Xenopus polo-like kinase Plx1

During mitosis the Xenopus polo-like kinase 1 (Plx1) plays key roles in the activation of Cdc25C, in spindle assembly, and in cyclin B degradation. Previous work has shown that the activation of Plx1 requires phosphorylation on serine and threonine residues. In the present work, we demonstrate that replacement of Ser-128 or Thr-201 with a negatively charged aspartic acid residue (S128D or T201D) elevates Plx1 activity severalfold and that replacement of both Ser-128 and Thr-201 with Asp residues (S128D/T201D) increases Plx1 activity approximately 40-fold. Microinjection of mRNA encoding S128D/T201D Plx1 into Xenopus oocytes induced directly the activation of both Cdc25C and cyclin B-Cdc2. In egg extracts T201D Plx1 delayed the timing of deactivation of Cdc25C during exit from M phase and accelerated Cdc25C activation during entry into M phase. This supports the concept that Plx1 is a "trigger" kinase for the activation of Cdc25C during the G(2)/M transition. In addition, during anaphase T201D Plx1 reduced preferentially the degradation of cyclin B2 and delayed the reduction in Cdc2 histone H1 kinase activity. In early embryos S128D/T201D Plx1 resulted in arrest of cleavage and formation of multiple interphase nuclei. Consistent with these results, Plx1 was found to be localized on centrosomes at prophase, on spindles at metaphase, and at the midbody during cytokinesis. These results demonstrate that in Xenopus laevis activation of Plx1 is sufficient for the activation of Cdc25C at the initiation of mitosis and that inactivation of Plx1 is required for complete degradation of cyclin B2 after anaphase and completion of cytokinesis.     

The spindle checkpoint,APC/CCdc20, and APC/CCdh1 play distinct roles in connecting mitosis to S phase

DNA replication depends on a preceding licensing event by Cdt1 and Cdc6. In animal cells, relicensing after S phase but before mitosis is prevented by the Cdt1 inhibitor geminin and mitotic cyclin activity. Here, we show that geminin, like cyclin B1 and securin, is a bona fide target of the spindle checkpoint and APC/C^Cdc20. Cyclin B1 and geminin are degraded simultaneously during metaphase, which directs Cdt1 accumulation on segregating sister chromatids. Subsequent activation of APC/C^Cdh1 leads to degradation of Cdc6 well before Cdt1 becomes unstable in a replication-coupled manner. In mitosis, the spindle checkpoint supports Cdt1 accumulation, which promotes S phase onset. We conclude that the spindle checkpoint, APC/C^Cdc20, and APC/C^Cdh1 act successively to ensure that the disappearance of licensing inhibitors coincides exactly with a peak of Cdt1 and Cdc6. Whereas cell cycle entry from quiescence requires Cdc6 resynthesis, our results indicate that proliferating cells use a window of time in mitosis, before Cdc6 is degraded, as an earlier opportunity to direct S phase.     

Regulation of the localization and stability of Cdc6 in living yeast cells

The Cdc6 protein is an essential regulator for initiation of DNA replication. Following the G1/S transition, Cdc6 is degraded through a ubiquitin-mediated proteolysis pathway. In this study, we tagged Cdc6 with green fluorescent protein (GFP) and used site-specific mutations to study the regulation of Cdc6 localization and degradation in living yeast cells. Our major findings are: (1). Cdc6-GFP distributes predominantly in the nucleus in all cell cycle stages, with a small increase in cytoplasmic localization in G2/M cells. (2). This nuclear localization is critical for Cdc6 degradation. When the N-terminal nuclear localization signal (NLS) was mutated, Cdc6-GFP no longer accumulated in the nucleus, and the mutant cdc6 was stabilized compared to wild type. (3). The putative CDK phosphorylation sites are not required for Cdc6 nuclear localization, but are important for protein stability. These observations suggest that the stability of Cdc6 protein is regulated by two factors: nuclear localization and phosphorylation by CDK1.     

Role for Cdk1 (Cdc2)/cyclin A in preventing the mammalian origin recognition complex's largest subunit (Orc1) from binding to chromatin during mitosis

The eukaryotic origin recognition complex (ORC) selects the genomic sites where prereplication complexes are assembled and DNA replication begins. In proliferating mammalian cells, ORC activity appears to be regulated by reducing the affinity of the Orc1 subunit for chromatin during S phase and then preventing reformation of a stable ORC-chromatin complex until mitosis is completed and a nuclear membrane is assembled. Here we show that part of the mechanism by which this is accomplished is the selective association of Orc1 with Cdk1 (Cdc2)/cyclin A during the G(2)/M phase of cell division. This association accounted for the appearance in M-phase cells of hyperphosphorylated Orc1 that was subsequently dephosphorylated during the M-to-G(1) transition. Moreover, inhibition of Cdk activity in metaphase cells resulted in rapid binding of Orc1 to chromatin. However, chromatin binding was not mediated through increased affinity of Orc1 for Orc2, suggesting that additional events are involved in the assembly of functional ORC-chromatin sites. These results reveal that the same cyclin-dependent protein kinase that initiates mitosis in mammalian cells also concomitantly inhibits assembly of functional ORC-chromatin sites.