The human origin recognition complex protein 1 dissociates from chromatin during S phase in HeLa cells

We investigated the association of human origin recognition complex (ORC) proteins hOrc1p and hOrc2p with chromatin in HeLa cells. Independent procedures including limited nuclease digestion and differential salt extraction of isolated nuclei showed that a complex containing hOrc1p and hOrc2p occurs in a nuclease-resistant compartment of chromatin and can be eluted with moderate high salt concentrations. A second fraction of hOrc2p that dissociates in vitro at low salt conditions was found to occur in a chromatin compartment characterized by its high accessibility to micrococcal nuclease. Functional differences between these two sites become apparent in HeLa cells that synchronously enter the S phase after a release from a double-thymidine block. The hOrc1p/hOrc2p-containing complexes dissociate from their chromatin sites during S phase and reassociate at the end of mitosis. In contrast, the fraction of hOrc2p in nuclease-accessible, more open chromatin remains bound during all phases of the cell cycle. We propose that the hOrc1p/hOrc2p-containing complexes are components of the human origin recognition complex. Thus, the observed cell cycle-dependent release of the hOrc1p/hOrc2p-containing complexes is in line with previous studies with Xenopus and Drosophila systems, which indicated that a change in ORC stability occurs after prereplication complex formation. This could be a powerful mechanism that prevents the rereplication of already replicated chromatin in the metazoan cell cycle.     

Human origin recognition complex large subunit is degraded by ubiquitin-mediated proteolysis after initiation of DNA replication

Eukaryotic cells possess overlapping mechanisms to ensure that DNA replication is restricted to the S phase of the cell cycle. The levels of hOrc1p, the largest subunit of the human origin recognition complex, vary during the cell division cycle. In rapidly proliferating cells, hOrc1p is expressed and targeted to chromatin as cells exit mitosis and prereplicative complexes are formed. Later, as cyclin A accumulates and cells enter S phase, hOrc1p is ubiquitinated on chromatin and then degraded. hOrc1p destruction occurs through the proteasome and is signaled in part by the SCF(Skp2) ubiquitin-ligase complex. Other hORC subunits are stable throughout the cell cycle. The regulation of hOrc1p may be an important mechanism in maintaining the ploidy in human cells.     

Noc3p,a bHLH protein,plays an integral role in the initiation of DNA replication in budding yeast

Initiation of eukaryotic DNA replication requires many proteins that interact with one another and with replicators. Using a yeast genetic screen, we have identified Noc3p (nucleolar complex-associated protein) as a novel replication-initiation protein. Noc3p interacts with MCM proteins and ORC and binds to chromatin and replicators throughout the cell cycle. It functions as a critical link between ORC and other initiation proteins to effect chromatin association of Cdc6p and MCM proteins for the establishment and maintenance of prereplication complexes. Noc3p is highly conserved in eukaryotes and is the first identified bHLH (basic helix-loop-helix) protein required for replication initiation. As Noc3p is also required for pre-rRNA processing, Noc3p is a multifunctional protein that plays essential roles in two vital cellular processes.     

Disruption of centrosome structure,chromosome segregation,and cytokinesis by misexpression of human Cdc14A phosphatase

In budding yeast, the Cdc14p phosphatase activates mitotic exit by dephosphorylation of specific cyclin-dependent kinase (Cdk) substrates and seems to be regulated by sequestration in the nucleolus until its release in mitosis. Herein, we have analyzed the two human homologs of Cdc14p, hCdc14A and hCdc14B. We demonstrate that the human Cdc14A phosphatase is selective for Cdk substrates in vitro and that although the protein abundance and intrinsic phosphatase activity of hCdc14A and B vary modestly during the cell cycle, their localization is cell cycle regulated. hCdc14A dynamically localizes to interphase but not mitotic centrosomes, and hCdc14B localizes to the interphase nucleolus. These distinct patterns of localization suggest that each isoform of human Cdc14 likely regulates separate cell cycle events. In addition, hCdc14A overexpression induces the loss of the pericentriolar markers pericentrin and gamma-tubulin from centrosomes. Overproduction of hCdc14A also causes mitotic spindle and chromosome segregation defects, defective karyokinesis, and a failure to complete cytokinesis. Thus, the hCdc14A phosphatase appears to play a role in the regulation of the centrosome cycle, mitosis, and cytokinesis, thereby influencing chromosome partitioning and genomic stability in human cells.     

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.     

Regulation of the anaphase-promoting complex by the dual specificity phosphatase human Cdc14a

Two forms of the anaphase-promoting complex (APC) mediate the degradation of critical cell cycle regulators. APC(Cdc20) promotes sister-chromatid separation by ubiquitinating securin, whereas APC(Cdh1) ubiquitinates mitotic cyclins, allowing the exit from mitosis. Here we show that phosphorylation of human Cdh1 (hCdh1) by cyclin B-Cdc2 alters the conformation of hCdh1 and prevents it from activating APC. A human homologue of yeast Cdc14, human Cdc14a (hCdc14a), dephosphorylates hCdh1 and activates APC(Cdh1). In contrast, hCdc14a does not affect the activity of APC(Cdc20). hCdc14a is a major phosphatase for hCdh1 and localizes to centrosomes in HeLa cells. Therefore, hCdc14a may promote the activation of APC(Cdh1) and exit from mitosis in mammalian cells.     

Cyclin A-dependent kinase activity affects chromatin binding of ORC, Cdc6, and MCM in egg extracts of Xenopus laevis.

The initiation of DNA replication in eukaryotes requires the loading of the origin recognition complex (ORC), Cdc6, and minichromosome maintenance (MCM) proteins onto chromatin to form the preinitiation complex. In Xenopus egg extract, the proteins Orc1, Orc2, Cdc6, and Mcm4 are underphosphorylated in interphase and hyperphosphorylated in metaphase extract. We find that chromatin binding of ORC, Cdc6, and MCM proteins does not require cyclin-dependent kinase activities. High cyclin A-dependent kinase activity inhibits the binding and promotes the release of Xenopus ORC, Cdc6, and MCM from sperm chromatin, but has no effect on chromatin binding of control proteins. Cyclin A together with ORC, Cdc6 and MCM proteins is bound to sperm chromatin in DNA replicating pseudonuclei. In contrast, high cyclin E/cdk2 was not detected on chromatin, but was found soluble in the nucleoplasm. High cyclin E kinase activity allows the binding of Xenopus ORC and Cdc6, but not MCM, to sperm chromatin, even though the kinase does not phosphorylate MCM directly. We conclude that chromatin-bound cyclin A kinase controls DNA replication by protein phosphorylation and chromatin release of Cdc6 and MCM, whereas soluble cyclin E kinase prevents rereplication during the cell cycle by the inhibition of premature MCM chromatin association.     

The human Cdc14 phosphatases interact with and dephosphorylate the tumor suppressor protein p53

The yeast Cdc14 phosphatase has been shown to play an important role in cell cycle regulation by dephosphorylating proteins phosphorylated by the cyclin-dependent kinase Cdc28/clb. We recently cloned two human orthologs of the yeast CDC14, termed hCDC14A and -B, the gene products of which share approximately 80% amino acid sequence identity within their N termini and phosphatase domains. Here we report that the hCdc14A and hCdc14B proteins interact with the tumor suppressor protein p53 both in vitro and in vivo. This interaction is dependent on the N termini of the hCdc14 proteins and the C terminus of p53. Furthermore, the hCdc14 phosphatases were found to dephosphorylate p53 specifically at the p34(Cdc2)/clb phosphorylation site (p53-phosphor-Ser(315)). Our findings that hCdc14 is a cyclin-dependent kinase substrate phosphatase suggest that it may play a role in cell cycle control in human cells. Furthermore, the identification of p53 as a substrate for hCdc14 indicates that hCdc14 may regulate the function of p53.     

The role and regulation of the preRC component Cdc6 in the initiation of premeiotic DNA replication

In all eukaryotes, the initiation of DNA replication is regulated by the ordered assembly of DNA/protein complexes on origins of DNA replication. In this report, we examine the role of Cdc6, a component of the prereplication complex, in the initiation of premeiotic DNA replication in budding yeast. We show that in the meiotic cycle, Cdc6 is required for DNA synthesis and sporulation. Moreover, similarly to the regulation in the mitotic cell cycle, Cdc6 is specifically degraded upon entry into the meiotic S phase. By contrast, chromatin-immunoprecipitation analysis reveals that the origin-bound Cdc6 is stable throughout the meiotic cycle. Preliminary evidence suggests that this protection reflects a change in chromatin structure that occurs in meiosis. Using the cdc28-degron allele, we show that depletion of Cdc28 leads to stabilization of Cdc6 in the mitotic cycle, but not in the meiotic cycle. We show physical association between Cdc6 and the meiosis-specific hCDK2 homolog Ime2. These results suggest that under meiotic conditions, Ime2, rather than Cdc28, regulates the stability of Cdc6. Chromatin-immunoprecipitation analysis reveals that similarly to the mitotic cell cycle, Mcm2 binds origins in G1 and meiotic S phases, and at the end of the second meiotic division, it is gradually removed from chromatin.     

Stability, chromatin association and functional activity of mammalian pre-replication complex proteins during the cell cycle

We have examined the behavior of pre-replication complex (pre-RC) proteins in relation to key cell cycle transitions in Chinese Hamster Ovary (CHO) cells. ORC1, ORC4 and Cdc6 were stable (T1/2 >2 h) and associated with a chromatin-containing fraction throughout the cell cycle. Green fluorescent protein-tagged ORC1 associated with chromatin throughout mitosis in living cells and co-localized with ORC4 in metaphase spreads. Association of Mcm proteins with chromatin took place during telophase, approximately 30 min after the destruction of geminin and cyclins A and B, and was coincident with the licensing of chromatin to replicate in geminin-supplemented Xenopus egg extracts. Neither Mcm recruitment nor licensing required protein synthesis throughout mitosis. Moreover, licensing could be uncoupled from origin specification in geminin-supplemented extracts; site-specific initiation within the dihydrofolate reductase locus required nuclei from cells that had passed through the origin decision point (ODP). These results demonstrate that mammalian pre-RC assembly takes place during telophase, mediated by post-translational modifications of pre-existing proteins, and is not sufficient to select specific origin sites. A subsequent, as yet undefined, step selects which pre-RCs will function as replication origins.     

ATR (AT mutated Rad3 related) activity stabilizes Cdc6 and delays G2/M-phase entry during hydroxyurea-induced S-phase arrest of HeLa cells

The Cdc6 protein, a key DNA replication initiation factor, contributes to the long-term maintenance of the S-phase checkpoint by anchoring the Rad3–Rad26 complex to chromatin. Here, we demonstrate that ATR (AT mutated and Rad3 related) activity is essential for maintaining high chromatin levels of the Cdc6 protein, thereby delaying entry into mitosis during hydroxyurea (HU)-induced S-phase arrest of HeLa cells. Downregulation of ATR (AT mutated and Rad3 related) (i.e., using ATR-siRNA) reduced the protein levels of chromatin Cdc6 and significantly increased the cellular levels of phospho-histone H3 (Ser 10), an index of mitosis. Downregulation of Cdc6 was completely restored by pretreatment with MG132, a proteasome inhibitor. Moreover, mitotic entry of MG132-pretreated cells was significantly downregulated. Our results also show that ATR (AT mutated and Rad3 related) kinase phosphorylates Cdc6 at serine residue 6. Thus, this ATR (AT mutated and Rad3 related)-mediated phosphorylation of Cdc6 is likely associated with stabilization of Cdc6 protein, thereby maintaining high levels of chromatin Cdc6 and delaying premature mitotic entry. This novel mechanism likely contributes to the functional regulation of chromatin Cdc6, which delays the cell cycle of hydroxyurea-induced cells to enter mitosis at the S-phase checkpoint.     

Cyclin E uses Cdc6 as a chromatin-associated receptor required for DNA replication

Using an in vitro chromatin assembly assay in Xenopus egg extract, we show that cyclin E binds specifically and saturably to chromatin in three phases. In the first phase, the origin recognition complex and Cdc6 prereplication proteins, but not the minichromosome maintenance complex, are necessary and biochemically sufficient for ATP-dependent binding of cyclin E--Cdk2 to DNA. We find that cyclin E binds the NH(2)-terminal region of Cdc6 containing Cy--Arg-X-Leu (RXL) motifs. Cyclin E proteins with mutated substrate selection (Met-Arg-Ala-Ile-Leu; MRAIL) motifs fail to bind Cdc6, fail to compete with endogenous cyclin E--Cdk2 for chromatin binding, and fail to rescue replication in cyclin E--depleted extracts. Cdc6 proteins with mutations in the three consensus RXL motifs are quantitatively deficient for cyclin E binding and for rescuing replication in Cdc6-depleted extracts. Thus, the cyclin E--Cdc6 interaction that localizes the Cdk2 complex to chromatin is important for DNA replication. During the second phase, cyclin E--Cdk2 accumulates on chromatin, dependent on polymerase activity. In the third phase, cyclin E is phosphorylated, and the cyclin E--Cdk2 complex is displaced from chromatin in mitosis. In vitro, mitogen-activated protein kinase and especially cyclin B--Cdc2, but not the polo-like kinase 1, remove cyclin E--Cdk2 from chromatin. Rebinding of hyperphosphorylated cyclin E--Cdk2 to interphase chromatin requires dephosphorylation, and the Cdk kinase-directed Cdc14 phosphatase is sufficient for this dephosphorylation in vitro. These three phases of cyclin E association with chromatin may facilitate the diverse activities of cyclin E--Cdk2 in initiating replication, blocking rereplication, and allowing resetting of origins after mitosis.     

Interaction of Xenopus Cdc2 x cyclin A1 with the origin recognition complex

The initiation of DNA replication in eukaryotes is regulated in a minimum of at least two ways. First, several proteins, including origin recognition complex (ORC), Cdc6 protein, and the minichromosome maintenance (MCM) protein complex, need to be assembled on chromatin before initiation. Second, cyclin-dependent kinases regulate DNA replication in both a positive and a negative way by inducing the initiation of DNA replication at G(1)/S transition and preventing further rounds of origin firing within the same cell cycle. Here we characterize a link between the two levels. Immunoprecipitation of Xenopus origin recognition complex with anti-XOrc1 or anti-XOrc2 antibodies specifically co-immunoprecipitates a histone H1 kinase activity. The kinase activity is sensitive to several inhibitors of cyclin-dependent kinases including 6-dimethylaminopurine (6-DMAP), olomoucine, and p21(Cip1). This kinase activity also copurifies with ORC over several fractionation steps and was identified as a complex of the Cdc2 catalytic subunit and cyclin A1. Neither Cdk2 nor cyclin E could be detected in ORC immunoprecipitations. Reciprocal immunoprecipitations with anti-Xenopus Cdc2 or anti-Xenopus cyclin A1 antibodies specifically co-precipitate XOrc1 and XOrc2. Our results indicate that Xenopus ORC and Cdc2 x cyclin A1 physically interact and demonstrate a physical link between an active cyclin-dependent kinase and proteins involved in the initiation of DNA replication.     

Site-specific loading of an MCM protein complex in a DNA replication initiation zone upstream of the c-MYC gene in the HeLa cell cycle

The MCM proteins participate in an orderly association, beginning with the origin recognition complex, that culminates in the initiation of chromosomal DNA replication. Among these, MCM proteins 4, 6, and 7 constitute a subcomplex that reportedly possesses DNA helicase activity. Little is known about DNA sequences initially bound by these MCM proteins or about their cell cycle distribution in the chromatin. We have determined the locations of certain MCM and associated proteins by chromatin immunoprecipitation (ChIP) in a zone of initiation of DNA replication upstream of the c-MYC gene in the HeLa cell cycle. MCM7 and its clamp-loading partner Cdc6 are highly specifically colocalized by ChIP and re-ChIP in G(1) and early S on a 198-bp segment located near the center of the initiation zone. ChIP and Re-ChIP colocalizes MCM7 and ORC1 to the same segment specifically in late G(1). MCM proteins 6 and 7 can be coimmunoprecipitated throughout the cell cycle, whereas MCM4 is reduced in the complex in late S and G(2), reappearing upon mitosis. MCM7 is not visualized by immunohistochemistry on metaphase chromosomes. MCM7 is recruited to multiple sites in chromatin in S and G(2), at which time it is not detected with ORC1. The rate of dissemination is surprisingly slow and is unlikely to be simply attributed to progression with replication forks. Results indicate sequence-specific loading of MCM proteins onto DNA in late G(1) followed by a recruitment to multiple sites in chromatin subsequent to replication.     

Assembly of a complex containing Cdc45p, replication protein A, and Mcm2p at replication origins controlled by S-phase cyclin-dependent kinases and Cdc7p-Dbf4p kinase

In Saccharomyces cerevisiae, replication origins are activated with characteristic timing during S phase. S-phase cyclin-dependent kinases (S-CDKs) and Cdc7p-Dbf4p kinase are required for origin activation throughout S phase. The activation of S-CDKs leads to association of Cdc45p with chromatin, raising the possibility that Cdc45p defines the assembly of a new complex at each origin. Here we show that both Cdc45p and replication protein A (RPA) bind to Mcm2p at the G(1)-S transition in an S-CDK-dependent manner. During S phase, Cdc45p associates with different replication origins at specific times. The origin associations of Cdc45p and RPA are mutually dependent, and both S-CDKs and Cdc7p-Dbf4p are required for efficient binding of Cdc45p to origins. These findings suggest that S-CDKs and Cdc7p-Dbf4p promote loading of Cdc45p and RPA onto a preformed prereplication complex at each origin with preprogrammed timing. The ARS1 association of Mcm2p, but not that of the origin recognition complex, is diminished by disruption of the B2 element of ARS1, a potential origin DNA-unwinding element. Cdc45p is required for recruiting DNA polymerase alpha onto chromatin, and it associates with Mcm2p, RPA, and DNA polymerase epsilon only during S phase. These results suggest that the complex containing Cdc45p, RPA, and MCMs is involved in origin unwinding and assembly of replication forks at each origin.     

Differential binding of replication proteins across the human c-myc replicator

The binding of the prereplication complex proteins Orc1, Orc2, Mcm3, Mcm7, and Cdc6 and the novel DNA unwinding element (DUE) binding protein DUE-B to the endogenous human c-myc replicator was studied by chromatin immunoprecipitation. In G(1)-arrested HeLa cells, Mcm3, Mcm7, and DUE-B were prominent near the DUE, while Orc1 and Orc2 were least abundant near the DUE and more abundant at flanking sites. Cdc6 binding mirrored that of Orc2 in G(1)-arrested cells but decreased in asynchronous or M-phase cells. Similarly, the signals from Orc1, Mcm3, and Mcm7 were at background levels in cells arrested in M phase, whereas Orc2 retained the distribution seen in G(1)-phase cells. Previously shown to cause histone hyperacetylation and delocalization of replication initiation, trichostatin A treatment of cells led to a parallel qualitative change in the distribution of Mcm3, but not Orc2, across the c-myc replicator. Orc2, Mcm3, and DUE-B were also bound at an ectopic c-myc replicator, where deletion of sequences essential for origin activity was associated with the loss of DUE-B binding or the alteration of chromatin structure and loss of Mcm3 binding. These results show that proteins implicated in replication initiation are selectively and differentially bound across the c-myc replicator, dependent on discrete structural elements in DNA or chromatin.     

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.     

Xenopus CDC7/DRF1 complex is required for the initiation of DNA replication

The Cdc7 kinase is essential for the initiation of DNA replication in eukaryotes. Two regulatory subunits of the Xenopus Cdc7 kinase have been identified: XDbf4 and XDrf1. In this study we determined the expression pattern of XDbf4 and XDrf1 and examined their involvement in DNA replication. We show that XDrf1 expression is restricted to oogenesis and early embryos, whereas XDbf4 is expressed throughout development. Immunodepletion from Xenopus egg extracts indicated that both proteins are only found in complexes with XCdc7 and there is a 5-fold molar excess of the XCdc7/Drf1 over SCdc7/Dbf4 complexes. Both complexes exhibit kinase activity and are differentially phosphorylated during the cell cycle. Depletion of the XCdc7/Drf1 from egg extracts inhibited DNA replication, whereas depletion of XCdc7/Dbf4 had little effect. Chromatin binding studies indicated that XCdc7/Drf1 is required for pre-replication complex activation but not their assembly. XCdc7/Dbf4 complexes bound to the chromatin in two steps: the first step was independent of pre-replication complex assembly and the second step was dependent on pre-replication complex activation. By contrast, binding of XCdc7/Drf1 complexes was entirely dependent on pre-replication complex assembly. Finally, we present evidence that the association of the two complexes on the chromatin is not regulated by ATR checkpoint pathways that result from DNA replication blocks. These data suggest that Cdc7/Drf1 but not Cdc7/Dbf4 complexes support the initiation of DNA replication in Xenopus egg extracts and during early embryonic development.     

Regulation of the Rab5 GTPase-activating protein RN-tre by the dual specificity phosphatase Cdc14A in human cells

The Cdc14 family of dual specificity phosphatases regulates key mitotic events in the eukaryotic cell cycle. Although extensively characterized in yeast, little is known about the function of mammalian Cdc14 family members. Here we report a genetic substrate-trapping system designed to identify substrates of the human Cdc14A (hCdc14A) phosphatase. Using this approach, we identify RN-tre, a GTPase-activating protein for the Rab5 GTPase, as a novel physiological target of hCdc14A. As a Rab5 GTPase-activating protein, RN-tre has previously been implicated in control of intracellular membrane trafficking. We find that RN-tre forms a stable complex with the catalytically inactive hCdc14A C278S mutant but not with the wild type protein in human cells, indicative of a substrate/enzyme interaction. In support, we show that RN-tre is regulated by cell cycle-dependent phosphorylation peaking at mitosis, which can be antagonized by hCdc14A activity in vitro as well as in vivo. Furthermore, we show that RN-tre phosphorylation is critical for efficient hCdc14A association and that RN-tre binding can be displaced by tungstate, a competitive inhibitor that binds to the active site of hCdc14A. Consistent with the preference of hCdc14A for phosphorylations mediated by proline-directed kinases, we find that RN-tre is a direct substrate of cyclin-dependent kinase. Finally, phosphorylation of RN-tre appears to finely modulate its catalytic activity. Our findings reveal a novel connection between the cell cycle machinery and the endocytic pathway.     

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.