DNA replication of mitotic chromatin in Xenopus egg extracts

Prereplication complexes are assembled at eukaryotic origins of DNA replication in the G1 phase of the cell cycle, and they are activated in S phase by cyclin-dependent kinase (Cdk)2/cyclin E and Cdk2/cyclin A. Previous experiments using Xenopus nuclear assembly egg extracts suggested that Cdk1/cyclin A, which is normally active in early mitosis, can replace the function of Cdk2 in driving DNA replication, whereas Cdk1/cyclin B, which functions later in mitosis, cannot. Here, we use a completely soluble replication system derived from Xenopus egg extracts to show that Cdk1/cyclin B also can support DNA replication. The ability of mitotic Cdks to drive DNA replication raises the question of whether DNA replication is possible in mitosis. To address this question, chromatin containing prereplication complexes was driven into mitosis with Cdk1/cyclin B. Strikingly, upon addition of a replication extract, the chromatin underwent a complete round of DNA replication. Replicating mitotic chromosomes became visibly decondensed, and, after DNA replication was complete, they recondensed. Our results indicate that there is extensive overlap in the substrate specificity of the major metazoan Cdk/cyclin complexes and that mitosis is not fundamentally incompatible with DNA replication. The results suggest that origins that fail to initiate DNA replication in S phase might still be able to do so in mitosis.     

Cell division cycle 6, a mitotic substrate of polo-like kinase 1, regulates chromosomal segregation mediated by cyclin-dependent kinase 1 and separase

Defining the links between cell division and DNA replication is essential for understanding normal cell cycle progression and tumorigenesis. In this report we explore the effect of phosphorylation of cell division cycle 6 (Cdc6), a DNA replication initiation factor, by polo-like kinase 1 (Plk1) on the regulation of chromosomal segregation. In mitosis, the phosphorylation of Cdc6 was highly increased, in correlation with the level of Plk1, and conversely, Cdc6 is hypophosphorylated in Plk1-depleted cells, although cyclin A- and cyclin B1-dependent kinases are active. Binding between Cdc6 and Plk1 occurs through the polo-box domain of Plk1, and Cdc6 is phosphorylated by Plk1 on T37. Immunohistochemistry studies reveal that Cdc6 and Plk1 colocalize to the central spindle in anaphase. Expression of T37V mutant of Cdc6 (Cdc6-TV) induces binucleated cells and incompletely separated nuclei. Wild-type Cdc6 but not Cdc6-TV binds cyclin-dependent kinase 1 (Cdk1). Expression of wild-type Plk1 but not kinase-defective mutant promotes the binding of Cdc6 to Cdk1. Cells expressing wild-type Cdc6 display lower Cdk1 activity and higher separase activity than cells expressing Cdc6-TV. These results suggest that Plk1-mediated phosphorylation of Cdc6 promotes the interaction of Cdc6 and Cdk1, leading to the attenuation of Cdk1 activity, release of separase, and subsequent anaphase progression.     

Multistep regulation of DNA replication by Cdk phosphorylation of HsCdc6

We have characterized HsCdc6, a human protein homologous to the budding yeast Cdc6p that is essential for DNA replication. We show that, unlike Cdc6p, the levels of HsCdc6 protein remain constant throughout the cell cycle in human cells. However, phosphorylation of HsCdc6 is regulated during the cell cycle. HsCdc6 is an excellent substrate for Cdk2 in vitro and is phosphorylated in vivo at three sites (Ser-54, Ser-74, and Ser-106) that are phosphorylated by Cdk2 in vitro, strongly suggesting that HsCdc6 is an in vivo Cdk substrate. HsCdc6 is nuclear in G1, but translocates to the cytoplasm at the start of S phase via Crm1-dependent export. An HsCdc6A1A2A3 mutant, which mimics unphosphorylated HsCdc6, is exclusively nuclear, and its expression inhibits initiation of DNA replication. An HsCdc6E1E2E3 mutant, which mimics phosphorylated HsCdc6, is exclusively cytoplasmic and is not associated with the chromatin/nuclear matrix fraction. Based on these results, we propose that phosphorylation of HsCdc6 by Cdks regulates DNA replication of at least two steps: first, by promoting initiation of DNA replication and, second, through nuclear exclusion preventing DNA rereplication.     

Binding of cyclin-dependent kinases to ORC and Cdc6p regulates the chromosome replication cycle

Cdc6p and the origin recognition complex (ORC) are essential for assembly of a pre-replicative complex (preRC) at origins of replication, before the initiation of DNA synthesis. In the absence of Cdc6p, cells fail to initiate DNA replication and undergo a "reductional" mitosis, in which the unreplicated chromosomes are randomly segregated to the spindle poles. We show here that the cells harboring a mutation in the essential Cdc6p Walker A-box arrest in late mitosis, probably at anaphase. This cell cycle block requires either the three Cdc28p phosphorylation sites within the N terminus of Cdc6p or a short region (aa 8-17) that contains a Cy (Cyclin) interaction sequence. These same two Cdc6p mutants that allow a reductional mitosis are defective in binding Cdc28p kinase. In addition to Cdc6p, ORC also binds to cyclin-dependent kinases (CDKs). Interestingly, Sic1p, a CDK inhibitor protein, blocked the S phase-specific Cdc28p-Clb5p kinase from interacting with ORC, but did not prevent the G(1)-specific Cdc28p-Cln2p kinase-ORC interaction. We suggest that ORC, Cdc6p, and Sic1p bind to different CDKs in a cell cycle-dependent manner to temporally regulate events that (i) allow preRC formation after mitosis, (ii) prevent mitosis before DNA replication can occur, and (iii) promote initiation of DNA replication.     

Stepwise assembly of initiation proteins at budding yeast replication origins in vitro

The initiation of DNA replication in the budding yeast Saccharomyces cerevisiae occurs in two sequential and mutually exclusive steps. Prereplicative complexes (pre-RCs) containing origin recognition complex (ORC), Cdc6p, and the MCM2-7 proteins assemble only under conditions of low cyclin-dependent kinase (Cdk) activity during G(1), whereas origin activation is driven by the increase in Cdk activity at the end of G(1). As a first step toward the reconstitution of this two-step process in vitro, we describe a system in which extracts prepared from G(1)-arrested cells promote sequential assembly of ORC, Cdc6p, and MCM2-7 proteins onto exogenously added origin-containing DNA. This reaction requires an intact ARS consensus sequence and requires ATP for two distinct steps. Extracts from cells arrested in mitosis also can support the binding of ORC but are unable to load either Cdc6p or MCM2-7 proteins. This system should be useful for studying the mechanism and regulation of pre-RC assembly.     

Analysis of Cdc6 function in the assembly of mammalian prereplication complexes

Eukaryotic DNA replication requires the previous formation of a prereplication complex containing the ATPase Cdc6 and the minichromosome maintenance (Mcm) complex. Although considerable insight has been gained from in vitro studies and yeast genetics, the functional analysis of replication proteins in intact mammalian cells has been lacking. We have made use of adenoviral vectors to express normal and mutant forms of Cdc6 in quiescent mammalian cells to assess function. We demonstrate that Cdc6 expression alone is sufficient to induce a stable association of endogenous Mcm proteins with chromatin in serum-deprived cells where cyclin-dependent kinase (cdk) activity is low. Moreover, endogenous Cdc6 is sufficient to load Mcm proteins onto chromatin in the absence of cdk activity in p21-arrested cells. Cdc6 synergizes with physiological levels of cyclin E/Cdk2 to induce semiconservative DNA replication in quiescent cells whereas cyclin A/Cdk2 is unable to collaborate with Cdc6. Cdc6 that cannot be phosphorylated by cdks is fully capable of inducing Mcm chromatin association and replication. Mutation of the Cdc6 ATP-binding site severely impairs the ability of Cdc6 to induce Mcm chromatin loading and reduces its ability to induce replication. Nevertheless, the ATPase domain of Cdc6 in the absence of the noncatalytic amino terminus is not sufficient for either Mcm chromatin loading or DNA replication, indicating a requirement for this domain of Cdc6.     

The Cdc6p nucleotide-binding motif is required for loading Mcm proteins onto chromatin

Cdc6p has an essential function in the mechanism and regulation of the initiation of DNA replication. Budding yeast Cdc6p binds to chromatin near autonomously replicating sequence elements in late M to early G1 phase through an interaction with Origin Recognition Complex or another origin-associated factor. It then facilitates the subsequent loading of the Mcm family of proteins near autonomously replicating sequence elements by an unknown mechanism. All Cdc6p homologues contain a bipartite Walker ATP-binding motif that suggests that ATP binding or hydrolysis may regulate Cdc6p activity. To determine whether these motifs are important for Cdc6p activity, mutations were made in conserved residues of the Walker A and B motifs. Substitution of lysine 114 to alanine (K114A) in the Walker A motif results in a temperature-sensitive phenotype in yeast and slower progression into S phase at the permissive temperature. A K114E mutation is lethal. The Cdc6^K114E protein binds to chromatin but fails to promote loading of the Mcm proteins, suggesting that ATP binding is essential for this activity. The mutant arrests with a G1 DNA content but retains the ability to restrain mitosis in the absence of DNA replication, unlike depletion of Cdc6p. In contrast, Cdc6p containing a double alanine mutation in the Walker B motif, DE(223, 224)AA, is functional, and the mutant exhibits an apparently normal S phase. These results suggest that Cdc6p nucleotide binding is important for establishing the prereplicative complex at origins of DNA replication and that the amino terminus of Cdc6p is required for blocking entry into mitosis.     

The p21(Cip1) protein, a cyclin inhibitor, regulates the levels and the intracellular localization of CDC25A in mice regenerating livers

Liver cells from p21(Cip1-/-) mice subjected to partial hepatectomy (PH) progress into DNA synthesis faster than those from wild-type mice. These cells also show a premature induction of cyclin E/cyclin-dependent kinase (CDK) 2 activity. We studied the mechanisms whereby cells lacking p21(Cip1) showed a premature induction of this activity. Whereas the levels of CDK2, cyclin E, and p27(Kip1) were similar in both wild-type and p21(Cip1-/-) mice, those of the activator CDC25A were much higher in p21(Cip1-/-) quiescent and regenerating livers than in wild-type animals. Moreover, p21(Cip1-/-) cells also showed a premature translocation of CDC25A from cytoplasm into the nucleus. The ectopic expression of p21(Cip1) into mice embryo fibroblasts from p21(Cip1-/-) mice decreased the levels of CDC25A and delayed its nuclear translocation. The levels of CDC25A messenger RNA in p21(Cip1-/-) cells were higher than in wild-type cells, suggesting that this increase might be responsible, at least in part, for the high levels of CDC25A protein in these cells. Thus, the results reported here indicate that p21(Cip1) regulates the levels and the intracellular localization of CDC25A. We also found a good correlation between CDC25A nuclear translocation and cyclin E/CDK2 activation. In conclusion, premature translocation of CDC25A to the nucleus might be involved in the advanced induction of cyclin E/CDK2 activity and DNA replication in cells from animals lacking p21(Cip1).     

Cloning and characterization of the Xenopus cyclin-dependent kinase inhibitor p27XIC1.

We have isolated a gene encoding Xic-1, a 27-kDa cyclin-dependent kinase (Cdk) inhibitor from Xenopus ovary that shares significant homology with both mammalian CIP1 and Kip1/Kip2. The N- and C-terminal halves of Xic-1 are sufficient for interacting with Cdks and proliferating cell nuclear antigen, respectively. Recombinant Xic-1 inhibits Xenopus cyclin E/Cdk2, cyclin A/Cdk2 and cyclin B/Cdc2 activities, although with quite different IC50 values. Truncation of the N terminus of Xic-1 increases the IC50 value for cyclin A/Cdk2 50-fold with no effect on the inhibition of cyclin E/Cdk2 or cyclin B/Cdc2.Xic-1 inhibits both single-stranded and nuclear DNA synthesis in egg extracts, an effect reversed by proliferating cell nuclear antigen or cyclin E/Cdk2, respectively. These results suggest a function for Xic-1 in the control of DNA synthesis by cyclin E/Cdk2.     

Cdc6p-dependent loading of Mcm proteins onto pre-replicative chromatin in budding yeast

The Cdc6 protein is essential for the assembly of pre-replicative complexes (pre-RCs) at origins of DNA replication in the budding yeast Saccharomyces cerevisiae. This reaction is blocked in vivo by the cyclin-dependent kinase Cdc28p, together with its regulatory subunits, the B type cyclins that are present throughout S, G₂, and M phases. Because the destruction of B type cyclins and the consequent inactivation of the kinase are essential for exit from mitosis, pre-RC formation can only occur after passage through mitosis. Therefore, pre-RC formation has been proposed to be essential for coupling S phase and mitosis and for limiting DNA replication to once per cell cycle. The Mcm2–7 family of proteins has been implicated in limiting replication to once per cell cycle from experiments with Xenopus egg extracts. Here we show that the Mcm proteins of budding yeast are abundant and are quantitatively found in a chromatin-enriched fraction specifically during the G₁ phase of the cell cycle. This chromatin binding depends on the de novo synthesis of Cdc6p, providing evidence that a conserved biochemical pathway plays a critical role in coordinating DNA replication with mitosis in both yeast and higher eukaryotes. Cdc6p and the origin recognition complex can be selectively removed from this chromatin-enriched fraction without removing the Mcm proteins. From these results, we propose that Cdc6p (and the origin recognition complex) nucleates the binding of Mcm proteins to chromatin, but once bound, the Mcm proteins appear to interact tightly with some other component of chromatin.     

The forkhead box M1 transcription factor contributes to the development and growth of mouse colorectal cancer

BACKGROUND & AIMS: In this study, we used Forkhead Box m1b (Foxm1b) transgenic mice and conditional Foxm1 knock-out mice to examine the role of Foxm1 in colon cancer development and proliferation. METHODS: To induce mouse colorectal cancer, we used a single intraperitoneal injection of azoxymethane (AOM) followed by three 1-week cycles of 2.5% dextran sodium sulfate (DSS) water, each cycle separated by 2 weeks. For these colon tumor studies, we used either Rosa26-Foxm1b transgenic mice that ubiquitously expressed the human Foxm1b complementary DNA or mice in which the Foxm1 fl/fl targeted allele was deleted in colonic epithelial cells using the gut-specific Villin-Cre recombinase transgene (Villin-Cre). Colorectal tumor number and bromodeoxyuridine labeling were determined in Rosa26-Foxm1b mice, Villin-Cre Foxm1-/-, mice and wild-type mice after 12 weeks of AOM/DDS exposure. We also used Foxm1 small interfering RNA-depleted human DLD1 and mouse CT26 colon cancer cell lines to examine DNA replication and anchorage-independent growth. RESULTS: After 12 weeks of treatment with AOM/DSS, Rosa26 Foxm1b transgenic mice showed an increase in the number and size of colorectal tumors compared with wild-type mice. Likewise, a significant reduction in the development and growth of colorectal tumors was found in Villin-Cre Foxm1-/- mice compared with Foxm1 fl/fl mice after AOM/DSS treatment, which was associated with decreased expression of cyclin A2, cyclin B1, survivin, and T-cell factor 4 genes. Moreover, Foxm1-depleted colon cancer cell lines showed reduced DNA replication and anchorage-independent growth. CONCLUSIONS: These studies suggest that Foxm1 is critical for the proliferation and growth of colorectal cancer.     

Multisite phosphoregulation of Cdc25 activity refines the mitotic entrance and exit switches

Cyclin-dependent kinase 1 (Cdk1) kinase dephosphorylation and activation by Cdc25 phosphatase are essential for mitotic entry. Activated Cdk1 phosphorylates Cdc25 and other substrates, further activating Cdc25 to form a positive feedback loop that drives the abrupt G2/mitosis switch. Conversely, mitotic exit requires Cdk1 inactivation and reversal of Cdk1 substrate phosphorylation. This dephosphorylation is mediated, in part, by Clp1/Cdc14, a Cdk1-antagonizing phosphatase, which reverses Cdk1 phosphorylation of itself, Cdc25, and other Cdk1 substrates. Thus, Cdc25 phosphoregulation is essential for proper G2-M transition, and its contributions to cell cycle control have been modeled based on studies using Xenopus and human cell extracts. Because cell extract systems only approximate in vivo conditions where proteins interact within dynamic cellular environments, here, we use Schizosaccharomyces pombe to characterize, both experimentally and mathematically, the in vivo contributions of Cdk1-mediated phosphorylation of Cdc25 to the mitotic transition. Through comprehensive mapping of Cdk1 phosphosites on Cdc25 and characterization of phosphomutants, we show that Cdc25 hyperphosphorylation by Cdk1 governs Cdc25 catalytic activation, the precision of mitotic entry, and unvarying cell length but not Cdc25 localization or abundance. We propose a mathematical model that explains Cdc25 regulation by Cdk1 through a distributive and disordered phosphorylation mechanism that ultrasensitively activates Cdc25. We also show that Clp1/Cdc14 dephosphorylation of Cdk1 sites on Cdc25 controls the proper timing of cell division, a mechanism that is likely due to the double negative feedback loop between Clp1/Cdc14 and Cdc25 that controls the abruptness of the mitotic exit switch.