The cyclin-dependent kinase (Cdk) inhibitor p21 is induced by the tumor suppressor p53 and is required for the G 1 -S block in cells with DNA damage. We report that there are two copies of a cyclin-binding motif in p21, Cy1 and Cy2, which interact with the cyclins independently of Cdk2. The cyclin-binding motifs of p21 are required for optimum inhibition of cyclin-Cdk kinases in vitro and for growth suppression in vivo. Peptides containing only the Cy1 or Cy2 motif partially inhibit cyclin-Cdk kinase activity in vitro and DNA replication in Xenopus egg extracts. A monoclonal antibody which recognizes the Cy1 site of p21 specifically disrupts the association of p21 with cyclin E-Cdk2 and with cyclin D1-Cdk4 in cell extracts. Taken together, these observations suggest that the cyclin-binding motif of p21 is important for kinase inhibition and for formation of p21-cyclin-Cdk complexes in the cell. Finally, we show that the cyclin-Cdk complex is partially active if associated with only the cyclin-binding motif of p21, providing an explanation for how p21 is found associated with active cyclin-Cdk complexes in vivo. The Cy sequences may be general motifs used by Cdk inhibitors or substrates to interact with the cyclin in a cyclin-Cdk complex.The periodic activation and inactivation of cyclin-dependent kinases (Cdks) is essential for the progression of a cell through each phase of the cell cycle (29, 39). The activity of a given Cdk is regulated by synthesis of the corresponding cyclin in specific stages of the cycle and by posttranslational modifications on the Cdk subunit. Inactivation of the kinase is regulated by destruction of the cyclin, by posttranslational modifications on the Cdk subunit, and by newly discovered inhibitors which associate with the cyclin-Cdk. There are two such related families of inhibitors. The first family includes p21 (7, 10, 14, 32, 45), p27 (16, 35, 36, 43), and p57 (23, 28) and acts on a wide range of Cdks. The second family of inhibitors consists of p15, p16, and p18 (11,13,37) and specifically inhibits the cyclin D-dependent G 1 kinases Cdk4 and Cdk6.

In a two-hybrid screen for proteins that interact with human PCNA, we identified and cloned a human protein (hCdc18) homologous to yeast CDC6/Cdc18 and human Orc1. Unlike yeast, in which the rapid and total destruction of CDC6/Cdc18 protein in S phase is a central feature of DNA replication, the total level of the human protein is unchanged throughout the cell cycle. Epitope-tagged protein is nuclear in G 1 and cytoplasmic in S-phase cells, suggesting that DNA replication may be regulated by either the translocation of this protein between the nucleus and the cytoplasm or the selective degradation of the protein in the nucleus. Mutation of the only nuclear localization signal of this protein does not alter its nuclear localization, implying that the protein is translocated to the nucleus through its association with other nuclear proteins. Rapid elimination of the nuclear pool of this protein after the onset of DNA replication and its association with human Orc1 protein and cyclin-cdks supports its identification as human CDC6/Cdc18 protein.The recent identification of multiple eukaryotic proteins that bind directly or indirectly to origins of DNA replication has set the stage for a thorough exploration of how DNA replication is initiated and regulated in eukaryotes. Central to the process is the origin recognition complex (ORC), a tight complex of six polypeptides identified initially in Saccharomyces cerevisiae because it binds in a sequence-specific manner to origins of DNA replication and is involved in the initiation of chromosomal DNA replication (1,3,27). Homologs of three of the subunits of the ORC (Orc1, Orc2, and Orc4) have been identified in humans (16,32) and in other eukaryotes (14,17,22,28).An additional molecule, CDC6 in S. cerevisiae and Cdc18 in Schizosaccharomyces pombe, is essential for the onset of DNA replication and known to associate physically with the ORC and cdc2 kinase (2,12,15,18,22,24,37). The yeast CDC6/ Cdc18 proteins decrease in concentration as cells proceed through S phase, with overexpression of Cdc18 in S. pombe resulting in the rereplication of DNA in G 2 (29). The Xenopus CDC6/Cdc18 and ORC are required for DNA replication and for the loading of the minichromosome maintenance (MCM) proteins onto the chromatin (6,11,13,19,34,35). Collectively, a model has emerged that emphasizes the central role of CDC6/Cdc18 in cooperating with ORC and MCM proteins to form a prereplication complex at origins of DNA replication in G 1 . Once replication begins, the concordant removal of CDC6/ Cdc18 prevents the loading of MCM proteins onto the originbound ORC in G 2 , thereby preventing rereplication of DNA. After mitosis, synthesis of new CDC6/Cdc18 protein allows the loading of MCM proteins on the chromatin, perhaps by forming a bridge between the chromatin-bound ORC and the MCM proteins (4). The prereplication initiation complex thus formed in G 1 is ready to initiate DNA replication upon the activation of S-phase-promoting factors like cyclin-cdk's and CDC7 kinase.We identified and cloned...

Fenl or maturation factor 1 is a 5'-3' exonuclease essential for the degradation of the RNA primer-

Cdc25A, a phosphatase essential for G 1 -S transition, associates with, dephosphorylates, and activates the cell cycle kinase cyclin E-cdk2. p21CIP1 and p27 are cyclin-dependent kinase (cdk) inhibitors induced by growth-suppressive signals such as p53 and transforming growth factor ␤ (TGF-␤). We have identified a cyclin binding motif near the N terminus of Cdc25A that is similar to the cyclin binding Cy (or RR LFG) motif of the p21 CIP1 family of cdk inhibitors and separate from the catalytic domain. Mutations in this motif disrupt the association of Cdc25A with cyclin E-or cyclin A-cdk2 in vitro and in vivo and selectively interfere with the dephosphorylation of cyclin E-cdk2. A peptide based on the Cy motif of p21 competitively disrupts the association of Cdc25A with cyclin-cdks and inhibits the dephosphorylation of the kinase. p21 inhibits Cdc25A-cyclin-cdk2 association and the dephosphorylation of cdk2. Conversely, Cdc25A, which is itself an oncogene up-regulated by the Myc oncogene, associates with cyclin-cdk and protects it from inhibition by p21. Cdc25A also protects DNA replication in Xenopus egg extracts from inhibition by p21. These results describe a mechanism by which the Myc-or Cdc25A-induced oncogenic and p53-or TGF-␤-induced growth-suppressive pathways counterbalance each other by competing for cyclin-cdks.The eukaryotic cell division cycle is regulated by various phosphorylation and dephosphorylation events. The key phosphorylation events during the cell cycle are carried out by cyclin-dependent kinases (cdks) (9,20,31,35,39). The cdks are positively regulated by the binding of appropriate cyclin molecules (7, 10, 32) and by stimulatory phosphorylation by cdk-activating kinase (CAK) at a conserved threonine residue (Thr-160 of cdk2) (37). The activities of cdks are also modulated by inhibitory mechanisms. There are two families of cdk inhibitors which negatively regulate kinase activities (29). The first family consists of p21, p27, and p57 and acts on a wide range of cyclin-cdk complexes. The second group includes p15, p16, and p18 and inhibits only cyclin D-dependent G 1 kinases cdk4 and cdk6. Phosphorylation of conserved threonine and tyrosine residues near the ATP binding sites of cdks (Thr-14 and Tyr-15 on cdk2) (14) by wee1 and mik1 protein kinases is another important mechanism employed to keep the cdks inactive. In the fission yeast Schizosaccharomyces pombe there is only one known type of phosphatase, Cdc25, which removes the inhibitory phosphate groups from Cdc2 (CDC28) during G 1 -S and G 2 -M transitions (28). In human and murine cells, there are three known CDC25 genes (CDC25A, CDC25B, and CDC25C) (11,30,34). The three phosphatases share approximately 40 to 50% homology at the amino acid level. CDC25C and CDC25A function at G 2 -M and G 1 -S transitions during the human cell cycle, respectively (17-19). Cdc25C dephosphorylates Cdc2 in the Cdc2-cyclin B complex and activates its histone H1 kinase. The phosphatase activities of Cdc25 proteins are, in turn, regulated by phosphorylation....

CDC6 is a protein essential for DNA replication, the expression and abundance of which are cell cycle-regulated in Saccharomyces cerevisiae. We have demonstrated previously that the subcellular localization of the human CDC6 homolog, HsCDC6, is cell cycledependent: nuclear during G 1 phase and cytoplasmic during S phase. Here we demonstrate that endogenous HsCDC6 is phosphorylated during the G 1 /S transition. The N-terminal region contains putative cyclin-dependent kinase phosphorylation sites adjoining nuclear localization sequences (NLSs) and a cyclin-docking motif, whereas the C-terminal region contains a nuclear export signal (NES). In addition, we show that the observed regulated subcellular localization depends on phosphorylation status, NLS, and NES. When the four putative substrate sites (serines 45, 54, 74, and 106) for cyclin-dependent kinases are mutated to alanines, the resulting HsCDC6A4 protein is localized predominantly to the nucleus. This localization depends upon two functional NLSs, because expression of HsCDC6 containing mutations in the two putative NLSs results in predominantly cytoplasmic distribution. Furthermore, mutation of the four serines to phosphate-mimicking aspartates (HsCDC6D4) results in strictly cytoplasmic localization. This cytoplasmic localization depends upon the C-terminal NES. Together these results demonstrate that HsCDC6 is phosphorylated at the G 1 /S phase of the cell cycle and that the phosphorylation status determines the subcellular localization.Little is known regarding the regulation of DNA replication initiation in mammalian cells. In yeast, the origin recognition complex, required for replication initiation, consists of six subunits and is associated with specific DNA sequences (replicators) (1-3). In addition, other factors are required for DNA replication initiation including CDC6, CDC45, and MCM (mini-chromosome maintenance) family proteins (4). Several lines of evidence suggest that ORC, 1 CDC6, and MCM may function together as the replication initiator complex (reviewed in Refs. 5 and 6). Recently several human proteins have been identified that seem to be structural homologs of proteins known to be directly involved in DNA replication in yeast (7)(8)(9)(10)(11)(12)(13)(14). CDC6 in Saccharomyces cerevisiae, Cdc18 in Schizosaccharomyces pombe, and XCDC6 in Xenopus are homologs and have been shown to be essential for DNA replication. For example, in S. cerevisiae the assembly of a prereplication initiation complex at origins of replication requires CDC6 (15). In both S. cerevisiae and S. pombe, the CDC6/Cdc18 protein is degraded at the G 1 /S transition after phosphorylation by cdk (16, 17). In S. pombe, overexpression of Cdc18 results in re-replication without mitosis (16), whereas specific mutations in S. cerevisiae CDC6 cause over-replication of DNA (18). Together these results demonstrate the importance of CDC6 in DNA replication and suggest that stringent regulation of CDC6 protein levels, such that it is active and/or available in G 1 but destroyed s...

The cell cycle regulator p21 interacts with and inhibits the DNA replication and repair factor proliferating cell nuclear antigen (PCNA). We have defined a 39 amino acid fragment of p21 which is sufficient to bind PCNA with high affinity (Kd 10-20 nM). This peptide can inhibit DNA replication in vitro and microinjection of a GST fusion protein containing this domain inhibited S phase in vivo. Despite its high affinity for PCNA, the free 39 amino acid peptide does not have a well-defined structure, as judged from circular dichroism and nuclear magnetic resonance measurements, suggesting an induced fit mechanism for the PCNA-p21 interaction. The association of the small peptide with PCNA was thermolabile, suggesting that portions of p21 adjoining the minimal region of contact stabilize the interaction. In addition, a domain containing 67 amino acids from the N-terminus of PCNA was defined as both necessary and sufficient for binding to p21.

A new member of human origin recognition complex (ORC) has been cloned and identified as the human homologue of Saccharomyces cerevisiae ORC4. HsORC4 is a 45-kDa protein encoded by a 2.2-kilobase mRNA whose amino acid sequence is 29% identical to ScORC4. Hs-ORC4 has a putative nucleotide triphosphate binding motif that is not seen in ScORC4. HsORC4P also reveals an unsuspected homology to the ORC1-Cdc18 family of proteins. HsORC4 mRNA expression and protein levels remain constant through the cell cycle. HsORC4P is coimmunoprecipitated from cell extracts with another subunit of human ORC, HsORC2P, consistent with it being a part of the putative human origin recognition complex.Initiation of eukaryotic DNA replication involves the controlled and simultaneous firing of numerous sites of initiation. In the budding yeast Saccharomyces cerevisiae, these sites are defined by specific sequences recognized by a multisubunit complex, the origin recognition complex (ORC) 1 (1-5). All six members of ORC identified in yeast are essential for cell viability (3, 6 -11). ORC, in its pre-replicative or in its post-replicative form, is bound to DNA throughout the cell-cycle (1, 2) and could act as a platform for the recruitment of other proteins involved in the replication machinery.One of the proteins believed to be recruited by ORC before the initiation of DNA replication is the CDC6/Cdc18 (S. cerevisiae or Schizosaccharomyces pombe) protein. CDC6/Cdc18 is closely related in sequence to one of the subunits of ORC, ORC1, over a region that includes a putative nucleotide binding motif. Yeast ORC has been demonstrated to utilize ATP for binding to DNA and to have an ATPase activity that is modulated by binding to the origin of DNA replication (1, 12), suggesting that like DNA replication initiator proteins in Escherichia coli (DnaA) or the simian virus 40 (T antigen), ATP binding and hydrolysis by the eukaryotic initiator protein will be an important regulator of the initiation process.Although DNA sequences defining an origin of replication have not yet been identified in higher eukaryotes, two members of a putative ORC complex homologous to yeast ORC1 and ORC2 have been identified so far in mammals, both in humans and in mice (13,14), suggesting a universal mechanism of initiation of DNA replication in eukaryotes. We report here the identification of a novel member of human ORC homologous to S. cerevisiae ScORC4. Cloning the gene for a third member of the human origin recognition complex is an important step toward the ultimate goal of reconstituting the entire human ORC in vitro. EXPERIMENTAL PROCEDURESCloning and Sequencing-In the Expressed Sequence Tag (EST) Data base (National Center for Biotechnology Information), the partial sequence of a mouse cDNA (AA168456) was deposited with significant homology to a portion of ScORC4 from S. cerevisiae. A BLAST search with the AA168456 sequence revealed a homologous sequence human EST W23942, which in its turn identified a mouse EST AA110785. A BLAST search with the latter identified ...

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