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PubMed2004

Cdt1 phosphorylation by cyclin A-dependent kinases negatively regulates its function without affecting geminin binding.

The current concept regarding cell cycle regulation of DNA replication is that Cdt1, together with origin recognition complex and CDC6 proteins, constitutes the machinery that loads the minichromosome maintenance complex, a candidate replicative helicase, onto chromatin during the G(1) phase. The actions of origin recognition complex and CDC6 are suppressed through phosphorylation by cyclin-dependent kinases (Cdks) after S phase to prohibit rereplication. It has been suggested in metazoan cells that the function of Cdt1 is blocked through binding to an inhibitor protein, geminin. However, the functional relationship between the Cdt1-geminin system and Cdks remains to be clarified. In this report, we demonstrate that human Cdt1 is phosphorylated by cyclin A-dependent kinases dependent on its cyclin-binding motif. Cdk phosphorylation resulted in the binding of Cdt1 to the F-box protein Skp2 and subsequent degradation. In contrast, in vitro DNA binding activity of Cdt1 was inhibited by the phosphorylation. However, geminin binding to Cdt1 was not affected by the phosphorylation. Finally we provide evidence that inactivation of Cdk1 results in Cdt1 dephosphorylation and rebinding to chromatin in murine FT210 cells synchronized around the G(2)/M phase. Taken together, these findings suggest that Cdt1 function is also negatively regulated by the Cdk phosphorylation independent of geminin binding.

Amino Acid Motifs↗
PubMed2007

Cdt1 and geminin: role during cell cycle progression and DNA damage in higher eukaryotes.

DNA replication in eukaryotic cells must be strictly regulated to ensure that the entire genome is duplicated only once in each cell cycle. For this purpose, the initiation of DNA replication is controlled by the "licensing" reaction, which is established by the formation of a pre-replicative complex (pre-RC) with the sequential assembly of the origin recognition complex (ORC), Cdc6, Cdt1 and Mcm2-7 onto origin regions. Among these, Cdt1 is likely the most important target for regulating licensing in higher eukaryotic cells, since illegitimate accumulation of Cdt1 causes multiple rounds of DNA replication without an intervening mitosis. Cdt1 is regulated over the course of the cell cycle mainly by the controlled expression of an inhibitor protein, geminin, and the level of Cdt1 periodically fluctuates due to ubiquitination and proteolysis. While the expression of geminin from S phase to metaphase of mitosis prevents licensing, Cdt1 accumulates from M to G1 phases and is degraded at the onset of S phase. Furthermore, Cdt1 is also proteolyzed in G1 phase in response to DNA damage, presumably providing a new checkpoint control.

Animals↗
PubMed2003

Interaction and assembly of murine pre-replicative complex proteins in yeast and mouse cells.

Eukaryotic cells coordinate chromosome duplication by the assembly of protein complexes at origins of DNA replication by sequential binding of member proteins of the origin recognition complex (ORC), CDC6, and minichromosome maintenance (MCM) proteins. These pre-replicative complexes (pre-RCs) are activated by cyclin-dependent kinases and DBF4/CDC7 kinase. Here, we carried out a comprehensive yeast two-hybrid screen to establish sequential interactions between two individual proteins of the mouse pre-RC that are probably required for the initiation of DNA replication. The studies revealed multiple interactions among ORC subunits and MCM proteins as well as interactions between individual ORC and MCM proteins. In particular CDC6 was found to bind strongly to ORC1 and ORC2, and to MCM7 proteins. DBF4 interacts with the subunits of ORC as well as with MCM proteins. It was also demonstrated that CDC7 binds to different ORC and MCM proteins. CDC45 interacts with ORC1 and ORC6, and weakly with MCM3, -6, and -7. The three subunits of the single-stranded DNA binding protein RPA show interactions with various ORC subunits as well as with several MCM proteins. The data obtained by yeast two-hybrid analysis were paradigmatically confirmed in synchronized murine FM3A cells by immunoprecipitation of the interacting partners. Some of the interactions were found to be cell-cycle-dependent; however, most of them were cell-cycle-independent. Altogether, 90 protein-protein interactions were detected in this study, 52 of them were found for the first time in any eukaryotic pre-RC. These data may help to understand the complex interplay of the components of the mouse pre-RC and should allow us to refine its structural architecture as well as its assembly in real time.

Animals↗
PubMed1996

Interaction of Cdc2 and Cdc18 with a fission yeast ORC2-like protein.

In fission yeast, Cdc2 kinase has both positive and negative roles in regulating DNA replication, being first necessary for the transition from G1 to S phase and later required to prevent the re-initiation of DNA replication during G2. We report here that Cdc2 interacts with Orp2, a protein similar to the Orc2 replication factor subunit of Saccharomyces cerevisiae origin recognition complex (ORC). ORC binds chromosomal origins and is essential for chromosomal replication initiation. Fission yeast Orp2 is required for DNA replication and interacts with the rate-limiting replication activator Cdc18. Cells lacking Orp2 undergo aberrant mitosis, indicating that Orp2 is involved in generating a checkpoint signal. These findings suggest that ORC functions are conserved among eukaryotes and provide evidence that Cdc2 controls DNA replication initiation by acting directly at chromosomal origins.

Amino Acid Sequence↗
PubMed2003

The ORC1 cycle in human cells: II. Dynamic changes in the human ORC complex during the cell cycle.

The origin recognition complex (ORC) plays a central role in regulating the initiation of DNA replication in eukaryotes. The level of the ORC1 subunit oscillates throughout the cell cycle, defining an ORC1 cycle. ORC1 accumulates in G1 and is degraded in S phase, although other ORC subunits (ORCs 2-5) remain at almost constant levels. The behavior of ORC components in human cell nuclei with respect to the ORC1 cycle demonstrates that ORCs 2-5 form a complex that is present throughout the cell cycle and that associates with ORC1 when it accumulates in G1 nuclei. ORCs 2-5 are found in both nuclease-insoluble and -soluble fractions. The appearance of nuclease-insoluble ORCs 2-5 parallels the increase in the level of ORC1 associating with nuclease-insoluble, non-chromatin nuclear structures. Thus, ORCs 2-5 are temporally recruited to nuclease-insoluble structures by formation of the ORC1-5 complex. An artificial reduction in the level of ORC1 in human cells by RNA interference results in a shift of ORC2 to the nuclease-soluble fraction, and the association of MCM proteins with chromatin fractions is also blocked by this treatment. These results indicate that ORC1 regulates the status of the ORC complex in human nuclei by tethering ORCs 2-5 to nuclear structures. This dynamic shift is further required for the loading of MCM proteins onto chromatin. Thus, the pre-replication complex in human cells may be regulated by the temporal accumulation of ORC1 in G1 nuclei.

Amino Acid Sequence↗
PubMed1997

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.

Cell Cycle↗
PubMed2002

Functions of sensor 1 and sensor 2 regions of Saccharomyces cerevisiae Cdc6p in vivo and in vitro.

Cdc6p is a key regulator of the cell cycle in eukaryotes and is a member of the AAA(+) (ATPases associated with a variety of cellular activities) family of proteins. In this family of proteins, the sensor 1 and sensor 2 regions are important for their function and ATPase activity. Here, site-directed mutagenesis has been used to examine the role of these regions of Saccharomyces cerevisiae Cdc6p in controlling the cell cycle progression and initiation of DNA replication. Two important amino acid residues (Asn(263) in sensor 1 and Arg(332) in sensor 2) were identified as key residues for Cdc6p function in vivo. Cells expressing mutant Cdc6p (N263A or R332E) grew slowly and accumulated in the S phase. In cells expressing mutant Cdc6p, loading of the minichromosome maintenance (MCM) complex of proteins was decreased, suggesting that the slow progression of S phase in these cells was due to inefficient MCM loading on chromatin. Purified wild type Cdc6p but not mutant Cdc6p (N263A and R332E) caused the structural modification of origin recognition complex proteins. These results are consistent with the idea that Cdc6p uses its ATPase activity to change the conformation of origin recognition complex, and then together they recruit the MCM complex.

Amino Acid Sequence↗
PubMed2002

Site-specific ORC binding, pre-replication complex assembly and DNA synthesis at Schizosaccharomyces pombe replication origins.

Previous studies have shown that the Schizo saccharomyces pombe Orc4 subunit is solely responsible for in vitro binding of origin recognition complex (ORC) to specific AT-rich sites within S.pombe replication origins. Using ARS3001, a S.pombe replication origin consisting of four genetically required sites, we show that, in situ as well as in vitro, Orc4 binds strongly to the Delta3 site, weakly to the Delta6 site and not at all to the remaining sequences. In situ, the footprint over Delta3 is extended during G(1) phase, but only when Cdc18 is present and Mcm proteins are bound to chromatin. Moreover, this footprint extends into the adjacent Delta2 site, where leading strand DNA synthesis begins. Therefore, we conclude that ARS3001 consists of a single primary ORC binding site that assembles a pre-replication complex and initiates DNA synthesis, plus an additional novel origin element (Delta9) that neither binds ORC nor functions as a centromere, but does bind an as yet unidentified protein throughout the cell cycle. Schizosaccharomyces pombe may be an appropriate paradigm for the complex origins found in the metazoa.

Base Composition↗
PubMed2001

Genome-wide distribution of ORC and MCM proteins in S. cerevisiae: high-resolution mapping of replication origins.

DNA replication origins are fundamental to chromosome organization and duplication, but understanding of these elements is limited because only a small fraction of these sites have been identified in eukaryotic genomes. Origin Recognition Complex (ORC) and minichromosome maintenance (MCM) proteins form prereplicative complexes at origins of replication. Using these proteins as molecular landmarks for origins, we identified ORC- and MCM-bound sites throughout the yeast genome. Four hundred twenty-nine sites in the yeast genome were predicted to contain replication origins, and approximately 80% of the loci identified on chromosome X demonstrated origin function. A substantial fraction of the predicted origins are associated with repetitive DNA sequences, including subtelomeric elements (X and Y') and transposable element-associated sequences (long terminal repeats). These findings identify the global set of yeast replication origins and open avenues of investigation into the role(s) ORC and MCM proteins play in chromosomal architecture and dynamics.

Binding Sites↗
PubMed1997

Regulation of replicon size in Xenopus egg extracts.

Once a specific number of cells have been produced in the early Xenopus laevis embryo, replicon size during the S phase of the cell cycle increases. Here, it is reported that similar increase in replicon size occurred when the concentration of nuclei in replication-competent Xenopus egg extracts exceeded a critical threshold. In this system, the origin recognition complex (ORC) did not become stoichiometrically limiting for initiation, and similar amounts of this complex bound to chromatin regardless of replicon size. These data suggest that in early development, an unidentified factor controls how many preformed ORC-DNA complexes initiate DNA replication.

Animals↗
PubMed2004

Dynamics of pre-replication complex proteins during the cell division cycle.

Replication of the human genome every time a cell divides is a highly coordinated process that ensures accurate and efficient inheritance of the genetic information. The molecular mechanism that guarantees that many origins of replication fire only once per cell-cycle has been the area of intense research. The origin recognition complex (ORC) marks the position of replication origins in the genome and serves as the landing pad for the assembly of a multiprotein, pre-replicative complex (pre-RC) at the origins, consisting of ORC, cell division cycle 6 (Cdc6), Cdc10-dependent transcript (Cdt1) and mini-chromosome maintenance (MCM) proteins. The MCM proteins serve as key participants in the mechanism that limits eukaryotic DNA replication to once-per-cell-cycle and its binding to the chromatin marks the final step of pre-RC formation, a process referred to as 'replication licensing'. We present data demonstrating how the MCM proteins associate with the chromatin during the G1 phase, probably defining pre-RCs and then anticipate replication fork movement in a precisely coordinated manner during the S phase of the cell cycle. The process of DNA replication must also be carefully coordinated with other cell-cycle processes including mitosis and cytokinesis. Some of the proteins that control initiation of DNA replication are likely to interact with the pathways that control these important cell-cycle transitions. Herein, we discuss the participation of human ORC proteins in other vital functions, in addition to their bona fide roles in replication.

Cell Cycle↗
PubMed2003

HP1/ORC complex and heterochromatin assembly.

We have used the highly conserved heterochromatin component, heterochromatin protein 1 (HP1), as a molecular tag for purifying other protein components of Drosophila heterochromatin. A complex of HP1 associated with the origin recognition complex (ORC) and an HP1/ORC-associated protein (HOAP) was purified from the maternally loaded cytoplasm of early Drosophila embryo. We propose that the DNA-binding activities of ORC and HOAP function to recruit underphosphorylatedisoforms of HP1 to sites of heterochromatin nucleation. The roles of highly phosphorylated HP1, other DNA-binding proteins known to interact with HP1, and histone modifying activities in heterochromatin assembly are also addressed.

Animals↗
PubMed2001

Triggering ubiquitination of a CDK inhibitor at origins of DNA replication.

To ensure proper timing of the G1-S transition in the cell cycle, the cyclin E-Cdk2 complex, which is responsible for the initiation of DNA replication, is restrained by the p21(Cip1)/p27(Kip1)/p57(Kip2) family of CDK (cyclin-dependent kinase) inhibitors in humans and by the related p27(Xic1) protein in Xenopus. Activation of cyclin E-Cdk2 is linked to the ubiquitination of human p27(Kip1) or Xenopus p27(Xic1) by SCF (for Skp1-Cullin-F-box protein) ubiquitin ligases. For human p27(Kip1), ubiquitination requires direct phosphorylation by cyclin E-Cdk2. We show here that Xic1 ubiquitination does not require phosphorylation by cyclin E-Cdk2, but it does require nuclear accumulation of the Xic1-cyclin E-Cdk2 complex and recruitment of this complex to chromatin by the origin-recognition complex together with Cdc6 replication preinitiation factors; it also requires an activation step necessitating cyclin E-Cdk2-kinase and SCF ubiquitin-ligase activity, and additional factors associated with mini-chromosome maintenance proteins, including the inactivation of geminin. Components of the SCF ubiquitin-ligase complex, including Skp1 and Cul1, are also recruited to chromatin through cyclin E-Cdk2 and the preinitiation complex. Thus, activation of the cyclin E-Cdk2 kinase and ubiquitin-dependent destruction of its inhibitor are spatially constrained to the site of a properly assembled preinitiation complex.

Animals↗
PubMed2002

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.

CDC2 Protein Kinase↗
PubMed1998

Regulation of DNA-replication origins during cell-cycle progression.

We have shown previously that chromosome VI of Saccharomyces cerevisiae contains nine origins of DNA replication that differ in initiation frequency and replicate sequentially during the S phase of the cell cycle. Here we show that there are links between activation of these multiple origins and regulation of S-phase progression. We study the effects of a DNA-damaging agent, methyl methane sulphonate (MMS), and of mutations in checkpoint genes such as rad53 on the activity of origins, measured by two-dimensional gel analysis, and on cell-cycle progression, measured by fluorescence-activated cell sorting. We find that when MMS slows down S-phase progression it also selectively blocks initiation from late origins. A rad53 mutation enhances late and/or inefficient origins and releases the initiation block by MMS. Mutation of rad53 also results in a late origin becoming early replicating. We conclude that rad53 regulates the timing of initiation of replication from late origins during normal cell growth and blocks initiation from late origins in MMS-treated cells. rad53 is, therefore, involved in the cell's surveillance of S-phase progression. We also find that orc2, which encodes subunit 2 of the origin-recognition complex, is involved in suppression of late origins.

Cell Cycle↗
PubMed2005

Chinese hamster ORC subunits dynamically associate with chromatin throughout the cell-cycle.

In yeast, the Origin Recognition Complex (ORC) is bound to replication origins throughout the cell-cycle, but in animal cells, there are conflicting data as to whether and when ORC is removed from chromatin. We find ORC1, 2 and ORC4 to be metabolically stable proteins that co-fractionate with chromatin throughout the cell-cycle in Chinese hamster fibroblasts. Since cellular extraction methods cannot directly examine the chromatin binding properties of proteins in vivo, we examined ORC:chromatin interactions in living cells. Fluorescence loss in photobleaching (FLIP) studies revealed ORC1 and ORC4 to be highly dynamic proteins during the cell-cycle with exchange kinetics similar to other regulatory chromatin proteins. In vivo interaction with chromatin was not significantly altered throughout the cell-cycle, including S-phase. These data support a model in which ORC subunits dynamically interact with chromatin throughout the cell-cycle.

Animals↗
PubMed2006

Genome-wide mapping of ORC and Mcm2p binding sites on tiling arrays and identification of essential ARS consensus sequences in S. cerevisiae.

BACKGROUND: Eukaryotic replication origins exhibit different initiation efficiencies and activation times within S-phase. Although local chromatin structure and function influences origin activity, the exact mechanisms remain poorly understood. A key to understanding the exact features of chromatin that impinge on replication origin function is to define the precise locations of the DNA sequences that control origin function. In S. cerevisiae, Autonomously Replicating Sequences (ARSs) contain a consensus sequence (ACS) that binds the Origin Recognition Complex (ORC) and is essential for origin function. However, an ACS is not sufficient for origin function and the majority of ACS matches do not function as ORC binding sites, complicating the specific identification of these sites. RESULTS: To identify essential origin sequences genome-wide, we utilized a tiled oligonucleotide array (NimbleGen) to map the ORC and Mcm2p binding sites at high resolution. These binding sites define a set of potential Autonomously Replicating Sequences (ARSs), which we term nimARSs. The nimARS set comprises 529 ORC and/or Mcm2p binding sites, which includes 95% of known ARSs, and experimental verification demonstrates that 94% are functional. The resolution of the analysis facilitated identification of potential ACSs (nimACSs) within 370 nimARSs. Cross-validation shows that the nimACS predictions include 58% of known ACSs, and experimental verification indicates that 82% are essential for ARS activity. CONCLUSION: These findings provide the most comprehensive, accurate, and detailed mapping of ORC binding sites to date, adding to the emerging picture of the chromatin organization of the budding yeast genome.

Algorithms↗
PubMed2003

Multiple ORC-binding sites are required for efficient MCM loading and origin firing in fission yeast.

In most eukaryotes, replication origins are composed of long chromosome regions, and the exact sequences required for origin recognition complex (ORC) and minichromosome maintenance (MCM) complex association remain elusive. Here, we show that two stretches of adenine/thymine residues are collectively essential for a fission yeast chromosomal origin. Chromatin immunoprecipitation assays revealed that the ORC subunits are located within a 1 kb region of ori2004. Analyses of deletion derivatives of ori2004 showed that adenine stretches are required for ORC binding in vivo. Synergistic interaction between ORC and adenine stretches was observed. On the other hand, MCM subunits were localized preferentially to a region near the initiation site, which is distant from adenine stretches. This association was dependent on adenine stretches and stimulated by a non-adenine element. Our results suggest that association of multiple ORC molecules with a replication origin is required for efficient MCM loading and origin firing in fission yeast.

Adenine↗