Securin is required for chromosomal stability in human cells.

Abnormalities of chromosome number are the most common genetic aberrations in cancer. The mechanisms regulating the fidelity of mitotic chromosome transmission in mammalian cells are therefore of great interest. Here we show that human cells without an hSecurin gene lose chromosomes at a high frequency. This loss was linked to abnormal anaphases during which cells underwent repetitive unsuccessful attempts to segregate their chromosomes. The abnormal mitoses were associated with biochemical defects in the activation of separin, the sister-separating protease, rendering it unable to cleave the cohesin subunit Scc1 efficiently. These results illuminate the function of mammalian securin and show that it is essential for the maintenance of euploidy.

Chromosome segregation and cancer: cutting through the mystery.

Mitosis is the most dramatic--and potentially dangerous--event in the cell cycle, as sister chromatids are irreversibly segregated to daughter cells. Defects in the checkpoints that normally maintain the fidelity of this process can lead to chromosomal instability (CIN) and cancer. However, CIN--a driving force of tumorigenesis--could be the cancer cell's ultimate vulnerability. An important goal is to identify novel anticancer compounds that directly target the mitotic errors at the heart of CIN.

sud1(+) targets cyclin-dependent kinase-phosphorylated Cdc18 and Rum1 proteins for degradation and stops unwanted diploidization in fission yeast.

In the fission yeast Schizosaccharomyces pombe, S phase is limited to a single round per cell cycle through cyclin-dependent kinase phosphorylation of critical replication factors, including the Cdc18 replication initiator protein. Because defects in Cdc18 phosphorylation lead to a hyperstable and hyperactive form of Cdc18 that promotes high levels of overreplication in vivo, we wished to identify the components of the Cdc18 proteolysis pathway in fission yeast. In this paper we describe one such component, encoded by the sud1(+) gene. sud1(+) shares homology with the budding yeast CDC4 gene and is required to prevent spontaneous re-replication in fission yeast. Cells lacking sud1(+) accumulate high levels of Cdc18 and the CDK inhibitor Rum1, because they cannot degrade these two key cell cycle regulators. Through genetic analysis we show that hyperaccumulation of Rum1 contributes to re-replication in Deltasud1 cells, but is not the cause of the defect in Cdc18 proteolysis. Rather, Sud1 itself is associated with the ubiquitin pathway in fission yeast and binds to Cdc18 in vivo. Most importantly, Sud1-Cdc18 binding requires prior phosphorylation of the Cdc18 polypeptide at CDK consensus sites. These results provide a biochemical mechanism for the phosphorylation-dependent degradation of Cdc18 and other cell cycle regulators, including Rum1. Evolutionary conservation of the Sud1/CDC4 pathway suggests that phosphorylation-coupled proteolysis may be a general feature of nearly all eukaryotic cell cycles.

Regulation of the replication initiator protein p65cdc18 by CDK phosphorylation.

Cyclin-dependent kinases (CDKs) promote the initiation of DNA replication and prevent reinitiation before mitosis, presumably through phosphorylation of key substrates at origins of replication. In fission yeast, the p65cdc18 protein is required to initiate DNA replication and interacts with the origin recognition complex (ORC) and the p34cdc2 CDK. Here we report that p65cdc18 becomes highly phosphorylated as cells undergo the G1 --> S phase transition. This modification is dependent on p34cdc2 protein kinase activity, as well as six consensus CDK phosphorylation sites within the p65cdc18 polypeptide. Genetic interactions between cdc18+ and the S-phase cyclin cig2+ suggest that CDK-dependent phosphorylation antagonizes cdc18+ function in vivo. Using site-directed mutagenesis, we show that phosphorylation at CDK consensus sites directly targets p65cdc18 for rapid degradation and inhibits its replication activity, as strong expression of a constitutively hypophosphorylated mutant form of p65cdc18 results in large amounts of DNA over-replication in vivo. Furthermore, the over-replication phenotype produced by this mutant p65cdc18 is resistant to increased mitotic cyclin/CDK activity, a known inhibitor of over-replication. Therefore, p65cdc18 is the first example of a cellular initiation factor directly regulated in vivo by CDK-dependent phosphorylation and proteolysis. Regulation of p65cdc18 by CDK phosphorylation is likely to contribute to the CDK-driven "replication switch" that restricts initiation at eukaryotic origins to once per cell cycle.

Cyclin-dependent kinase and initiation at eukaryotic origins: a replication switch?

A growing body of evidence indicates that cyclin-dependent kinases (CDKs) regulate the activity of eukaryotic origins of replication both positively and negatively. Although the details of this control remain unclear, recent work suggests that CDKs act directly at origins, where they associate with and phosphorylate several key initiator proteins. These data suggest that a CDK-regulated replication switch operates at each origin to ensure that initiation occurs precisely once per cell cycle.

Interaction of the S phase regulator cdc18 with cyclin-dependent kinase in fission yeast.

The fission yeast gene cdc18(+) is required for entry into S phase and for coupling mitosis to the successful completion of S phase. Cdc18 is a highly unstable protein that is expressed only once per cell cycle at the G1/S boundary. Overexpression of Cdc18 causes a mitotic delay and reinitiation of DNA replication, suggesting that the inactivation of Cdc18 plays a role in preventing rereplication within a given cell cycle. In this paper, we present evidence that Cdc18 is associated with active cyclin-dependent kinase in vivo. We have expressed Cdc18 as a glutathione S-transferase fusion in fission yeast and demonstrated that the fusion protein is functional in vivo. We find that the Cdc18 fusion protein copurifies with a kinase activity capable of phosphorylating histone H1 and Cdc18. The activity was identified by a variety of methods as the cyclin-dependent kinase containing the product of the cdc2(+) gene. The amino terminus of Cdc18 is required for association with cyclin-dependent kinase, but the association does not require the consensus cyclin-dependent kinase phosphorylation sites in this region. Additionally, both G1/S and mitotic forms of cyclin-dependent kinase phosphorylate and interact with Cdc18. These interactions between Cdc18 and cyclin-dependent kinases suggest mechanisms by which cyclin-dependent kinases could activate the initiation of DNA replication and could prevent rereplication.

Rum1 and Cdc18 link inhibition of cyclin-dependent kinase to the initiation of DNA replication in Schizosaccharomyces pombe.

Eukaryotic cells have evolved regulatory mechanisms to ensure the strict alternation of DNA replication and mitosis. Recent work has suggested that the mitotic form of cyclin-dependent kinase (Cdc2/cyclin B) has a role in preventing re-replication of the genome before mitosis, but the relevant targets of this inhibition are unknown. In this report we present evidence that the mitotic cyclin-dependent kinase affects DNA replication by inhibiting the accumulation and function of Cdc18, a critical regulator of S-phase entry. We found that the ruml+ gene efficiently suppresses the lethality of a conditional cdc18 mutant. Conversely, deletion of ruml+ increases the severity of the cdc18 mutant phenotype, resulting in inappropriate cell division and a rapid loss of viability. Biochemical experiments indicate that Ruml potently inhibits Cdc2 phosphorylation of histone H1 or a Cdc18 fusion protein by directly interacting with the Cdc2/cyclin B complex. Overexpression of Ruml under conditions that promote re-replication of the genome induces a striking accumulation of Cdc18 protein by a largely post-transcriptional mechanism. Overexpression of SIC1, an unrelated cyclin-dependent kinase inhibitor from budding yeast, causes a similar accumulation of Cdc18 and also leads to re-replication. Our data link a potent inhibitor of Cdc2 kinase to a key protein required for the initiation of DNA replication and strongly suggest that inhibition of Cdc18 by cyclin-dependent kinases has an important role in ensuring that the genome is duplicated precisely once each cell cycle.

Replication and transcription. Silence of the ORCs.

The origin recognition complex, a multi-protein complex known to bind to replication origins, has now been implicated in transcriptional silencing, providing another link between DNA replication and transcription.