Control of DNA rereplication via Cdc2 phosphorylation sites in the origin recognition complex

Cdc2 kinase is a master regulator of cell cycle progression in the fission yeast Schizosaccharomyces pombe. Our data indicate that Cdc2 phosphorylates replication factor Orp2, a subunit of the origin recognition complex (ORC). Cdc2 phosphorylation of Orp2 appears to be one of multiple mechanisms by which Cdc2 prevents DNA rereplication in a single cell cycle. Cdc2 phosphorylation of Orp2 is not required for Cdc2 to activate DNA replication initiation. Phosphorylation of Orp2 appears first in S phase and becomes maximal in G(2) and M when Cdc2 kinase activity is required to prevent reinitiation of DNA replication. A mutant lacking Cdc2 phosphorylation sites in Orp2 (orp2-T4A) allowed greater rereplication of DNA than congenic orp2 wild-type strains when the limiting replication initiation factor Cdc18 was deregulated. Thus, Cdc2 phosphorylation of Orp2 may be redundant with regulation of Cdc18 for preventing reinitiation of DNA synthesis. Since Cdc2 phosphorylation sites are present in Orp2 (also known as Orc2) from yeasts to metazoans, we propose that cell cycle-regulated phosphorylation of the ORC provides a safety net to prevent DNA rereplication and resulting genetic instability.     

Fission yeast Cdc23/Mcm10 functions after pre-replicative complex formation to promote Cdc45 chromatin binding

Using a cytological assay to monitor the successive chromatin association of replication proteins leading to replication initiation, we have investigated the function of fission yeast Cdc23/Mcm10 in DNA replication. Inactivation of Cdc23 before replication initiation using tight degron mutations has no effect on Mcm2 chromatin association, and thus pre-replicative complex (pre-RC) formation, although Cdc45 chromatin binding is blocked. Inactivating Cdc23 during an S phase block after Cdc45 has bound causes a small reduction in Cdc45 chromatin binding, and replication does not terminate in the absence of Mcm10 function. These observations show that Cdc23/Mcm10 function is conserved between fission yeast and Xenopus, where in vitro analysis has indicated a similar requirement for Cdc45 binding, but apparently not compared with Saccharomyces cerevisiae, where Mcm10 is needed for Mcm2 chromatin binding. However, unlike the situation in Xenopus, where Mcm10 chromatin binding is dependent on Mcm2-7, we show that the fission yeast protein is bound to chromatin throughout the cell cycle in growing cells, and only displaced from chromatin during quiescence. On return to growth, Cdc23 chromatin binding is rapidly reestablished independently from pre-RC formation, suggesting that chromatin association of Cdc23 provides a link between proliferation and competence to execute DNA replication.     

CDK phosphorylation of Drc1 regulates DNA replication in fission yeast

Cyclin-dependent kinases (CDKs) are absolutely required for DNA replication in eukaryotic cells. CDKs are thought to activate one or more replication factors, but the identities of these proteins are unknown. Here we describe fission yeast Drc1, a protein required for DNA replication that is phosphorylated by Cdc2. Drc1 depletion leads to catastrophic mitotic divisions with incompletely replicated DNA, indicating that Drc1 is required for DNA synthesis and S-M replication checkpoint control. Drc1 associates with Cdc2 and is phosphorylated at the onset of S phase when Cdc2 is activated. Mutant Drc1 that lacks CDK phosphorylation sites is nonfunctional and fails to interact with Cut5 replication factor. These data suggest that Cdc2 promotes DNA replication by phosphorylating Drc1 and regulating its association with Cut5.     

Cdc6 chromatin affinity is unaffected by serine-54 phosphorylation, S-phase progression, and overexpression of cyclin A

Ectopically expressed Cdc6 is translocated from the nucleus during S phase in a cyclin A-Cdk2-dependent process, suggesting that reinitiation of DNA replication is prevented by removal of phosphorylated Cdc6 from chromatin after origin firing. However, whether endogenous Cdc6 translocates during S phase remains controversial. To resolve the questions regarding regulation of endogenous Cdc6, we cloned the cDNA encoding the Chinese hamster Cdc6 homolog and specifically focused on analyzing the localizations and chromatin affinities of endogenous and exogenous proteins during S phase and following overexpression of cyclin A. In agreement with other reports, ectopically expressed Cdc6 translocates from the nucleus during S phase and in response to overexpressed cyclin A. In contrast, using a combination of biochemical and immunohistochemical assays, we show convincingly that endogenous Cdc6 remains nuclear and chromatin bound throughout the entire S period, while Mcm5 loses chromatin affinity during S phase. Overexpression of cyclin A is unable to alter the nuclear localization of Cdc6. Furthermore, using a phosphospecific antibody we show that phosphoserine-54 Cdc6 maintains a high affinity for chromatin during the S period. Considering recent in vitro studies, these data are consistent with a proposed model in which Cdc6 is serine-54 phosphorylated during S phase and functions as a chromatin-bound signal that prevents reformation of prereplication complexes.     

NetPhosYeast: prediction of protein phosphorylation sites in yeast

We here present a neural network-based method for the prediction of protein phosphorylation sites in yeast--an important model organism for basic research. Existing protein phosphorylation site predictors are primarily based on mammalian data and show reduced sensitivity on yeast phosphorylation sites compared to those in humans, suggesting the need for an yeast-specific phosphorylation site predictor. NetPhosYeast achieves a correlation coefficient close to 0.75 with a sensitivity of 0.84 and specificity of 0.90 and outperforms existing predictors in the identification of phosphorylation sites in yeast. AVAILABILITY: The NetPhosYeast prediction service is available as a public web server at http://www.cbs.dtu.dk/services/NetPhosYeast/.     

CDK phosphorylation of a novel NLS-NES module distributed between two subunits of the Mcm2-7 complex prevents chromosomal rereplication

Cyclin-dependent kinases (CDKs) use multiple mechanisms to block reassembly of prereplicative complexes (pre-RCs) at replication origins to prevent inappropriate rereplication. In Saccharomyces cerevisiae, one of these mechanisms promotes the net nuclear export of a pre-RC component, the Mcm2-7 complex, during S, G2, and M phases. Here we identify two partial nuclear localization signals (NLSs) on Mcm2 and Mcm3 that are each necessary, but not sufficient, for nuclear localization of the Mcm2-7 complex. When brought together in cis, however, the two partial signals constitute a potent NLS, sufficient for robust nuclear localization when fused to an otherwise cytoplasmic protein. We also identify a Crm1-dependent nuclear export signal (NES) adjacent to the Mcm3 NLS. Remarkably, the Mcm2-Mcm3 NLS and the Mcm3 NES are sufficient to form a transport module that recapitulates the cell cycle-regulated localization of the entire Mcm2-7 complex. Moreover, we show that CDK regulation promotes net export by phosphorylation of the Mcm3 portion of this module and that nuclear export of the Mcm2-7 complex is sufficient to disrupt replication initiation. We speculate that the distribution of partial transport signals among distinct subunits of a complex may enhance the specificity of protein localization and raises the possibility that previously undetected distributed transport signals are used by other multiprotein complexes.     

Extensive chromatin fragmentation improves enrichment of protein binding sites in chromatin immunoprecipitation experiments

Extensive sonication of formaldehyde-crosslinked chromatin can generate DNA fragments averaging 200 bp in length (range 75–300 bp). Fragmentation is largely random with respect to genomic region and nucleosome position. ChIP experiments employing such extensively fragmented samples show 2- to 4-fold increased enrichment of protein binding sites over control genomic regions, when compared to samples sonicated to a more conventional size range (300–500 bp). The basis of improved fold enrichments is that immunoprecipitation of protein-bound regions is unaffected by fragment size, whereas immunoprecipitation of control genomic regions decreases progressively along with reduced fragment size due to fewer nonspecific binding sites. The use of extensively sonicated samples improves mapping of protein binding sites, and it extends the dynamic range for quantitative measurements of histone density. We show that many yeast promoter regions are virtually devoid of histones.     

The RLF-B component of the replication licensing system is distinct from Cdc6 and functions after Cdc6 binds to chromatin

Replication licensing factor (RLF) is an essential initiation factor that can prevent re-replication of DNA in a single cell cycle [1] [2]. It is required for the initiation of DNA replication, binds to chromatin early in the cell cycle, is removed from chromatin as DNA replicates and is unable to re-bind replicated chromatin until the following mitosis. Chromatography of RLF from Xenopus extracts has shown that it consists of two components termed RLF-B and RLF-M [3]. The RLF-M component consists of complexes of all six Xenopus minichromosome maintenance (MCM/P1) proteins (XMcm2-7), which bind to chromatin in late mitosis and are removed as replication occurs [3] [4] [5] [6] [7]. The identity of RLF-B is currently unknown. At least two factors must be present on chromatin before licensing can occur: the Xenopus origin recognition complex (XORC) [8] [9] and Xenopus Cdc6 (XCdc6) [10]. XORC saturates Xenopus sperm chromatin at approximately one copy per replication origin whereas XCdc6 binds to chromatin only if XORC is bound first [9] [10] [11]. Although XORC has been shown to be a distinct activity from RLF-B [9], the relationship between XCdc6 and RLF-B is currently unclear. Here, we show that active XCdc6 is loaded onto chromatin in extracts with defective RLF, and that both RLF-M and RLF-B are still required for the licensing of XCdc6-containing chromatin. Furthermore, RLF-B can be separated from XCdc6 by immunoprecipitation and standard chromatography. These experiments demonstrate that RLF-B is both functionally and physically distinct from XCdc6, and that XCdc6 is loaded onto chromatin before RLF-B function is executed.     

Location of the ethidium binding sites of high affinity in chromatin

The influence of H1 and H5 histones proteins upon the accessibility of ethidium bromide into chromatin is studied by steady-state fluorescence anisotropy in the range of r-values ([Dye]/[Phosphate]) smaller than 0.01. This corresponds to the very strong binding process. When H1 and H5 are present, the DNA segment which contains the binding sites is 25–30 base pairs long, even if H1 and H5 are digested by trypsin or by natural proteolysis, but presumably still interacting with the DNA chromatin. On the contrary, when H1 or H5 are separated from chromatin by an increase of the ionic strength, ethidium binds to a segment of DNA about 55–60 base pairs long. We may explain the results by assuming that the ethidium sites are located on a continuous segment constituting about one half of the linker, the other half interacting with H1 and H5. When chromatin is depleted from these proteins, the high affinity sites are distributed all along the linker.     

Cdc2 activation: the interplay of cyclin binding and Thr161 phosphorylation

The mitotic kinase cdc2 must bind to a regulatory subunit--a cyclin--to be active. Cyclin binding controls the timing of activation of the kinase subunit, by modulating its interaction with upstream regulatory enzymes, and it also determines subcellular localization and substrate specificity. In this article, I summarize our present knowledge of the mechanisms that control cdc2 activation.     

Regulation of the localization and stability of Cdc6 in living yeast cells

The Cdc6 protein is an essential regulator for initiation of DNA replication. Following the G1/S transition, Cdc6 is degraded through a ubiquitin-mediated proteolysis pathway. In this study, we tagged Cdc6 with green fluorescent protein (GFP) and used site-specific mutations to study the regulation of Cdc6 localization and degradation in living yeast cells. Our major findings are: (1). Cdc6-GFP distributes predominantly in the nucleus in all cell cycle stages, with a small increase in cytoplasmic localization in G2/M cells. (2). This nuclear localization is critical for Cdc6 degradation. When the N-terminal nuclear localization signal (NLS) was mutated, Cdc6-GFP no longer accumulated in the nucleus, and the mutant cdc6 was stabilized compared to wild type. (3). The putative CDK phosphorylation sites are not required for Cdc6 nuclear localization, but are important for protein stability. These observations suggest that the stability of Cdc6 protein is regulated by two factors: nuclear localization and phosphorylation by CDK1.     

Interaction between yeast Cdc6 protein and B-type cyclin/Cdc28 kinases.

During purification of recombinant Cdc6 expressed in yeast, we found that Cdc6 interacts with the critical cell cycle, cyclin-dependent protein kinase Cdc28. Cdc6 and Cdc28 can be coimmunoprecipitated from extracts, Cdc6 is retained on the Cdc28-binding matrix p13-agarose, and Cdc28 is retained on an affinity column charged with bacterially produced Cdc6. Cdc6, which is a phosphoprotein in vivo, contains five Cdc28 consensus sites and is a substrate of the Cdc28 kinase in vitro. Cdc6 also inhibits Cdc28 histone H1 kinase activity. Strikingly, Cdc6 interacts preferentially with B-type cyclin/Cdc28 complexes and not Cln/Cdc28 in log-phase cells. However, Cdc6 does not associate with Cdc28 when cells are blocked at the restrictive temperature in a cdc34 mutant, a point in the cell cycle when the B-type cyclin/Cdc28 inhibitor p40Sic1 accumulates and purified p40Sic1 inhibits the Cdc6/Cdc28 interaction. Deletion of the Cdc28 interaction domain from Cdc6 yields a protein that cannot support growth. However, when overproduced, the mutant protein can support growth. Furthermore, whereas overproduction of wild-type Cdc6 leads to growth inhibition and bud hyperpolarization, overproduction of the mutant protein supports growth at normal rates with normal morphology. Thus, the interaction may have a role in the essential function of Cdc6 in initiation and in restraining mitosis until replication is complete.     

Analysis of the role of phosphorylation in fission yeast Cdc13p/cyclinB function

The Cdk1p-cyclin B complex drives entry into mitosis in all eukaryotes. Cdc13p is the single essential cyclin in Schizosaccharomyces pombe and a member of the cyclin B family. Cdc13p abundance rises during G(2)-phase and falls as cells progress through mitosis and G(1). Cdc13p degradation, mediated by the anaphase-promoting complex, is an important mechanism of Cdk1p inhibition and mitotic exit. Cdk1p-cyclin B1 complexes shuttle between the nucleus and cytoplasm, and preventing nuclear accumulation of Cdk1p-cyclin B1 in mammalian cells appears to be one mechanism of preventing entry into mitosis during a DNA damage-induced checkpoint delay. In vertebrates, phosphorylation plays a key role in regulating the intracellular distribution of cyclins. Previous mass spectrometric analysis identified sites of Cdc13p phosphorylation. Here, we have confirmed that these sites are the sole in vivo Cdc13p phosphorylation sites and have studied the role that phosphorylation plays in Cdc13p localization and function. Our data indicate that Cdc13p accumulates in the nucleolus in response to G(2) checkpoint delays, rather than in the cytoplasm, and that phosphorylation plays no role in Cdc13p localization or function.     

Differential susceptibility of yeast S and M phase CDK complexes to inhibitory tyrosine phosphorylation

BACKGROUND: Several checkpoint pathways employ Wee1-mediated inhibitory tyrosine phosphorylation of cyclin-dependent kinases (CDKs) to restrain cell-cycle progression. Whereas in vertebrates this strategy can delay both DNA replication and mitosis, in yeast cells only mitosis is delayed. This is particularly surprising because yeasts, unlike vertebrates, employ a single family of cyclins (B type) and the same CDK to promote both S phase and mitosis. The G2-specific arrest could be explained in two fundamentally different ways: tyrosine phosphorylation of cyclin/CDK complexes could leave sufficient residual activity to promote S phase, or S phase-promoting cyclin/CDK complexes could somehow be protected from checkpoint-induced tyrosine phosphorylation. RESULTS: We demonstrate that in Saccharomyces cerevisiae, several cyclin/CDK complexes are protected from inhibitory tyrosine phosphorylation, allowing Clb5,6p to promote DNA replication and Clb3,4p to promote spindle assembly, even under checkpoint-inducing conditions that block nuclear division. In vivo, S phase-promoting Clb5p/Cdc28p complexes were phosphorylated more slowly and dephosphorylated more effectively than were mitosis-promoting Clb2p/Cdc28p complexes. Moreover, we show that the CDK inhibitor (CKI) Sic1p protects bound Clb5p/Cdc28p complexes from tyrosine phosphorylation, allowing the accumulation of unphosphorylated complexes that are unleashed when Sic1p is degraded to promote S phase. The vertebrate CKI p27(Kip1) similarly protects Cyclin A/Cdk2 complexes from Wee1, suggesting that the antagonism between CKIs and Wee1 is evolutionarily conserved. CONCLUSIONS: In yeast cells, the combination of CKI binding and preferential phosphorylation/dephosphorylation of different B cyclin/CDK complexes renders S phase progression immune from checkpoints acting via CDK tyrosine phosphorylation.     

Roles of antiestrogen binding sites in human endometrial cancer cells

Estrogen-noncompatible antiestrogen binding sites (AEBS) as well as estrogen receptors (ER), and the growth-inhibitory effect of tamoxifen were investigated in two human endometrial cancer cell lines, IK-90 and HEC-IA cells. IK-90 cells contained specific AEBS, but no ER was found in these cells. Scatchard plot analysis of AEBS in 12,000 g supernatant from IK-90 cells showed a high affinity binding site for tamoxifen (Kd:5.6 +/- 1.0 nM) with the maximum binding site of 457 +/- 47 fmol/mg protein. However, no measurable ER or AEBS was found in HEC-IA cells. The effect of tamoxifen on the growth of cells was found to be identical in both cell lines; the addition of 10 microM tamoxifen to culture medium was cytocidal whereas tamoxifen at lower concentrations (1 nM-1 microM) did not significantly affect the growth of both IK-90 and HEC-IA cells. These results demonstrate for the first time the presence of AEBS in human endometrial cancer cells. The present results also suggest that AEBS does not play a fundamental role in mediating the growth-inhibitory effect of tamoxifen in endometrial cancer cells.