Control of pre-replicative complex during the division cycle in Chlamydomonas reinhardtii.

DNA replication is fundamental to all living organisms. In yeast and animals, it is triggered by an assembly of pre-replicative complex including ORC, CDC6 and MCMs. Cyclin Dependent Kinase (CDK) regulates both assembly and firing of the pre-replicative complex. We tested temperature-sensitive mutants blocking Chlamydomonas DNA replication. The mutants were partially or completely defective in DNA replication and did not produce mitotic spindles. After a long G1, wild type Chlamydomonas cells enter a division phase when it undergoes multiple rapid synchronous divisions ('multiple fission'). Using tagged transgenic strains, we found that MCM4 and MCM6 were localized to the nucleus throughout the entire multiple fission division cycle, except for transient cytoplasmic localization during each mitosis. Chlamydomonas CDC6 was transiently localized in nucleus in early division cycles. CDC6 protein levels were very low, probably due to proteasomal degradation. CDC6 levels were severely reduced by inactivation of CDKA1 (CDK1 ortholog) but not the plant-specific CDKB1. Proteasome inhibition did not detectably increase CDC6 levels in the cdka1 mutant, suggesting that CDKA1 might upregulate CDC6 at the transcriptional level. All of the DNA replication proteins tested were essentially undetectable until late G1. They accumulated specifically during multiple fission and then were degraded as cells completed their terminal divisions. We speculate that loading of origins with the MCM helicase may not occur until the end of the long G1, unlike in the budding yeast system. We also developed a simple assay for salt-resistant chromatin binding of MCM4, and found that tight MCM4 loading was dependent on ORC1, CDC6 and MCM6, but not on RNR1 or CDKB1. These results provide a microbial framework for approaching replication control in the plant kingdom.

PP2ACdc55 dephosphorylates Pds1 and inhibits spindle elongation in S. cerevisiae.

PP2ACdc55 (the form of protein phosphatase 2A containing Cdc55) regulates cell cycle progression by reversing cyclin-dependent kinase (CDK)- and polo-like kinase (Cdc5)-dependent phosphorylation events. In S. cerevisiae, Cdk1 phosphorylates securin (Pds1), which facilitates Pds1 binding and inhibits separase (Esp1). During anaphase, Esp1 cleaves the cohesin subunit Scc1 and promotes spindle elongation. Here, we show that PP2ACdc55 directly dephosphorylates Pds1 both in vivo and in vitro Pds1 hyperphosphorylation in a cdc55 deletion mutant enhanced the Pds1-Esp1 interaction, which played a positive role in Pds1 nuclear accumulation and in spindle elongation. We also show that nuclear PP2ACdc55 plays a role during replication stress to inhibit spindle elongation. This pathway acted independently of the known Mec1, Swe1 or spindle assembly checkpoint (SAC) checkpoint pathways. We propose a model where Pds1 dephosphorylation by PP2ACdc55 disrupts the Pds1-Esp1 protein interaction and inhibits Pds1 nuclear accumulation, which prevents spindle elongation, a process that is elevated during replication stress.

A new cell cycle checkpoint that senses plasma membrane/cell wall damage in budding yeast.

In nature, cells face a variety of stresses that cause physical damage to the plasma membrane and cell wall. It is well established that evolutionarily conserved cell cycle checkpoints monitor various cellular perturbations, including DNA damage and spindle misalignment. However, the ability of these cell cycle checkpoints to sense a damaged plasma membrane/cell wall is poorly understood. To the best of our knowledge, our recent paper described the first example of such a checkpoint, using budding yeast as a model. In this review, we will discuss this important question as well as provide hypothetical explanations to be tested in the future.

Plasma membrane/cell wall perturbation activates a novel cell cycle checkpoint during G1 in Saccharomyces cerevisiae.

Cellular wound healing or the repair of plasma membrane/cell wall damage (plasma membrane damage) occurs frequently in nature. Although various cellular perturbations, such as DNA damage, spindle misalignment, and impaired daughter cell formation, are monitored by cell cycle checkpoint mechanisms in budding yeast, whether plasma membrane damage is monitored by any of these checkpoints remains to be addressed. Here, we define the mechanism by which cells sense membrane damage and inhibit DNA replication. We found that the inhibition of DNA replication upon plasma membrane damage requires GSK3/Mck1-dependent degradation of Cdc6, a component of the prereplicative complex. Furthermore, the CDK inhibitor Sic1 is stabilized in response to plasma membrane damage, leading to cell integrity maintenance in parallel with the Mck1-Cdc6 pathway. Cells defective in both Cdc6 degradation and Sic1 stabilization failed to grow in the presence of plasma membrane damage. Taking these data together, we propose that plasma membrane damage triggers G1 arrest via Cdc6 degradation and Sic1 stabilization to promote the cellular wound healing process.

A yeast GSK-3 kinase Mck1 promotes Cdc6 degradation to inhibit DNA re-replication.

Cdc6p is an essential component of the pre-replicative complex (pre-RC), which binds to DNA replication origins to promote initiation of DNA replication. Only once per cell cycle does DNA replication take place. After initiation, the pre-RC components are disassembled in order to prevent re-replication. It has been shown that the N-terminal region of Cdc6p is targeted for degradation after phosphorylation by Cyclin Dependent Kinase (CDK). Here we show that Mck1p, a yeast homologue of GSK-3 kinase, is also required for Cdc6 degradation through a distinct mechanism. Cdc6 is an unstable protein and is accumulated in the nucleus only during G1 and early S-phase in wild-type cells. In mck1 deletion cells, CDC6p is stabilized and accumulates in the nucleus even in late S phase and mitosis. Overexpression of Mck1p induces rapid Cdc6p degradation in a manner dependent on Threonine-368, a GSK-3 phosphorylation consensus site, and SCF(CDC4). We show evidence that Mck1p-dependent degradation of Cdc6 is required for prevention of DNA re-replication. Loss of Mck1 activity results in synthetic lethality with other pre-RC mutants previously implicated in re-replication control, and these double mutant strains over-replicate DNA within a single cell cycle. These results suggest that a GSK3 family protein plays an unexpected role in preventing DNA over-replication through Cdc6 degradation in Saccharomyces cerevisiae. We propose that both CDK and Mck1 kinases are required for Cdc6 degradation to ensure a tight control of DNA replication.

Cdc6 is sequentially regulated by PP2A-Cdc55, Cdc14, and Sic1 for origin licensing in S. cerevisiae.

Cdc6, a subunit of the pre-replicative complex (pre-RC), contains multiple regulatory cyclin-dependent kinase (Cdk1) consensus sites, SP or TP motifs. In Saccharomyces cerevisiae, Cdk1 phosphorylates Cdc6-T7 to recruit Cks1, the Cdk1 phospho-adaptor in S phase, for subsequent multisite phosphorylation and protein degradation. Cdc6 accumulates in mitosis and is tightly bound by Clb2 through N-terminal phosphorylation in order to prevent premature origin licensing and degradation. It has been extensively studied how Cdc6 phosphorylation is regulated by the cyclin-Cdk1 complex. However, a detailed mechanism on how Cdc6 phosphorylation is reversed by phosphatases has not been elucidated. Here, we show that PP2ACdc55 dephosphorylates Cdc6 N-terminal sites to release Clb2. Cdc14 dephosphorylates the C-terminal phospho-degron, leading to Cdc6 stabilization in mitosis. In addition, Cdk1 inhibitor Sic1 releases Clb2·Cdk1·Cks1 from Cdc6 to load Mcm2-7 on the chromatin upon mitotic exit. Thus, pre-RC assembly and origin licensing are promoted by phosphatases through the attenuation of distinct Cdk1-dependent Cdc6 inhibitory mechanisms.

Tryptophan confers resistance to SDS-associated cell membrane stress in Saccharomyces cerevisiae.

Sodium dodecyl sulfate is a detergent that disrupts cell membranes, activates cell wall integrity signaling and restricts cell growth in Saccharomyces cerevisiae. However, the underlying mechanism of how sodium dodecyl sulfate inhibits cell growth is not fully understood. Previously, we have shown that deletion of the MCK1 gene leads to sensitivity to sodium dodecyl sulfate; thus, we implemented a suppressor gene screening revealing that the overexpression of TAT2 tryptophan permease rescues cell growth in sodium dodecyl sulfate-treated Δmck1 cells. Therefore, we questioned the involvement of tryptophan in the response to sodium dodecyl sulfate treatment. In this work, we show that trp1-1 cells have a disadvantage in the response to sodium dodecyl sulfate compared to auxotrophy for adenine, histidine, leucine or uracil when cells are grown on rich media. While also critical in the response to tea tree oil, TRP1 does not avert growth inhibition due to other cell wall/membrane perturbations that activate cell wall integrity signaling such as Calcofluor White, Congo Red or heat stress. This implicates a distinction from the cell wall integrity pathway and suggests specificity to membrane stress as opposed to cell wall stress. We discovered that tyrosine biosynthesis is also essential upon sodium dodecyl sulfate perturbation whereas phenylalanine biosynthesis appears dispensable. Finally, we observe enhanced tryptophan import within minutes upon exposure to sodium dodecyl sulfate indicating that these cells are not starved for tryptophan. In summary, we conclude that internal concentration of tryptophan and tyrosine makes cells more resistant to detergent such as sodium dodecyl sulfate.

Cyclin and cyclin-dependent kinase substrate requirements for preventing rereplication reveal the need for concomitant activation and inhibition.

DNA replication initiation in S. cerevisiae is promoted by B-type cyclin-dependent kinase (Cdk) activity. In addition, once-per-cell-cycle replication is enforced by cyclin-Cdk-dependent phosphorylation of the prereplicative complex (pre-RC) components Mcm2-7, Cdc6, and Orc1-6. Several of these controls must be simultaneously blocked by mutation to obtain rereplication. We looked for but did not obtain strong evidence for cyclin specificity in the use of different mechanisms to control rereplication: both the S-phase cyclin Clb5 and the mitotic cyclins Clb1-4 were inferred to be capable of imposing ORC-based and MCM-based controls. We found evidence that the S-phase cyclin Clb6 could promote initiation of replication without blocking reinitiation, and this activity was highly toxic when the ability of other cyclins to block reinitiation was prevented by mutation. The failure of Clb6 to regulate reinitiation was due to rapid Clb6 proteolysis, since this toxic activity of Clb6 was lost when Clb6 was stabilized by mutation. Clb6-dependent toxicity is also relieved when early accumulation of mitotic cyclins is allowed to impose rereplication controls. Cell-cycle timing of rereplication control is crucial: sufficient rereplication block activity must be available as soon as firing begins. DNA rereplication induces DNA damage, and when rereplication controls are compromised, the DNA damage checkpoint factors Mre11 and Rad17 provide additional mechanisms that maintain viability and also prevent further rereplication, and this probably contributes to genome stability.

Disruption of mechanisms that prevent rereplication triggers a DNA damage response.

Eukaryotes replicate DNA once and only once per cell cycle due to multiple, partially overlapping mechanisms efficiently preventing reinitiation. The consequences of reinitiation are unknown. Here we show that the induction of rereplication by mutations in components of the prereplicative complex (origin recognition complex [ORC], Cdc6, and minichromosome maintenance proteins) causes a cell cycle arrest with activated Rad53, a large-budded morphology, and an undivided nucleus. Combining a mutation disrupting the Clb5-Orc6 interaction (ORC6-rxl) and a mutation stabilizing Cdc6 (CDC6(Delta)NT) causes a cell cycle delay with a similar phenotype, although this background is only partially compromised for rereplication control and does not exhibit overreplication detectable by fluorescence-activated cell sorting. We conducted a systematic screen that identified genetic requirements for the viability of these cells. ORC6-rxl CDC6(Delta)NT cells depend heavily on genes required for the DNA damage response and for double-strand-break repair by homologous recombination. Our results implicate an Mre11-Mec1-dependent pathway in limiting the extent of rereplication.

Cdc6 degradation requires phosphodegron created by GSK-3 and Cdk1 for SCFCdc4 recognition in Saccharomyces cerevisiae.

To ensure genome integrity, DNA replication takes place only once per cell cycle and is tightly controlled by cyclin-dependent kinase (Cdk1). Cdc6p is part of the prereplicative complex, which is essential for DNA replication. Cdc6 is phosphorylated by cyclin-Cdk1 to promote its degradation after origin firing to prevent DNA rereplication. We previously showed that a yeast GSK-3 homologue, Mck1 kinase, promotes Cdc6 degradation in a SCF(Cdc4)-dependent manner, therefore preventing rereplication. Here we present evidence that Mck1 directly phosphorylates a GSK-3 consensus site in the C-terminus of Cdc6. The Mck1-dependent Cdc6 phosphorylation required priming by cyclin/Cdk1 at an adjacent CDK consensus site. The sequential phosphorylation by Mck1 and Clb2/Cdk1 generated a Cdc4 E3 ubiquitin ligase-binding motif to promote Cdc6 degradation during mitosis. We further revealed that Cdc6 degradation triggered by Mck1 kinase was enhanced upon DNA damage caused by the alkylating agent methyl methanesulfonate and that the resulting degradation was mediated through Cdc4. Thus, Mck1 kinase ensures proper DNA replication, prevents DNA damage, and maintains genome integrity by inhibiting Cdc6.

The interaction between mitotic checkpoint proteins, CENP-E and BubR1, is diminished in epothilone B-resistant A549 cells.

Centromere associated protein-E (CENP-E), a mitotic checkpoint protein, is required for efficient, stable microtubule capture at kinetochores during mitosis. Absence of CENP-E results in misaligned chromosomes leading to metaphase arrest. Microtubule-interacting agents such as Taxol and epothilone B (EpoB), at concentrations that induce mitotic arrest, transiently increase expression of CENP-E in a variety of cancer cell lines. The CENP-E level in an EpoB-resistant A549 cell line, EpoB40, is ~ 2-fold higher than in A549 cells. CENP-E overexpression, after transfection with CENP-E cDNA into drug sensitive cells, does not alter Taxol or EpoB sensitivity. However, suppression of CENP-E expression by CENP-E siRNA results in a moderate increase in drug sensitivity, suggesting that a minimal quantity of CENP-E is required for maintaining its function. It is known that CENP-E binds to BubR1 and enhances its recruitment to each unattached kinetochore. Suppression of CENP-E results in a decrease in BubR1 levels in EpoB40 cells. During metaphase, both targeting of CENP-E and BubR1 to the kinetochores and the interaction between CENP-E and BubR1 are significantly reduced in EpoB40 cells, compared to A549 cells. In addition, the distance between the two centrosomes during metaphase is shorter in EpoB40 than in A549 cells, suggesting that defects in the spindle-assembly checkpoint have occurred in EpoB40 cells during the development of drug resistance. These results indicate that defects in the mitotic checkpoint may have a role in, or be the result of, the development of EpoB resistance.

Specific genetic interactions between spindle assembly checkpoint proteins and B-Type cyclins in Saccharomyces cerevisiae.

The B-type cyclin Clb5 is involved primarily in control of DNA replication in Saccharomyces cerevisiae. We conducted a synthetic genetic array (SGA) analysis, testing for synthetic lethality between the clb5 deletion and a selected 87 deletions related to diverse aspects of cell cycle control based on GO annotations. Deletion of the spindle checkpoint genes BUB1 and BUB3 caused synthetic lethality with clb5. The spindle checkpoint monitors the attachment of spindles to the kinetochore or spindle tension during early mitosis. However, another spindle checkpoint gene, MAD2, could be deleted without ill effects in the absence of CLB5, suggesting that the bub1/3 clb5 synthetic lethality reflected some function other than the spindle checkpoint of Bub1 and Bub3. To characterize the lethality of bub3 clb5 cells, we constructed a temperature-sensitive clb5 allele. At nonpermissive temperature, bub3 clb5-ts cells showed defects in spindle elongation and cytokinesis. High-copy plasmid suppression of bub3 clb5 lethality identified the C-terminal fragment of BIR1, the yeast homolog of survivin; cytologically, the BIR1 fragment rescued the growth and cytokinesis defects. Bir1 interacts with IplI (Aurora B homolog), and the addition of bub3 clb5-ts significantly enhanced the lethality of the temperature-sensitive ipl1-321. Overall, we conclude that the synthetic lethality between clb5 and bub1 or bub3 is likely related to functions of Bub1/3 unrelated to their spindle checkpoint function. We tested requirements for other B-type cyclins in the absence of spindle checkpoint components. In the absence of the related CLB3 and CLB4 cyclins, the spindle integrity checkpoint becomes essential, since bub3 or mad2 deletion is lethal in a clb3 clb4 background. clb3 clb4 mad2 cells accumulated with unseparated spindle pole bodies. Thus, different B-type cyclins are required for distinct aspects of spindle morphogenesis and function, as revealed by differential genetic interactions with spindle checkpoint components.

Low concentrations of taxol cause mitotic delay followed by premature dissociation of p55CDC from Mad2 and BubR1 and abrogation of the spindle checkpoint, leading to aneuploidy.

Taxol is widely used for the treatment of human cancer. Its mechanism of action in cells is dependent on drug concentration. At low concentrations of Taxol (5-10 nM), cells exhibit aberrant mitosis, including aneuploidy, in the absence of mitotic arrest. At higher concentrations of Taxol (>20 nM), the cell cycle is blocked at metaphase by spindle checkpoint activation. Here we demonstrate that low concentrations of Taxol cause mitotic delay, and result in an aneuploid population of cells after exit from mitosis. Low concentrations of Taxol dissociated p55CDC-Mad2 or p55CDC-BubR1 complexes after mitosis, whereas high concentrations of Taxol sustained the protein complex formation leading to mitotic block. The induction of apoptosis and aneuploidy by low concentrations of Taxol may result from chromosome missegregation caused by spindle checkpoint defects.

Control of localization of a spindle checkpoint protein, Mad2, in fission yeast.

To ensure accurate chromosome segregation, the spindle checkpoint delays the onset of sister chromatid separation when the spindle is not attached to a kinetochore. Mad2, a component of the checkpoint, targets fission yeast Slp1/budding yeast Cdc20/human p55CDC and prevents it from promoting proteolysis, which is a prerequisite to sister chromatid separation. The protein is localized to unattached kinetochores in higher eukaryotes, and it is thought to be required for activation of the checkpoint as well. In this study, Mad2 and its target Slp1 were visualized in a tractable organism, fission yeast Schizosaccharomyces pombe. When cells were arrested at a prometaphase-like stage, the Mad2-Slp1 complex was stable and the two proteins were colocalized to unattached kinetochores. When the spindle attachment was completed, the complex was no longer detectable and only Mad2 was found associated to the spindle. These results would suggest that unattached kinetochores provide sites for assembly of the Mad2-Slp1 complex. During interphase, Mad2 was localized to the nuclear periphery as well as to the chromatin domain. This localization was abolished in a yeast strain lacking Mad1, a protein that physically interacts with Mad2. Mad1 may anchor Mad2 to the nuclear membrane and regulate its entry into the nucleus.