Loss of Ubp3 increases silencing,decreases unequal recombination in rDNA,and shortens the replicative life span in Saccharomyces cerevisiae

Ubp3 is a conserved ubiquitin protease that acts as an antisilencing factor in MAT and telomeric regions. Here we show that ubp3∆ mutants also display increased silencing in ribosomal DNA (rDNA). Consistent with this, RNA polymerase II occupancy is lower in cells lacking Ubp3 than in wild-type cells in all heterochromatic regions. Moreover, in a ubp3∆ mutant, unequal recombination in rDNA is highly suppressed. We present genetic evidence that this effect on rDNA recombination, but not silencing, is entirely dependent on the silencing factor Sir2. Further, ubp3∆ sir2∆ mutants age prematurely at the same rate as sir2∆ mutants. Thus our data suggest that recombination negatively influences replicative life span more so than silencing. However, in ubp3∆ mutants, recombination is not a prerequisite for aging, since cells lacking Ubp3 have a shorter life span than isogenic wild-type cells. We discuss the data in view of different models on how silencing and unequal recombination affect replicative life span and the role of Ubp3 in these processes.     

Cell cycle-dependent nuclear export of Cdh1p may contribute to the inactivation of APC/C(Cdh1)

Cdh1p is a substrate-specific subunit of the anaphase-promoting complex (APC/C), which functions as an E3 ubiquitin ligase to degrade the mitotic cyclin Clb2p and other substrates during the G(1) phase of the cell cycle. Cdh1p is phosphorylated and thereby inactivated at the G(1)/S transition predominantly by Cdc28p-Clb5p. Here we show that Cdh1p is nuclear during the G(1) phase of the cell cycle, but redistributes to the cytoplasm between S phase and the end of mitosis. Nuclear export of Cdh1p is regulated by phosphorylation and requires active Cdc28p kinase. Cdh1p binds to the importin Pse1p and the exportin Msn5p, which is necessary and sufficient to promote efficient export of Cdh1p in vivo. Although msn5delta cells are viable, they are sensitive to Cdh1p overexpression. Likewise, a mutant form of Cdh1p, which is constitutively nuclear, prevents accumulation of Clb2p and leads to cell cycle arrest when overexpressed in wild-type cells. Taken together, these results suggest that phosphorylation-dependent nuclear export of Cdh1p by Msn5p contributes to efficient inactivation of APC/C(Cdh1).     

The Budding Yeast Ubiquitin Protease Ubp7 Is a Novel Component Involved in S Phase Progression

DNA damage must be repaired in an accurate and timely fashion to preserve genome stability. Cellular mechanisms preventing genome instability are crucial to human health because genome instability is considered a hallmark of cancer. Collectively referred to as the DNA damage response, conserved pathways ensure proper DNA damage recognition and repair. The function of numerous DNA damage response components is fine-tuned by posttranslational modifications, including ubiquitination. This not only involves the enzyme cascade responsible for conjugating ubiquitin to substrates but also requires enzymes that mediate directed removal of ubiquitin. Deubiquitinases remove ubiquitin from substrates to prevent degradation or to mediate signaling functions. The Saccharomyces cerevisiae deubiquitinase Ubp7 has been characterized previously as an endocytic factor. However, here we identify Ubp7 as a novel factor affecting S phase progression after hydroxyurea treatment and demonstrate an evolutionary and genetic interaction of Ubp7 with DNA damage repair pathways of homologous recombination and nucleotide excision repair. We find that deletion of UBP7 sensitizes cells to hydroxyurea and cisplatin and demonstrate that factors that stabilize replication forks are critical under these conditions. Furthermore, ubp7Δ cells exhibit an S phase progression defect upon checkpoint activation by hydroxyurea treatment. ubp7Δ mutants are epistatic to factors involved in histone maintenance and modification, and we find that a subset of Ubp7 is chromatin-associated. In summary, our results suggest that Ubp7 contributes to S phase progression by affecting the chromatin state at replication forks, and we propose histone H2B ubiquitination as a potential substrate of Ubp7.     

The yeast mitotic cyclin Clb2 cannot substitute for S phase cyclins in replication origin firing

Cyclin-dependent kinases (CDKs) drive the cell cycle, central to which is the accurate control of chromosome replication. In Saccharomyces cerevisiae, six closely related B-type cyclins (Clb1–6) drive the events of S phase and mitosis. Either Clb5 or Clb6 can activate early-firing replication origins, whereas only Clb5 can activate late origins. Clb1–4 are expressed later in the cell cycle. Whether Clb cyclins differ only in timing of expression, or else impart different kinase specificities is under ongoing investigation. This study shows that the expression of Clb2 during S phase in cells lacking Clb5 failed to rescue late origin activation. Early expression of Clb2 in cells lacking both Clb5 and Clb6 did not activate early origins on schedule to restore the correct S phase entry time. Therefore, Clb2 cannot drive timely activation of either early or late replication origins, demonstrating that Clb2-directed CDK has a specificity distinct from that driven by Clb5 and Clb6.     

Clb2 and the APC/CCdh1 regulate Swe1 stability

Swe1/Wee1 regulates mitotic entry by inhibiting Clb2-Cdk1 and its accumulation is involved in stress induced G2 arrest. The APC/C^Cdh1 substrates Cdc5, Clb2 and Hsl1 regulate Swe1 degradation. We observed that clb2Dcdh1D double mutant S. cerevisiae does not express any detectable levels of Swe1, presumably due to its constitutive degradation. This effect of Cdh1 inactivation is due to stabilization of Cdc5 and Hsl1, as expression of the non-degradable Cdc5^T29A in clb2D cells prevented Swe1 accumulation. Strikingly, expression of non-degradable Hsl1^mdb/mkb prevented Swe1 accumulation even in wild type Clb2 cells. Interestingly Swe1 accumulation could be reconstituted in all these mutants by eliciting a replication fork stress with hydroxyurea. Cells expressing the Clb2^ME mutant, that cannot bind Swe1, behaved like clb2D cells, and failed to accumulate Swe1 in the absence of Cdh1 or the presence of Cdc5^T29A. This suggests that for Swe1 to accumulate it must interact with Clb2. We further show that in the absence of Clb2, Hsl1 is no longer essential for Swe1 degradation. We hypothesize that Clb2-Cdk1 protects Swe1 from premature degradation until its Hsl1 mediated de-protection, which enables its Cdc5 mediated degradation. Swe1 levels are thus regulated by monitoring the levels of three major mitotic regulators.     

Phosphorylation and dephosphorylation regulate APC/CCdh1 substrate degradation

The Anaphase Promoting Complex/Cyclosome (APC/C) ubiquitin ligase activated by its G1 specific adaptor protein Cdh1 is a major regulator of the cell cycle. The APC/C^Cdh1 mediates degradation of dozens of proteins, however, the kinetics and requirements for their degradation are largely unknown. We demonstrate that overexpression of the constitutive active CDH1^m11 mutant that is not inhibited by phosphorylation results in mitotic exit in the absence of the FEAR and MEN pathways, and DNA re-replication in the absence of Cdc7 activity. This mode of mitotic exit also reveals additional requirements for APC/C^Cdh1 substrate degradation, which for some substrates such as Pds1 or Clb5 is dephosphorylation, but for others such as Cdc5 is phosphorylation.     

Two different modes of cyclin clb2 proteolysis during mitosis in Saccharomyces cerevisiae

Sister chromatid separation and mitotic exit are triggered by the anaphase-promoting complex (APC/C) which is a multi-subunit ubiquitin ligase required for proteolytic degradation of various target proteins. Cdc20 and Cdh1 are substrate-specific activators of the APC/C. It was previously proposed that Cdh1 is essential for proteolysis of the yeast mitotic cyclin Clb2. We show that Clb2 proteolysis is triggered by two different modes during mitosis. A fraction of Clb2 is degraded during anaphase in the absence of Cdh1. However, a second fraction of Clb2 remains stable during anaphase and is degraded in a Cdh1-dependent manner as cells exit from mitosis. Most of cyclin Clb3 is degraded independently of Cdh1. Our data imply that degradation of mitotic cyclins is initiated by a Cdh1-independent mechanism.     

Negative regulation of G1 and G2 by S-phase cyclins of Saccharomyces cerevisiae.

Cell cycle progression in the budding yeast Saccharomyces cerevisiae is controlled by the Cdc28 protein kinase, which is sequentially activated by different sets of cyclins. Previous genetic analysis has revealed that two B-type cyclins, Clb5 and Clb6, have a positive role in DNA replication. In the present study, we show, in addition, that these cyclins negatively regulate G1- and G2-specific functions. The consequences of this negative regulation were most apparent in clb6 mutants, which had a shorter pre-Start G1 phase as well as a shorter G2 phase than congenic wild-type cells. As a consequence, clb6 mutants grew and proliferated more rapidly than wild-type cells. It was more difficult to assess the role of Clb5 in G1 and G2 by genetic analysis because of the extreme prolongation of S phase in clb5 mutants. Nevertheless, both Clb5 and Clb6 were shown to be responsible for down-regulation of the protein kinase activities associated with Cln2, a G1 cyclin, and Clb2, a mitotic cyclin, in vivo. These observations are consistent with the observed cell cycle phase accelerations associated with the clb6 mutant and are suggestive of similar functions for Clb5. Genetic evidence suggested that the inhibition of mitotic cyclin-dependent kinase activities was dependent on and possibly mediated through the CDC6 gene product. Thus, Clb5 and Clb6 may stabilize S phase by promoting DNA replication while inhibiting other cell cycle activities.     

Activity of the APC(Cdh1) form of the anaphase-promoting complex persists until S phase and prevents the premature expression of Cdc20p.

Cell cycle progression is driven by waves of cyclin expression coupled with regulated protein degradation. An essential step for initiating mitosis is the inactivation of proteolysis mediated by the anaphase-promoting complex/cyclosome (APC/C) bound to its regulator Cdh1p/Hct1p. Yeast APC(Cdh1) was proposed previously to be inactivated at Start by G1 cyclin/cyclin-dependent kinase (CDK). Here, we demonstrate that in a normal cell cycle APC(Cdh1) is inactivated in a graded manner and is not extinguished until S phase. Complete inactivation of APC(Cdh1) requires S phase cyclins. Further, persistent APC(Cdh1) activity throughout G1 helps to ensure the proper timing of Cdc20p expression. This suggests that S phase cyclins have an important role in allowing the accumulation of mitotic cyclins and further suggests a regulatory loop among S phase cyclins, APC(Cdh1), and APC(Cdc20).     

APC/CCdh1 Targets Brain-Specific Kinase 2 (BRSK2) for Degradation via the Ubiquitin-Proteasome Pathway

Studies of brain-specific kinase 2 (BRSK2), an AMP-activated protein kinase (AMPK)-related kinase, and its homologs suggest that they are multifunctional regulators of cell-cycle progression. BRSK2, which contains a ubiquitin-associated (UBA) domain, is polyubiquitinated in cells. However, the regulatory mechanisms and exact biological function of BRSK2 remain unclear. Herein, we show that BRSK2 co-localizes with the centrosomes during mitosis. We also demonstrate that BRSK2 protein levels fluctuate during the cell cycle, peaking during mitosis and declining in G1 phase. Furthermore, Cdh1, rather than Cdc20, promotes the degradation of BRSK2 in vivo. Consistent with this finding, knock-down of endogenous Cdh1 blocks BRSK2 degradation during the G1 phase. The conserved KEN box of BRSK2 is required for anaphase-promoting complex/cyclosome-Cdh1 (APC/C^Cdh1)-dependent degradation. Additionally, overexpression of either BRSK2(WT) or BRSK2(ΔKEN) increases the percentage of cells in G2/M. Thus, our results provide the first evidence that BRSK2 regulates cell-cycle progression controlled by APC/C^Cdh1 through the ubiquitin-proteasome pathway.     

A new linear cyclin docking motif that mediates exclusively S‐phase CDK‐specific signaling

Cyclin‐dependent kinases (CDKs), the master regulators of cell division, are activated by different cyclins at different cell cycle stages. In addition to being activators of CDKs, cyclins recognize various linear motifs to target CDK activity to specific proteins. We uncovered a cyclin docking motif, NLxxxL, that contributes to phosphorylation‐dependent degradation of the CDK inhibitor Far1 at the G1/S stage in the yeast Saccharomyces cerevisiae. This motif is recognized exclusively by S‐phase CDK (S‐CDK) Clb5/6‐Cdc28 and is considerably more potent than the conventional RxL docking motif. The NLxxxL and RxL motifs were found to overlap in some target proteins, suggesting that cyclin docking motifs can evolve to switch from one to another for fine‐tuning of cell cycle events. Using time‐lapse fluorescence microscopy, we show how different docking connections temporally control phosphorylation‐driven target degradation. This also revealed a differential function of the phosphoadaptor protein Cks1, as Cks1 docking potentiated degron phosphorylation of RxL‐containing but not of NLxxxL‐containing substrates. The NLxxxL motif was found to govern S‐cyclin‐specificity in multiple yeast CDK targets including Fin1, Lif1, and Slx4, suggesting its wider importance.     

A process independent of the anaphase-promoting complex contributes to instability of the yeast S phase cyclin Clb5

Proteolytic destruction of many cyclins is induced by a multi-subunit ubiquitin ligase termed the anaphase promoting complex/cyclosome (APC/C). In the budding yeast Saccharomyces cerevisiae, the S phase cyclin Clb5 and the mitotic cyclins Clb1-4 are known as substrates of this complex. The relevance of APC/C in proteolysis of Clb5 is still under debate. Importantly, a deletion of the Clb5 destruction box has little influence on cell cycle progression. To understand Clb5 degradation in more detail, we applied in vivo pulse labeling to determine the half-life of Clb5 at different cell cycle stages and in the presence or absence of APC/C activity. Clb5 is significantly unstable, with a half-life of approximately 8-10 min, at cell cycle periods when APC/C is inactive and in mutants impaired in APC/C function. A Clb5 version lacking its cyclin destruction box is similarly unstable. The half-life of Clb5 is further decreased in a destruction box-dependent manner to 3-5 min in mitotic or G(1) cells with active APC/C. Clb5 instability is highly dependent on the function of the proteasome. We conclude that Clb5 proteolysis involves two different modes for targeting of Clb5 to the proteasome, an APC/C-dependent and an APC/C-independent mechanism. These different modes apparently have overlapping functions in restricting Clb5 levels in a normal cell cycle, but APC/C function is essential in the presence of abnormally high Clb5 levels.     

Orthologues of the anaphase-promoting complex/cyclosome coactivators Cdc20p and Cdh1p are important for mitotic progression and morphogenesis in Candida albicans

The conserved anaphase-promoting complex/cyclosome (APC/C) system mediates protein degradation during mitotic progression. Conserved coactivators Cdc20p and Cdh1p regulate the APC/C during early to late mitosis and G(1) phase. Candida albicans is an important fungal pathogen of humans, and it forms highly polarized cells when mitosis is blocked through depletion of the polo-like kinase Cdc5p or other treatments. However, the mechanisms governing mitotic progression and associated polarized growth in the pathogen are poorly understood. In order to gain insights into these processes, we characterized C. albicans orthologues of Cdc20p and Cdh1p. Cdc20p-depleted cells were blocked in early or late mitosis with elevated levels of Cdc5p and the mitotic cyclin Clb2p, suggesting that Cdc20p is essential and has some conserved functions during mitosis. However, the yeast cells formed highly polarized buds in contrast to the large doublets of S. cerevisiae cdc20 mutants, implying a distinct role in morphogenesis. In comparison, cdh1Δ/cdh1Δ cells were viable but showed enrichment of Clb2p and Cdc5p, suggesting that Cdh1p may influence mitotic exit. The cdh1Δ/cdh1Δ phenotype was pleiotropic, consisting of normal or enlarged yeast, pseudohyphae, and some elongated buds, whereas S. cerevisiae cdh1Δ yeast cells were reduced in size. Thus, C. albicans Cdh1p may have some distinct functions. Finally, absence of Cdh1p or Cdc20p had a minor or no effect on hyphal development, respectively. Overall, the results suggest that Cdc20p and Cdh1p may be APC/C activators that are important for mitosis but also morphogenesis in C. albicans. Their novel features imply additional variations in function and underscore rewiring in the emerging mitotic regulatory networks of the pathogen.     

Two redundant oscillatory mechanisms in the yeast cell cycle

The cell division cycle requires oscillations in activity of B-type cyclin (Clb)-Cdk1 kinases. Oscillations are due to periodic cyclin degradation by the anaphase-promoting complex (APC) activated by Cdc20 or Cdh1, and to cyclical accumulation of the Sic1 inhibitor. The results presented here suggest that the regulatory machinery controlling Clb kinase levels embeds two distinct oscillatory mechanisms. One, a "relaxation oscillator," involves alternation between two meta-stable states: Clb high/inhibitors (Sic1/APC-Cdh1) low, and Clb low/inhibitors high. The other, a "negative feedback oscillator," involves Clb kinase activation of APC-Cdc20, leading to Clb degradation. Genetic analysis suggests that these two mechanisms can function independently, and inactivation of both mechanisms is required to prevent mitosis. Computational modeling confirms that two such mechanisms can be linked to yield a robust cell cycle control system.     

Multiple mechanisms determine the order of APC/C substrate degradation in mitosis

The ubiquitin protein ligase anaphase-promoting complex or cyclosome (APC/C) controls mitosis by promoting ordered degradation of securin, cyclins, and other proteins. The mechanisms underlying the timing of APC/C substrate degradation are poorly understood. We explored these mechanisms using quantitative fluorescence microscopy of GFP-tagged APC/C^Cdc20 substrates in living budding yeast cells. Degradation of the S cyclin, Clb5, begins early in mitosis, followed 6 min later by the degradation of securin and Dbf4. Anaphase begins when less than half of securin is degraded. The spindle assembly checkpoint delays the onset of Clb5 degradation but does not influence securin degradation. Early Clb5 degradation depends on its interaction with the Cdk1–Cks1 complex and the presence of a Cdc20-binding “ABBA motif” in its N-terminal region. The degradation of securin and Dbf4 is delayed by Cdk1-dependent phosphorylation near their Cdc20-binding sites. Thus, a remarkably diverse array of mechanisms generates robust ordering of APC/C^Cdc20 substrate destruction.