Alternation of chromosome replication and segregation is essential for successful completion of the cell cycle and it requires an oscillation of Cdk1 (cyclin-dependent kinase 1)–CycB (cyclin B) activity. In the present review, we illustrate the essential features of checkpoint controlled and uncontrolled cell-cycle oscillations by using mechanical metaphors. Despite variations in the molecular details of the oscillatory mechanism, the underlying network motifs responsible for the oscillations are always well-conserved. The checkpoint-controlled cell cycles are always driven by a negative-feedback loop amplified by double-negative feedbacks (antagonism).
- cell cycle
Alternation of chromosome replication and segregation
During the cell division cycle, cells must replicate their DNA first and segregate the end-products of the replication process (sister chromatids) later. In order to preserve the genetic information encoded by chromosomes, these two processes, chromosome replication and segregation, must alternate with each other during the cell cycle . This strict alternation of chromosome replication and segregation is ensured by biochemical regulatory mechanisms (Figure 1). Eukaryotic chromosome replication is triggered by Cdk (cyclin-dependent kinase) activity. Cdks are active in complex with their cyclin partners  and in lower eukaryotes Cdk1–CycB (cyclin B) dimers initiate S-phase. In order to avoid over-replication of the genome, a licensing control system ensures that each replication origin is activated only once per cell cycle . Origin firing and licensing are co-ordinated via Cdk activity, which besides activating origin firing, also inactivates components in the licensing system . Therefore replication origins are licensed in a low Cdk activity (G1) phase of the cell cycle (Figure 1).
DNA replication produces two identical sister chromatids which are held together by cohesin complexes . The rule of strict alternation requires that DNA replication is followed by chromosome segregation (anaphase) which depends on removal of cohesins from chromosomes. Cohesins are cleaved by a specific protease, separase, when the inhibitor of separase, securin, is degraded . Chromosome segregation (anaphase) is initiated by activation of APC/C (anaphase-promoting complex /cyclosome ) ubiquitin-ligase which targets securin for proteasome-dependent degradation, thereby activating separase. The other essential substrate of APC/C is the CycB subunit of the Cdk1–CycB complex. The concomitant degradation of CycB during anaphase inactivates Cdk and thereby allows replication origins to re-license.
In summary, the alternation of DNA replication and chromosome segregation requires a fluctuation in Cdk activity between low and high values. The oscillation in Cdk activity is provided by systems-level feedback loops. The simplest Cdk oscillator is operating in early embryos (such as Xenopus and Drosophila) and it is based on the negative-feedback loop between Cdk and APC/C . In the following discussion we present simple mechanical models to illustrate the main features of eukaryotic cell-cycle controls and we direct interested readers to the original publications for the corresponding mathematical models [10–19].
The Cdk–APC/C negative-feedback loop
A good illustration of the Cdk–APC/C negative-feedback loop is the Japanese deer chaser Shishi Odoshi (Figure 2). The open end of the hollow arm corresponds to Cdk activity, which is lifted up by the heavy closed end of the arm (CycB synthesis) and the rising water level in the hollow arm corresponds to APC/C activation. The arm tips over once the weight of the water exceeds a threshold determined by the weight at the closed end. At this point the water flows out, the arm is emptied and it rises again. The arm oscillates between low and high values similar to Cdk activity during the cell cycle. Observe that APC/C activity (water in the arm) oscillates as well: when the arm is in the upper position (high Cdk) APC/C activity increases, whereas APC/C decreases when the arm is in the lower position (low Cdk). The rising and falling water levels in the arm introduce important time-delays which are essential for oscillators based on negative-feedback only .
This Cdk–APC/C negative-feedback oscillator operates in such an ‘uncontrolled’ way only in some early embryos such as Xenopus . In non-embryonic cells the oscillator is controlled by checkpoint mechanisms that stop the oscillator in low (before DNA replication) and high (before chromosome segregation) Cdk activity states.
The G1 checkpoint
The G1 checkpoint blocks the cell-cycle clock in the low Cdk activity (G=) state by keeping APC/C active which promotes CycB degradation. Since the form of APC/C (called APCCdc20) promoting chromosome segregation is inactivated at low Cdk activity, the G1 checkpoint uses another form of APC/C (called APCCdh1). APCCdh1 is active at low Cdk activity, but it is inactivated by Cdk-dependent phosphorylation [22,23]. Since APCCdh1 promotes CycB degradation by ubiquitination, it has an antagonistic relationship with Cdk activity . As a consequence APCCdh1 can be represented as a heavy ball (see Figure 3) in our mechanical metaphor . When the ball is rolled out, APCCdh1 is active and Cdk activity is kept low (arm down). On the other hand, when Cdk activity is high (arm is up) the ball is back to the hinge and APCCdh1 is inactive.
In fact, the G1 checkpoint also uses a stoichiometric CKI (Cdk inhibitor; called Sic1 and Rum1 in budding and fission yeasts respectively) to keep Cdk activity low in G1 phase [25,26]. The CKI also behaves like a ball in the mechanism because its relationship with Cdk activity is also antagonistic: the Cdk1–CycB complex inhibits the transcription [27,28] and promotes the degradation of its inhibitor (CKI) in yeast [29,30].
In order to terminate the G1 checkpoint, cells require SK (starter kinase) molecules that inactivate APCCdh1 and the CKI . Usually cells use Cdk–cyclin complexes (such as Cdk1–Cln complexes in budding yeast) as SKs which are resistant to inhibition by APCCdh1 and the CKI. SKs can be represented as a force that pushes back the ball to the hinge where its exerted torque is zero (Figure 3C). Inactivation of APCCdh1 and the CKI by SKs allow CycB synthesis to raise Cdk activity and to initiate S- and M-phases. Once the ball is pushed back to the upright support and Cdk activity is self-maintaining, the SK has to be inactivated. This is important for subsequent cell-cycle transition, because permanent activation of the SK could inhibit the rolling out of the ball at the time of APCCdc20 activation . For this reason Cdk1–CycB activity represses the transcription of cyclin components of the SK .
Once the G1 checkpoint is in place, the chromosome cycle is dictated by the timing of SK activation rather than by the free-running cell-cycle clock . Since this is often linked to reaching a critical cell size , the G1 checkpoint allows co-ordination of the chromosome cycle with cytoplasmic growth.
The mitotic checkpoint
The high Cdk activity state is stabilized by the mitotic checkpoint that blocks APCCdc20 activation (water flow) until all of the chromosomes have a bipolar attachment to the spindle . Once the mitotic checkpoint is lifted, the activation of APCCdc20 (water in the hollow arm) promotes complete destruction of CycB and thereby drives mitotic exit. The drop in Cdk activity caused by APCCdc20 can activate APCCdh1 and CKI (rolling out of the ball) with passive help from a phosphatase. Interestingly, in budding yeast APCCdc20 can trigger anaphase by activating separase via complete degradation of securin, but it fails to destroy all of the CycB and, therefore, it does not allow rolling out of the ball as well. Therefore mitotic exit in budding yeast requires an active role from the Cdc14 phosphatase which corresponds to a force that pushes the ball to the right on Figure 3(B) . Cdc14 phosphatase activates APCCdh1 directly and CKI indirectly (through activation of its transcription factor, Swi5), by dephosphorylation of their Cdk phosphorylation sites .
Since the Cdc14 phosphatase represents a force pushing the ball to the right, it becomes inhibitory at the time of G1 checkpoint inactivation, when the ball needs to be pushed to the left. Therefore Cdc14 has to be inactivated after exit from mitosis, which is achieved by a negative-feedback loop. The release of Cdc14 from the nucleolus is a separase-dependent process which also requires both Cdk1–CycB  and Polo-kinase  activities. Since Cdc14 activates APCCdh1 which promotes the degradation of both CycB  and Polo-kinase [39,40], which in turn, are both required for Cdc14 to be released, the phosphatase gets re-sequestered in the nucleolus [41,42].
Bistable switch in the budding yeast cell cycle
During the budding yeast cell cycle, both activation and inactivation of Cdk are initiated by helper molecules (SKs and Cdc14 phosphatase) which push the ball back and forth along the arm on Figure 3. As a consequence the budding yeast cell cycle has lost all the clock-like characteristics of the Cdk-APCCdc20 negative-feedback loop. The budding yeast cell-cycle control rather centres around the bistable switch based on the antagonism between Cdk1–CycB and its negative regulators (APCCdh1 and CKI) [13,18,43]. This antagonism creates two alternative steady states (see Figure 3) and the transitions between these two states are triggered by helper molecules (SK and Cdc14) which are down-regulated after the transition they are enforcing.
The bistable switch in budding yeast cell-cycle control is supported by a lot of experimental evidence. Cdc20-deprived budding yeast cells are blocked with high Cdk1–CycB activity and inactive APCCdh1 and CKI . Notice that this situation corresponds to the absence of water flow in the Shishi Odoshi model. This high Cdk activity steady-state is stable against ectopic (e.g. Gal promoter induced) overexpression of Cdh1 (cadherin 1) or CKI [44,45]. These Cdk1–CycB complex inhibitors cannot work at high Cdk, because high Cdk activity promotes the inactivation and degradation of its negative regulators. Ectopic expression of these CKIs represents a heavier ball in the Shishi Odoshi, which cannot roll down because the arm is completely pulled up.
On the other hand, ectopic expression of non-phosphorylable Cdh1 and CKI (ball components) can bypass all Cdk controls, and destabilize the high Cdk activity state and induce mitotic exit [44,45]. Non-phosphorylable Cdh1 (Cdh1 constitutively active, Cdh1CA) and CKI (non-degradable CKI, CKInd) are resistant to Cdk inhibition and should be considered as forces which push down the arm of Shishi Odoshi by down-regulating Cdk activity. As a consequence, the ectopic expression of either of these Cdk inhibitory molecules forces the ball (endogenous Cdh1 and CKI) to roll out which further inhibits Cdk activity. The mitotic exit transition becomes irreversible once the rolling out ball stabilizes the low Cdk activity G1 state even in the absence of the initial stimulus (Cdh1CA or CKInd). For instance, it has been shown recently, that Cdh1CA-induced mitotic exit becomes irreversible after 50 min .
Mitotic exit induced by non-phosphorylable Cdk inhibitors (Cdh1 or CKI) takes place in the absence of anaphase because separase is not activated in the absence of Cdc20-dependent securin degradation. Since Cdc14 activation is separase-dependent, Cdc14 release from the nucleolus is compromised and the activation of endogenous Cdh1 and CKI is slow, i.e. the ball rolls out slowly. The mitotic exit process can be accelerated by overexpression of Cdc14 phosphatase  or inducing anaphase .
Cycling without APC/C
The cells forced to exit from mitosis in the absence of Cdc20 are inviable because APCCdc20 is essential for securin degradation and thereby for anaphase. This Cdc20 requirement of anaphase can be easily bypassed by securin (called Pds1 in budding yeast) deletion and cdc20Δpds1Δ double mutants undergo normal anaphase [46,47]. Although Cdc14 phosphatase is activated , it is not strong enough to overcome the high Cdk activity (Cdc14 cannot push the ball out from the hinge). This block of mitotic exit can be overcome by either reduction of Cdk activity or overexpression of CKI (called Sic1 in budding yeast). The Cdk activity can be reduced by deleting one of the early-acting cyclin genes such as Clb5, and cdc20Δpds1Δclb5Δ triple-mutant cells can exit from mitosis . Overexpression of the stoichiometric CKI makes the ball heavier which, with the help of Cdc14, can down-regulate Cdk activity as well . The combination of these two effects (reduced cyclin level and CKI overexpression) in the cdc20Δcdh1Δpds1Δclb5Δsic1op mutant makes APC/C altogether dispensable for budding yeast cells . The viability of this quintuple mutant shows that alternation of chromosome replication and segregation only requires oscillation in Cdk activity, but not APC/C. In the absence of APC/C-dependent cyclin degradation, the Cdk1–CycB activity oscillations are driven by fluctuating CKI levels at more or less constant CycB levels. At high Cdk1–CycB activity, Sic1 gets up-regulated through Cdc14-dependent activation of its transcription factor, Swi5 and inhibits Cdk1–CycB complexes. However down-regulation of Cdk1–CycB activities up-regulates SK (Cdk1–Clns) which are not inhibited by Sic1 and they promote Sic1 degradation. The oscillating Cdk1–CycB activity co-ordinates DNA replication with anaphase as well, because cohesin cleavage is regulated not only by separase, but also at the substrate (cohesion) level. Cohesin cleavage is dependent on prior phosphorylation of cohesin by Polo-kinase , the activity of which is dependent on Cdk1–CycB .
The oscillation of Cdk1–CycB activity required for the successful completion of the cell cycle can be achieved by different biochemical mechanisms. Although the molecular details could be different, the underlying network motifs are well-conserved. The Cdk oscillation in checkpoint-controlled cell cycles are driven by a negative-feedback loop amplified by double-negative feedback (antagonism).
This work was supported by the Biotechnology and Biological Sciences Research Council (OCISB grant); and Unicellsys [European Commission Framework Programme 7 number 201142].
We are grateful to Hisao Moriya (Okayama University, Okayama, Japan) for pointing out that the mechanical device in our publication  is called Shishi Odoshi in Japan.
Signalling and Control from a Systems Perspective: A Biochemical Society Focused Meeting held at University of York, U.K., 22–24 March 2010, as part of the Systems Biochemistry Linked Focused Meetings. Organized and Edited by David Fell (Oxford Brookes, U.K.), Hans Westerhoff (Manchester, U.K., and Amsterdam, The Netherlands) and Michael White (Liverpool, U.K.).
Abbreviations: APC/C, anaphase-promoting complex/cyclosome; Cdh1, cadherin 1; Cdk, cyclin-dependent kinase; CKI, Cdk inhibitor; CycB, cyclin B; SK, starter kinase
- © The Authors Journal compilation © 2010 Biochemical Society