A study on premature segregation of unreplicated chromosomes 5

Chapter 5 A search for Cdc34-mediated up- or down- regulated proteins that mediate spindle elongation 5.1 Background As described above, we have observed that Cdc34 can induce prematur

Chapter 5 A search for Cdc34-mediated up- or down-

regulated proteins that mediate spindle elongation

5.1 Background

As described above, we have observed that Cdc34 can induce premature spindle elongation

by regulating spindle dynamics Although both cdc34-1 and cdc6Δ MET-CDC6 cells arrest

in G1 phase and cannot undergo S phase, they both demonstrate very different spindle

phenotypes cdc34-1 mutant (with functional Cdc6) cannot assemble a spindle and duplicated spindle poles remain connected by a bridge; however cdc6Δ MET-CDC6 cells

(with functional Cdc34) not only assemble a spindle but also untimely extend it to cause segregation of unreplicated chromosomes This raised the possibility that Cdc34 plays a central role in inducing spindle elongation In Chapter 3, we have presented evidence suggesting that Cdc34 stimulates spindle elongation by stabilizing microtubule associated proteins To elucidate the nature of Cdc34’s role in spindle extension, we employed a strategy which involved labeling of cells with stable amino acid isotopes (SILAC)(Ong et al 2002) which would help to identify proteins that are up- or down-regulated through quantitative mass spectrometry (de Godoy et al 2008) SILAC is a metabolic labeling method that utilizes a cell’s machinery to incorporate exogenous heavy isotopes of amino acid residues into expressed proteins This labeling strategy facilitates subsequent mass spectrometric analysis to differentiate between peptide signals from labeled or unlabeled source

5.2 Results

5.2.1 Cdc34 promotes up-regulation of the polo-like kinase Cdc5 during

premature spindle elongation

For SILAC mass spectrometry, cdc34-1 cdc6Δ MET-CDC6 lys1Δ strain (US7048) was used

Two overnight cultures were grown; one in medium supplemented with normal unlabelled lysine (refer to Section 2.2.15 for more details) and the other in medium supplemented with labeled H-lysine The next day, both cultures were diluted and synchronized in mitosis using

nocodazole and methionine was added to repress and deplete CDC6 Subsequently, the

unlabelled lysine cells were released into medium supplemented with methionine at 36ºC Three hours later, these cells were collected for protein extraction In contrast, the labeled H-lysine cells were released into medium containing methionine at 24˚C for three hours Cells were again collected for the preparation of total protein extracts Subsequently, both ‘total protein extracts’ from 36˚C and 24˚C samples were subjected to mass spectrometric analysis

as described in Section Error! Reference source not found

As shown in Figure 33A, the spectra produced by mass spectrometry analysis identified 2781 proteins Of these, 187 were up-regulated, 2390 were unchanged and 204 were down-regulated Cdc5, which was up-regulated two fold upon restoration of Cdc34 function, specifically drew our attention because when overexpressed, Cdc5 has been shown

to promote, spindle formation in spindle assembly-defective cdc28Y19E cells Additionally,

Cdc5 is also reported to play a synergistic role with Cdk1 to phosphorylate Cdh1 at multiple sites leading to its inactivation (Crasta et al 2008) This allows accumulation of microtubule associated proteins and, consequently spindle formation

Up-regulated Proteins:

Protein

Gene Names

Molecular Weight (kDa)

Ratio H/L Normalized

Ratio H/L Significance

Molecular Weight (kDa)

Ratio H/L Normalized

Ratio H/L Significance

Table 6: Cdc34 mediated Up- or Down-regulated proteins with functions relevant to SCF and spindle dynamics

To confirm the above observations, we attempted to monitor the stability of Cdc5 in

the absence and presence of Cdc34 function We treated two separate cultures of cdc34-1 cdc6Δ MET-CDC6 cdh1Δ cells (US6938) with nocodazole to arrest them in mitosis in medium supplemented with methionine at 24˚C to repress CDC6 transcription and to ensure

complete Cdc6 depletion Subsequently, both cultures were released into medium containing methionine at 36˚C to inactivate Cdc34 and arrest at Cdc34-execution point until 210 minutes

At this juncture, one culture was kept in 34˚C while the other was shifted to 24˚C to allow restoration of Cdc34 function Samples were taken every 30 minutes for Western Blot analysis Similar to microtubule associated proteins such as Ase1 and Cin8, Cdc5 is unstable

in the absence of Cdc34, even when CDH1 is deleted This suggests that, Cdc5 may be

targeted for proteolysis via the ubiquitin-independent pathway Upon restoration of Cdc34, Cdc5 is stabilized (Figure 33B)

G6PD Cdc5

Two-fold increase in Cdc5

B

A

Figure 33 Cdc34 promotes up-regulation of the polo-like kinase Cdc5 during premature

spindle elongation

A For SILAC mass spectrometry, strain was used Two

overnight cultures were grown; one in medium supplemented with normal unlabelled lysine

(refer to Section 2.2.15 for more details) and the other in medium supplemented with labeled

H-lysine The next day, both cultures were diluted and synchronized in mitosis using

nocoda-zole and methionine was added to repress and deplete Subsequently, the unlabelled

lysine cells were released into medium supplemented with methionine at 36ºC Three hours

later, these cells were collected for protein extraction In contrast, the labelled H-lysine cells

collected for the preparation of total protein extracts Subsequently, both ‘total protein extracts’

Section 2.2.15 As shown is the spectrum of Cdc5 produced by mass spectrometry analysis

B To confirm the above observations, we attempted to monitor the stability of Cdc5 in the

absence and presence of Cdc34 function We treated two separate cultures of

cells with nocodazole to arrest them in mitosis in medium supplemented

transcription and to ensure complete Cdc6 depletion

-Samples were taken every 30 minutes for Western Blot analysis.

5.2.2 Ectopic expression of Cdc5 can induce spindle formation and

elongation

Our initial investigations in cdc34-1 and cdc34-1 cdc6Δ MET-CDC6 cdh1Δ cells have

provided evidence that ectopic expression of Ase1, Cin8 and Kip1 can induce spindle formation and spindle elongation in a large majority of cells, although not in all cells Since Cdc5 stabilization is mediated by Cdc34, similar to that observed for microtubule associated proteins, and its stabilization coincides with spindle elongation, we tested if Cdc5 over-

expression can affect the fate of spindles To test this possibility, cdc34-1 and cdc34-1cdh1Δ cells each carrying GAL-CDC5 and GAL-CDC5 (N209A-kinase dead) (US7044, US7046,

US7045 and US7047) were first synchronized in G2-M by nocodazole treatment in raffinose medium supplemented with methionine These cells were then subjected to second synchronization in the subsequent G1 phase by α-factor treatment in medium containing raffinose and methionine The cells were pre-induced with galactose for 1 hour to express Cdc5 or kinase-dead Cdc5 and then released into medium containing raffinose and galactose

at 34˚C As shown in Figure 34, over-expression of Cdc5 allowed 30% of cdc34-1 cells to

form short spindles at non-permissive temperature Moreover, over-expression of Cdc5 can

induce spindle elongation in 60% of cdc34-1 cdh1Δ cells It should be noted that these cells

can assemble short spindles without Cdc5 over-expression due to lack of Cdh1 function As

a control, we have overexpressed kinase-dead Cdc5 and have observed that kinase-dead Cdc5 fails to promote any spindle formation or spindle elongation The data suggests that Cdc5 can contribute to the regulation of spindle dynamics and can promote spindle elongation

Nomarski DAPI Anti-tubulin

cdc34-1 GALCDC5(N209A)

Nomarski DAPI Anti-tubulin

Nomarski DAPI Anti-tubulin

cdc34-1 GALCDC5

Nomarski DAPI Anti-tubulin

100% no spindles

70% no spindles 40% short spindles

30% short spindles 60% long spindles

5.2.3 Cdc5 is unstable in the absence of Cdc34 function

Our previous investigations showed that Ase1 and Cin8 are highly unstable in cdc34-1 cells compared to that in cdc6Δ cells This explains the inability of cdc34-1 cells to assemble a spindle Both Ase1 and Cin8 become more stable when CDH1 is deleted in cdc34-1 cells

and they contribute to short spindle assembly Since Cdc5 overexpression cannot induce

spindle formation and spindle elongation in all the cdc34-1 and cdc34-1 cdh1Δ cells, it is

possible that Cdc5 is in low abundance in Cdc34 deficient cells due to enhanced proteolysis

To test this, cdc34-1, cdc6Δ MET-CDC6, cdc34-1 cdh1Δ and cdc6Δ MET-CDC6 cdh1Δ cells carrying myc6-tagged CDC5 under the control of the GAL1 promoter (US7044, US7046,

US7049 and US7050) were grown overnight in medium lacking methionine The overnight cultures were washed and inoculated into raffinose and galactose medium supplemented with methionine at 36˚C Cells were kept in this medium for 2 hours to pre-induce Cdc5 and then released into their respective non-permissive conditions in glucose medium with 0.1mg/ml cycloheximide (to inhibit de novo synthesis of Cdc5) and the fate of the protein pulse was

monitored As shown in Figure 35, protein pulse was not detected in cdc34-1 cells indicating that Cdc5 is extremely unstable However, Cdc5 was remarkably stable in cdc6Δ cells, correlating with premature spindle elongation Moreover, Cdc5 was more stable in cdc34-1 cdh1Δ cells as compared to cdc34-1 cells (Figure 35, lower panels) This suggests that Cdc5

may be degraded via an ubiquitin-independent pathway in the absence of Cdc34 Overall,

Cdc5 protein pulse appeared most stable in cdc6Δ cells due to the presence of Cdc34 This

strongly supports the notion that Cdc34 is involved in regulating the stability of spindle elongation effectors such as Ase1, Cin8 and Cdc5

(Cdc34 +) (Cdc34 -)

Figure 35 Cdc5 is unstable in the absence of Cdc34 function

-cdc34-1

-5.3 Discussion

Using SILAC mass spectrometry, we have identified a few up- or down-regulated proteins with functions closely related to spindle elongation upon restoration of Cdc34 function Amongst those proteins, Cdc5 seems to be a plausible candidate as it has been reported to play a synergistic role with Cdk1 to inactivate Cdh1, thus allowing the accumulation and up-regulation of microtubule associated proteins such as Cin8 and Kip1 These proteins presumably generate a ‘sliding force’ to induce spindle elongation regardless of which cell cycle stage the cells are in Cdc34 appears to be also responsible for stabilization and up-regulation of Cdc5 Once stabilized in G1 phase, Cdc5 can contribute to spindle elongation

in cells that fail to undergo S phase This is because prolonged delay in G1 phase will allow Cdc5 to accumulate; consequently Cdh1 is inactivated, thus resulting in the accumulation of microtubule associated proteins and triggering premature spindle elongation Our mass spectrometric analysis has also identified Kip3 as one the proteins that is, unlike Cdc5, down-regulated upon the restoration of Cdc34 function Kip3, a kinesin-8 family member, undergoes plus-end directed motility and can perform plus-end microtubule depolymerization (Du et al.), (Walczak 2006), (Gupta et al 2006) It is possible that Kip3 is subjected to negative regulation by Cdc5 This may contribute to premature spindle elongation due to stabilization of the spindle dynamics

Polo-like kinases (e.g Cdc5) are evolutionarily conserved proteins that belong to a subfamily of Ser/Thr protein kinases In the budding yeast, polo-like kinase Cdc5 is well known for its multiple roles in mitosis (Lee et al 2005) During mitosis, Cdc5 regulates transcription of mitotic genes via phosphorylation of Ndd1, a subunit of the Mcm1-Fkh2-Ndd1 transcription factor (Darieva et al 2006) In anaphase, Cdc5 phosphorylates the subunits of condensin to promote chromosome condensation (St-Pierre et al 2009) In addition, Cdc5 mediated phosphorylation during metaphase to anaphase transition leads to

cleavage of cohesins by separase (Alexandru et al 2001) Cdc5 has also been implicated in regulation of APC/C and in the degradation of cyclin B during anaphase (Charles et al 1998; Shirayama et al 1998) As a component of the FEAR pathway, Cdc5 mediates the release of Cdc14 phosphatase in early anaphase through phosphorylation (Visintin et al 2003; Rahal et

al 2008) Furthermore, Cdc5 phosphorylates Bfa1, the negative regulator of the MEN pathway to promote mitotic exit (Hu et al 2001) During cytokinesis, Cdc5 is also involved

in Rho1 activation and contractile actin ring (CAR) assembly (Yoshida et al 2006)

In cultured mammalian cells, Plk1 facilitate the establishment of a centrosomal scaffold for microtubule nucleation by phosphorylating and displacing the centrosomal protein, Nlp (Casenghi et al 2003) Plk1 also appears to regulate microtubule dynamics by either positively or negatively regulation of various microtubule associated components While Plk1 has been reported to phosphorylate and diminish the microtubule-stabilizing activity of TCTP (Yarm 2002), Plx1 was reported to stabilize microtubules via negative

regulation of microtubule-destabilizing protein, Stathmin/Op18 in Xenopus (Budde et al

2001)

Thus polo kinase(s) participate in a myriad of cellular functions While polo kinase’s role in the regulation of spindle dynamics has long been known in vertebrate cells, its involvement in aspects of spindle biogenesis in budding yeast has emerged only in the last few years The present study specifically underscores its role in the premature spindle elongation which can lead to extreme genomic instability in cells that are either delayed or failed completely to initiate DNA replication Cdc34 emerges in these investigations as the master regulator of spindle dynamics in G1 phase by promoting stability of microtubule associated proteins and Cdc5 to induce premature spindle elongation should cell cycle committed (post START) cells fail to execute S phase

Chapter6 Perspective and future directions

Since 19th century, abnormal chromosome number – also known as aneuploidy – has been recognized as a near ubiquitous feature of human cancers Study of colorectal cancers has shown that approximately 85% display aneuploidy errors, and contain cells with an average

of 60 to 90 chromosomes (Pellman 2001) Moreover, high clinical grades tumours are closely associated with greater degrees of aneuploidy In normal cells, the constancy of chromosome number is maintained through a highly coordinated progression through the cell division cycle The cellular coordination is imposed by checkpoint controls to ensure that, if

a certain event is executed erroneously or interrupted, the initiation of the subsequent phase

of the cell cycle is delayed Budding yeast Saccharomyces s cerevisiae has served as an

excellent model to uncover the regulatory circuits underlying these controls While replication, DNA damage and spindle assembly checkpoints have been extensively investigated, G1-M checkpoint has remained under explored In this study we have attempted to uncover the mechanism underlying the G1-M checkpoint; deficiency of which leads to premature segregation of the unreplicated chromosomes; a phenotype that represents

an extreme form of chromosome instability

An Overview and the Emerging Model

It has long been thought that precocious segregation of chromosomes in checkpoint deficient mutants is due to a premature entry into mitosis However, recent evidence suggests that, at least in case of the replication checkpoint deficient mutants, premature segregation of unreplicated chromosomes is not due to premature entry into mitosis but because spindle dynamics is deregulated (Krishnan et al 2004) Tight regulation of the spindle dynamics is particularly necessary in the early part of the division cycle when chromosomes have not attained biorientation and so are not able to resist the tendency of the spindle to elongate

Cells that are unable to initiate DNA replication (cdc6, cdc7 and cdc45 mutants for instance)

represent the danger posed by the spindle to unreplicated chromosomes It has long been

thought that precocious segregation of unreplicated chromosomes in cdc6 mutants is due to

premature entry into mitosis This has led to the postulation of a G1-M checkpoint of which Cdc6 is considered to be a prominent component (Toyn et al 1995) However, the exact nature of this checkpoint and Cdc6’s role in it has remained a mystery

So far, Cdc6 is the only known component of the G1-M checkpoint Although unable

to initiate S phase, Cdc6 deficient cells undergo precocious partitioning of the unreplicated chromosomes Therefore, Cdc6 has been ascribed the role of a checkpoint protein whose function is to prevent onset of mitosis when cells fail to initiate DNA replication In a somewhat extended version of this notion, the signals emerging from the pre-replication complex activates Cdc6’s checkpoint function which prevents premature initiation of mitosis (hence, segregation of unreplicated chromosomes) (Piatti et al 1995) This model is conceptually attractive as it suggests a long-range coordination control that prevents cell cycle division-committed G1 cells from initiating mitosis prematurely Our study documented in this thesis brings this entire notion of G1/M checkpoint into question First, our results show that premature segregation of chromosomes in Cdc6 deficient cells does not result from untimely onset of mitosis We also find that premature segregation of unreplicated chromosomes is a phenotype exhibited not only by Cdc6 deficient cells but also

other mutants (cdc7Δ and cdc45Δ) that fail to initiate DNA replication, despite their ability to

assemble a pre-replication complex This suggested that untimely segregation of unreplicated chromosomes is perhaps a common property of G1 cells that traverse START but are unable

to initiate DNA replication

However, cdc34 mutant appears to be an exception to this tentative rule Cells

deficient in Cdc34 function traverse START and are unable to initiate DNA replication but

yet do not undergo precocious chromosome segregation It can be argued that this is so

because Cdc6 is functional in cdc34 mutant and prevents untimely partitioning of chromosomes The fact that cdc34 cdc6Δ double mutant also fails to segregate chromosomes

precociously implies that it is not the deficiency of Cdc6 per se that causes untimely

chromosome segregation This is in fact not surprising because cdc34 cdc6Δ mutant cells are

unable to assemble a spindle that can mediate chromosome segregation Short spindle

assembly can be induced in the cdc34 cdc6Δ double mutant by deletion of CDH1 (which

controls the stability of microtubule binding proteins Cin8, Kip1 and Ase1) Surprisingly,

cdc34 cdc6Δ cdh1Δ triple mutant assembles a short spindle yet is unable to extend it to

segregate unreplicated chromosomes These observations together with the previous report that premature chromosome segregation in replication checkpoint deficient mutants is due to the deregulation of spindle dynamics (Krishnan et al 2004) prompted us to direct our efforts

to the exploration of spindle regulation in cdc34 cdc6Δ cells

Once cells traverse START in late G1 , they are irreversibly committed to undertake the progression through the division cycle and exhibit two conspicuous morphological features before initiation of S phase: emergence of a bud and duplicated spindle poles connected by an inter SPB bridge The short spindle assembly requires breaking of the bridge that connects the duplicated SPBs It has been previously shown that plus-end directed kinesin motors Cin8 (homologue of Eg5) and Kip1 and microtubule associated protein Ase1 mediate SPB separation by breaking the bridge, thus leading to short spindle assembly (Crasta et al 2006) Like mitotic cyclins, Cin8, Kip1 and Ase1 are targeted for proteolytic destruction by E3 ubiquitin ligase APCCdh1 (anaphase promoting complex activated by Cdh1) that is active during G1 through to S phase To be able to separate SPBs and assemble a short spindle, it is imperative for cells to accumulate these microtubule associated proteins The premature extension of the spindle in G1 can be viewed not as an unscheduled entry into mitosis per se

but as a disconnection of the spindle cycle from the rest of the cell cycle machinery Under normal circumstances, short spindle forms only at the end of S phase However, the failure to initiate S phase causes an extended stay in G1 phase This results in the accumulation of elongation-conducive microtubule binding proteins (Cin8, Kip1, Ase1) and eventually premature extension of the spindle Since at this stage, chromosomes are unreplicated, kinetochore duplication, bi-orientation and cohesins are unable to resist the dramatic spindle extension, as they collectively do during normal S phase, G2 and metaphase, hence spindles form and prematurely elongate (i.e premature chromosome segregation) Thus initiation of DNA replication that allows duplication of kinetochores, chromosome biorientation and tethering of chromosomes by cohesins provide a major deterrent to premature spindle extension As our results suggest, the mechanics of MAPs accumulation in G1 is controlled

by a complex regulatory network Previous studies had shown that Cdh1 is a potent inhibitor

of spindle assembly in that the expression of a constitutively active allele (cdh1-m11A)

causes cells to arrest in G2 with unseparated SPBs (Crasta et al 2006) Failure to break the intra-SPB bridge can be attributed to very low levels of Cin8, Kip1and Ase1 Further analysis revealed that Cdc28/Cdk1-Clb kinase and Cdc5 acted in synergy to fully inactivate Cdh1 via phosphorylation at multiple sites during normal cell cycle to allow accumulation of MAPs and the assembly of a short spindle (Crasta et al 2008) As noted before, deletion of

CDH1 in cdc34-1 cdc6Δ cells only allows biogenesis of short spindles, but spindle elongation

is still prohibited However, restoration of Cdc34 function results in further accumulation of Cin8 and Ase1, triggering spindle elongation and premature chromosome segregation This suggests that the limiting factor that prevents spindle elongation is the further accumulation

of microtubule associated proteins These results also imply that the MAPs are targeted for destruction not only by Cdh1 but also by another machinery that is negatively regulated by Cdc34 (or SCF by implication)

Based on our observations, we propose that Cdc34 has a dual role in mediating premature spindle elongation in cells that fail to undergo S phase (Figure 36) Firstly, Cdc34 catalyzes degradation of Sic1 with the help of Cdc28-Cln kinase, leading to the activation of Cdc28/Clb kinase required for Cdh1 inactivation This results in accumulation of microtubule associated proteins to an extent such that only biogenesis of short spindles is permitted Cdc34 also appears to be involved in the stabilization and up-regulation of microtubule associated proteins such as Ase1 and Cin8, and Cdc5 by protecting them against the yet-to-be-identified degradation pathway This further augments the accumulation of Ase1 and Cin8 to a sufficiently high level that is necessary to provide sufficient force to elongate the spindle The up-regulated Cdc5 also works in synergy with Cdk1 to enhance the inactivation of Cdh1 In addition, Cdc5 is also required for proper spindle dynamics and growth (Park et al 2008) Intriguingly, Cdc5 can phosphorylate several spindle pole body (SPB) or spindle associated proteins, in vitro, raising the possibility that Cdc5 regulates spindle functions via phosphorylation of these proteins directly Cdc5 has also been implicated in negatively regulating microtubule-destabilizing proteins (Park et al 2008) Hence, yeast microtubule destabilizing protein Kip3 may also participate in this regulatory circuit

Figure 36 The emerging model proposed that Cdc34 has a dual role in mediating premature spindle elongation in cells that fail to undergo S phase.

Finally, this regulatory scheme suggests that Cdc34, and not Cdc6, is a key player in the premature spindle elongation and untimely segregation of unreplicated chromosomes Cdc6 does not directly participate in this process; instead, its deficiency prevents the initiation of DNA replication and allows the manifestation of the underlying circuitry Cells that have traversed START and completed the ‘Cdc34-execution point’ also set in motion the spindle assembly and elongation machinery It is through the initiation of DNA replication (via kinetochore duplication, cohesin-loading and biorientation) that cells restrain spindle elongation and avoid premature chromosome segregation In other words, cells that have traversed START and completed Cdc34 execution step must initiate DNA replication or face extreme chromosomal instability

Future Directions

While this study has contributed to a better understanding of premature spindle elongation and has introduced a new perspective where both spindle cycle and cell cycle require precise and timely coordination especially in cells with unreplicated chromosomes, much remains to

be done in order to achieve a full understanding of the mechanisms underlying the entire regulatory network that safeguards genomic integrity by avoiding premature spindle elongation Our experimental outcomes, together with mass spectrometric analysis, suggest that Cdc34 is the most upstream master regulator of spindle dynamics We postulate that Cdc34 catalyzes short spindle biogenesis by targeting Cdh1 inactivation involving a number

of regulators and simultaneously boosts the abundance of microtubule associated proteins by protecting them against ubiquitin-independent degradation pathway Cdc34 also promotes stability of Cdc5 that plays an important role in complete inactivation of Cdh1 and negative regulation of microtubule depolymerization factors such as Kip3 In order to strengthen this hypothesis, it is imperative to investigate the following:

(i) Since Cdc34 is component of the SCF complex, it will be necessary to identify the participating F-box proteins, which determines the substrate specificity of the SCF complex

in the present context This will help in the identification of proteins whose destruction is required for the stabilization of both microtubule associated proteins and Cdc5 polo kinase

(ii) Since Chinese hamster ovary cells can assemble a mitotic spindle and progress through M-phase with unreplicated chromosomes, it will be interesting to determine if Cdc34 function is required for the unscheduled progression into mitosis A positive answer to this query may suggest the conservation across species of the regulatory scheme we have proposed

(iii) As Kip3 was identified as one of the proteins down-regulated during premature spindle elongation, it will be useful to determine if the Cdc34-mediated accumulation of Cdc5 is responsible for the negative regulation of Kip3

(iv) It will be interesting to ascertain if the function we have ascribed to Cdc34 in inducing premature spindle elongation is via its requirement for the activity of SCF complex

or independent of SCF

In closing, this study has attempted to attain a better understanding of the regulation of

spindle dynamics in G1 and its coordination with S phase initiation in Saccharomyces cerevisiae It is hoped that by gaining further insights into the spindle biogenesis and spindle

elongation process regulated by Cdc34, Cdks, Polo kinases, APCCdh1, our understanding of how proliferating cells manage to segregate their chromosomes with high fidelity between daughter cells will be enhanced Insights gained from the studies on model organisms may eventually be useful in understanding the cell cycle coordination and chromosome stability in vertebrate cells

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