A study on premature segregation of unreplicated chromosomes 3

Chapter3 Premature Chromosome Segregation in
cells with Unreplicated Chromosomes

3.1 Background
The essence of a successful mitosis is to transmit identical set of chromosomes to
each of the two progeny cells. To achieve this, many eukaryotic cells assemble a
bipolar apparatus, the mitotic spindle - that mediates equal partitioning of the
duplicated chromosomes between the two daughters. Central to this precise
segregation of the chromosomes is the amphitelic attachment of each sister-chromatid
pair to the spindle such that each member of the sister-kinetochore pair is attached to
the opposite pole (Dewar et al. 2004). This arrangement ensures the movement of
each chromosome-set in the opposite direction, fueled by the dramatic extension of
the mitotic spindle during anaphase.
However, chromosome segregation is also governed by complex dynamics at
another level. Prior to anaphase onset, spindle extension is resisted by the sister-
chromatid cohesion mediated by the cohesin complex holding duplicated sister
chromatids together. The cohesin complex in the budding yeast is composed of four
subunits: Smc1, Smc3, Scc1 and Scc3 (Nasmyth et al. 2005). These subunits are
assembled in a ‘ring-shaped’ structure that encircles the sister chromatids along the
entire length of the chromosomes. At the onset of anaphase, sister-chromatid
cohesion is dissolved by proteolytic cleavage of cohesin subunit Scc1 by separase, a
caspase-like protease encoded by the ESP1 gene in budding yeast (Uhlmann et al.
1999; Uhlmann et al. 2000). However, separase Esp1 continues to be inhibited by
securin, a protein encoded by the PDS1 gene (Morgan 1999; Shirayama et al. 1999)
till metaphase. During metaphase to anaphase transition, the E3 ubiquitin ligase

APC
Cdc20
mediates the proteolytic destruction of securin, freeing the separase from the
inhibitory effects of securin (Visintin et al. 1997; Fang et al. 1998). This leads to the

cleavage of the cohesin subunit Scc1, loss of cohesion between sister chromatids
culminating in progressive separation (mediated by the mitotic spindle) of the
duplicated chromosomes.
Being the central act of mitosis, chromosome segregation is coordinated with other
major events of the cell cycle. For instance, chromosome segregation is not initiated
until DNA replication is complete. It is also transiently suppressed if cells incur DNA
damage during S phase. The suppression is lifted only after the DNA lesions have
been repaired. Chromosome segregation or more specifically, cohesin cleavage is
also delayed when cells are unable to establish amphitelic attachment between the
sister-chromatids and the mitotic spindle. Such negative regulations are imposed by
various checkpoint controls that are operative during S phase and mitosis (DNA
replication-, DNA damage- and spindle assembly checkpoints) (Hartwell et al. 1989;
Nyberg et al. 2002). These surveillance systems ensure that chromosomes do not
segregate prematurely until the prior events are successfully completed. If these
checkpoint controls fail or function sluggishly, cells initiate chromosome separation
precociously, resulting often in unequal segregation of sister chromatids and genomic
instability leading to aneuploidy. Aneuploidy is observed in many cancer cells and is
thought to be due to either loss or malfunctioning of the checkpoint controls.
While malfunctioning of the DNA damage checkpoint and spindle assembly
checkpoint (which normally cause arrest in G2 and prometaphase, respectively) result
in mis-segregation of chromosomes, untimely chromosome segregation perhaps
represents the most dramatic manifestation of a defect in replication checkpoint
(Krishnan et al. 2004; Krishnan et al. 2005). Wild type yeast cells treated with

replication inhibitor hydroxyurea (HU) arrest in early S phase with a single nucleus,
unreplicated chromosomes and a short spindle. Checkpoint defective mec1 or rad53
cells also arrest in early S phase in response to HU treatment but proceed to rapidly
extend the spindle and unequally partition the largely unreplicated chromosomes
(Krishnan et al. 2004). Since chromosome segregation is very conspicuously
associated with mitosis (M phase), It has long been thought that precocious

chromosome segregation in these cells is due a premature onset of mitosis in the
absence of checkpoint control. However, it has now been shown that this unnatural
chromosome segregation is not due to checkpoint deficient cells’ premature entry into
mitosis but because of a combination of two events: (i) absence of chromosome
biorientation due to a failure to replicate the centromeric regions caused by replication
fork collapse (Krishnan et al. 2004) and (ii) deregulation of spindle dynamics in the
absence of the checkpoint (Krishnan et al. 2004). These findings have put the
understanding of replication checkpoint on a mechanistic footing.
Temperature sensitive mutants defective in the replication protein Cdc6 also
exhibit a similarly dramatic phenotype. At non-permissive temperature, the mutant
cells traverse START, construct a bud but are unable to assemble a functional
replication complex and therefore fail to initiate DNA replication (Piatti et al. 1995).
Despite their inability to enter S phase, these cells proceed to segregate the
unreplicated chromosomes unequally and undergo what has been sometimes been
referred to as ‘reductional division’. Once again, because of the intimate association
between chromosome segregation and mitosis, it has been proposed that Cdc6 is a
component of the G1-M checkpoint whose function is to prevent onset of mitosis
when cells fail to initiate DNA replication (Piatti et al. 1995). Thus far, no other
component of this checkpoint pathway has been identified. This notion has gained

strength from the observation that Cdc6 protein can inhibit Cdk1/Clb kinase and may
facilitate mitotic exit (Calzada et al. 2001; Lau et al. 2006). However, the origin of
the signal which activates the checkpoint function of Cdc6 or the exact mechanism by
which it prevents mitotic entry is not understood. Clearly, the inability to initiate
replication is not sensed by the replication checkpoint pathway (which detects stalled
replication forks); since it is not activated in cdc6 mutant and cells are unable to
prevent precocious chromosome segregation. In this chapter, we characterize the
behaviour of Cdc6 deficient cells more thoroughly and explore the mechanistic
underpinnings of Cdc6’s role in preventing untimely partitioning of unreplicated
chromosomes.

3.2 Results
3.2.1 Cells depleted of Cdc6 undergo premature nuclear division in
the absence of DNA replication
Premature chromosome segregation has been reported in replication checkpoint
defective mutants (such as mec1) treated with DNA replication inhibitors
(hydroxyurea) (Krishnan et al. 2004). Similarly, it has also been reported that Cdc6
deficient cells fail to initiate DNA replication but proceed to segregate the
unreplicated chromosomes prematurely (“reductional anaphase”) (Piatti et al. 1995;
Toyn et al. 1995). Recent evidence showed that the DNA replication checkpoint
thwarts untimely chromosome separation by directly regulating spindle dynamics.
The role of Cdc6 or G1-M checkpoint has remained unclear since the observation was
first documented (Piatti et al. 1995). The phenotype in both mec1 and cdc6 mutants
suggests that the mechanisms by which the DNA replication checkpoint and Cdc6
prevent premature chromosome segregation may be similar. To pursue this question,
we first re-examine if cdc6 mutant indeed exhibits premature chromosome separation.
Yeast strain carrying a CDC6 deletion (cdc6Δ) and one copy of the galactose-
inducible GAL-ubiCDC6 construct (US4275) was grown in YEP medium
supplemented with raffinose and galactose (YEP+raff+gal). Cells were arrested in
G2/M with nocodazole in YEP medium supplemented with glucose (YEPD) to
repress CDC6 transcription, washed free of nocodazole and then released into
YEP+Glu (YEPD) medium containing α-factor. The double synchronization afforded
by the G2 and subsequent G1 (α-factor) arrest precludes assembly of pre-replication
complexes (required for DNA replication) and depletes any pre-existing Cdc6. Under
these conditions, Cdc6 depleted cells uniformly arrest in G1 and are unable to initiate

DNA replication. Samples were collected at various time points for
immunofluorescence staining and FACs analysis. As expected, all the cells arrested
in G1 phase with a large bud, a long spindle, and a random segregation of
unreplicated chromosome (1C DNA) (Figure 7). The presence of cells with long
spindles showed that premature chromosome segregation had taken place. The wild-
type cells (US1363) treated in similar manner progressed into mitosis with a large bud,
a long spindle and equal segregation of duplicated sister chromatids. The kinetics of
spindle elongation between wild type and cdc6Δ cells were similar with spindle
elongation observed between 60 to 105 minutes after release from α factor. Moreover,
the Western blot analysis confirmed that Cdc6 is completely degraded in YEPD
medium. This phenotype is similar to that of the temperature-sensitive cdc6-1
mutants arrested at 37˚C (data not shown). These results confirmed that cells lacking
Cdc6 are unable to replicate DNA yet they prematurely partition the unreplicated
chromosomes, implying a role for Cdc6, in the G1/M checkpoint, in preventing
premature mitosis or chromosome segregation.

Nomarski
DAPI Anti-tubulin

60min
90min
105min
180min
0
20
40
60
80
100
0

15
30
45
60
75
90
105
120
135
150
165
180
budding index
cd c6 long spindl
e
wt budding index
w
t

l
o
n
g

sp
i
n
d
l
e

Cdc6
G6PD
Glu
RG
cyc
noc

180min
120min
60min
2N
1N
Figure 7. Cells depleted of Cdc6 undergo premature nuclear division in the absence
of DNA replication.
-
inducible GAL-ubiCDC6 construct was grown in YEP medium supplemented with
-
-
         



conditions, Cdc6 depleted cells uniformly arrest in G1 and are unable to initiate
DNA replication. Samples were collected at various time points for immunofluo-
rescence staining, FACs and Western Blot analysis.
% cells with anaphase spindles
Time (mins)

3.2.2 Premature Nuclear Division in Cdc6 Depleted Cells is
Associated with Major Mitotic Events
As mentioned earlier, major mitotic events such as APC activation, destruction of
securin (Pds1) and cleavage of the cohesin subunit (Scc1) precede anaphase or
chromosome segregation.
To determine if the premature chromosome segregation in Cdc6 deficient cells
is due to premature entry into mitosis, we monitored securin (Pds1) degradation and
cohesin (Scc1) cleavage. We first compared the kinetics of Pds1 degradation in a
cdc6Δ mutant (US4364) and wild type cells (US3538). Both strains carrying the
native promoter-driven PDS1-HA3 were synchronized as described in the previous
experiment and released into YEPD medium at 25˚C. As depicted in Figure 8A, both
strains showed Pds1 degradation from 105 minutes onwards. The wild type cells
degraded Pds1 almost completely before entering the next G1 phase. However, Pds1
was not degraded to the same extent in cdc6Δ mutant and a significant residual
amount persisted until 210 minutes (Figure 8A). Since the degree of synchrony in
both strains is comparable, this indicates that the APC
Cdc20
activity may not be
operating at its full capacity.
Next we determined if nuclear division in both wild type (US3335) and cdc6Δ
cells (US4344) is accompanied by Scc1 cleavage. Both strains carrying the native
promoter-driven SCC1-myc
18
gene were synchronized as described previously to
ensure that the cdc6 mutant did not undergo S phase but instead proceeded to
premature chromosome segregation. In the wild type strain, Scc1 cleavage was
observed from 75 minutes onwards. However, detectable Scc1 cleavage was only
noticeable after 105 minutes in cdc6Δ mutant (Figure 8B). Moreover, the abundance
of Scc1 cleaved product in cdc6Δ mutant was lower compared to that in the wild type

cells. This may be because Pds1 degradation is less pronounced (Figure 8A), leading
to fewer available active Esp1 molecules.
Besides Pds1 degradation and Scc1 cleavage, Clb2 degradation also serves as
an indicator of cell cycle progression. We monitored Clb2 degradation by Western
blotting in both wild type (US1363) and cdc6Δ (US4275) cells. While Clb2
degradation was prominent from 105 minutes onwards in wild type cells and
diminished after 150 min as cells entered the next cycle, the Clb2 proteolysis in cdc6Δ
mutant was very sluggish (Figure 8C). Once again, this may be due to insufficient
activation of APC in Cdc6 deficient cells.
Taken together, these observations suggest that cellular events (Pds1
destruction, Clb2 proteolysis) that accompany chromosome segregation in normal
cycle are significantly less pronounced in cdc6Δ cells.


3
Wildtype 
3
RG cyc
210
195
180
165
150
135
120
105
90
75
60
45

30
15

Glu noc
RG cyc
210
195
180
165
150
135
120
105
90
75
60
45
30
15

Glu noc
Pds1-HA
3
G6PD
Pds1-HA
3
G6PD
Wildtype 



RG cyc
180
165
150
135
120
105
90
75
60
45
30
15

Glu noc
 


RG cyc
180
165
150
135
120
105
90
75
60
45
30

15

Glu noc
Scc1-myc
18
Scc1-myc
18
G6PD
G6PD
*Scc1 cleaved product
*Scc1 cleaved product
Figure 8. Premature Nuclear Division in Cdc6 Depleted Cells is Associated with
Major Mitotic Events.

type cells. Both strains carrying the native promoter-driven PDS1-HA
3
were grown
in YEP medium supplemented with raffinose and galactose (YEP+raff+gal). Cells
were arrested in G2-M with nocodazole in YEPD medium to repress CDC6 transcrip-
tion, washed free of nocodazole and then released into YEPD medium containing
            
collected at various time points for Western Blot analysis.

cells. Both strains carrying the native promoter-driven SCC1-myc
18
were grown in
YEP medium supplemented with raffinose and galactose (YEP+raff+gal). Cells were
arrested in G2-M with nocodazole in YEPD medium to repress CDC6 transcription,



various time points for Western Blot analysis.
A
B
Glucose
Glucose
Glucose
Glucose


Wildtype
RG cyc
180
165
150
135
120
105
90
75
60
45
30
15

RG cyc
180
165
150
135
120

105
90
75
60
45
30
15

Clb2
Cdc28
Clb2
Cdc28
C
Figure 8. Premature Nuclear Division in Cdc6 Depleted Cells is Associated with
Major Mitotic Events.
C. Clb2 degradation also serves as an indicator of cell cycle progression. We moni-
     
grown in YEP medium supplemented with raffinose and galactose (YEP+raff+gal).
-



analysis.
Glucose
Glucose



3.2.3 Precocious Nuclear Division in Cdc6 Depleted Cells Does Not
Require Onset of Mitosis

APC activity is critical for progression through mitosis. As Pds1 and Clb2 are
degraded via APC-dependent ubiquitylation, the amount of Pds1 and Clb2 reflects the
activation status of APC. Destruction of Pds1 allows activated Esp1 to cleave Scc1,
thus dissolving chromosome cohesion leading to partition of sister chromatids. In the
preceding section we observed that although cdc6Δ cells cannot replicate DNA, they
seem to activate, albeit sluggishly, the essential events associated with normal
mitosis, such as Pds1 and Clb2 degradation as well as Scc1 cleavage. However,
despite the sluggish pace, chromosome segregation, though precocious, is remarkably
robust. Therefore we asked if APC activity is at all necessary for the precocious
chromosome segregation in Cdc6 depleted cells. We introduced a temperature
sensitive cdc23-1 allele in cdc6Δ cells expressing SCC1-myc
18
. Cdc23 is an
indispensable component of the APC; cdc23-1 mutation renders the APC inactive at
33˚C and causes cells to arrest at metaphase with short spindles and undivided sister
chromatids. Both cdc6Δ GAL-CDC6 SCC1-myc
18
(US4344) and cdc6Δ GAL-CDC6
cdc23-1 SCC1-myc
18
(US4262) cells were first synchronized in YEPD medium
containing nocodazole to deplete Cdc6. Cells were then synchronized in the next G1
phase in YEPD medium containing α factor. Finally, cells were released into YEPD
medium at the restrictive temperature (33˚C). As shown in Figure 9, while Scc1
cleavage was observed in cdc6Δ GAL-CDC6 SCC1-myc
18
cells, no detectable Scc1
cleavage product was observed in cdc6Δ GAL-CDC6 cdc23-1 SCC1-myc
18
strain. To

confirm that these cdc6Δ cells indeed undergo premature chromosome segregation,
we performed immunofluorescence staining of the spindles and images were captured
under microscope. The cdc6Δ GAL-CDC6 cdc23-1 SCC1-myc
18
cells extended their

mitotic spindle and divide their nuclei despite the lack of Cdc23 function. This
strongly implies that lack of APC activity does not prevent spindle elongation or
nuclear division in cdc6Δ mutant.



Nomarski
DAPI Anti-tubulin









180
160
140
120
100
80
60

40
20

Glu noc
180
160
140
120
100
80
60
40
20

Glu noc
G6PD
G6PD
Scc1-myc
18
Scc1-myc
18
*Scc1
cleaved product
*Scc1
cleaved product
33ºC
33ºC
33ºC at 180min
RG cyc
GluNoc

Glu 
30min
180min
90min
RG cyc
GluNoc
Glu 
30min
180min
90min
Figure 9. APC activity is not required for the precocious chromosome segregation
in Cdc6 depleted cells

18

18

cells were first synchronized in YEPD medium containing nocodazole to deplete
Cdc6. Cells were then synchronized in the next G1 phase in YEPD medium contain-
             
        
FACS and Western Blot analysis.
 

1N
2N
1N
2N

We set out to test whether APC is indeed required for chromosome segregation. APC

activity can be inhibited through over-expression of Pds1. Excessive Pds1
overwhelms the APC machinery and continues to inhibit the separase and hence Scc1
cleavage. Thus, wild type cell over-expressing Pds1 arrest in metaphase short
spindles and undivided nuclei (data not shown). Two strains were used: cdc6Δ
mutant with or without GAL-HA
3
-PDS1 (US6527 and US6537). The cdc6Δ mutant
without Pds1 over-expression served as a control. Here the cdc6Δ were kept alive by
MET3 promoter-driven wild type CDC6. Cdc6 can be depleted by growing these
cells in methionine containing medium that represses the MET3 promoter. Cells
grown in the absence of methionine were synchronized in metaphase by nocodazole
treatment in +Met medium to shut off CDC6 transcription and prevent pre-RC
assembly and DNA replication. Subsequently, the cells were subjected to a second
synchronization step in the ensuing G1 phase with α-factor, as before. Finally, cells
were released into YEP medium containing methionine, raffinose and galactose to
suppress Cdc6 expression and to induce over-expression of Pds1. As shown in Figure
10, Pds1 overexpression is induced as early as 60 minutes after release from α-factor
and peaks at around 160 minutes in cdc6Δ GAL-HA
3
-PDS1 strain. No trace of Pds1
was detected in cdc6Δ mutant control. Immunofluoresence analysis clearly showed
premature chromosome segregation accompanied by spindle elongation in both cdc6Δ
and cdc6Δ GAL-HA3-PDS1 strains (Figure 10). These results clearly suggest that
premature nuclear division in cdc6Δ mutant can occur in the absence of APC activity.
Here, APC activity is suppressed through overexpression of Pds1; Esp1 separase is
inhibited and cohesin cleavage is prevented. Thus, premature chromosome
segregation in Cdc6 deficient cells does not require APC activity and is most likely
not due to premature entry into mitosis.

Nomarski DAPI Anti-tubulin




GAL-HA
3
-PDS1
-met cyc
160
140
120
100
80
60
40
20

+met noc
-met cyc
160
140
120
100
80
60
40
20

+met noc
+met RG
160min

+met RG
G6PD
Pds1-HA
3
Pds1-HA
3
G6PD


3
-PDS1
Figure 10. APC activity is not required for the precocious chromosome segrega-
tion in Cdc6 depleted cells.
     
3
-PDS1. The

mutant was kept alive by MET3 promoter-driven wild type CDC6. Cdc6 can be
depleted by growing these cells in methionine containing medium which
represses the MET3 promoter. Cells grown in the absence of methionine were
synchronized in metaphase by nocodazole treatment in +Met medium to shut off
CDC6 transcription and to prevent pre-RC assembly and DNA replication. Sub-
sequently, the cells were subjected to a second synchronization step in the ensu-
            
-
-
rescence and Western Blot analysis.


3.2.4 Precocious Nuclear Division in Cdc6 Depleted Cells Can Be

Prevented by Dicentric Chromosomes
In a normal mitosis, chromosome segregation can be prevented by restraining cohesin
cleavage. This is possible because while the microtubules emanating from opposite
SPBs exert a poleward pulling force on the sister kinetochores to which they are
attached, this force is opposed by the cohesins holding the two sister chromatids
together, thus preventing premature chromosome segregation. In contrast, cdc6Δ
mutant cells cannot undergo DNA replication and have only unreplicated
chromosomes with one kinetochore monotelically attached to one SPB. Although
these chromosomes have cohesion associated with them, unlike a sister-chromatid
pair, they are unable to resist poleward pull exerted by any untimely extension of the
spindle. It is therefore possible that premature chromosome segregation in Cdc6
deficient cells is due to unscheduled extension of the spindle. Since we have shown
that premature nuclear division in cdc6Δ mutant does not appear to require the major
signature-events’ of mitosis, we ask if the premature nuclear division is due to
deregulation of spindle dynamics. To address this, we artificially introduced dicentric
chromosomes into cdc6Δ MET-CDC6 mutant. The dicentric chromosomes comprise
a circular minichromosome carrying two centromeres, one which is constitutively
expressed and can support the assembly of a kinetochore and the other conditionally
expressed in that it cannot support kinetochore assembly in the presence of galactose
because of its juxtaposition to GAL1-10 promoter. However, in the presence of
glucose that represses GAL1-10 promoter, the conditional centromere can now
support kinetochore assembly (Tanaka et al., 2004). The conditional and the
constitutive centromeres constitute a system that mimics the establishment of a

bipolar attachment. However, since both centromeres are on a single circular vector,
the bioriented minichromosomes cannot be segregated away from each other;
consequently it resists spindle elongation. Therefore we introduced
minichromosomes into cdc6Δ MET-CDC6 (US7007) cells where it can be propagated
stably by growing cells in the absence of methionine (for Cdc6 expression) and the
presence of galactose (conditional centromere inactive). In a parallel strain, we

introduced a CEN4 plasmid (carrying only one functional copy of CEN4) into cdc6Δ
MET-CDC6 mutant (US7008) as a control. Both strains were first arrested in
+Met+galactose medium containing nocodazole to synchronize in metaphase and
repress transcription of CDC6, followed by release into medium containing α-factor
to impose a subsequent arrest in G1. This synchronization protocol ensures that at
this juncture, both the CEN4 plasmid and dicentric chromosome have not undergone
replication. Cells were then released into medium containing glucose to activate the
second centromere in the dicentric plasmid and thus allow minichromosome to
establish bi-orientation. We monitored the state of the nucleus and the spindle using
DAPI and immunofluorescence staining, respectively. The cdc6Δ MET-CDC6
mutant carrying CEN4 plasmid equipped with only one centromere underwent
premature nuclear division (Fig 11, accompanied by dramatic spindle elongation).
However, the cdc6Δ MET-CDC6 mutant carrying the dicentric chromosome plasmid
with two active centromeres assembled short spindles and failed to undergo premature
nuclear division. From these observations, we conclude that the presence of two
centromeres in the minichromosomes promotes bipolar attachment that mimics
biorientation in normal duplicated chromosomes and restrains spindle elongation.
Hence, it is very likely that precocious segregation of unreplicated chromosomes in

cdc6Δ mutant is due to misregulation of the spindle leading to premature elongation.
In the next section we explore this notion further.

Nomarski
DAPI Anti-tubulin
-

CEN4 plasmid
-
CDC6 Dicen-
tric plasmid

Centromere1
active
GAL OFF
Centromere 2
active
Centromere1
active
CEN4
URA3
Spindle elongation
No spindle elongation
180min
Dicentric plasmid
Monocentric plasmid
cyc
noc

180min
120min
60min
2N
1N
cyc
noc

180min
120min
60min
2N
1N


Dicentric plasmid

 plasmid
Figure 11. Precocious Nuclear Division in Cdc6 Depleted Cells Can Be Prevented by
Dicentric Chromosomes.

-
   

             
+Met+galactose medium containing nocodazole to synchronize in metaphase and


juncture, both the CEN4 plasmid and dicentric chromosome have not undergone repli-

          





3.2.5 Precocious Nuclear Division in Cdc6 Depleted Cells Is Due to
Deregulation of Spindle Dynamics
It had also been shown previously that the DNA replication checkpoint prevents
precocious segregation of largely unreplicated chromosomes by regulating spindle
dynamics. We suspected that Cdc6, a putative G1-M checkpoint protein, may be
involved in regulating spindle dynamics through an unknown mechanism. Since the
BimC family kinesins such as Cin8, Kip1 and microtubule associated proteins, Ase1
are important for the dynamic behaviour of the mitotic spindle, we compared the

levels of these proteins in wild type, cdc6Δ and cdc34-1 strains. We used cdc34-1
strain as a control because, like cdc6Δ mutant, it also arrest in G1 phase at the
restrictive temperature and is unable to initiate DNA replication. However, unlike
cdc6Δ mutant, cdc34-1 cells do not assemble a spindle although centrosomes are
duplicated. To perform this study, we tagged Cin8, Kip1 and Ase1 with HA
3
in both
wild type (US4122, US4677 and US7009) and cdc34-1 (US5239, US6342 and
US7011) strains, Cin8 and Kip1 with HA
3
in cdc6Δ GAL-CDC6 strain (US4366 and
US6343) and Ase1 with HA
3
in cdc6Δ MET-CDC6 strain (US7010). As described
earlier, all the strains were arrested in mitosis in the presence of nocodazole and
subsequently in G1 by α factor treatment to ensure good synchrony as all three strains
may recover from nocodazole arrest at different rate. Finally, all three strains were
released into fresh medium at 36˚C and Cin8, Kip and Ase1 levels were monitored by
Western Blotting. As shown in Figures 12, 13 and 14, Cin8, Kip1 and Ase1 protein
levels were elevated in cdc6Δ cells compared to cdc34-1 cells. The abundance of
these proteins peaked around 60 minutes after release from α-factor coinciding with
that of wild type cells. The kinetics of Cin8, Kip1 and Ase1 abundance in both wild
type and cdc6Δ cells are comparable, suggesting that both strains were undergoing

spindle elongation and nuclear division at about the same rate after release from α-
factor. The fact that Cin8, Kip1 and Ase1 levels were upregulated in cdc6Δ mutant
compared to cdc34-1 cells correlates with premature nuclear division observed in
cdc6Δ cells since a high abundance of Cin8, Kip1 and Ase1 has been closely
associated with precocious spindle elongation (Krishnan et al. 2004). These results
imply that spindle dynamics in Cdc6 depleted cells may be deregulated due to an

excessive accumulation of Cin8, Kip1 and Ase1. This may implicate Cdc6’s role in
regulating microtubule associated proteins and thus, in controlling spindle dynamics
and spindle elongation.