A study on premature segregation of unreplicated chromosomes 3

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 onse

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

APCCdc20 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

G6PD

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

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 APCCdc20 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

RG cyc Glu noc 15 30 45 60 75 90 105 120 135 150 165 180 195 210

RG cyc Glu noc 15 30 45 60 75 90 105 120 135 150 165 180 195 210

Pds1-HA3G6PD

Pds1-HA3G6PD

Wildtype

Scc1-myc18

Scc1-myc18G6PD

type cells Both strains carrying the native promoter-driven PDS1-HA3 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-myc18 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

Wildtype

Clb2 Cdc28

Clb2 Cdc28

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

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

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