Kinetochore Function in Saccharomyces cerevisiae

SCF?

APCSpindle Checkpoint

Cohesins

CENMotors

Cohesins

Cohesins

Cohesins

Components Inner Outer SpindleKinetochore

KINETOCHORE ORGANIZATION

Adapted from Kitagawa and Hieter, Nature Reviews Mol.Cell Biology,Sept 2001, Vol2.

Model of kinetochore oscillations, alignment and segregation in vivo

Adapted fromPearson et al, JCB, Vol 152 2001.

Pre- anaphase centromere separation, and alignment at metaphase.

Spindle elongation coincident with Anaphase A

Chromosome arm separation resulting inChromatin recoil towards the SPB

Endogenous CEN3GALCEN3

GAL1-10 promoter45 Kb

Conditional Dicentric chromosomes

50%

Consequence of dicentric chromosome activation in wild type cells

OR

50% chance to break in next division

Alignment

RAD52 dependent process

Endogenous CEN3GALCEN3

GAL1-10 promoter 45 Kb

Breakage and Recombination

Monocentric Derivative

•Can quantitate the effect of dicentric chromosome activation in different mutant backgrounds

Novel PCR product

2 ± 0.2-rad5272 ± 5.0+WT

% Viability(Mean ± SD)RAD52

RelevantGenotype

74.5 ± 1.0+ndc10-2ts (33C) a

31.0 ± 10.0-mcm21 rad5278.0 ± 1.0+mcm21

58.0 ± 3.0-mcm19 rad52

81.0 ± 6.0+mcm19

80.0 ± 7.0-chl4 rad52

81.0 ± 2.0+chl4

ndc10-2ts rad52 (33C)

+ 36.0 ± 2.0

kip3 + 28.0 ± 6.0

dhc + 21.0 ± 4.0

stu210-2ts (35C) + 31.5 ± 6.0

bim1 + 2.0 ± 6.0

Possible models for the differences in cellular viability following dicentric chromosome activation in various mutants

1. Both kinetochores are equally defective resulting in partial suppression of dicentricchromosome breakage

OROR

25%

25% chanceto break in next division

Alignment 50%

•Core kinetochore components have been shown to partially suppress dicentricchromosome breakage (ndc10 and mcm21)

•Intermediate levels of suppression

2. One kinetochore is defective resulting in complete suppression of dicentricchromosome breakage

Stable segregation

•Complete suppression is observed in chl4 dicentric chromosome containing cells

3. Reduction in cellular viability following dicentric chromosome activation due to a failure to detect errors in attachment

SPB

SPB

(A) Monocentric chromosomeBipolar attachment- TENSION Monopolar attachment – NO TENSION

SPB

SPB

SPB

SPB

(B) Dicentric chromosome

Bipolar attachment -TENSION Monopolar attachment- TENSIONSP

B

SPB

bim1 is synthetic lethal with checkpoint genes

Expectations (AIM1)

RAD52

High viability(wt levels)

Lowered viability(<25%)

•Lowered viability<25%

rad52

•High viability(>70% suppression)• no physical breakage

(AIM2)•Increased breakage

•Reduced repair

•Reduced breakageIncreased loss

(AIM3)

Naked CEN DNA

De novo Kinetochoreassembly

Replication /Propagation Template Directed

Kinetochore Assembly

CHL4 independent

CHL4 dependent

Hypothesis for the basis of Complete Suppression in chl4:

Endogenous CEN3GALCEN3

GAL1-10 promoter45 Kb

Experimental design

Segregation proficient

Tf. CEN plasmid+Chl4p

Tf. CEN plasmid+Chl4p Segregation proficientoutgrowth

Tf. CEN plasmid-Chl4p Segregation deficient

outgrowth

Deplete Chl4 Are they segregationproficient ?

outgrowth

Centromere Plasmid

Wild typeStable Unstable(80%-90%)

chl4 plasmidStable Unstable (0.08%-5%)

Wt+plasmidchl4Stable Unstable(60-90%) (0.08%-5%)

pYe (CEN3) B 10/10 0/10 0/15 15/15 12/27 15/27

pYe (CEN3) 30 10/10 0/10 0/15 15/15 20/36 16/36

pYe (CEN3) 41 10/10 0/10 0/15 15/15 17/32 15/32

• Mitotic stability depends on the timing of Chl4p loss relative to introduction of centromere DNA.

• Centromere plasmids were faithfully segregated in 49 out of 95 chl4 transformants

Promoter replacement

GAL1-10

Multi UB tagN-terminal ARG residue

CHL4 ORF

Galactose : Chl4p Glucose : No Chl4p Turner et al 2000

Wild type chl4

Galalctose (+Chl4p) Glucose (-Chl4p)

Plasmids are unstable in the absence of Chl4p

Tf. CEN plasmidon Gal (+Chl4p)

Segregation proficient

Deplete Chl4 Are they segregationproficient ?

Gal Glu(-Chl4p) Glu Glu

Centromere plasmids are stable upon loss of Chl4p

WT chl4 Gal(+Chl4p)

Glu(-Chl4p)

Gal Glu

Centromere plasmids are stably segregated for over 35 generations following depletion of Chl4p

% lo

ss p

er g

ener

atio

n

Does a small fraction of cells with mitotically unstable plasmids accumulate following loss of Chl4p

 Established centromeres switch at a frequency of 2-3% per generation in the absence of Chl4p

CONCLUSION

SUMMARY

•Centromere plasmids introduced in the absence of Chl4p fail to segregate. - no de novo kinetochore function.

• Established centromere plasmids segregate with high fidelity in the absence of Chl4p - Propagation of kinetochores is not affected

Is the failure of “new” centromeres to direct chromosome segregation in chl4 due to a defect in kinetochore assembly

CONCLUSION

The quantitative increase in accessibility of GALCEN3 indicates that Chl4p is essential for assembly of proteins at newly introduced centromeres.

Model for the role of CHL4

Specific Aim 2.

Determine the mechanism that underlies complete suppression of dicentricchromosome breakage

APPROACH AND EXPECTATION

1. CHIP to ask whether kinetochore components interact differentially with new and old centromeres in wild type and chl4 cells.

Core components will be reduced or absent from new centromeres

OR Components of different outer complexes will be reduced or absent from new vs old centromeres

2. Gel-shift to determine if Chl4p binds CEN DNA

Chl4p alone will bind CEN DNA Chl4p complex will bind DNA Will not bind DNA

CONCLUSIONSChl4p is a CEN- DNA binding protein

3. To obtain and identify components of the Chl4p complex utilizing TAP-TAG

Preparation of extracts

Tandem Affinity Purification (TAP)

Protein analysis / Identification Functional assays (Gel mobility shift)

Strategy overview

Construction of recombinant cells or organisms expressingthe TAP-tagged target protein

(CHL4-CBP-spacer-TEV cleavage site-spacer-ProtA)

4. To determine if Chl4p function is cell cycle regulated

Cells containing a degron allele of CHL4 (GAL-UBI-CHL4)

Factor arrest

Deplete Chl4p

Map chromatin structure of the centromere

HU arrest

Strategy overview

CONCLUSION:

Chl4p is required during replication and/or for maintenance of kinetochore structure

Specific Aim 3.

Investigate the mechanisms that underlie cellular lethality following activationof a dicentric chromosome

Reduced cellular viability following activation of the dicentric chromosome could be due to :

1. Synthetic lethality

2. Defect in DNA repair

3. Increased/Decreased dicentric breakage

4. Increased dicentric chromosome loss

- MMS and gamma sensitivity

- Dicentric Plasmid analysis

- Colony Color assay

- 40Kb circular derivative

Are bim1 cells defective in repair

Do bim1 cells exhibit increased/ decreased dicentric breakage

Wild type

rad52

bim1

bim1 cells are not MMS sensitive

27/50 dicentric plasmids derived from bim1 cells have both centromeres intact as compared to 0/50 that are intact in wild type cells.

W ild type ch l4 b im 1

Do bim1 cells exhibit increased chromosome loss

SUMMARY of bim1 results

• bim1 cells exhibit severely reduced viability following activation of the dicentric chromosome

• bim1 suppresses dicentric breakage

• bim1 cells are not MMS sensitive

• bim1 exhibits elevated chromosome loss rates

Hypothesis and Model for increased dicentric chromosome loss in bim1SP

B

SPB

(A) Monocentric chromosomeBipolar attachment- TENSION Monopolar attachment – NO TENSION

SPB

SPB

SPB

SPB

(B) Dicentric chromosome

Bipolar attachment -TENSION Monopolar attachment- TENSIONSP

B

SPB

APPROACH AND EXPECTATION

1. To determine if there is increased dicentric chromosome loss utilizing a colony color assay

bim1 cells will exhibit elevated dicentric chromosome loss rates

2. Visualize dicentric chromosome segregation utilizing the LacO-LacI labelling system

The two Lac-O spots will segregate together to one cell

To determine the effect of bim1 on monocentric chromosome segregation

Decreased centromere separation

Increased CEN separation Decreased oscillations

Defect in metaphase alignment

APPROACH AND EXPECTATION

Visualize CEN3 Lac-O spots in bim1 cells

CONCLUSION

Bim1p plays a distinct role in centromere motility and attaining bipolar attachments

ACKNOWLEDGEMENTS

KERRY BLOOM

Elaine YehLeanna TopperDale BeachPaul MaddoxChad PearsonJeff MolkDavid BouckJennifer Stemple

GOLDSTEIN LAB

SALMON LAB

Jean Claude LabbeJen Yi Lee

Jennifer Deluca