Kinetochore Function in Saccharomyces cerevisiae

1. Kinetochore Function in Saccharomyces cerevisiae

2. KINETOCHORE ORGANIZATION Cohesins SCF? Cohesins Inner Outer Spindle CEN Kinetochore Components Cohesins APC Motors Spindle Check point Cohesins Adapted from Kitagawa and Hieter, Nature Reviews Mol.Cell Biology, Sept 2001, Vol2.

3. Pre- anaphase centromere separation, and alignment at metaphase. Spindle elongation coincident with Anaphase A Chromosome arm separation resulting in Chromatin recoil towards the SPB Adapted from Pearson et al, JCB, Vol 152 2001. Model of kinetochore oscillations, alignment and segregation in vivo

4. Conditional Dicentric chromosomes GAL1-10 promoter 45 Kb GALCEN3 Endogenous CEN3 Consequence of dicentric chromosome activation in wild type cells OR Alignment 50% 50% chance to break in next division

5. 45 Kb GAL1-10 promoter GALCEN3 Endogenous CEN3 Breakage and Recombination Monocentric Derivative RAD52 dependent process Novel PCR product • Can quantitate the effect of dicentric chromosome activation in different • mutant backgrounds

6. Relevant Genotype RAD52 % Viability (Mean ± SD) WT + 72 ± 5.0 rad52 - 2 ± 0.2 chl4 + 81.0 ± 2.0 chl4 rad52 - 80.0 ± 7.0 mcm19 + 81.0 ± 6.0 mcm19 rad52 - 58.0 ± 3.0 mcm21 + 78.0 ± 1.0 mcm21 rad52 - 31.0 ± 10.0 ndc10-2ts(33C) a + 74.5 ± 1.0 ndc10-2ts rad52 (33C) 36.0 ± 2.0 + dhc + 21.0 ± 4.0 kip3 + 28.0 ± 6.0 bim1 + 2.0 ± 6.0 stu210-2ts(35C)+ 31.5 ± 6.0

7. Both kinetochores are equally defective resulting in partial suppression of dicentric chromosome breakage OR OR 25% 50% Alignment 25% chance to break in next division Possible models for the differences in cellular viability following dicentric chromosome activation in various mutants • Intermediate levels of suppression • Core kinetochore components have been shown to partially suppress dicentric • chromosome breakage (ndc10 and mcm21)

8. 2. One kinetochore is defective resulting in complete suppression of dicentric chromosome breakage Stable segregation • Complete suppression is observed in chl4 dicentric chromosome containing cells

9. 3. Reduction in cellular viability following dicentric chromosome activation due to a failure to detect errors in attachment (A) Monocentric chromosome Bipolar attachment- TENSION Monopolar attachment – NO TENSION SPB SPB SPB SPB (B) Dicentric chromosome Monopolar attachment- TENSION Bipolar attachment -TENSION SPB SPB SPB SPB bim1 is synthetic lethal with checkpoint genes

10. RAD52 High viability (wt levels) Lowered viability (<25%) rad52 • Lowered viability • <25% • High viability • (>70% suppression) • no physical breakage • Increased • breakage • Reduced • repair • Reduced breakage • Increased loss (AIM3) Expectations (AIM1) (AIM2)

11. Hypothesis for the basis of Complete Suppression in chl4: Replication / Propagation Template Directed Kinetochore Assembly De novo Kinetochore assembly Naked CEN DNA GAL1-10 promoter 45 Kb GALCEN3 Endogenous CEN3 CHL4 independent CHL4 dependent

12. Tf. CEN plasmid +Chl4p outgrowth Segregation proficient Tf. CEN plasmid -Chl4p outgrowth Segregation deficient Tf. CEN plasmid +Chl4p outgrowth Are they segregation proficient ? Segregation proficient Experimental design Deplete Chl4

13. Centromere Plasmid Wild type Stable Unstable (80%-90%) chl4plasmid Stable Unstable (0.08%-5%) Wt+plasmidchl4 Stable 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 • Centromere plasmids were faithfully segregated in 49 out of 95 chl4transformants • Mitotic stability depends on the timing of Chl4p loss relative to introduction • of centromere DNA.

14. CHL4 ORF Promoter replacement GAL1-10 Multi UB tag N-terminal ARG residue Galactose : Chl4p Glucose : No Chl4p Turner et al 2000

15. Galalctose (+Chl4p) Glucose (-Chl4p) Wild type chl4 Plasmids are unstable in the absence of Chl4p

16. Deplete Chl4 Are they segregation proficient ? Gal Glu(-Chl4p) Glu Glu Tf. CEN plasmid on Gal (+Chl4p) Segregation proficient Centromere plasmids are stable upon loss of Chl4p

17. Gal Glu Centromere plasmids are stably segregated for over 35 generations following depletion of Chl4p % loss per generation Gal (+Chl4p) Glu (-Chl4p) WT chl4

18. CONCLUSION Established centromeres switch at a frequency of 2-3% per generation in the absence of Chl4p Does a small fraction of cells with mitotically unstable plasmids accumulate following loss of Chl4p

19. 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

20. CONCLUSION The quantitative increase in accessibility of GALCEN3 indicates that Chl4p is essential for assembly of proteins at newly introduced centromeres. Is the failure of “new” centromeres to direct chromosome segregation in chl4 due to a defect in kinetochore assembly

21. Model for the role of CHL4

22. Specific Aim 2. Determine the mechanism that underlies complete suppression of dicentric chromosome breakage

23. Components of different outer complexes will be reduced or absent from new vs old centromeres Core components will be reduced or absent from new centromeres OR Will not bind DNA Chl4p alone will bind CEN DNA Chl4p complex will bind DNA CONCLUSIONS Chl4p is a CEN- DNA binding protein APPROACH AND EXPECTATION • CHIP to ask whether kinetochore components interact differentially with • new and old centromeres in wild type and chl4 cells. 2. Gel-shift to determine if Chl4p binds CEN DNA

24. Strategy overview Construction of recombinant cells or organisms expressing the TAP-tagged target protein (CHL4-CBP-spacer-TEV cleavage site-spacer-ProtA) Preparation of extracts Tandem Affinity Purification (TAP) Functional assays (Gel mobility shift) Protein analysis / Identification • To obtain and identify components of the Chl4p complex utilizing TAP-TAG

25. Strategy overview Cells containing a degron allele of CHL4 (GAL-UBI-CHL4) HU arrest  Factor arrest Deplete Chl4p Map chromatin structure of the centromere CONCLUSION: Chl4p is required during replication and/or for maintenance of kinetochore structure 4. To determine if Chl4p function is cell cycle regulated

26. Specific Aim 3. Investigate the mechanisms that underlie cellular lethality following activation of a dicentric chromosome

27. Synthetic lethality 2. Defect in DNA repair 3. Increased/Decreased dicentric breakage 4. Increased dicentric chromosome loss Reduced cellular viability following activation of the dicentric chromosome could be due to : - 40Kb circular derivative - MMS and gamma sensitivity - Dicentric Plasmid analysis - Colony Color assay

28. Wild type rad52 bim1 Are bim1 cells defective in repair bim1 cells are not MMS sensitive Do bim1 cells exhibit increased/ decreased dicentric breakage 27/50 dicentric plasmids derived from bim1 cells have both centromeres intact as compared to 0/50 that are intact in wild type cells.

29. Do bim1 cells exhibit increased chromosome loss

30. SUMMARY of bim1 results • bim1 cells exhibit severely reduced viability following activation of the dicentric • chromosome • bim1 cells are not MMS sensitive • bim1 suppresses dicentric breakage • bim1 exhibits elevated chromosome loss rates

31. (A) Monocentric chromosome Bipolar attachment- TENSION Monopolar attachment – NO TENSION SPB SPB SPB SPB (B) Dicentric chromosome Monopolar attachment- TENSION Bipolar attachment -TENSION SPB SPB SPB SPB Hypothesis and Model for increased dicentric chromosome loss in bim1

32. To determine if there is increased dicentric chromosome loss utilizing a colony color assay bim1 cells will exhibit elevated dicentric chromosome loss rates • Visualize dicentric chromosome segregation utilizing the LacO-LacI labelling system The two Lac-O spots will segregate together to one cell APPROACH AND EXPECTATION

33. APPROACH AND EXPECTATION Visualize CEN3Lac-O spots in bim1 cells Decreased centromere separation Defect in metaphase alignment Decreased oscillations Increased CEN separation CONCLUSION Bim1p plays a distinct role in centromere motility and attaining bipolar attachments To determine the effect of bim1 on monocentric chromosome segregation

34. ACKNOWLEDGEMENTS KERRY BLOOM GOLDSTEIN LAB Jean Claude Labbe Jen Yi Lee Elaine Yeh Leanna Topper Dale Beach Paul Maddox Chad Pearson Jeff Molk David Bouck Jennifer Stemple SALMON LAB Jennifer Deluca