1. Kinetochore Function in Saccharomyces cerevisiae

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

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

4. Endogenous CEN3GALCEN3 GAL1-10 promoter 45 Kb Conditional Dicentric chromosomes 50% Consequence of dicentric chromosome activation in wild type cells OR 50% chance to break in next division Alignment

5. 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

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

7. 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 dicentric chromosome breakage OR 25% 25% chance to break in next division Alignment 50% Core kinetochore components have been shown to partially suppress dicentric chromosome breakage (ndc10 and mcm21) Intermediate levels of suppression

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 SPB (A) Monocentric chromosome Bipolar attachment- TENSION Monopolar attachment – NO TENSION SPB (B) Dicentric chromosome Bipolar attachment -TENSION Monopolar attachment- TENSION SPB bim1 is synthetic lethal with checkpoint genes

10. 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 breakage Increased loss (AIM3)

11. Naked CEN DNA De novo Kinetochore assembly Replication / Propagation Template Directed Kinetochore Assembly CHL4 independent CHL4 dependent Hypothesis for the basis of Complete Suppression in chl4 : Endogenous CEN3GALCEN3 GAL1-10 promoter 45 Kb

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

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) B10/10 0/10 0/15 15/1512/27 15/27 pYe (CEN3) 3010/10 0/100/15 15/1520/36 16/36 pYe (CEN3) 4110/10 0/100/15 15/1517/32 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

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

15. Wild typechl4 Galalctose (+Chl4p) Glucose (-Chl4p) Plasmids are unstable in the absence of Chl4p

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

17. WTchl4 Gal (+Chl4p) Glu (-Chl4p) Gal Glu Centromere plasmids are stably segregated for over 35 generations following depletion of Chl4p % loss per generation

18. 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

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. 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.

21. Model for the role of CHL4

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

23. 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 CONCLUSIONS Chl4p is a CEN- DNA binding protein

24. 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 expressing the TAP-tagged target protein ( CHL4-CBP-spacer-TEV cleavage site-spacer-ProtA )

25. 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

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

27. 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

28. 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.

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 suppresses dicentric breakage bim1 cells are not MMS sensitive bim1 exhibits elevated chromosome loss rates

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

32. 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

33. 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

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