The document discusses two mechanisms by which DNA damage checkpoints inhibit mitotic exit in yeast cells. First, the Rad53 checkpoint kinase prevents mitotic exit by inhibiting the Mitotic Exit Network (MEN). Second, the FEAR pathway promotes limited release of the phosphatase Cdc14 from the nucleolus early in anaphase. The study finds that Rad53 acts through the Dun1 kinase to regulate the MEN more directly, while FEAR provides an alternate mechanism for temporary Cdc14 release and a delay in full mitotic exit. Experiments visualize budding, Cdc14 localization, and DNA content in various yeast strains to illustrate the two inhibitory mechanisms.

1. Fengshan Liang and Yanchang Wang DNA Damage Checkpoints Inhibit Mitotic Exit by Two Different Mechanisms MOLECULAR AND CELLULAR BIOLOGY, July 2007, p. 5067–5078 Presenter: MilapThaker Proseminar in Molecular and Cellular Biology Harvard University Date 11/29/2007

2. Key Point 1: Two Different Mechanisms Milap Thaker - Graduate Proseminar - Molecular and Cellular Biology 2

3. Milap Thaker - Graduate Proseminar - Molecular and Cellular Biology 3 Key Point 1: Two Different Mechanisms

4. Milap Thaker - Graduate Proseminar - Molecular and Cellular Biology 4

5. Relevant context: How to activate Mitotic Exit Network? Milap Thaker - Graduate Proseminar - Molecular and Cellular Biology 5

6. Mitotic Exit Network: Take 2 MilapThaker - Graduate Proseminar - Molecular and Cellular Biology 6

7. Alternate Mechanism: The FEAR Pathway:FEAR: CDC14 Early Anaphase Release Milap Thaker - Graduate Proseminar - Molecular and Cellular Biology 7

8. Mitotic Exit Network vs. CDC14 Release: Specific Temporal Differences Milap Thaker - Graduate Proseminar - Molecular and Cellular Biology 8

9. Materials and Methods Yeast Strains grown in YPD (standard yeast-extract-peptone-dextrose) medium Arrested in G1 by 5 µg/ml α-factor After 2 hours, washed 2x with water Cytology: Cells fixed with 3.7% formaldehyde for 5 min at RT, then washed twice with PBS buffer (phosphate buffered saline) and resuspended in PBS Milap Thaker - Graduate Proseminar - Molecular and Cellular Biology 9

10. DAPI Staining Technique Cells fixed with 100% MeOH at -20 Celsius for 30 mins. Cells pelleted, resuspended in DAPI at a final concentration of 2.5µg/ml for 1 min at RT Cells finally resuspended in 1X PBS Fluorescence microscopy used to examine GFP and DNA Milap Thaker - Graduate Proseminar - Molecular and Cellular Biology 10

12. RIPA = Radioimmunoprecipitation Assay

13. Incubated with Anti-HA (hemagglutinin) antibody

14. Washed in SDS (sodium dodecyl sulfate) protein-loading buffer

16. Results 2 – short spindling Milap Thaker - Graduate Proseminar - Molecular and Cellular Biology 13 Fig 1b

17. Results 3 – Rebudding Milap Thaker - Graduate Proseminar - Molecular and Cellular Biology 14 Fig 1c

18. Re-Incubated Cells Milap Thaker - Graduate Proseminar - Molecular and Cellular Biology 15 Fig 1c

19. Results 4 – cdc 14 Release Milap Thaker - Graduate Proseminar - Molecular and Cellular Biology 16 Fig 2a

20. Cdc 14 Localization - Images Milap Thaker - Graduate Proseminar - Molecular and Cellular Biology 17 Fig 2a(2)

21. Cdc 14 Release Milap Thaker - Graduate Proseminar - Molecular and Cellular Biology 18 Fig 2B

22. Rebudding of Cells (2) Milap Thaker - Graduate Proseminar - Molecular and Cellular Biology 19 Fig 3a

23. Rebudding of Cells (3) Milap Thaker - Graduate Proseminar - Molecular and Cellular Biology 20 Fig 3a(2)

24. Cdc 14 Release (2) Milap Thaker - Graduate Proseminar - Molecular and Cellular Biology 21 Fig 3B

25. Budding, CDC 14 Release (3) Milap Thaker - Graduate Proseminar - Molecular and Cellular Biology 22 Fig 4a,b

26. Microcolonies (2) Milap Thaker - Graduate Proseminar - Molecular and Cellular Biology 23 Fig 4C

27. Budding, CDC 14 (3) Milap Thaker - Graduate Proseminar - Molecular and Cellular Biology 24 Fig 5a

28. Budding, Cdc 14 Release (4) Milap Thaker - Graduate Proseminar - Molecular and Cellular Biology 25 Fig 5B

29. Microcolonies (3) Milap Thaker - Graduate Proseminar - Molecular and Cellular Biology 26 Fig 5C

30. Budding, Cdc14 Release (5) Milap Thaker - Graduate Proseminar - Molecular and Cellular Biology 27 Fig 6a

31. Budding, Cdc 14 Release (6) Milap Thaker - Graduate Proseminar - Molecular and Cellular Biology 28 Fig 6B

32. Colonization across states Milap Thaker - Graduate Proseminar - Molecular and Cellular Biology 29 Fig 6C

33. Nuclear Localization, Segregation Milap Thaker - Graduate Proseminar - Molecular and Cellular Biology 30 Fig 7a

34. Budding, cdc 14 Localization (6) Milap Thaker - Graduate Proseminar - Molecular and Cellular Biology 31 Fig 7b

35. Cdc 5-1 mutation vs. Cdc5-1RAD53-21 Milap Thaker - Graduate Proseminar - Molecular and Cellular Biology 32 Fig 7c

36. Western Blot, States Milap Thaker - Graduate Proseminar - Molecular and Cellular Biology 33 Figs 8a, 8b

37. Master Conclusion Set (1) Milap Thaker - Graduate Proseminar - Molecular and Cellular Biology 34 Fig 8c

38. Cell cycle checkpoint path: Milap Thaker - Graduate Proseminar - Molecular and Cellular Biology 35

40. This is because cdc13-1, rad53021 mutants exhibited cdc14 release

41. Rad 53 kinase known to phosphorylate Dun1 directly, known activator of Dun1

42. Dun1 plays a more direct role in regulating MEN than Rad53 (downstream)

43. Authors contend possibility that Dun1 kinase phosphorylates Bfa1 and prevents Bfa1/Tem1 dissociation.