Specific areas of note: Cdc13-1 has high probability of short spindles Also, note that cdc13-1 rad 53 mutants look “most healthy” of mix

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

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

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

4 Milap Thaker - Graduate Proseminar - Molecular and Cellular Biology

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

Mitotic Exit Network: Take 2 6 Milap Thaker - Graduate Proseminar - Molecular and Cellular Biology

Alternate Mechanism: The FEAR Pathway 7 Milap Thaker - Graduate Proseminar - Molecular and Cellular Biology

MEN VS. FEAR: Specific Temporal Differences 8 Milap Thaker - Graduate Proseminar - Molecular and Cellular Biology

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 9 Milap Thaker - Graduate Proseminar - Molecular and Cellular Biology

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 10 Milap Thaker - Graduate Proseminar - Molecular and Cellular Biology

Protein Techniques For Immunoprecipitation, cells harvested (50 ml) and protein extracts are prepared with RIPA RIPA = Radioimmunoprecipitation Assay Incubated with Anti-HA (hemagglutinin) antibody Washed in SDS (sodium dodecyl sulfate) protein-loading buffer Boiled 5 mins, resolved with 8% SDS gel

Results 1a KEY ELEMENTS TO NOTE: WILD TYPE HIGH COLONIZATION LANE 2 NO COLONIZATION ALSO, NOTE LANE 6 HIGH COLONIZATION (rad53) 12 Milap Thaker - Graduate Proseminar - Molecular and Cellular Biology

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

Men & Fear: Proposed Mechanism 35 Milap Thaker - Graduate Proseminar - Molecular and Cellular Biology

36 Master Conclusion Set (2) Rad53 key player in mitotic exit prevention This is because cdc13-1, rad53021 mutants exhibited cdc14 release Rad 53 kinase known to phosphorylate Dun1 directly, known activator of Dun1 Dun1 plays a more direct role in regulating MEN than Rad53 (downstream) Authors contend possibility that Dun1 kinase phosphorylates Bfa1 and prevents Bfa1/Tem1 dissociation. Support: Less Bfa1-Tem1 complex in Rad53-21 when DNA damaged Milap Thaker - Graduate Proseminar - Molecular and Cellular Biology

Master Conclusion Set (3) 37 Milap Thaker - Graduate Proseminar - Molecular and Cellular Biology