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Fengshan liang and yanchang wang l.jpg

DNA Damage Checkpoints Inhibit Mitotic Exit by

Two Different Mechanisms

Fengshan Liang and Yanchang Wang

MOLECULAR AND CELLULAR BIOLOGY, July 2007, p. 5067–5078

Presenter: MilapThaker

Proseminar in Molecular and Cellular Biology

Harvard University

Date 11/29/2007


Key point 1 two different mechanisms l.jpg
Key Point 1: Two Different Mechanisms

Milap Thaker - Graduate Proseminar - Molecular and Cellular Biology


Slide3 l.jpg

Key Point 1: Two Different Mechanisms

Milap Thaker - Graduate Proseminar - Molecular and Cellular Biology



Relevant context how to activate mitotic exit network l.jpg
Relevant context: BiologyHow to activate Mitotic Exit Network?

Milap Thaker - Graduate Proseminar - Molecular and Cellular Biology


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Mitotic Exit Network: Take 2 Biology

MilapThaker - Graduate Proseminar - Molecular and Cellular Biology


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Alternate Mechanism: The FEAR Pathway Biology

Milap Thaker - Graduate Proseminar - Molecular and Cellular Biology


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MEN VS. FEAR: Specific Temporal Differences Biology

Milap Thaker - Graduate Proseminar - Molecular and Cellular Biology


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Materials and Methods Biology

  • 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


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DAPI Staining Technique Biology

  • 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


Protein techniques l.jpg

Protein Techniques Biology

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 l.jpg
Results 1a Biology

KEY ELEMENTS TO NOTE:

WILD TYPE HIGH COLONIZATION

LANE 2 NO COLONIZATION

ALSO, NOTE LANE 6 HIGH COLONIZATION (rad53)

Milap Thaker - Graduate Proseminar - Molecular and Cellular Biology


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Results 2 – short spindling Biology

Fig 1b

Milap Thaker - Graduate Proseminar - Molecular and Cellular Biology


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Results 3 – BiologyRebudding

Fig 1c

Milap Thaker - Graduate Proseminar - Molecular and Cellular Biology


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Re-Incubated Cells Biology

Fig 1c

Milap Thaker - Graduate Proseminar - Molecular and Cellular Biology


Results 4 cdc 14 release l.jpg
Results 4 – Biologycdc 14 release

Fig 2a

Milap Thaker - Graduate Proseminar - Molecular and Cellular Biology


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Cdc Biology 14 Localization - Images

Fig 2a(2)

Milap Thaker - Graduate Proseminar - Molecular and Cellular Biology


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Cdc Biology 14 Release

Fig 2B

Milap Thaker - Graduate Proseminar - Molecular and Cellular Biology


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Rebudding Biology of Cells (2)

Fig 3a

Milap Thaker - Graduate Proseminar - Molecular and Cellular Biology


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Rebudding Biology of Cells (3)

Fig 3a(2)

Milap Thaker - Graduate Proseminar - Molecular and Cellular Biology


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Cdc Biology 14 Release (2)

Fig 3B

Milap Thaker - Graduate Proseminar - Molecular and Cellular Biology


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Budding, CDC 14 Release (3) Biology

Fig 4a,b

Milap Thaker - Graduate Proseminar - Molecular and Cellular Biology


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Microcolonies Biology (2)

Fig 4C

Milap Thaker - Graduate Proseminar - Molecular and Cellular Biology


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Budding, CDC 14 (3) Biology

Fig 5a

Milap Thaker - Graduate Proseminar - Molecular and Cellular Biology


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Budding, BiologyCdc 14 Release (4)

Fig 5B

Milap Thaker - Graduate Proseminar - Molecular and Cellular Biology


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Microcolonies Biology (3)

Fig 5C

Milap Thaker - Graduate Proseminar - Molecular and Cellular Biology


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Budding, Cdc14 Release (5) Biology

Fig 6a

Milap Thaker - Graduate Proseminar - Molecular and Cellular Biology


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Budding, BiologyCdc 14 Release (6)

Fig 6B

Milap Thaker - Graduate Proseminar - Molecular and Cellular Biology


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Colonization across states Biology

Fig 6C

Milap Thaker - Graduate Proseminar - Molecular and Cellular Biology


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Nuclear Localization, Segregation Biology

Fig 7a

Milap Thaker - Graduate Proseminar - Molecular and Cellular Biology


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Budding, Biologycdc 14 Localization (6)

Fig 7b

Milap Thaker - Graduate Proseminar - Molecular and Cellular Biology


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Cdc Biology 5-1 mutation vs. Cdc5-1RAD53-21

Fig 7c

Milap Thaker - Graduate Proseminar - Molecular and Cellular Biology


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Western Blot, States Biology

Figs 8a, 8b

Milap Thaker - Graduate Proseminar - Molecular and Cellular Biology


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Master Conclusion Set (1) Biology

Fig 8c

Milap Thaker - Graduate Proseminar - Molecular and Cellular Biology


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Men & Fear: Proposed Mechanism Biology

Milap Thaker - Graduate Proseminar - Molecular and Cellular Biology


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Master Conclusion Set (2) Biology

  • 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 l.jpg
Master Conclusion Set (3) Biology

Milap Thaker - Graduate Proseminar - Molecular and Cellular Biology


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