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~2 lectures ~25 slides. Maybe 3 hr to prepare. Done Monday and to Charley…

~2 lectures ~25 slides. Maybe 3 hr to prepare. Done Monday and to Charley… Other example of treatment- 23-44 and 23-45 DNA damage & spindle checkpoints & genomic instability DNA damage checkpoints (1-2) Spindle checkpoints (1-2, plus key experiments)

stephanie-ashley
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~2 lectures ~25 slides. Maybe 3 hr to prepare. Done Monday and to Charley…

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  1. ~2 lectures ~25 slides. Maybe 3 hr to prepare. Done Monday and to Charley… Other example of treatment- 23-44 and 23-45 DNA damage & spindle checkpoints & genomic instability DNA damage checkpoints (1-2) Spindle checkpoints (1-2, plus key experiments) Genome instability and cancer (23-24,27,28,29,17-43 telomeres),23-40,41,33,36,42,45. # mutations needed. Relationship to age. Mechanisms of genome instability- telomeres as prime suspect

  2. A second success story of cancer treatment based on basic research… STI-571 inhibits Bcr-Abl protein kinase

  3. Recall this figure: Towards the molecular controls that govern the cell cycle 1. Essential proteins that activate events: A. CDK B. APC and SCF proteolysis 2. Non-essential proteins that ensure events are complete (acting at “checkpoints”) DNA checkpoints Spindle checkpoints Other… * p53+ *DNA damage G2 checkpoint

  4. DNA damage checkpoints Active in all cell cycle phases Prevent cell killing and genomic instability Conserved in all eucaryotic cells Not-essential, typically: ensure fidelity An early observation: The DNA replication (& damage) checkpoint is caffeine-sensitive How do these checkpoint controls work?

  5. Overview of DNA replication and damage checkpoints Normal cell cycle Replication bubble Cell cycle with damage Replication bubble Stalled replication fork.. Delays replication from other origins Keeps this fork stable to allow repair..

  6. When DNA replication and damage checkpoints fail… Normal cell cycle Replication bubble Cell cycle with damage Replication bubble Replicates broken DNA Acentric fragment lost… Stalled fork breaks BIG TROUBLE…..

  7. How DNA damage and replication checkpoints normally work… A model 1 doublestrand DNA break !!… Proteins assemble on singlestranded DNA Repair Recognizes damage Recruits “sliding clamp” Protein kinase (ATM) Recruits protein kinase and its substrates.. P P P (p53+ in G1) ATM protein kinase is caffeine-sensitive!

  8. Test of Model: GFP fusions RFP-1 GFP-2 Recognizes damage Recruits “sliding clamp” Damage mutant 1 cell Damage mutant 2 cell Damage normal cell No damage RFP GFP

  9. The Spindle Checkpoint “Repair” of wayward chromosome: then anaphase Delay with wayward chromosome Normal mitosis

  10. Key, incredible experiment from B. Nicklas Grasshopper spermatocytes have weird sex chromosomes XXY, which pair 90% of the time correctly, and 10% of the time incorrectly. Correct alignment (90%) No delay Incorrect alignment (10%) 4-5 hour delay Occasional correction 4-5 hr delay Error and dead

  11. Key, incredible experiment from B. Nicklas Grasshopper spermatocytes have weird sex chromosomes XXY, which pair 90% of the time correctly, and 10% of the time incorrectly. Correct alignment (90%) No delay Incorrect alignment (10%) 4-5 hour delay pull Occasional correction Pull on wayward chromosome.. Conclusion: At least in meiosis I, lack of tension signals delay (see previous..)

  12. Mad2 protein binds to unattached chromosomes, leading to inhibition of APC Pink- anti-Mad2 on unattached chromosome Lighter pink- recently attached chromosome (I can’t quite see the attachments, but I take their word for it…)

  13. Controversy persists: Does Tension or Microtubule Occupancy sSgnal? Mitosis-Occupancy? Mitosis- Occupancy? Centromeres not constrained- may even face same pole yet be occupied- so tension more reasonable… Centromeres constrained to be facing opposite centrosomes.

  14. Cancer, Genome Instability, Telomeres, Checkpoints.. Incidence of age….’nuff said ~3-6 mutations required. Accumulation of mutations

  15. Structure of mutations • How do mutations arise? • Mismatch repair defect (inherited form of colon cancer) • DNA breaks(in repair-defective cells, in cells subjected to frequent breaks, in older cells with shorter telomeres)

  16. Breakage-Fusion-Bridge Cycle

  17. How are DNA breaks generated??? 1. Stalled replication forks (??) 2. Shortening telomeres Stalled fork breaks BIG TROUBLE….. “DNA break” BFB cycle

  18. Two ways at looking at genome instability and telomere length. high Activation of telomerase Genome Stability crisis low p53- Cell divisions

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