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

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

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) A.DNA checkpoints B.Spindle checkpoints C.Other… *DNA damage G2 checkpoint * p53+

DNA damage checkpoints 1.Active in all cell cycle phases 2.Prevent cell killing and genomic instability 3.Conserved in all eucaryotic cells 4.Not-essential, typically: ensure fidelity An early observation: The DNA replication (& damage) checkpoint is caffeine-sensitive How do these checkpoint controls work?

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

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

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

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

The Spindle Checkpoint Normal mitosis Delay with wayward chromosome Repair of wayward chromosome: then anaphase

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 4-5 hr delay Error and dead Occasional correction

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 on wayward chromosome.. Occasional correction pull Conclusion: At least in meiosis I, lack of tension signals delay (see previous..)

Pink- anti-Mad2 on unattached chromosome Lighter pink- recently attached chromosome (I cant quite see the attachments, but I take their word for it…) Mad2 protein binds to unattached chromosomes, leading to inhibition of APC

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

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

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)

Breakage-Fusion-Bridge Cycle

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

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