Post on 27-Mar-2015
~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
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. CDKB. APC and SCF proteolysis
2. Non-essential proteins that ensure events are complete
(acting at “checkpoints”)A. DNA checkpointsB. Spindle checkpointsC. 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 originsKeeps 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 model1 doublestrand DNA break !!…
Proteins assemble on singlestranded DNA
Recognizes damage
Recruits “sliding clamp”
Recruits protein kinase and
its substrates..
Protein kinase (ATM)
P
PP
(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 damageDamage normal cell
Damage mutant 1 cell
RFP
GFP
Damage mutant 2 cell
The Spindle Checkpoint
Normal mitosisDelay 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 can’t 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? 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)
How are DNA breaks generated???
1. Stalled replication forks (??) 2. Shortening telomeres
Stalled fork breaks
BIG TROUBLE…..
“DNA break”
BFB cycle