~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)

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.