CHECKPOINTS

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CHECKPOINTS
regulatory pathways that control the order and
timing of cell cycle transitions and ensure that
critical events such as DNA replication and
chromosome segregation are completed with high
fidelity

• Specific point(s) in the cell cycle?
• Operative in normal cell cycle or only when
  perturbed?
• Essential?

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Eukaryotic Checkpoint Systems

• DNA Damage
• Replication Inhibition
• Spindle Assembly
• Nuclear Migration

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Checkpoints - A Signal Transduction Pathway

• Sensors
• Signal Transducers
• Targets

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S. cerevisiae DNA Damage Checkpoint Pathway
Sensors
Transducers
Targets
DNA Damage
Mec1-Ddc2, Tel1
Swi6, Dun1
9-1-1 complex, Rad24, RPA
Pds1, Stalled Forks, Primase
Rad9
Replication Arrest
Chk1, Rad53
Pds1, Cdc5
Pole, Rfc5, Dpb11, Rpa
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Response to DNA Damage or Replication Inhibiiton

• G1/S Checkpoint - Inhibition of initiation of S
  in response to DNA damage
• Inter-S Checkpoint - Retardation of DNA
  replication in response to DNA damage Inhibition
  of late origins Prevention of replication fork
  collapse
• G2/M Checkpoint - Inhibition of initiation of
  mitosis in response to DNA damage
• Meiotic Prophase Checkpoint - Inhibition of
  meiosis I by DNA damage
• Transcriptional induction of repair genes

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What is Recognized by the Checkpoint Sensor?

• DS Breaks?
• Tel1/ATM dependent not active in G1
• DNA adducts/dimers?
• Excision repair mutants dont activate
  checkpoint
• Unreplicated DNA?
• cdc6 (Sc), cdc18 (Sp) dont activate checkpoint
• SS DNA?
• Rad17 looks like an exonuclease cdc13 elicits
  checkpoint MRX (Xrs2/Mre11/Rad50) complex
  required for DSB checkpoint activation RPA
  required for recruitment of ATR

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S. cerevisiae DNA Damage Checkpoint Pathway
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S. cerevisiae DNA Damage Checkpoint Pathway
Recognition and Processing of Initial Damage
aka the 9-1-1 complex
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S. cerevisiae DNA Damage Checkpoint Pathway
G1 Phase
Rad24
Ddc2
Mec1
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Dun1
Swi6
Cln Trans.
Repair Trans.
Swi4
Crt1
Repair Genes
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S. cerevisiae dsDNA Break Checkpoint Pathway
Mre11-Rad50 -Xrs2
Tel1
Sae2
H2A
Exo
g-H2A
Rpa
Rad24-Rfc2-5
Mec1-Ddc2
Ddc1-Mec3 -Rad17
Rad52
Rad51 filament
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S. cerevisiae DNA Damage Checkpoint Pathway
Pds1
S Phase
Cdc5
9
Chk1
Rad53
Cdc7
Mec1
Late Origins
9-1-1 Group
Rpa1
Pri1
Rfc5
Pol e Dbp11Rfc5
Rad53
Mec1
Cdc5
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S. cerevisiae Replication Arrest Checkpoint
Elicited by ssDNA RPA
leading strand
Normal Replication Fork
Helicase Dissociated from Polymerases
helicase
RPA
lagging strand
Polymerases Dissociated from Each other
Stable Stalled Fork
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Fork Collapse in the Absence of DNA Checkpoint
Replicating DNA isolated from HU treated rad53
cells
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Resolution of Collapsed Forks
Resection of nascent chains
Hemicatenane runoff four-way junctions
Double strand break formation
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Response to Replication Stress
Replication Stress
Passive replication
Template switch
Fork Stall
Fork collapse
Recombination- mediated restart
Mec1 activation and recruitment
Rad24 9-1-1 complex
Block late origin firing
Replisome stabilization
Suppression of recombination
Damage bypass Mutagenesis
Replication restart
Replication resumption
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S. cerevisiae DNA Damage Checkpoint Pathway
G2 Phase
Mec1
Pds1
Rad53
Chk1
dNTPs
Dun1
Repair
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DNA Damage Arrest Through Chk1
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Regulation of Cdks by Tyr-15 Phosphorylation
Active
Cdc2
Nim1
Cdc25
Wee1
Wee1
Cdc2
PP2A?
Inactive
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Regulation of Cdks by Tyr-15 Phosphorylation
Active
Cdc2
Nim1
Cdc25
Cdc25
Wee1
Wee1
Cdc2
Chk1
PP2A?
Inactive
Cdc25
14-3-3
Inactive
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DNA Damage Arrest Through Chk1

• Cdc2(Y15A) mutants fail to arrest
• weets mik1- strains still exhibit damage-induced
  reduction in rate of Y15 dephosphorylation in
  response to damage gt cdc25 mediates arrest
• Chk1 phosphorylates cdc25 at S216
• cdc25S216A mutants fail to arrest
• Phosphorylated but not unphosphorylated cdc25
  binds 14-3-3 (humans but perhaps not pombe)
• Chk1 acts downstream of Rad3 (ATM/Mec1)

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Consequences of a Loss of DNA Checkpoint

• Entry into S phase with a double strand break?
  a thymidine dimer or mismatch?
• Exit from S phase with a double strand break? a
  thymidine dimer or mismatch? a collapsed
  replication fork?
• Passage through mitosis with a double strand
  break? a thymidine dimer or mismatch?

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Spindle Assembly Checkpoint
Arrest of Metaphase - Anaphase Transition Until
Spindle Assembly is Complete
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APC and the Exit from Mitosis
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Regulation of Cohesin Degradation
At Anaphase APC degrades Pds1, releasing Esp1 to
disrupt cohensins
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Sequential Activities During Mitosis
Clb3,4,5
Clb1,2
Cdc20
Hct1
H1 Kinase Activity
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Sequential Activities During Mitosis
Clb3,4
Clb1,2
Cdc20
Hct1
H1 Kinase Activity
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Spindle Assembly Checkpoint
What Elicits a Spindle Assembly Checkpoint?

• Spindle Depolymerization / Stabilization
• Multiple minichromosomes
• Defects in the spindle pole body
• Defects in microtubules
• Defects in the kinetochore (ctf13, e.g.)
• Centromere mutations
• Defects in microtubule motors

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Spindle Assembly Checkpoint Pathway
What Does the Pathway Recognize?

• Kinetochore tension
• Externally applied tension release arrest caused
  by unattached sex chromosome in grasshopper cells
• 3F3/2 epitope correlates with arrest and is
  eliminated by externally applied tension
• Haploid mitosis elicits only partial checkpoint
  (cdc6 exp)
• Unattached Kinetochore
• Laser ablation of the last unattached
  kinetochore releases arrest
• Taxol releases tension without eliciting arrest

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Spindle Assembly Checkpoint
Isolation of mutants defective in the checkpoint

• Sensitivity to high benomyl mad1, mad2,
  mad3
• Sensitivity to sublethal benomyl bub1,
  bub2, bub3
• Serendipity mps1
• Synthetic lethality with ctf13 cdc55

Sensitivity rescued by halting cell cycle
progression
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Spindle Assembly Checkpoint
Effect of spindle checkpoint mutants on Clb
accumulation and H1 kinase activity
Clbs
H1 kinase
cdc55 CDC28-FV
wt
mad, bub
cdc55
a-factor arrest and then release into Nocodazole
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Spindle Assembly Checkpoint Proteins
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Spindle Checkpoint Targets Cdc20

• Mad1, 2, 3 bind to Cdc20
• (2-hybrid, Co-IP)
• Cdc20 bypasses spindle, DNA damage
  checkpoint (not DNA replication checkpoint)
• Dominant alleles of CDC20 render cells
  resistant to spindle checkpoint (mutant
  protein no longer binds Mad2)

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X-Mad2 Binds to Unattached Kinetochores
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Spindle Assembly Checkpoint Pathway
X-Mad2 binds to unattached kinetochores
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Spindle Assembly Checkpoint Pathway
Mad2 leaves kinetochores as they become attached
to microtubules
Blue DNA Green Microtubules Pink Mad2
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mBub1 Binds to Unattached Kinetchores
Pro- metaphase
Prophase
Metaphase
Anaphase
Bub1
Kinetochores
DAPI
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mBub1 Binds to Unattached Kinetchores
Nocodazole
- Nocodazole
a-Bub1
DAPI
Merged
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Spindle Assembly Checkpoint Pathway

• BUB1-5 (dominant negative mutation) arrests in
  mitosis
• BUB1-5 arrest requires MAD1-3, MPS1, BUB3
• Bub1 and Bub3 physically and genetically
  interact
• Mad1 hyperphosphorylated in mitosis and on
  arrest
• Mad1 hyperphosphorylation requires BUB1,3 MAD2
• Mps1 phosphorylates Mad1, even in bub1

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Spindle Assembly Checkpoint Pathway
Spindle defects
Bub1/Bub3
Mad2
Mps1/Mad1
Bub2/Mad3
(Pds1)
G1
S
G2/M
Anaphase
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Spindle Assembly Checkpoint Pathway
Problems with a linear checkpoint pathway

• Increase Mps1 or Mad1 expression causes mitotic
  arrest
• BUB1-5 causes mitotic arrest
• All three cases require all the other MAD, BUB
  genes
• Mad1 is hyperphosphorylated by Mps1
  overexpression but not by BUB1-5

gt Codependent Pathway, i.e. a complex
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Spindle Assembly Checkpoint Pathway
Constitutive Interactions
Bub3
Bub3
Mad2
Mad3
Bub1
Mad1
Checkpoint Induced Interactions
Bub3
Bub3
Mad3
Mad2
Cdc20
Bub1
Mad2
Mad1
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Spindle Assembly Checkpoint Pathway
Mad1 or Cdc20 Induces A Significant
Conformational Change in Mad2
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What Initiates the Checkpoint Signal?
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Second Spindle Checkpoint Pathway
Bub2 is on a separate but parallel checkpoint
pathway from Mad2
Kinetics of bud formation of a-factor arrested
cells released into nocodazole medium
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Second Spindle Checkpoint Pathway
The mitotic exit network (MEN) prevents mitosis
if the spindle is not properly aligned
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MEN Location, Location, Location
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Two Spindle Checkpoint Pathways