Lecture 10 Checkpoints Outline: Review G1/S DNA damage/replication checkpoint spindle assembly...
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Lecture 10
Checkpoints
Outline:Review G1/SDNA damage/replication checkpoint spindle assembly checkpointspindle position checkpoint
Paper: Centrosomes enhance the fidelity of cytokinesis in vertebrates and are required for cell cycle progression
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Control of G1 progression in budding yeast
Mitotic exit: Cdk inactivationG1/S-Cdk activity increases - not susceptible to Sic1S cyclin synthesis inducedS-Cdk inactive until phosphorylation of Sic1 and Cdh1
by G1/S-CdksDNA replication
(Cdh1)
Cdc14SCF
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Possible mechanism to coordinate cell growth and cell cycle progression
Cln3 synthesized in parallel with cell growthHow is threshold level reached?Cells inherit fixed amount of inhibitor (DNA?)
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Control of G1 progression in mammalian cells
G1-Cdk induced by growth factor, phosphorylates RbE2F induces more of itself, and S phase Cyclins (E, A)S-Cdks further phosphorylate RbMore S-Cdks accumulate - DNA replication
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chromosome condensation during S-phase
S
M
DNA damage:
chemicalsradiationnormal DNA metabolism
TOAST
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Chromosome non-disjunction:
aneuploidy
TOAST
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MPF
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1974
Irradiate tissue culture cells
First clue about feedback control
caffeine treatmentno delay:lethal chromosomedamage
DNA damage
Delayed entry into M-phase until damage repaired
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How to identify genes involved in checkpoint function?
YEAST
Identify mutants that cannot recover from DNA damage
2 classes:repair deficientarrest deficient
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repair deficient
arrest deficient
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Conserved elements of DNA damage and replication checkpoints
I. Sensors Proteins with functional analogs in DNA replication
recognize damageload onto DNA
II. Transducers:= kinases
ATM and ATR
Chk1 and Chk2
phosphorylate substrates affecting protein activity or stability
cell cycle arrestactivate DNA repairmaintain arrest until repair completere-initiate cell cycle progressionor APOPTOSIS
III. Effector output:
inhibited by caffeine
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DNA damage pathway in budding yeast
ionizing radiationRAD9
MEC1 (ATR kinase)
RAD53
CDC5 (polo kinase)
CLB/CDC28
mitosis
CHK1
PDS1
cohesins
anaphase entry
G2 or M arrest
Examples of pathways that block the cell cycle:
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Another feedback pathway in fission yeastDNA damage G2 arrest
Two genes identified:chk1, rad24
Both act through cdc25
Cdc25Y15 ppase
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DNA damage
14,3,3 proteinwith NES
Mechanism: Sequester Cdc25 away from Cdc2
Cdc25 phosphorylated by Chk1
P-Cdc25 recognized by Rad24
Rad24 transports P-Cdc25 out of nucleus
Cdc25 cannot dephosphorylate nuclear Cdc2
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translocation has not been confirmed in other organisms
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DNA damage checkpoint in mammalian cells
G1 arrest mediated by p53
p53 = tumor suppressor/transcription factor:stabilized in damaged cells
induces expression of Cdk inhibitor p21CIP
induces apoptosis
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Irradiation induces arrest in G1 and G2
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G1 checkpoint is non-functional in absence of p53
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Also a faster response recently identified, independent of p53
Cyclin D degradation and “inhibitor swap”
ATMkinase
Cyclin D-Cdk4,6Cyclin E-Cdk2
transcription
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APC activated toward Cyclin D
p21CIP released
p21CIP inhibits Cyclin E-Cdk2
ionizing radiation
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Spindle Checkpoints
Kinetochore-mediated
MTOC-mediated
senses when all chromosomes have been attached and properly aligned
regulates sister separation
senses proper spindle position
regulates exit from mitosis
cross-talk
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Budding yeast
mutants that fail to arrest in the presence of microtubule depolymerizing drugs
Hoyt labbub:budding uninhibited by benomyl
Murray labmad:mitotic arrest deficient
Bub1, Bub2, Bub3
Mad1, Mad2, Mad3
Mps1
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Kinetochore checkpoint
Activator of APC-mediated destruction of Pds1, B Cyclins chromosome segregation
unattachedkinetochore
Pds1, cyclins
APC
Cdc20
diffusible signal
Cdc20Target:
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focus on Mad2
associates only with unattached kinetochores
inhibits Cdc20-APC
mouse knockout embryonic lethal
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Model
unattached kinetochores “activate” Mad2
Mad2* diffuses away and inhibits Cdc20-APC
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FRAP experiment: Mad2 turns over rapidly
Howell et al. (2000)
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Questions
What activates/inactivates Mad2?
binding partners: Mad1, Mad3, BubR1, Bub3?
What is Mad2*
complex? oligomer?
What generates and stops the signal???
somatic cells - MT attachmentmeiotic cells - tension
How transduced???
dynein-dependent transport to spindle polesmay turn it off
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Different checkpoint proteins in higher eukaryotes
No Bub2
Mad3 homolog = BubR1, acquired kinase domain
CENP-E
ZW10/Rod (dynein interactors)
All behave like Mad2: associate with unattached kinetochoresRequired for checkpoint signaling
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Motor-dependent mechanism for checkpoint signaling?QuickTime™ and aTIFF (Uncompressed) decompressor
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CENP-E activates BubR1 kinase activity until attached to microtubules - creates “wait” signal
After attachment, complexes are carried off the kinetochore by dynein
Mao, Desai and Cleveland, JCB 170, 873-80 (2005)
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spindle position checkpoint
Activator of APC-mediated destruction of B Cyclins mitotic exit
spindle positiondefect
B cyclins
APC
Cdh1= Hct1
Cdc14 pathway
Cdh1 = Hct1Target:
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Cfi1 sequesters Cdc14 phosphatase in the nucleolus
Release of Cdc14 allows it to dephosphorylate Cdh1 targets APC to cyclinsSic1 inhibits Cdk-Cyclin activity
What causes release of Cdc14?
Cdc14 pathway
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Two upstream factors identified that constitute aspindle position sensor
Tem1 (small GTPase)
Lte1 (GEF)
Together serve as activators for Cdc14 release
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Tem1 (GTPase) is only found on daughter spindle pole body (SPB)
taggedTem1 Bardin, Visintin and Amon
Cell 102, 21-31
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Lte1 (GEF) is only found in the bud
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Spindle position defect induced by dynein disruption
Release of nucleolar Cdc14 and mitotic exitonly occurs in cells with daughter nucleus in bud
anaphasearrest
telophase
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Similar spatial clues in vertebrate cells??
Piel et al. Science 291, 1550 (2001):
Mother centriole often moves to intercellular bridge (midbody) prior to final step in cytokinesis
A role for the centrosome in completion of cytokinesis:
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EM sectionsshowing mothercentriole nearmidbody