1. Lecture 10 Checkpoints Outline: Review G1/S
DNA damage/replication checkpoint
spindle assembly checkpoint
spindle position checkpoint
Paper: Centrosomes enhance the fidelity of
cytokinesis in vertebrates and are required for
cell cycle progression

2. Control of G1 progression in budding yeast
SCF
Cdc14
(Cdh1)
Mitotic exit: Cdk inactivation
G1/S-Cdk activity increases - not susceptible to Sic1
S cyclin synthesis induced
S-Cdk inactive until phosphorylation of Sic1 and Cdh1
by G1/S-Cdks
DNA replication

3. Possible mechanism to coordinate cell growth
and cell cycle progression
Cln3 synthesized in parallel with cell growth
How is threshold level reached?
Cells inherit fixed amount of inhibitor (DNA?)

4. Control of G1 progression in mammalian cells
G1-Cdk induced by growth factor, phosphorylates Rb
E2F induces more of itself, and S phase Cyclins (E, A)
S-Cdks further phosphorylate Rb
More S-Cdks accumulate - DNA replication

5. M  TOAST S chromosome condensation during S-phase DNA damage:
chemicals
radiation
normal DNA metabolism

6. Chromosome
non-disjunction:
aneuploidy
 TOAST

7. MPF

8. First clue about feedback control
1974
First clue about feedback control
Irradiate tissue culture cells
DNA damage
caffeine treatment
no delay:
lethal chromosome
damage
Delayed entry into M-phase
until damage repaired

9. How to identify genes involved in checkpoint function?
YEAST
Identify mutants that cannot recover from
DNA damage
2 classes:
repair deficient
arrest deficient

11. repair deficient
arrest deficient

12. Conserved elements of DNA damage and replication checkpoints
I. Sensors
Proteins with functional
analogs in DNA replication
recognize damage
load onto DNA
inhibited by caffeine
II. Transducers:
= kinases
ATM and ATR
Chk1 and Chk2
phosphorylate substrates affecting protein activity or stability
cell cycle arrest
activate DNA repair
maintain arrest until repair complete
re-initiate cell cycle progression
or APOPTOSIS
III. Effector output:

13. Examples of pathways that block the cell cycle:
DNA damage pathway in budding yeast
 G2 or M arrest
ionizing radiation
RAD9
MEC1 (ATR kinase)
CHK1
PDS1
cohesins
anaphase entry
RAD53
CDC5 (polo kinase)
CLB/CDC28
mitosis

14. Another feedback pathway in fission yeast DNA damage  G2 arrest
Cdc25
Y15 ppase
Two genes identified:
chk1, rad24
Both act through cdc25

15. Mechanism: Sequester Cdc25 away from Cdc2
DNA damage
Cdc25 phosphorylated by Chk1
P-Cdc25 recognized by Rad24
14,3,3 protein
with NES
Rad24 transports P-Cdc25 out of nucleus
Cdc25 cannot dephosphorylate nuclear Cdc2

16. translocation has not been confirmed in other organisms

17. p53 = tumor suppressor/transcription factor:
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

18. Irradiation induces arrest in G1 and G2

19. G1 checkpoint is non-functional in absence of p53

20. Also a faster response recently identified,  independent of p53
Cyclin D-Cdk4,6
Cyclin E-Cdk2
ATM
kinase
transcription
Also a faster response recently identified,
 independent of p53
 Cyclin D degradation and “inhibitor swap”

21. ionizing radiation
APC activated toward Cyclin D
p21CIP released
p21CIP inhibits Cyclin E-Cdk2

23. Spindle Checkpoints Kinetochore-mediated
 senses when all chromosomes have been
attached and properly aligned
cross-talk
 regulates sister separation
MTOC-mediated
 senses proper spindle position
 regulates exit from mitosis

24. Budding yeast mutants that fail to arrest in the presence
of microtubule depolymerizing drugs
Hoyt lab
bub:
budding uninhibited by benomyl
Bub1, Bub2, Bub3
Murray lab
mad:
mitotic arrest deficient
Mad1, Mad2, Mad3
Mps1

25. Kinetochore checkpoint
Cdc20
Target:
Activator of APC-mediated destruction of
Pds1, B Cyclins  chromosome segregation
unattached
kinetochore
Cdc20
diffusible signal
APC
Pds1, cyclins

26. focus on Mad2
associates only with unattached kinetochores
inhibits Cdc20-APC
mouse knockout embryonic lethal

27. Model  unattached kinetochores “activate” Mad2
 Mad2* diffuses away and inhibits Cdc20-APC

28. FRAP experiment: Mad2 turns over rapidly
Howell et al. (2000)

31. Questions How transduced??? What activates/inactivates Mad2?
binding partners: Mad1, Mad3, BubR1, Bub3?
What is Mad2*
complex? oligomer?
What generates and stops the signal???
How
transduced???
somatic cells - MT attachment
meiotic cells - tension
dynein-dependent transport to spindle poles
may turn it off

32. 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 kinetochores
Required for checkpoint signaling

33. Motor-dependent mechanism for checkpoint signaling?
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, (2005)

34. spindle position checkpoint
Cdh1 = Hct1
Target:
Activator of APC-mediated destruction of
B Cyclins  mitotic exit
spindle position
defect
Cdh1= Hct1
Cdc14 pathway
APC
B cyclins

35. Cdc14 pathway Cfi1 sequesters Cdc14 phosphatase in the nucleolus
Release of Cdc14 allows it to dephosphorylate
Cdh1  targets APC to cyclins
Sic1  inhibits Cdk-Cyclin activity
What causes release of Cdc14?

36. Two upstream factors identified that constitute a
spindle position sensor
Tem1 (small GTPase)
Lte1 (GEF)
Together serve as activators for Cdc14 release

37. is only found on daughter spindle pole body (SPB)
Tem1 (GTPase)
is only found on daughter spindle pole body (SPB)
tagged
Tem1
Bardin, Visintin and Amon
Cell 102, 21-31

38. Lte1 (GEF) is only found in the bud

40. Spindle position defect induced by dynein disruption
telophase
anaphase
arrest
Release of nucleolar Cdc14 and mitotic exit
only occurs in cells with daughter nucleus in bud

42. Similar spatial clues in vertebrate cells??
Piel et al. Science 291, 1550 (2001):
A role for the centrosome in completion of cytokinesis:
Mother centriole often moves to intercellular bridge
(midbody) prior to final step in cytokinesis

43. EM sections
showing mother
centriole near
midbody