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

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

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?)

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

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

Chromosome non-disjunction: aneuploidy  TOAST

MPF

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

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

repair deficient arrest deficient

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:

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

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

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

translocation has not been confirmed in other organisms

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

Irradiation induces arrest in G1 and G2

G1 checkpoint is non-functional in absence of p53

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”

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

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

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

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

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

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

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

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

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

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, 873-80 (2005)

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

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?

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

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

Lte1 (GEF) is only found in the bud

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

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

EM sections showing mother centriole near midbody