Cell Cycle Regulation

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Cell Cycle Regulation



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Continuous Duplication
Cytoplasmic Cycle
Cell Growth
Cytokinesis
2
1
0
Quantum Duplication
Chromosome Cycle
DNA Replication
Mitosis
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1
0
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The Basic Problem
S phase
G2
G1
Mitosis
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Cell Cycle Stages
G1(G0) - S - G2 - M
How do we determine which stage of the cycle a
cell is in?
1. FACS analysis
Fluorescence Activated Cell Sorting
2. Incorporation of radioactive or epitope
tagged nucleotides (BrdU)
3. Landmarks
Nuclear envelope breakdown
Condensed chromosomes
Spindle elongation
Determining G0 vs G1 can be difficult.
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G1
4
Cells sense their environment
- nutritional
- geometrical (cell-cell contact)
- physical (size am I big enough)
- regulatory (growth factors)
If all is well, the cell will commit to a new
cell cycle and pass START in yeast or the
R-point (restriction) in mammals.
The later stages of G1 involve preparation
for S phase and mitosis. These include
induction of gene expression and
duplication of centrosomes (MTOC).
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5
S phase
Cells initiate DNA synthesis.
They fire early origins early and late origins
late.
Early
Late
Cell growth continues.
During S phase cells must have a mechanism to
prevent activation of mitosis until DNA
replication is completed.
In addition, chromosomes must be replicated once
and only
once per S phase. Origins fire only once except
in special cases.
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6
G2
No major cytological events
Probably a sensing period for
accumulation of information
leading to the commitment to
Mitosis.
Proteins needed for Mitosis
are synthesized in G2.
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Mitosis
Prophase
Seven parts.
Prometaphase
Metaphase
Anaphase A
Anaphase B
Telophase
Cytokinesis
Each of these is carefully regulated
so as to occur in the proper order.
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Prophase
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Prometaphase
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Metaphase
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Anaphase
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Telophase
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Cytokinesis
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Interphase
Late Prophase
Prometaphase
Early Prophase
Metaphase
Anaphase A
Anaphase B
Telophase
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Cell Cycle Transitions
State A
State B
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Cell Cycle Transitions
State A
State B
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Cell Cycle Transitions
State A
State B
Metastable States Mutual Incompatibility
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Cell Cycle Transitions
State A
State B
State C
Inhibitory Barriers
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Cell Cycle Transitions
State A
State B
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Cell Cycle Transitions Cdc Mutants
X
State A
State B
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Cell Cycle Transitions
X
State A
State B
X
A Checkpoint Pathway Creates a Dependency
Relationship
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Cell Cycle Transitions
State A
State B
Self-Reinforcement
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Cell Cycle Transitions
State A
State B
Self-Reinforcement
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Cell Cycle Transitions
Mitosis
G1
S
Meta
Ana
Telo
G2
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Cell Cycle Transitions
G1
S
Meta
Ana
Telo
G2
Cdk1 SCF
Esp1
APC
Cdk1 SCF
APC
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Cell Cycle Transitions
G1
S
Meta
Ana
Telo
G2
Cdk
Esp1
APC/C
Cdk
CKI
Wee1
Pds1
Cdk1
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Cell Cycle Genetics
YEAST The genetic system.
Advantages
1. Eukaryotic cell cycle
2. Haploid---Diploid
3. Transformable

4. Reverse genetics
CDC Mutants

1. Conditional lethal mutants
cdc mutant
2. Arrest the cell cycle at a unique position.
Can be used for four purposes
1) Make a molecular map of the order of
24C
function of cdc protein and landmark
events.
2) Make a determination of whether certain
processes are dependent or not.
3) Identify genes important for a particular
37C
process.
4) Identify other genes in the process by
reversion analysis.
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Cdk1 cdc2

Cdc28
cdc2 was identified in S. pombe as a mutant
that arrests in G2 and gives rise to long cells.
A critical experiment identified a dominant
allele of cdc2 that cause the cell cycle to
accelerate rather than stop, yielding shorter
cells.
Why is this so important?
CDC28 was identified in S. cerevisiae
-protein kinase
Two stop points G1 and G2 (the original allele
had only one arrest point in G1)
How does one protein regulate two completely
different processes?
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The Relationship Between Cyclins and MPF
mitosis
mitosis
interphase
interphase
Cyclin A
Cyclin B
RNR
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Cyclins
Cyclins are periodically
accumulated during the cycle,
then rapidly destroyed.
M
I
M
I
M
I
3
1
2
MPF
MPF activity peaked with cyclin levels.

Cyclins are a regulatory component of MPF.
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Cyclins are the regulatory subunit of
cyclin-dependent kinases (Cdk)
Cyclins bind to Cdks and activate the kinase and,
in some circumstances, control their substrate
specificity
Activation of Cdks controls certain cell cycle
transitions
G1
S phase
Mitotic
Cyclin/Cdk
Cyclins/Cdk
Cyclins/Cdk
Mitotic
G1
S
G2
M
Exit
G1 Cdk
S Cdk
M Cdk
All Cdks
ON
ON
ON
OFF
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S. cerevisiae Cyclin/Cdk Activity
CLN1
CLB5
CLB3
CLB1
CLN3
CLN2
CLB6
CLB4
CLB2
2 Classes of Cyclins
G1 Cyclins
CLN
Cln1, 2, 3
S phase
CLB
Clb5, 6
Mitotic
CLB
Clb, 1, 2, 3, 4,
G1
G2
S
M
Redundancy among cyclins

Deletion of one Cln or Clb has little effect
cln1, 2, 3, ------ G1 Arrest
D
clb1, 2, 3, 4 ------ G2 Arrest
D
Cyclins gain functions later in the cycle.
Clbs can carry out the function of Clns in some
circumstances
Clb1-4 can carryout the functions of Clb5,6 but
Clb5,6 cannot function
in place of Clb1-4.
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The Start of START
Auto-activation Loop
Cell size
SBF
?
?
(Swi4/6)
Sic1
?
?
MBF
S phase
Nutrients
CLN3
uORF
Auto-activation Loop
What exactly is START?
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The end of START and the start of S phase
Budding Centrosome Duplication
Sic1 is made during the previous mitosis
Sic1
SCF
SBF
Clb1-4
MBF
Cdc28
Nutrients
G1
S
G2
M
Sic1 ensures that S is dependent upon G1 cyclins
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Ubiquitin Conjugation Cascade
E1
S
Ub Activating Enzyme
S
E2
Ub Conjugating Enzyme
E3
Ub Ligase
substrate
substrate
Destruction by the 26S Proteasome
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Sic1 degradation through the SCFCdc4 pathway
Rbx1
Signal
E2
C
dc
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Cln/Cdc28
C
dc
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Sic1
Sic1
S
kp
1

Cdc4
SCFCdc4
E1-S-Ub
Ub
Rbx1
N
S-Ub
Sic1
C
dc
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C
dc
53
Ub
Ub
N
S
kp
1
Proteasome
Sic1
S phase
Cdc4
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The F-box Hypothesis
Rbx1
Cdc34
Function
Substrate
F
Sic1
Cdk Inhibitor
Cdc53
Cdc4
Cln2
Cyclin
F
S
kp
1
S
kp
1
Grr1
F
Unknown
Targets
Other F-box
Proteins
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Tumorigenesis
Cell Cycle Control
Bud site selection
G1 cyclins (Cln1)
p27
Cdk Inhibitors (Sic1)
Cyclin E
DNA replication (Cdc6)
Grr1
Skp2
Fbw7
Plant flowering
Cdc4
UFO
Auxin response in plants
Circadian rhythms in plants
F-box Proteins
FKF1
TIR1
Sel-10
Met30
Cell Fate (Notch)
Aminoacid biosynthesis (Met4)
b-TRCP
Dactylin
NFkB Activation (IkB)
Limb development
Wnt Pathway (b-catenin)
Hedgehog (Ci)
Signaling/ Transcription
Development
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RING-Finger Based Ubiquitin Ligases
E1
E1
Rbx1
Apc11
Cdc34
E2
Apc2
Cdc53/ Cul1
E2
substrate
substrate
Skp1
P
P
RING
F
Simple RING-E3s
Anaphase Promoting Complex
SCF, VCB
F-box Proteins BC-box Proteins 5 different
Cullins
MDM2 Cbl BRCA1 Parkin
Cdc20 Cdh1
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Two Major Classes of E3s
HECT Family
RING Superfamily
E6-AP (Angelmans Syndrome) Smurf1 (Smad
destruction) Itch (Notch destruction) Rsp5
(membrane protein endocytosis)
SCFs
APC
Simple RING E3s
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Cell Cycle Logic
Making the cycle go forward

Grr1
SCF
SBF
Cdc4
SCF
(Swi4/6)
Sic1
Cdc4
SCF
S phase
MBF
Time
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Mitotic Entry Activation of Mitotic
Cyclin-Dependent Kinases
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Cdk Regulation
Synthesis
Destruction
SCF Complexes
Phosphatase
Kap1
Kinases
cyclin
T161
CAK
Civ
Synthesis
Destruction
Cdk
Cks
SCF Complexes
T14 Y15
CKIs
Cdk Inhibitors
Kinases
Phosphatases
Sic1
Wee1
Cdc25


Far1
Mik1
Pyp3
Rum1
Myt1
p21, p27, p57
p16, p15, p18, p19
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Mitotic Entry in S. pombe and Mammals
Phosphorylation Regulation of Cdc2 during Mitosis
Cdc2 (Cdk1)
(Cdc13) cyclin B
cyclin B/Cdc2

CAK
cyclin B/Cdc2 (T160-P) ACTIVE KINASE
-
Tyrosine
Wee1
Kinases
Mik1
cyclin B/Cdc2 Y-P (Inactive Y15-P)

Phosphotyrosine
Cdc25
Phosphatase
cyclin B/Cdc2 ACTIVE KINASE
G2
M
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Cell Cycle Logic
Autoactivation of Cdc2 makes mitosis irreversible
Cdc2 (Cdk1)
(Cdc13) cyclin B
cyclin B/Cdc2

CAK
cyclin B/Cdc2 (T160-P)
-
Tyrosine
Wee1
Kinases
Mik1
cyclin B/Cdc2 Y-P (Inactive Y15-P)
-

Phosphotyrosine
Cdc25
Phosphatase

cyclin B/Cdc2 (active)
G2
M
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APC/Cyclosome
The APC is a complex ubiquitin ligase that is
required for anaphase entry and mitotic
exit. Like the SCF, it has substrate specificity
components called Cdc20 and Cdh1/Hct1, 2 WD40
repeat proteins. The regulation of these
specificity components is critical.
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Anaphase Entry and Exit
APC
APC
Clb5
Chromosomes
Cdh1
Cdc20
Clb2
OK ?
Pds1
Cohesion
Clbs
Factors
Cdk1
Mitotic
Exit
Anaphase
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Chromosome Cohesion
Ub
Ub
Ub
Destruction by the
Pds1
Cdc20
APC
26S Proteosome
Pds1 has a destruction box which allows
Pds1
it to be recognized by the APC
Securin
Esp1
Separin
Esp1
Cohesin
Separin
Anaphase
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Cohesion in Mammals
Securin Pds1 Separin Esp1 Cohesin Scc1
APC
Separin (Esp1)
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Mitotic Exit
After anaphase is complete, in order to exit
mitosis and
initiate cytokinesis, cells must inactivate
B-type cyclin/Cdks.
Mitosis
Mitotic Exit
Cytokinesis G1 Entry
High
Low
Cyclin B/Cdk Activity
Cyclin B/Cdk Activity
Low
High
Cdc14 Phosphatase
Cdc14 Phosphatase
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Mitotic Exit
After anaphase is complete, in order to exit
mitosis and
initiate cytokinesis, cells must inactivate
B-type cyclin/Cdks.
This involves activation of the Cdh1 form of the
APC.
Active
Cdh1
Cdh1
Cdh1
Clb
Cdc14
Cdk1
(Phosphatase)
Inactive
Cdh1
APC
MEN
Cdc14
Cdc14
Inactive
Active
Clb/Cdk1
Cdc14
Sic1
Swi5
Swi5
Swi5
Transcription Factor
Clb
Cytoplasmic
Nuclear
Cdk1
Active
Inactive
Mitotic
Exit
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Cdc14 Activation for Mitotic Exit
The activation of Cdc14 is the key event in
execution of
mitotic exit. During S, G2 and Pre-anaphase,
Cdc14 is held
tethered in an inactive complex in the nucleolus.
When Anaphase is executed, Cdc14 is released and
goes
throughout the nucleus and cytoplasm to
dephosphorylate
key Cdk1 substrates.
Nucleolar
Spindle
Cdc14
MEN
Cfi1
Cfi1
Cdc14
Cdc14
(Net1)
(Net1)
Inactive
Active
The mitotic exit network (MEN) consists of
several protein kinases
and a G-protein Tem1. How it MEN regulated is
not known.
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How Mitotic Exit is Coupled to Anaphase
Spindle Pole Body
Tem1-GDP
Bfa1/Bub2 (GAP)
Lte1 (GEF)
Tem1-GTP
Mitotic Exit Network
Cdc15
Dbf2, Mob1
Clb2
Cdc14 (Nucleolus)
Cdc14 (Released)
Mitotic Exit
Sic1
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The Tem1-bearing SPB migrates into the daughter
cell to encounter Lte1
Tem1
Lte1
Tem1
Lte1
D
Tem1
M
Spindle in Green
Lte1 in Red
Tem1 in Red
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Mitotic Exit Summary
1. When anaphase occurs, Tem1 on the SPB is
thrust into the daughter cell where it
encounters the GEF, Lte1, Tem1 is converted to
the active GTP form. 2. Active Tem1 activates
Cdc15 and MEN, which causes the release of the
Cdc14 phosphatase from the nucleolus where it
is inhibited. 3. Cdc14 dephosphorylates Cdh1 to
activate the APC to destroy Clbs, it also
activates the synthesis of Sic1, a Cdk
inhibitor. 4. Together, the APC and SIC1 turn
off Cdk activity to initiate mitotic exit. Cell
move from high CDK, low CDC14 state to a Low CDK,
high Cdc14 state. To re-enter the next cell
cycle they need to turn off Cdc14 to
re-establish the null state, CDK off, Cdc14 off,
APC off, making cells permissive for Clb
activation of S phase.
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How does the cycle move forward? - Positive
amplification loops - Feedback inhibition
1) Clns activate their own transcription 2)
Once Clns provide sufficient activity to pass
START, they activate a ubiquitin proteolysis
pathway that destroys an inhibitor of Clb kinase
activity, Sic1. 3) Clb/Cdc28 kinase activate Clb
transcription and repress Cln transcription. 4)
Clb/kinase activate S phase. 5) Once S phase is
complete, Clb kinases activate mitosis. 6) Once
chromosomes are properly aligned at the metaphase
plate, a ubiquitin proteolysis pathway is
activated that destroys Clbs but not Clns and
resets the cycle. 7)Clb destruction allows PRC
complexes to form. 8) Cln kinase activity is
required to shut off the Clb proteolysis pathway
to allow S entry in the next cell cycle. This
allows Pds1 to be synthesized again which
recruits Esp1 into the nucleus. In
Mammals Cyclin B/Cdc2 can help activate itself by
turning on an activating phosphatase and turning
off an inhibitory kinase
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The Rao and Johnson Cell Fusion Experiments
Cell Cycle Regulation
M cells G1, S, or G2 cells
M
- Mitotic state is dominant.
S cells G1
G1 cells enter S
S cells G2
G2 cells do not enter S, but do not
enter mitosis until the S-phase
nucleus has entered G2.
- Block to re-replication
- Inhibitor of mitosis produced by
S phase cells
G1 cells G2
Like S above.
- G1 cells also block mitosis
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Cell Cycle Checkpoints
Definition
"A checkpoint is a biochemical pathway that
ensures
dependence of one process on another process that
is
otherwise biochemically unrelated."
B
C
Intrinsic
A
Mechanism
E
D
Extrinsic
Mechanism
Damage
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Why are checkpoints important?
Checkpoints control the order and timing of
events. In
some cases the natural timing of events can allow
the
proper order of events in the absence of a
checkpoint.
However, the fidelity is often compromised.
The accumulation of errors, whether due to
entering
DNA replication in the presence of damage, or mis-
segregating a chromosome is deleterious to the
reproductive fitness of unicellular organisms,
and in
multicellular organisms may lead to uncontrolled
cell
proliferation and cancer.
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Checkpoints in S. c.
DNA Damage Checkpoints
Spindle Assembly Checkpoints
S phase Checkpoints
Size Checkpoints
G1/M Checkpoint
Morphology Checkpoint
Meiotic Checkpoints
Checkpoints are defined by loss of function
mutations that relieve the dependency of two
events.
cdc13 ts
mutants
cdc13 rad9
mutants
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The Spindle Assembly Checkpoint
The proper assembly of a spindle is sensed by a
group of proteins called Mad or Bub located on
the kinetochore. These proteins send a signal to
inhibit the APC.
Mutant Hunt - benomyl sensitive mutants
that continue to cycle in the presence of
benomyl.
ben
WT
ben
mad or bub mutants
Anaphase A/B
Metaphase
Misaligned Chromosomes
Scc1
Mps1 Mad1,2,3 Bub1,2,3
Esp1
Cdc20
APC
Pds1
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The Spindle Assembly Checkpoint
What is being sensed?
Kinetochore - Microtubule Attachment
Tension and bipolar attachment
When tension is not present at sister chromatids,
a Mad/Bub- dependent phosphorylation occurs on
the kinetochore. This is thought to be part of
the signal used to turn off the APC.
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Signal Transduction
Signal
Sensor
Transducer
Effector
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DNA Damage Response Pathways
SIGNALS
Conserved Families
SENSORS
PCNA- RFC-like Proteins
Mediators (BRCT proteins, Mrc1/Claspin)
TRANSDUCERS
Kinases PIK ATM ATR
EFFECTORS
PK CHK1 and CHK2
STOP
Transcription
Cell Cycle Arrest
Apoptosis
DNA Repair
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The DNA Damage Response in Humans
Hus1
P
Rad17
P
RFC
ATRIP
Rad1
Rad9
PC
Claspin
P
P
P
P
BRCA1 BLM NBS1 Repair Proteins
Chk1
Chk2
P
P
P
P
p53
Cdc25
p21
DNA replication proteins?
Cdc2/Cyclin B
G1
M
G2
S
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DNA Damage Checkpoints - Sensing Damage
P
P
ATRIP
Rad17
RFC
ATP
P
P
ATRIP
Hus1
Hus1
Rad17
Rad1
Rad1
RFC
Rad9
Rad9
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ATR and RC-PC Engagement Activates Checkpoint
P
Hus1
Hus1
P
ATRIP
Rad17
Rad1
Rad1
RFC
Rad9
Rad9
P
P
P
P
P
P
Mediators
Nbs1
Chk1
Chk2
BRCA1
Checkpoint Responses
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G1 Arrest in Mammals
Cdk activity is rate limiting for S phase entry
and is the target for checkpoint control.
DNA Damage
Chk1,2
Cdc25A
ATM/ATR
?
Chk2
G1 Cyclin
p21
Mdm2
p53
p53
Cdks
or
Apoptosis
p53 levels increase in response to DNA
damage and activate transcription of p21
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How is p53 activated? - Relief of repression.
MDM2 binds p53 and targets it for
ubiquitin-mediated proteolysis.
p53 transcriptionally regulates MDM2 to make a
feedback loop.
RING Finger Ubiquitin Ligase
Mdm2
p53
Mdm2 transcription
In response to DNA damage, both p53 and Mdm2 are
phosphorylated, causing a disruption in Mdm2
binding, thereby allowing p53 to both increase
in abundance and become transcriptionally
active.
During activation, p53 increases the amount of
Mdm2 protein to return to low p53 levels when
the signal is eventually turned off.
This also explains why p53 levels are so high in
tumors in which p53 is mutant, no Mdm2 is made.
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G2 Arrest in Mammals
Cdc25 is regulated by Chk1 phosphorylation
DNA Damage
Inactive
ATR
Active
Chk1
Ser216
Ser216
14-3-3
P
Cdc25C
Cdc25C
OFF
ON
Nuclear
Cytoplasmic
S/G2
Mitosis
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G2 Arrest in S. pombe Mammals
Cdk activity is rate limiting for entry into
Mitosis
and is the target for checkpoint control.
DNA Damage
14-3-3
Cytoplasm OFF
Cdc25
ATM or ATR
P
Chk1
Chk1
Nucleus ON
Chk2
Chk2
Cdc25
Wee1
OFF
ON
cyclin B
cyclin B
Cdc2 Y-P
Cdc2
OFF
ON
p21
p53
Mitosis
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Mechanism of pre-anaphase arrest in response to
DNA damage
S. pombe Mammals
S. cerevisiae
ATR
MEC1 DDC2
rad3 rad26
Mediator
crb2
RAD9
RAD53
CHK1
Chk Kinases
chk1
Effectors


PDS1
cdc25
CDK
ESP1
cdc2
Anaphase Entry
Mitotic Exit
Mitotic Entry
Chk1 phosphorylation of Pds1 protects it from
degradation by the APCCdc20
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Overall Organization of the Cell Cycle
Replication Checkpoint
?
G1
G2
S
AnaA
AnaB
Tele
Meta
M Cdk
S Cdk
M Cdk
Pds1
B/Cdk1
Sic1
SCF
APC
APC
Spindle
Cdc20
Cdh1
Cdc14
G1 Cdk
Checkpoint
Mitosis
Cdc20
Cdh1
APC
APC
APC
SCF
APC
APC
ON
ON
ON
ON
OFF
B/Cdk1
B/Cdk1
B/Cdk1
OFF
OFF
ON
78
General Points
Cells need to do only a few things absolutely
right
1. They must duplicate their chromosomes
precisely,
i.e. completely but only once per cycle.
2. They must segregate their chromosomes
precisely.
3. They must divide their cell in two.
General Properties of Cell Cycle Transitions
1. Amplification mechanisms.
2. Out with the old, in with the new.
3. Overcoming inhibitory barriers- Checkpoints.
Checkpoints allow the coordination of events.