Download BC4 – The Cell Cycle

 BC4 – The Cell Cycle Boris Pfander Max Planck Ins9tute of Biochemistry [email protected] +49-­‐89-­‐8578-­‐3050 www.biochem.mpg.de/pfander 1 Topics Overview on the Cell Cycle The discovery of CDK – the master regulator of the cell cycle S-­‐phase and DNA replica9on Checkpoints that sense DNA damage M-­‐phase – mitosis and cytokinesis Chromosome condensa9on & cohesion 2 Cell cycle stages and transitions
G1>S
START
G2>M
metaphase > anaphase
& mitotic exit
3 Mitosis 4 5 5 Phases of Mitosis
6 6 Changes in chromosome morphology during mitosis
Condensin
Cohesin
7 Condensin shapes mitotic chromosomes
Condensin
Condensin
DNA
Condensin
Cohesin
P
Condensin
Houlard et al., NCB, 2015
8 Condensin might trap loops of DNA.
Condensin
Cdk1-Cyclin B target
9 Cohesin mediates sister chromatid cohesion.
Cohesin
Cohesin
Cohesin
The cohesin ring model
10 Metaphase > anaphase transition
11 Cyclin levels drop at the metaphase to anaphase
transition
Cdk1 activity drops!
12 Cyclin B levels and (kinase) activity of MPF
change in parallel in cycling Xenopus egg extracts
13 Metaphase to anaphase transition
14 -> exit from mitosis requires degradation of cyclin B
by what mechanism?
Glotzer, Murray & Kirschner, 1991:
1) radio-label cyclin B
↓
incubation in Xenopus egg extracts,
which are in anaphase or interphase
↓
SDS-PAGE and auto-radiography
anaphase
cyclin B
T [min.]:
interphase
↓
extract in:
0
30
0
30
15 2) radio-label cyclin B or ubiquitin
↓
incubation in Xenopus egg extracts,
which are in anaphase or interphase
↓
after 10 min.: SDS-PAGE and auto-radiography
(overexposure!!!)
I125-labeled:
cyclin B
ubiquitin
A
A
x4
x3
ubiquitin x 1
cyclin B
cyclin B
↓ ↓
x2
extract in:
I
I
(A = anaphase; I = interphase)
at the time of its degradation
cyclin B is covalently modified by addition of ubiquitin
è
16 Proteolysis controls late mitotic events
Exit from mitosis requires inactivation of Cdk1 by
degradation of mitotic cyclin
The degradation is mediated by the anaphase-promoting-complex
(APC), an E3 ubiquitin ligase
APC activation requires
Cdk1 activity
(ensures correct order of events)
Cdk1 facilitates its own
inactivation
(mitotic checkpoint and unknown
mechanisms ensure delay
to give Cdk1 enough time to act)
17 Anaphase-promoting complex/cyclosome
or
Cdc20
An atomic model of APC/C determined by cryo-EM.
By David Barford
APC – a gigantic E3 ubiquitin ligase
18 Metaphase to anaphase transition
and mitotic exit
19 20 APCCdc20
???
Are really both dependent on APCCdc20?
M cyclin
stable cyclin B
anaphase
mitotic exit
21 amount of DB-peptide
è
experimental evidence for existence of another APC substrate,
which inhibits anaphase (Holloway & Murray, 1993)
APCCdc20
anaphase
inhibitor
anaphase
destruction-box (DB) peptide
M cyclin
= APC recognition sequence (RxxL);
competitive inhibitor of APC
mitotic exit
22 What inhibits anaphase?
What keeps the replicated chromosomes together?
Metaphase spread
Cohesin blocks chromosome
segregation.
23 Cohesin cleavage promotes sister separation
Anaphase
Cohesin Scc1 subunit
Scc1
Protease
Scc1 Ct fragment
Uhlmann and Nasmyth, 1999
APC
Protease inhibitor
24 Sister chromatid cohesion & separation
Nasmyth lab
25 Sister chromatid cohesion
Emergence of sister chromatid cohesion
ê
memory of which chromatids
belong to each other
ê
"division of labor" made possible:
timely separation of duplication and
segregation of chromosomes
ê
evolution of large genomes with many chromosomes
26 APC controls (all) late mitotic events
Spindle assembly checkpoint (SAC)
27 Summary metaphase-to-anaphase transitions
1.  Maximal activity of Cdk-mitotic cyclin (cyclin B)
2.  Chromosomes align on metaphase plate, attached
and ready to segregate into the daughter cells.
3.  Cdk-cyclin B activates APC by phosphorylation;
4.  APC is a large E3 ubiquitin ligase complex, for its
activity in mitosis it requires a co-factor Cdc20
5.  Phosphorylated APC ubiquitylates cyclin B –
degradation and decrease of Cdk activity.
6.  APC ubiquitylates SECURIN, which frees SEPARASE
– sister chromatid cohesion is removed from sister
chromatids – onset of anaphase
7.  Multiple different proteins are degraded through APC
activity.
8.  Cdk activity decreases at the end of mitosis, proteins
are dephosphorylated by phosphatases.
28 Transition into the next G1
29 Two antagonistic oscillators control the cell cycle.
CDK off
CDK on
CDK off
APC on
APC off
APC on
30 The G1 phase of somatic cells cycles
is a state of stable Cdk inactivity
How do you establish a G1 (a prolonged, stable state of Cdk inactivity)?
Problem?
Cdk-cyclin B activate APCCdc20 – only phosphorylated APCCdc20 is active
APCCdc20 degrades cyclin B – decrease in Cdk activity
Less Cdk activity – less APCCdc20 phosphorylated – cyclin concentrations start
to increase again – cell cannot move out of mitosis to reach G1
31 The G1 phase of somatic cells cycles
is a state of stable Cdk inactivity
How do you establish a G1 (a prolonged, stable state of Cdk inactivity)?
Cdh1
1) keep APC active after mitosis
How? > 2nd APC-activator: Cdh1;
APCCdh1 is inhibited by CDK > becomes
active only upon decrease of CDK activity
followed by dephosphorylation of Cdh1
2) activate a CKI (e.g. Sic1 in S. cerevisiae)
32 Two flavors of APC/cyclosome
APCCdc20
Becomes active at mitotic entry upon activation by
M-cyclin/CDK –
Cdc20 binds only phosphorylated APC
APCCdh1
Active from the anaphase onset to the end of G1 phase
- Ensured by at least 2 different mechanisms
1. Inactivating phosphorylation of Cdh1 by M-cyclin/CDK
2. Inhibitors of Cdh1 activated during interphase
ONLY unphosphorylated Cdh1 binds unphosphorylated APC
The two versions of APC are active at different times during cell
cycle, they are differently regulated (and target different
substrates)
Finishing mitosis, one step at a time
Matt Sullivan & David O. Morgan
Nature Reviews Molecular Cell Biology 8, 894-903 (November 2007)
33 The G1 phase of somatic cells cycles is a state of stable
Cdk inactivity
34 Test yourself! 1. 
2. 
3. 
4. 
5. 
6. 
7. 
8. 
Which a2ributes of the cell cycle are conserved throughout eukaryotes? Order of phases? Length of phases? Presence of alternaGng S-­‐ and M-­‐
phases? Presence of alternaGng G1 and G2 phases? Mutants of Cdc28 in budding yeast arrest in G1; mutants of Cdc2 in fission yeast arrest in M-­‐phase? Both genes encode for CDK, how is this possible? Why are fission yeast wee mutants small? Explain! Describe two mechanisms that contribute to the G1/S cell cycle switch at start! The introducGon of many replicaGon origins brings about a specific challenge for eukaryotes. Name it and describe how cells regulate replicaGon iniGaGon in order to avoid this problem! Can the DNA damage checkpoint be arGficially acGvated in the absence of DNA damage? How? How are sister chromaGds held together? By which mechanism is this linkage removed at the metaphase-­‐to-­‐anaphase transiGon? Two different forms of the APC are acGve during the cell cycle. Describe similariGes and differences and why cells rely on two forms of the APC. 35 Thank you and good luck with the cell cycle!
36 Q&A session II
Dr. Sara Batelli
[email protected]
16.01. 2017
Review Session
 Cytoskeletal components give structure and movement to cells
Actin, Tubulin, Intermediate filaments
Motor proteins
 Cell Cycle Regulation – How and when a cell divides
How cyclins control the cell cycle
How check points insure that the cell cycle is progressing properly
Replication of a cell and its components to undergo mitosis
How cytoskeletal components help with the segregation of genetic
material and the division of one cell into two daughter cells
The three filament networks
tubulin
Intermediate
filaments
Size?
Nucleotide binding?
Polarity?
Organization?
f-actin
Actin cytoskeleton
Actin filaments allow cells to adopt different shapes and perform different functions
Actin filaments are important for the morphology of the cell
Villi
Contractile
bundles
Contractile
ring
Generation of
mechanical forces
Sheet-like and
finger-like
protrusions
Cell motility
Actin protein and fibers
Inside cells, actin exists in two states, the monomeric protein, called G-actin (for globular actin) and the 7
nm diameter filament, called F-actin (for filamentous actin). The factor that determines the relative
proportions of F-actin and G-actin is the concentration of actin protein
G- actin (globular)
Polar
molecule
 the actin molecule
has a plus (+) end
and a minus (-) end
 the actin molecule
has a nucleotide
binding site (for ADP
or ATP)
F- actin (filamentous)
 Actin polymerization involved
three steps:
nucleation
elongation
steady-state (equilibrium)
Actin polymerization: 3 steps
 Actins has a characteristic concentration, called the "critical concentration," below which the
monomer state is favored and above which the polymer state is favored. Increasing the
subunit concentration favors filament building, and decreasing it favors filament
deconstruction. This property allows the cell to rapidly control cytoskeleton structure.
 the resulting actin filament has a polarity: fast-growing (+) end slow-growing (-) ends
Actin polymerization – rate limiting step

Actin nucleation is the
rate-limiting step and
takes the longest

Elongation of an existing
actin filament is fast

Three actin monomers
form a nucleus


The actin monomer is
bound to ATP; upon
polymerization, actin
ATPase activity cleaves
ATP to ADP
The rate of polymerization
(kon) depends on the
concentration of free actin
monomers

The depolymerization rate
(koff) does not depend on
the concentration of the
free subunit

As the filament grows, a
critical concentration
(Cc) will be reached at
which subunits addition
equals subunit
dissociation
Actin treadmilling
Treadmilling is a phenomenon observed in many cellular cytoskeletal filaments, especially in
actin filaments and microtubules. It occurs when one end of a filament grows in length
while the other end shrinks resulting in a section of filament "moving"
 if the concentration is above Cc for the (+) end but below Cc of the (-) end, filaments
will undergo an assembly at the (+) end and disassembly at the (-) end
 despite the constant assembly and disassembly the length of the filament remains
constant – this is called treadmilling
Actin architecture and function is governed by actin-binding proteins
Actin dynamics – regulation of the actin network (I)
1) How does the cell maintain a pool of unpolymerized actin?
Regulation of monomeric actin concentration
(Profilin, Thymosin)
Profilin prevents nucleation but
promotes + end elongation
Thymosin prevents polymerization
Actin dynamics – regulation of the actin network (II)
2) How does the cell control actin polymerization?
Actin nucleation by the Arp2/3 complex
The activated Arp2/3 complex bypasses the rate-limiting step of actin nucleation
Actin dynamics – regulation of the actin network (III)
3) How does the cell control actin polymerization?
Actin elongation by formins
Actin dynamics – regulation of the actin network (IV)
4) How can actin networks be stabilized?
Capping, crosslinking
actin-crosslinking proteins are characterized by
actin-binding domains
Actin dynamics – regulation of the actin network (V)
5) How can F-actin be disassembled?
Cofilin
Binding to F-actin induces a twist in the filament, which increases the chance
of severing
Actin dynamics – regulation of the actin network
Actin network is spatio-temporally regulated (Rho-GTPases)
Actin – questions
How does the cell maintain a pool of unpolymerized actin?
How are the actin networks stabilized?
How can f-actin fibers quickly be removed?
How can cell signalling modulate the actin cytoskeleton?
What step of the actin polymerization process is the slowest?
What end of the actin polymer is more likely to grow and which one will
shrink?
What is treadmilling? Why does it occur?
Which are the main functions of the actin cytoskeleton?
Tubulin protein and tubulin network

α-tubulin and β-tubulin form
a heterodimer

both subunits have a GTPbinding site

β-tubulin can be in a GDP-or
GTP-bound form

Tubulin heterodimers
form protofilaments,
which have a polarity

Microtubules are very
stiff due to multiple
molecular interactions
between subunits
Assembly of microtubules: three steps
if the rate of subunit addition is faster than hydrolysis, a GTP-cap is formed
(true also for F-actin → ATP-cap)
Tubulin polymerization and treadmilling

Subunits with bound nucleoside triphosphate (T) polymerize at both ends and then undergo
hydrolysis in the filament (D)

As the filament grows, elongation is faster than hydrolysis at the (+) end

However, hydrolysis is faster than elongation at the (-) end

The critical concentration for polymerization on the (+) end in T form is lower than for the (-) end in
the D form. If the subunit concentration is between these two values, the (+) end grows while the (-)
end shrinks, resulting in treadmilling

If the GTP-cap is lost, a filament may start to shrink (catastrophe) / (rescue)
Tubulin catastrophe and rescue: dynamic instability
Microtubules stability depends on the levels od GTP/GDP
Tubulin dynamics – regulation of the tubulin network (I)
1) How does the cell prevent spontaneous tubulin polymerization?
Stathmin
stathmin-bound tubulin heterodimers can not polymerize
Tubulin dynamics – regulation of the actin network (II)
2) Microtubule nucleation
γ- Tubulin/centrosome
Polar
Filament
Tubulin dynamics – regulation of the tubulin network (III)
3) How do cells stabilize microtubules?
Microtubules- associated protein (MAPs)
DIRECT BINDING
or τ proteins, after the
Greek letter by that name
Tubulin dynamics – regulation of the tubulin network (IV)
4) How do cells disassemble microtubules?
Katanin, some kinesins
Break bonds between tubulin heterodimers
Tubulin network is spatio-temporally regulated (as for actin network !!!)
Microtubules (MT) biological functions
Cell division
Intracellular transport
Tubulin – questions
How does the cell prevent tubulin polymerization?
How is microtubule nucleation regulated?
How do cells stabilize microtubules?
How do cells break or disassemble microtubules?
Why are microtubules so important?
How is the structure of actin and tubulin different?
What causes catastrophe?
Where does the nucleation of microtubules occur?
How are intermediate filaments different from actin and tubulin?
Intermediate filaments
(Diameter)
MT (25nm)
Int. Filaments
(10 nm)
F-actin (7 nm)
Intermediate filaments
Intermediate filaments have a diameter of about 10 nm, which is intermediate
between the diameters of the two other principal elements of the cytoskeleton,
actin filaments (about 7 nm) and microtubules (about 25 nm). In contrast to actin
filaments and microtubules, the intermediate filaments are not directly involved in
cell movements. Instead, they appear to play basically a structural role by
providing mechanical strength to cells and tissues. They do not have a polarity and
they do not bind nucleotides
Cytoskeletal motor proteins
Motor proteins bind to a polarized cytoskeletal filament and use the energy
derived from repeated cycles of ATP hydrolysis to move steadily along it.
Dozens of different motor proteins coexist in every eukaryotic cell. They differ in
the type of filament they bind to (either actin or microtubules), the direction in
which they move along the filament, and the “cargo” they carry. Many motor
proteins carry membrane-enclosed organelles such as mitochondria, or
secretory vesicles to their appropriate locations in the cell. Other motor proteins
cause cytoskeletal filaments to slide against each other, generating the force
that drives such phenomena as muscle contraction, ciliary beating, and cell
division
The three cytoskeletal motor protein families
ACTIN
MICROTUBULE
The cycle of myosin-II
Structural changes are used by myosin to walk along an actin filament → ATP
hydrolysis induces a conformational change
Conformational
change
The cycle of kinesin -1
cargo binding
domain
stalk
motor domain
Exchange of ADP to ATP in the leading
head (light green) causes a structural
change in the neck region; detachment
of the lagging head (dark green) is
facilitated by release of Pi
most kinesins move toward the
microtubule (+) end but some kinesins can
move toward the (-) end
Comparison of the cycles of kinesin and myosin II
The cycle of dynein
motor
domain
associated
peptides
microtubule
binding domain
dyneins moves toward
the microtubule (-) end
The cycle of dynein
Cytoplasmic dynein is a multi-protein motor complex responsible for all minus-end
directed microtubule transport in most eukaryotic cells. Dynein carries out a large
number of functions in the cell and is subject to complicated regulatory control by
accessory factors. In relation to the other microtubule motor family, the kinesins,
dynein is much larger and complicated. Despite being identified 20 years earlier
than kinesin, dynein’s complexity has made it relatively intractable to studies of its
mechanism of movement.
Dynein functions
All of the myosins except one move toward the plus end of an actin filament, although they
do so at different speeds. The exception is myosin VI, which moves toward the minus end.
Most of kinesins have the motor domain at the N-terminus of the heavy chain and walk
toward the plus end of the microtubule. A particularly interesting family has the motor
domain at the C-terminus and walks in the opposite direction, toward the minus end of
the microtubule.
The dyneins are a family of minus-end-directed microtubule motors, but they are
unrelated to the kinesin superfamily.
Motor proteins – questions
Some one is diagnosed with Alzheimer…..which cytoskeletal component
could be disrupted in the neurons?
What are cytoskeletal motors?
What are some of the roles of myosin?
How is myosin activity regulated?
How are the structures of kinesin and myosin similar and different?
Why do we need vesicle transport?
What is the main role of dyneins?
Which motor proteins mostly move things in the + directions which in the direction?
The cell cycle and its regulation
S-phase = DNA synthesis
G2 phase = gap S/M
G1 phase = gap M/S
M-phase = mitosis and cytokinesis
The cell cycle and its regulation
Is the length of the cell cycle phases the same between
different organisms?
… but there is always an
alternation of S and M
phases !!!
How is the progression of the cell cycle regulated?
Cyclin dependent kinases (CDKs)






Small family of serine-threonine kinases
Highly conserved
Cyclin-binding is ESSENTIAL for their activity
Highly regulated
Can phosphorylate hundreds of distinct proteins
Are constant throughout the cell cycle
CDKs are the MASTER REGULATORS of the cell cycle
The term “master regulator” or “master regulatory gene” was
first coined by Susumu Ohno over 30 years ago for a “gene
that occupies the very top of a regulatory hierarchy,” which, by
its definition, should not be under the regulatory influence of
any other gene.
How is the progression of the cell cycle regulated?
Cyclin dependent kinases (CDKs)






Small family of serine-threonine kinases
Highly conserved
Cyclin-binding is ESSENTIAL for their activity
Highly regulated
Can phosphorylate hundreds of distinct proteins
Are constant throughout the cell cycle
CDKs are the MASTER REGULATORS of the cell cycle
Cyclins
 A diverse family of proteins
 All contain conserved cyclin box
(≈ 100 amino acids Cdk interaction site)
 Cyclins are oscillating – different types are produced at different
cell-cycle stages
 All cyclins associate with CDKs
 Cyclins affect the substrate specificity of CDKs and their localization
 (BUT Not all cyclins are cycling during the cell cycle)
 (AND Not all cyclins act in cell cycle (e.g. in transcription))
Experiments in model organisms for cell cycle analysis
Which model organism have been used in order to
study the cell cycle?
Budding and Fission Yeast
Simple system
Early embryos – synchronized cells
Mammalian cell lines – similarity with humans
Experiments in model organisms for cell cycle analysis
Which are the two main features of the cell cycle?
The cell cycle is DIRECTIONAL and ORDERED
Timer theory
From embryos experiments
Domino theory
From yeast experiments
Experiments in model organisms for cell cycle analysis
Yeast cell cycle mutants (easy model)
Forward genetic screen for temperature
sensitive mutants that arrest in a specific
cell cycle phase
The morphology is an indication
of the specific cell cycle phase
Forward genetics (or a forward genetic screen) is an approach used to identify genes
responsible for a particular phenotype of an organism. Reverse genetics (or a reverse
genetic screen), on the other hand, analyzes the phenotype of an organism following the
disruption of a known gene. In short, forward genetics starts with a phenotype and moves
towards identifying the gene(s) responsible, where as reverse genetics starts with a known
gene and assays the effect of its disruption by analyzing the resultant phenotypes. Both
forward and reverse genetic screens aim to determine gene function.
Experiments in model organisms for cell cycle analysis
Yeast cell cycle mutants (easy model)
Forward genetic screen for temperature
sensitive mutants that arrest in a specific
cell cycle phase
WT
Arrested
The morphology is an indication
of the specific cell cycle phase
Among the different mutants, cdc28 was very
interesting because caused an arrest in G1
Which protein is the yeast cdc28 ???
Experiments in model organisms for cell cycle analysis
Yeast cell cycle mutants – discovery of CDK
Cdc28 = Cdk of budding yeast
Cdc2 = Cdk of fission yeast
→ Reduced activity of Cdc2
(cycle slower, cell bigger)
→ Increased activity of Cdc2
(cycle faster, cell smaller)
Oocytes and embryos – discovery of cyclins
Experiments in model organisms for cell cycle analysis
The different cyclins confer the specificity of the cell cycle
No cyclins
No cyclins
Principles of cell cycle regulation
and Checkpoints
Intra S checkpoint
G0
At the end of mitosis, all cyclins are degraded and there is NO ACTIVITY of CDKs
G1 cyclin(s) must appear to drive the G1>S transition
Principles of cell cycle regulation
 The regulation of the cell cycle is conserved among eukaryotes
 The cell cycle contains checkpoints (= control mechanisms which ensure
proper division of the cell. Each checkpoint serves as a potential halting point
along the cell cycle, during which the conditions of the cell are assessed, with
progression through the various phases of the cell cycle occurring when
favorable conditions are met)
 The cell cycle is directional (= each phase depends on the previous one) and
ordered (= no phase is ever left out and occurs in a temporal order but its
length differs between organisms)
G1 > S transition (STARTing the cell cycle)
G1: external stimuli
Can the cell divide?
Synthesis of new G1 cyclin(s)
(activation of Cdk/G1 cyclin complex)
1
Restriction point
(no way back!)
Phosphorylation of targets
(transcription factors)
2
Transcription of G1- cyclin(s)
and
3
Transcription of S-phase cyclin(s)
Transition G1 > S
Main components
Only S-phase cyclins promote
DNA replication
G1 > S transition (STARTing the cell cycle)
G1: external stimuli
Can the cell divide?
Synthesis of new G1 cyclin(s)
(activation of Cdk/G1 cyclin complex)
1
Phosphorylation of targets
(transcription factors)
2
Sic1
Transcription of G1- cyclin(s)
and
3
Transcription of S-phase cyclin(s)
Transition G1 > S
What is Sic1?
Sic1 is an inhibitor of Cyclin Dependent Kinases (CKI). It blocks the activity of Cdk in the
presence of S-phase cyclins (It binds to S-Cdk but not G1-Cdk)
CKI has to be removed before S-phase !!!
Multisite phosphorylation of Sic1
S-Cdk
complex
inactive
G1-Cdk
complex
S-Cdk complex
active
Multisite phosphorylation of Sic1
 Multiple phosphorylation of Sic1 (from G1/S-Cdk complexes) is required
for its efficient degradation
 Cooperativity
 Creation of a switch -> proper length of G1 ensured
Multisite phosphorylation of Sic1
feedback
Summary G1
 There is nearly no Cdk activity in G1 phase because all cyclins get
degraded at the end of mitosis
 New cyclins need to be synthesized in order to start the next cell cycle
 The G1 cyclins are synthesized in response to EXTERNAL factors
 G1 cyclins are resistant to the degradation
 G1 cyclins/CDKs trigger transcription of many genes required for
cellular proliferation – including S-phase cyclins
 Before S-phase can start, CKI (Cdk Kinase Inhibitors) needs to be
removed by degradation.
S phase – genome replication
Semiconservative replication describes the mechanism by which DNA is replicated in all known cells.
This mechanism of replication was one of three models originally proposed for DNA replication:
- Semiconservative replication would produce two copies that each contained one of the original
strands and one new strand.
- Conservative replication would leave the two original template DNA strands together in a double helix
and would produce a copy composed of two new strands containing all of the new DNA base pairs.
- Dispersive replication would produce two copies of the DNA, both containing distinct regions of DNA
composed of either both original strands or both new strands.
S phase – genome replication
The majority of organisms use
multiple replication origins
PROBLEM
The different ORIs need to be
synchronized because the DNA must be
duplicated exactly once per cell cycle
How?
There are 2 steps
S phase – genome replication
1) Licensing = Process in which ORIs acquire competence to replicate through the loading
of inactive helicase precursors; occurs prior to S phase (in G1) through formation of
pre-replicative complexes (pre-RCs)
LOADING
2) Firing = Process in which the replication starts (in S phase)
ACTIVATION
S phase – genome replication
LOADING – CDK off
Phosphorylation (-)
ACTIVATION – CDK on
Phosphorylation (+)
S phase summary
 DNA replication is semi-conservative and initiates at many sites in
order to be fast
 Pre-RC can be established only from unphosphorylated proteins – end
of the M phase - G1
 For replication initiation, the pre-RC needs to be phosphorylated by
Cdk-S phase cyclins
 No pre-RC can be established when there is Cdk activity –
PREVENTION OF RE-REPLICATION
Checkpoints
1
2
3
4
Checkpoints – kinase cascade
Control mechanisms which ensure proper division of the cell. Each checkpoint serves as a
potential halting point along the cell cycle, during which the conditions of the cell are
assessed, with progression through the various phases of the cell cycle occurring when
favorable conditions are met.
Different Signals of DNA alteration
The expression of
sensor + effector
kinases are
sufficient to activate
the checkpoint,
even without DNA
damage
Local Sensor
kinases
Phosphorylation
Effector kinases
Effects
Checkpoints – kinase cascade
How does the checkpoint induce cell cycle arrest?
Inhibition of CDK (in mammals)
Inhibition of CDK targets (in yeast)
Different regulation of Cdk activity:



Activation by cyclins
Inhibition by CKI
Inhibitory and stimulatory phosphorylation
M phase and chromosome condensation/cohesion
First step: preparation (from prophase to metaphase)
Metaphase > Anaphase transition
Second step: real cell division (from anaphase to cytokinesis)
M phase and chromosome condensation/cohesion
Chromosomes need to condense!!!
Condensin
Sister
chromatid
Cohesin
1) Cohesin forms a ring that mediates sister chromatid cohesion (it keeps the 2
chromatids together). It starts to be deposited during the S phase
2) Condensin also forms a ring and helps chromosomes condensation. It has a
similar structure compared to cohesin
Metaphase > anaphase transition. Exit from mitosis
Exit from mitosis (between metaphase and anaphase) requires inactivation
of Cdk by degradation of mitotic cyclin
The degradation is mediated by the anaphase-promoting-complex (APC),
an E3 ubiquitin ligase
Metaphase > anaphase transition. Exit from mitosis
 APC activation requires both MPF activity (M-cyclin/Cdk) and the presence of the
Cdc20 activating subunit
 Cdk facilitates its own inactivation (mitotic checkpoint and unknown mechanisms
ensure delay to give Cdk enough time to act)
What keeps the replicated chromosomes together?
Sister chromatid cohesion (= protein complex that forms a ring around the
two chromatids)
1
2
Two different events drive the mitotic exit !!!
1)
2)
Active APC complex degrades securin with activation of separase (that cleaves cohesin)
Active APC complex also induces the degradation of cyclin with subsequent inactivation of Cdk
Transition from M phase to the next G1
Problem:
Cdk-Mcyclin phosphorylates/activates the APC complex
(only phosphorylated APC/Cdc20 is functional)
APC/Cdc20 complex degrades cyclin > decrease in Cdk activity
Less Cdk activity induces a reduction in the phosphorylation of the APC complex
Cyclin concentration starts to increase again > the cell cannot move out of
mitosis to reach a new G1 phase
Metaphase > anaphase transition. Exit from mitosis
Solution (to keep the level of M-cyclin low):
The APC complex has to stay active after mitosis
There is a second APC activator: Cdh1 (green line in figure below)
APC/Cdh1 is inhibited by MPF (= Cyclin M/Cdk complex) and becomes active
only upon drop of MPF activity → M-cyclins degradation → exit from mitosis
Cdh1-
Two APC complexes