Chapter 17 – The cell cycle; Molecular Biology of THE CELL

Mitosis is the process of nuclear division. Cytokinesis is the process of cell division. Both happen in
the M phase. The interphase is much longer, this includes S phase and interphases. At the transition
from metaphase to anaphase an abrupt change in the biochemical state of the cell occurs. After it
passes this point the cell carries on to the end of cytokinesis into the interphase.

Chromosomes are duplicated in the S phase while most other cell components are duplicated
continuously throughout the cycle.

If extracellular conditions are favourable and signals to grow and

divide are present, cells in early G1 or G0 progress through a
commitment point near the end of G1 known as Start.

The cell-cycle control system triggers the major

events of the cell cycle. It depends on cyclically
activated cyclin-dependent protein kinases,
Cdks. When cyclin forms a complex with Cdk,
the protein kinase is activated to trigger specific
cell-cycle events. Without cyclin, Cdk is inactive.

The concentrations of the three

major cyclin types oscillate during
the cell cycle, while the
concentrations of Cdks do not
change and exceed cyclin
amounts.
A separate regulatory protein
complex, the APC/C, initiates the
metaphase-to-anaphase
transition.
There are three major classes of cyclins, each defined by the stage of the cell cycle at which they bind
Cdks and function.
- G1/S-cyclins activate Cdks in late G1 and thereby help trigger progression through Start,
resulting in a commitment to cell-cycle entry. Their levels drop in S phase.
- S-cyclins bind Cdks soon after progression through Start and help stimulate chromosome
duplication. S-cyclin levels remain elevated until mitosis, and these cyclins also contribute to
the control of some early mitotic events.
- M-cylins activate Cdks that stimulate entry into mitosis at the G2/M transition. M-cyclin
levels drop in mid-mitosis.
 In the inactive state, without cyclin bound, the
active site is blocked by a region of the protein
called the T-loop. The binding of cyclin causes
the T-loop to move out of the active site,
resulting in partial activation of the Cdk.
Phosphorylation of Cdk by Cdk-activating kinase,
CAK, at a threonine residue in the T-loop further
activates the enzyme by changing the shape of
the T-loop, improving the ability of the enzyme to bind its protein substrates.

The Cdks activity is regulated by phosphorylation. The

active cyclin-Cdk complex is turned off when the kinase
Wee1 phosphorylates two closely spaced sites above the
active site. Removal of these phosphates by the
phosphatase Cdc25 activates the cyclin-Cdks complex. Cdk
activity can be suppressed by inhibitory phosphorylation
and Cdk inhibitor proteins, CKIs.

The p27 binds to both the cyclin and Cdk in the complex
distorting the active site of the Cdk. It also inserts into the
ATP-binding site, further inhibiting the enzyme activity.

Regulated proteolysis triggers the metaphase-

to-anaphase transition. APC/C, anaphase
promoting complex, or cyclosome is the key
regulator of the metaphase-to-anaphase
transition. It is a ubiquitin ligase family of
enzymes.

The APC/C is activated in mitosis by association

with Cdc20, which recognizes specific amino
acid sequences on M-cyclin and other target
proteins. With the help of two additional
proteins called E1 and E2, the APC/C assembles
polyubiquitin chains on the target protein. The
polyubiquitylated target is then recognized and
degraded in a proteasome.

The activity of the ubiquitin ligase SCF depends

on substrate-binding subunit called F-box
proteins, of which there are many different
types. The phosphorylation of a target protein,
such as the CKI shown, allows the target to be
recognized by specific F-box subunit.
Preparations for DNA replication begin in late mitosis
and G1, when DNA helicases are loaded by multiple
proteins at the replication origin, forming the
prereplicative complex (preRC).
S-Cdk activation leads to activation of the DNA
helicases to initiate DNA replication. After duplication of the chromosomes, they are segregated in M
phase.
S-Cdk activation in S phase also prevents assembly of new prereplicative complexes at any origin
until the following G1 – thereby ensuring that each origin is activated only once in each cell cycle.

A key player in the initiation of DNA replication is a large

multiprotein complex called origin of recognition complex,
ORC.
The replication origin is bound by the ORC throughout the
entire cell cycle.
In early G1, Cdc6 associates with the ORC, and these proteins
bind the DNA helicase, which contains six closely related
subunits called Mcm proteins. The helicase also associates
with a protein called Cdt1.
Using energy provided by ATP hydrolysis, the ORC and Cdc6
proteins load two copies of the DNA helicase, in an inactive
form, around the DNA next to the origin, thereby forming the
preRC.
At the onset of S-phase, S-Cdks stimulates the assembly of
several initiator proteins on each DNA helicase. While another
protein kinase, DDK, phosphorylates subunits of the DNA
helicase. As a result, the DNA helicases are activated and DNA
replication beings.
S-Cdks and other mechanisms also inactivate the preRC
components ORC, Cdc6 and Cdt1, thereby preventing
formation of new preRCs at origins until the end of mitosis.

M-Cdk drives the entry into mitosis.

Dephosphorylation activates M-Cdk
at the onset of mitosis.
Cdk associates with M-cyclin as the
levels of M-cyclin gradually rise.
The kinases CAK and Wee1
phosphorylate several sites of the
inactive M-Cdk. At the end of G2,
the M-Cdk complex is activated by the phosphatase Cdc25. Cdc25 is further stimulated by positive
feedback. And Wee1 is inhibited by positive feedback.
Principle stages of M phase (mitosis and cytokinesis)
 Cohesins hold sister
chromatids together. 

Condensin helps configure

duplicated chromosomes for
separation. 

The mitotic spindle is a

microtubule-based
machine. The plus ends of the microtubules
project away from the spindle poles, which in this
example are organized by centrosomes. One pair
of centrioles is located in each centrosome.
Kinetochore microtubules connect the spindle
poles with the kinetochores of sister chromatids,
while interpolar microtubules from the two poles
interact with each other. Astral microtubules
radiate out from the poles into the cytoplasm.

Microtubule-dependent motor
proteins govern spindle assembly
and function.
Kinesine related proteins usually
move towards the plus end of
microtubules.
Dyneins usually move toward the
minus end.

Centrosome duplication occurs early in the cell cycle. The

centrosome consists of a centriole pair and associated
pericentriolar matrix. At a certain point in G1, the two centrioles
of the pair separate. During S phase, a daughter centriole begins
to grow near the base of each mother centriole and at a right
angle to it. The elongation of the daughter centriole is completed in G2. The two centriole pairs
remain close together in a single centrosomal complex until the beginning of M phase, then the
complex splits and the two daughter centrosomes begin to separate.
In late prophase the mitotic spindle poles have moved to opposite sides of the nuclear envelope.
After the nuclear envelope breakdown, the sister-chromatids pairs are exposed to the large number
of dynamic plus ends of microtubules. In most cases, the kinetochores are first attached to the sides
of these microtubules, while at the same time the arms of the chromosomes are pushed outward
from the spindle interior, preventing the arms from blocking microtubule access to the kinetochores.
Eventually the laterally-attached sister chromatids are arranged in a ring around the outside of the
spindle. Most microtubules are concentrated in this ring, so that the spindle is relatively hollow
inside. When the kinetochores are captured in an end-on orientation with the microtubules,
additional microtubules are attached to the kinetochores.
Alternative forms of kinetochores attachment to
the spindle poles can lead to unstable
configurations. When microtubule from the same
pole attach to both kinetochores the result is
unstable. When microtubule from different poles
attach to the same kinetochores the result is
unstable. When the microtubules are oriented in
an unstable way, one of the microtubules tends
to dissociate. Only when microtubule from
different poles attach to different kinetochores it
results in stable binding which will increase the
affinity.

The APC/C triggers sister-chromatid

separation and the completion of
mitosis.
The activation of APC/C by Cdc20
leads to the ubiquitylation and
destruction of securing, which
normally holds seperase in an
inactive state.
The destruction of securing allows
seperase to cleave Scc1, a subunit of
the cohesion complex holding the
sister chromatids together.
The pulling forces if the mitotic
spindle then pull the sister
chromatids apart.
Phosphorylation by Cdks also inhibits
seperase. Thus, Cdk inactivation in anaphase, resulting from cyclin destruction, also promotes
separase activation by allowing its dephosphorylation.
The spindle assembly checkpoint is when unattached chromosomes block sister-chromatid
separation. This system depends on a sensor mechanism that monitors the strength of icrotubule
attachment. Any kinetochores that is not properly attached to the spindle send out a diffusible
negative signal that blocks Cdc20 and APC/C activation throughout the cell and thus blocks the
metaphase-to-anaphase transition.
Mad2 and other proteins are recruited to unattached kinetochores. The unattached kinetochores act
likes an enzyme that catalyzes a conformational change in Mad2 which can bind and inhibit Cdc20
and APC/C.

In anaphase A, the separated

sister chromatids move toward
the poles.
In anaphase B, the two spindle
poles move apart.

M-Cdk triggers the events of early mitosis, including chromosome condensation, assembly of the
mitotic spindle, and bipolar attachment of the sister-chromatid pairs to microtubules of the spindle.
Spindle formation in animal cells depends largely on the ability of mitotic chromosomes to stimulate
local microtubule nucleation and stability, as well as on the ability of motor proteins to organize
microtubules into a bipolar array.
Many cells also use centrosomes to facilitate spindle assembly.
Anaphase is triggered by the APC/C, which stimulates the destruction of the proteins that hold the
sister chromatids together. APC/C also promotes cyclin destruction and thus the inactivation of M-
Cdk. The resulting dephosphorylation of Cdk targets is required for the events that complete mitosis,
including the disassembly of the spindle and the re-formation of the nuclear envelope.

Cytokinesis is the division of the cytoplasm

in two.
The actin-myosin bundles of the contractile
ring are oriented as shown, so that their
contraction pulls the membrane inward.

The contractile ring is regulated by the

GTPase RhoA. RhoA is activated by a
RhoGEF protein and inactivated by a Rho
GTPase-activating protein, RhoGAP. The
active GTP-bound form of RoA is focused at
the future cleavage site. By binding formins,
activated RhoA promotes the assembly of actin filaments in the
contractile ring. By activating Rho-activated protein kinases, such as
Rock, it stimulates myosin II filament formation and activity, thereby
promoting contraction of the ring.
 Meiosis is the sexual reproduction of cells. It produces
haploid cells carrying only a single copy of each
chromosome.
There are two rounds of chromosome segregation in
meiosis.
The structure formed by two
closely aligned duplicated
homologs is called a bivalent. The
centromere is where the two sister
chromatids are fixed together.
Chiasma is where two
chromosomes cross-over.

Mitogens bind to cell-surface receptors to initiate

intracellular signaling pathways. This results in the
expression of the transcription regulatory protein Myc. Myc
increases the expression of many delayed response genes,
including some that lead to increased G1-Cdk activity. This
triggers the phosphorylation of members of the Rb family
of proteins, which inactivates these proteins. This results in
the freeing of the gene regulatory protein E2F to activate
the transcription of G1/S genes, including the genes for
G1/S-cyclin and S-cyclin. The resulting complexes with Cdks
form a positive feedback loop by further phosphoylating Rb
protein. E2F itself stimulates the transcription of their own
genes, another positive feedback loop.

DNA damage blocks cell division and arrests the

cell cycle in G1. When DNA is damages, various
protein kinases are recruited to the site of
damage and initiate a signaling pathway.
The first kinase is either ATM or ATR. Additional
protein kinases, Chk1 and Chk2, are then
recruited and activated. This results in the
phosphorylation of the transcription regulatory
protein p53. Mdm2 normally binds to p53 and
promotes its ubiquitylation and destruction.
Phosphorylation of p53 blocks its binding to
Mdm2. As a result, p53 accumulates to high
levels and stimulates transcription on several
genes, including the gene that encodes the CKI
protein p21.This protein binds and inactivates
G1/S-Cdk and S-Cdk complexes, arresting the
cell in G1.
 Cell-cycle arrest or apoptosis is induced by excessive
stimulation of mitogenic pathways. Abnormally high
levels of Myc cause the activation of Arf, which binds
and inhibits Mdm2 and thereby increasing p53 levels.
This either causes apoptosis or cell-cycle arrest.

The occupation of cell-surface receptors by growth

factors leads to the activation of PI 3-kinase, which
promotes protein synthesis through a complex
signaling pathway that leads to the activation of the
protein kinase TOR. Extracellular nutrients also help
activate TOR. TOR phosphorylates multiple proteins to
stimulate protein synthesis, it also inhibits protein
degradation