 DNA molecule not only carries genetic information, but also

undergoes conformational change

 Chromosomes exist through the cycle as Mitotic and interphase

chromosome

 Single chromosome can only be visible during mitosis

The Basis
of Mitosis
The Cell Cycle
 Coordination of cell division
A multicellular organism needs
to coordinate cell division
across different tissues &
organs
critical for normal growth,
 development & maintenance

 coordinate timing of

 cell division
 coordinate rates of cell
division
 not all cells can have the
same cell cycle

AP Biology
 CONTROL OF THE CELL CYCLE
If it wasn't controlled, your cells would continue to grow
and divide...over and over again!

A number of proteins regulate and control the cell cycle.

Tell the cell when is the proper time to grow and divide,

Stop the cell when the time's not right.

Clinically, cancer can be described as uncontrolled cell growth

and proliferation

Understanding cell cycle control has many implications for

cancer, especially for the development of therapeutics.
CELL CYCLE CONTROL CAN BE DISSECTED GENETICALLY BY ANALYSIS
OF YEAST MUTANTS
• Cell cycle control remarkably similar to humans
• Reproduce rapidly as bacteria
• Genome size < 1% of mammals
• Rapid molecular genetic manipulation
• Ability to proliferate in haploid state - easy to study mutation
• Cdc (cell-division cycle genes)
 CELL CYCLE REGULATION IN FISSION YEAST
Start promoting complex (SPF):
S form of P34cdc2 + cig1,cig2
(Cdk) (G1 cyclin)

MPF:
M form of P34cdc2 + cdc13
(Cdk) (M cyclin)

l
Inactive cyclin-Cdk complex
l
phosphorylation of tyr15 of p34cdc2
l
Activation of Cdk
l
Phosphorylation of Thr161
residue of Cdk by CaK
l
Dephosphorylation of Tyr
residue by phosphatase
CELL CYCLE REGULATION IN BUDDING YEAST
Chromosome cycle,
l

centrosome cycle, cytoplasmic

cycle

Cdk cdc28 interract with

l

different cyclins:
l
G1 – Cln1 and
Cln2 –
phosphorylates
APC to inactivate it
l
- Cln3
phosphorylates and
activates SPF and
MPF
l
S phase – Clb5
and Clb2 induce
chromosome
seggregation and
nuclear division
 BIOCHEMICAL ANALYSIS OF CELL CYCLE IN ANIMAL
EMBRYOS

• Xenopus egg contain 100,000 times more cytoplasm than human cell
• Cleavage division - rapid division of fertilised eggs
• Early embryonic cycle - simplified
CELL CYCLE CONTROL STUDIES IN CULTURED
MAMMALIAN CELLS

• Mutated to proliferate indefinitely in culture - “immortalised cell

lines”

• Unlimited source of genetically homogeneous cells

• Cytological study & biochemical analysis of proteins

• Examining molecular mechanisms in multicellular organisms

• Cancer biology
 Checkpoint control system

Checkpoints
cell cycle controlled by STOP & GO
chemical signals at critical points
signals indicate if key cellular
processes have been
completed correctly

AP Biology
G1/S cell cycle checkpoint
controls the passage of eukaryotic cells from the first 'gap' phase
(G1) into the DNA synthesis phase (S).
Checks:
That the size is CORRECT
That the environment is CORRECT

Major proteins involved:

Cyclins (proteins) - level fluctuate in the cell cycle
Cyclin dependent KINASES* (Cdks)

*Kinases – add a phosphate group (phosphorylate)

They add phosphate groups to proteins that control processes in
the cell cycle.
They only do this when the cyclins are present.
 G2/M DNA damage checkpoint
Prevents the cell from entering mitosis (M phase) if the
genome is damaged.

Checks if the cell is big enough (i.e. has the resources to

undergo mitosis)

Almost exclusively, internally controlled

M checkpoint
The M checkpoint is where the attachment of the spindle fibres
to the centromeres is assessed.

Only if this is correct can mitosis proceed.

Failure to attach spindle fibres correctly would lead to failure to

separate chromosomes
Regulation of the Cell Cycle: Cell Cycle Checkpoints

Differentiating cells
 Cell cycle signals
Cell cycle controls
 cyclins
 regulatory proteins
 levels cycle in the cell
 Cdk’s
 cyclin-dependent kinases
 phosphorylates cellular proteins
 activates or inactivates proteins
 Cdk-cyclin complex
 triggers passage through different stages of
cell cycle

AP Biology
Synthesis and Degradation of Cyclin correlates with MPF Activity
 Spindle checkpoint
G2 / M checkpoint
Chromosomes attached
l Replication at metaphase plate
completed
l DNA integrity Inactive
Active Active
Inactive
Cdk / G2 M APC cytokinesis
cyclin (MPF) C
G2 mitosis

G1

S
Cdk / G1
cyclin
Inactive
MPF = Mitosis Active
Promoting Factor G1 / S checkpoint l Growth factors
APC = Anaphase l Nutritional state of cell
AP BiologyComplex
Promoting l Size of cell
 Major players

l Cyclins , cdk’s (cyclin – dependant kinases)

l MPF (Maturation Promoting Factor) = cyclin B- cdc2
(yeast) or cdk1 kinase (“mitotic cdk”)
l CAK (Cdk Activating Kinases) regulators of cdks
l CKI – cdk inhibitory subunits
l APC (Anaphase Promoting Complex) and cdc34/SCF
ubiquitination pathways which activate proteolysis via
proteosomes.
SUPPRESSION OF Cdk ACTIVITY
CYCLICAL PROTEOLYSIS CONTROLS CELL CYCLE
 Functions of MPF (cyclin b-cdc2/cdk1)
lReversible breakdown of the NE & role of nuclear lamin proteins
via DNA transfection experiments.

lGenetic studies on S.cerevisiae identified a family of proteins

that are required for normal chromosome segregation and are
termed SMC ( Structural Maintenance Complex) proteins. They
are found in organisms up to humans and are typically called
“condensins”.

lPhosphorylation of condensins by MPF or another protein kinase

regulated by MPF is necessary for maintaining the condensed
state of the chromosomes via DNA binding.

lPhosphorylation of APC results in initiation of anaphase.

 Cohesin

Condensin
Activation of M-CdK
 Polyubiquitination of Mitotic Cyclins
lRegulated degradation of mitotic cyclins occurs in late anaphase via
the APC-ubiquitin pathway which makes use of a “destruction box”
sequence common to the N-T of mitotic cyclins.
lThis ubiquitin pathway involves E1 (ubiquitin activating enzyme) , E2
(ubiquitin conjugating enzyme) & E3 (ubiquitin ligase or APC).
l Mutant genes that code for non-degradable cyclins have been
produced by site directed mutagenic deletion of the destruction box.
OVERVIEW OF CELL CYCLE CONTROL SYSTEM
CONTROL OF CHROMOSOME DUPLICATION
 G1
checkpoint
lControlled by G1 Cdk-cyclin
lG1 cyclin levels also vary with the cell cycle
lMany additional levels of phosphorylation,dephosphorylation
lDamaged DNA stimulates transcription of many genes which
encodes protein that bind to S-Cdk, inhibits their activity &
blocks entry into mitosis
l In mammals p53 gene delay due to damaged DNA by
blocking Cdks
Control of Cell
cycle entry and S-
phase initiation in
animal cells
G1 to S phase transition
Control of initiation of DNA
replication
 G1
checkpoint
 G2 Checkpoint Control by MPF
lActive MPF = Mitotic Cdk + mitotic cyclin
lCdk is cyclin-dependant kinase
lMPF controls G2  M by phosphorylating
and activating proteins involving in:
l Chromosome condensation
l Nuclear envelope breakdown
l Spindle assembly
l It’s own self-destruction
G2
checkpoint
 G2
checkpoint
l
DNA replication/ DNA damage checkpoint
l
Triggered by MPF

Promotes mitosis by phosphorylating a variety of other

l

protein kinases
l
In yeast delay caused by rad 9 gene

Damaged DNA sends signal of protein kinases that

l

blocks the dephosphorylation & activation of M-Cdk

blocking entry into mitosis
 Cell Cycle Regulatory Components

lS. pombe genetic studies especially complementation and

recombination studies of the cdc temperature sensitive mutants
led to the identification of cdc2 and cdc 13 as cdc2 kinase and
cyclin b.
lBoth of them are linked in a complex as MPF or cyclin b-cdc2
kinase complex.
lIt also identified other regulatory components such as wee 1,
cdc 25 and CAK (cdc2 activating enzyme).
lMultiple pathways are involved in regulating the mitotic-cdk
complexes which are cdc2 in yeast and cdk1 in mammals.
lAll these cell cycle regulatory proteins are typically well
conserved from yeast to humans.
 M checkpoint
Spindle attachment checkpoint
l

l
Signal originates at kinetochore – inhibits attachment of spindle –
inactive APC

MAD genes in mammals; BUB genes in yeast

l

Activation of M-
Cdk
 The Regulatory Aspects of DNA Replication
Genetic studies with S.cerevisiae have dissected the
regulatory aspects of DNA replication:
a)Control of S-phase by regulated proteolysis of sic 1:
i)Sic 1 is a CKI specific for the S-phase cdk-cyclin complex.
ii)This proteolysis is initiated by the G1 phase cdk-cyclin which
phosphorylates sic1 making it a target for cdc34 ubiquitin
conjugating enzyme and a trimeric ubiquitin ligase called SCF.
The Regulatory Aspects of DNA Replication contd…

(b) Regulation of pre

replication complexes:
cdk’s simultaneously
activate initiation of
replication and prevent re-
initiation at origin sites by
phosphorylation &
disassociation of McM’s
from the ORCs (origin P
recognition complex) which
are permanently associated P
with replication origins.
For the McM’s to
reassemble at the origins
they need to be
dephosphorylated which
does not occur until mitosis.
 Checkpoints in Cell Cycle Regulation

To ensure orderly passage of the cells through cell cycle there

are four defined “checkpoints” where certain events need to
occur before the cell will proceed in the cell cycle; otherwise the
cell will be “arrested” at that checkpoint: G1 arrest, S arrest, G2
arrest and M arrest.
There are specific checkpoint protein kinases (chks) that are
directly involved in arresting the cells.
 Role of E2F and Rb Protein in Regulation of RP Passage
l The E2F transcription factor regulates the cyclin A & D and cdk2 genes.

l Rb (retinoblastoma) protein inhibits E2F transcription factor.

l Rb is the protein product of the tumor supressor gene RB which if mutated

enhances the probability of retinoblastoma and other cancers.
lNormally this protein is not active until mid/late G1 phase when the buildup of
cdk2-cyclin E and cdk4/6-cyclin D leads to phosphorylation of Rb and release
from supression of the E2F transcription factor.
l After mitosis Rb is dephosphorylated due to the low concentrations of the G1
cdks and cyclins. Rb will undergo phosphorylation as the cyclin levels rise in
G1. If not, the cell cycle goes into Go (i.e., it does not pass RP).
Mid-G1 Late-G1

+ Cyclin A
Cdk2

release from RP
MPF and its role in regulation of cell cycle
Creation of G1 phase
Initiation of S phase
 Passage through G2 phase

 Accumulation of M cyclins and M-Cdk complex

 M-Cdk inactive due to phosphorylation of tyr by wee1 kinase

Late G2:
dephosphorylation of tyr by phosphatase activity of cdc25

Cak mediated phosphorylation of thr

Active M-Cdk
Entry into M phase
 Different Forms of Cdc2-Cyclin in the Stages of Cell Cycle

M phase:
•Cdc2/Cdc13 (Try-15 –p; Thr-161
+p)
•At late mitosis: Cdc13 is degraded

START:
•Cdc2/Cdc13 (Try-15 + p; Thr-161
+p)

S to M phase:
•Cdc2/Cdc13 is activated (Try-15 –
p; Thr-161 +p) by
dephosphorylation
The Cdc25 Phosphatase & Wee1 Kinase Control
Cdc2 Activity
Cdc25:
•Phosphatase
•Remove inhibitory phosphate
from Tyr-15 of Cdc2
Wee1:
•Kinase
•Phosphorylate Tyr-15 of
Cdc25

S. pombe
Cdc18 Controls S phase Rum1 Inactivates MPF

S. cerevisiae
DNA Damage Triggers a Checkpoint

Prevents a defect in genetic

information

Blocks the cell cycle progression

Damaged DNA is repaired

DNA damage
→ ATM kinase
→ phosphorylates Chk2
→ phosphorylates/ inactivate Cdc25
Control of Mitosis in Animal Cells Requires Phosphorylation
and Dephosphorylation of MPF

Cdc2:
•Thr-161 is phosphorylated by
Cdc2-activating kinase (CAK)
= Wee1
Cdc25:
•Phosphatase
•Remove inhibitory phosphate
from Tyr-15 of Cdc2
Several Cdk-Cyclin Complexes Are Active at G1 and S Phases

Cdk2, Cdk4, Cdk5:

•Thr-161 is phosphorylated by Cdc2-
activating kinase (CAK) = Wee1

D cyclins:
•Active at G0/G1 phase
•Controlled by synthesis/ degradation

E cyclins:
•Active at G1/S phase
•Controlled by synthesis/ degradation

A cyclins:
•Active at S phase and G2/M phases (only)
•Controlled by synthesis/ degradation
MPF REGULATION BY PHOSPHORYLATION AND DEPHOSPHORYLATION
MPF REGULATION BY PHOSPHORYLATION AND
DEPHOSPHORYLATION
MPF REGULATION BY PHOSPHORYLATION AND DEPHOSPHORYLATION
Which Protein Is Activated by Cdk-Cyclin Complex?

RB:
•Is a target of cdk-G1 cyclin (D)
complexes
•Is a tumor suppressor
RB-E2F → transcription inhibited
RB-p E2F → transcription
activated

Cdk-cyclin complex
phosphorylates RB
Target of CKI to Protein Degradation Controls G1/S
Progression

•A CKI in S. cerevisiae
•Binds to CDC28-CLB in G1 phase
•Sic1 is degraded when cells enter
S phase
•Sic1 is degraded by proteolysis
•SCF (Cdc53-Skp1-Cdc4) E3 ligase
confers the proteolysis of Sic1
•p27, p21, and cyclinE are also
substrates of SCF E3 ligase
Protein Degradation Also Regulates G2/M Progression

Cyclin A:
•Is degraded in metaphase

Cyclin B:
•Is degraded in anaphase

Pds1p:
•Is degraded in anaphase
•Ensures sister chromatids to
seperate
Anaphase Promoting Complex/
Cyclosome (APC/C) Regulates
Proteolysis at G2/M Phase

E3 ubiquitin ligase:
•Activated by phosphorylation

Degrade:
•B-type cyclin, Pds1p, and the
molecules unnecessary for mitosis

Nucleotide synthesis enzyme

DNA replication complex
subunit
The Proteolytic Activity of
APC/C Ensure the Proper
Segregation of Sister
Chromatids

Pds1p/Securin:
•Is degraded by APC/C

Esp1/Separin :
•Is activated by degradation of
securin
The APC/C Is Maintained at
Inactive State until All
Kinetochores Are Attached

Mad proteins:
•Inhibit the activity of APC/C
by binding to CDC20
Bub proteins :
•Inhibit the activity of APC/C
by binding to CDH1
Exit from Mitosis Is Controlled by
Cdc14

Cdc14:
•A phosphatase
dephosphorylates Cdh1 →
activate APC/C
•A phosphatase
dephosphorylates Sic1 →
inactivate mitotic cyclins
•In nucleolus at interphase
 Cell Structure Is
Reorganized at Mitosis
 Spindle Assembly &
Microtubule Nucleation
 Contractile ring

Regulation of contractile ring

Microtubules of anaphase spindle generates signals that influence the
positioning of the contractile ring
CYTOKINESIS IN PLANT CELL
CONTROL OF CELL DIVISION & CELL GROWTH

 Mitogens: stimulate cell division, primarily by triggering a wave of

G1/S-Cdk activity that relieves intracellular negative controls that
otherwise
block progress through the cell cycle.

Growth factors: which stimulate cell growth (an increase in cell mass)
by promoting the synthesis of proteins and other macromolecules and
by inhibiting their degradation.

 Survival factors: which promote cell survival by suppressing the form

of programmed cell death known as apoptosis.
Mechanisms controlling
cell-cycle entry and S-phase
initiation in animal cells
Arrest of the cell cycle in
G1 due to DNA damage
Cell-cycle arrest or apoptosis induced by excessive stimulation of mitogenic
pathways
Stimulation of cell growth
by extracellular growth
factors and nutrients
Growth Factor Signaling Through the Ras
Pathway  crossing of G1 checkpoint

Ras*, Raf*

MAPK
cascade

Activation of
nuclear TFs

Activation of
G1 Cdk cyclin
genes: G1  S