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The Cell Cycle
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In unicellular organisms, have only 1 cell = organism
Cell duplicates if extracellular environment allows it (nutrition, energy etc.)
ex: Yeast cell, on a grape, someone picks up the grape and mashes it up for wine  yeast now in
warm, glucose-filled grape juice  yeast will rapidly divide and make ethanol
o In yeast and bacteria cell cycle progresses rapidly (new cell every 20min)
Cell cycle is highly conserved: ALL cells go through it (same basic principle)
Once development is over, cell cycle is arrested and cells sit in G0 and do whatever they were
made to do (muscle cells contract moving muscles etc.)
o In our system there is not much cell cycle process going on, except for in blood vessels
or if cells were damaged and need to be replaced
Quiescence: G0 state; exit G1 dismantling their cell cycle machinery and make proteins involved
in their respective functions; if there is a problem with the cell, then gets back into G1 phase to
undergo cell cycle again
Under the microscope you can visualize mitosis and cytokinesis (genetic info is pulled into 2
pieces, cell is pinched off, new membranes surround forming 2 separate cells)
S phase: DNA replication/synthesis, longest phase (esp. in mammalian cells where the DNA is
1.5m long and decondensed into the cell)
Have a cut by a protease from the metaphase to the anaphase step allowing DNA to move to
either side of the cell
Gap phases: only present in certain cells (after fertilization want embryo to develop quickly so
skip G1 and G2 phases); they are where the cell can take a break (cell cycle is a program that
cannot be stopped) and sense whether everything is okay
G1: extracellular environment sensing (food, energy, environment), if all clear goes immed. to S
o Mutations in proteins that control this phase, cell no longer can sense whether it should
stop or can start cycle; involved in cancer
G2: DNA-damage sensing; once DNA is repaired, cell can continue and engage in mitosis
o G1 also control DNA-damage, because you do not want to go into S phase and replicate
damaged DNA
Damage control/checkpoints are absent in cancer cells
Unicellular Division: Fission & Budding
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Yeast, one cell, pinches off a daughter cell (a bud); daughter will get bigger and become a normal
cell – how is this controlled?
Fission yeast: has all the phases of
the cell cycle that we have; takes only
20 minutes and not well controlled
(doesn’t matter if the daughter has
mutations)
Genome is only 1% of mammalian
genome – has been sequenced
Is a haploid organism, works with
only 1 copy so can be manipulated
very easily (can have yeast strains
with everything knocked out)
Add a carcinogen to yeast culture which will mutate DNA  when proteins are mutated (AA
exchanged) they become temperature sensitive (can work at a low temp, but in high temp can no
longer work)  mutant proteins that are involved in cell cycle control, will no longer work (G1-S
transition will be arrested)
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Reintroduce yeast genome (wild type), cloning genome into a library of plasmids (small rings of
DNA); each plasmid contains 1 wild type gene of yeast  transfect library back into the yeast and
see which can recue the phenotype
o One of the genes responsible for this phenotype, when reintroduced into the yeast, can
pinch off and continue cytokinesis
Multicellular Division
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C. Elegans, Drosophilia, Xenopus, Frog, Zebrafish are studied
Another tool to study cell cycle: xenopus (frog oocyte); large, very specialized oogenesis
o Frog puts eggs into water (wants to make sure it doesn’t get eaten) SO oocyte grows to
a large egg (~1mm: 100,000-fold larger) without dividing, and the monster cell is then
ready for rapid cell division with just S and M phases (happens in hours)
Harvest 100 eggs + frog sperm + ATP put in test tube at a certain temperature  cell division (S
& M phases) every hour (biochemical process)
o Can manipulate: take away extract, separate components over columns, can purify
proteins or take some away and see if the process still works
o Drugs can inhibit parts of the process as well
If it does not progress properly  cancer (not a problem for unicellular organisms like yeast,
because they are in essense cancers)
Cell culture: petri dish + buffers + AA and glucose
Radiactive assay, during S phase gets incorporated into the DNA (3h thymidine); can then take
tissue slice and see black dots = cells in S phase (can detect proliferation)
o Monoclonal antibody linked to a flourescence molecule (shines pink) and all the cells in
S phase are pink
Flow cytometry (FACS): cytometry = cell measurement (can measure relative amount of DNA per
cell); take cells, add flourescent dye that binds DNA; put laser on cell making cells shine light, can
detect light (laser counts number of cells and measures the intensity of the flouresence staining
the DNA); then plot results
o If you have 1x DNA per cell  cell in is G1
o More than 1x DNA  S phase
o 2x DNA  G2/M phase
If you take away glucose, cells will arrest in G1 and there will be no cells in S, G2 or M until you
reprovide nutrients
Damage DNA, G1 will go up, the arrest in cell cycle
allows cells to repair damage
Irradiation/sunburn: cells die, all DNA is
fragmented (apoptotic peak: cells digest their DNA
and die)  accumulation of cells with less than 1
set of DNA
Cell Cycle Control System
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Checks that certain cycles are completed before it
goes to the next phase
Checkpoints: there is one at the end of G1
triggering DNA replication; after G2 to trigger
mitosis machinery; and at the exit of M phase to
trigger anaphase  cytokinesis completing cell
division
Tumor cells are missing these checkpoints allowing
damaged cells to enter S phase
o
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p53: often mutated gene in cancerous cells, cells with this mutation can no longer sense
DNA damage
Once organism is developed, they have G1 and G2; most organisms are not in the business of
cycling; 99% of cells are in G0 having exited M to function at their targetted location
Regulation Proteins
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Discovered from yeast (their cell cycle and proteins regulating it are the same)
CDKs: kinases that phosphorylate different proteins in the cell to allow them to move from phase
to phase; yeast has only 1
The kinase interacts with different cyclins (M, S, etc.)  the cyclins bring kinases to their
substrates
M-cyclin binding to Cdk  drives kinase to M phase substrates (is a localization protein)
S-Cdk are only available during S phase cannot be phosphorylated in other phases
Different cyclins bind to the same kinases bringing them to the correct substrate available during
that phase
Cyclins: when observed during cell cycle, are synthesized before their respective phase, binds
Cdk activating it to phosphorylate substrates; destroyed by proteolysis (by proteasome complex)
after functions
o Proteolysis leaves the cyclin in inactive form until another Cdk (like the next phase one)
comes in and repeats the cycle
G1 and G2 have inactive Cdks
Why is S-cyclin-Cdk complex active past S phase? Because you don’t want the cell to re-replicate
the DNA, when complex is on, it means you only get DNA replication once
How Do the Cdks get Activated?
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Solved by solving the crystal structure of proteins
ATP binds to the kinase active site  phosphate is transferred onto substrate
o Active site is embedded inside how to get at it?
Upon cyclin binding get rearrangement of ATP forming a T-loop exposing the active site  Cdkactivating kinase (CAK) transfers the phosphate  Fully active
Regulated by a kinase or phosphatase (Wee1) puts another phosphate on the Cdk (at another
area) inactivating it
Cdc25 removes this extra phosphate activating the kinase again  CONTROLLED REGULATION
Takes cell a while to make enough G1/S cyclin
Cyclins alone are not enough to activate the kinase
Inhibitory phosphorylation promoted by Wee1 kinase which can be taken out quickly by Cdc25
phosphatase
p27: a CKI inhibiting activity by blocking phosphorylation site; gets degraded constantly in cancer
cells making them no longer stop in G1
Control of Proteolysis
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Cell needs to get rid of cyclins to get ready for the next set for the next phase in the cycle
How to degrade proteins? Proteolysis
Proteasome: sits in cytosol, large complex that chews up proteins upon receiving the ubiquitin
signal (polyubiquitin chain)
Ubiquitin: super-conserved protein; causes an enzyme cascade attaching it to proteins that will
get degraded by proteasome
How to make sure not every protein
gets degraded? E1 (ubiquitin-activating
enzyme), E2 (conjugating enzyme takes active ubiquitin and transports it
to E3) and E3 (a ligase, binding ubiquitin
to substrate)
SCF: exists in yeast and mammalian
cells; important for cell cycle; is a ligase
complex of 3 subunits
o Has a substrate-recognition
protein (F-box proteins): steady, can be exchanged to bind to certain units based on
their specific AA sequence
o Substrates get phosphorylated regulating their activity
APC: anaphase-promoting complex; in an inactive state usually unless there in an activating
subunit bound to it (ex: Cdc20); once active it polyUb M-cyclin  degradation of cyclin
Different E3 Ub ligases do different things (why there are so many we don’t know)
Regulation of DNA Replication
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Cell gets into S phase, replicates DNA, and
ensures no repeat of replication will occur
 Idea: certain regions on DNA serve as start
points for replication to start (ORC: origin
recognition complex)
 ORCs are seen in DNA before it replicates (lots
of them) – each complex replicates a small
amount of DNA
Collects and assembles other proteins serving as an anchor
Initiated by synthesis of Cdc6: binds to ORC and with many proteins (Mcm proteins)  prereplicative complex
Pre-replicative complex: still inactive after assembly until S-Cdk cyclin phosphorylates it
Mcm proteins: all kinds of proteins; some are helicases are able to unwind DNA strands and
assembled upon Cdc binding
When DNA replication is complete, Mcm proteins come off, but ORC is still phosphorylated
because cell wants to prevent re-replication
When M phase is completed, origin of recognition complex gets dephosphorylated and can start
up again
Active M-Cdk triggers mitosis (fast process, 1hr)
Potentiates signal by phosphorylating more Cdc25; all you need is one active molecule to very
quickly make more active molecules through these two types of reactions
Checkpoints
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G2/M: important checkpoint, because if there is a problem with replication (incomplete) cell
should not go into mitosis and make 2 cells with damaged replicated
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Sensor makes a negative signal upon DNA damage detection preventing progression
past G2 (cell stops here) until DNA repaired
o Kinase inactivates Cdc25 by the negative sensor which inhibits M-Cdk until replication is
complete
Caffeine can suppress G2/M checkpoint and cells go into M phase even though DNA is not
completely replicated
M Phase
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Condensin and cohesin make dimers; has an ATP and DNA binding domain
Cohesin hinges two sister chromatids together in M phase (MTs attach on either end when the
chromatids are lined up and with cohesin hold them together)
Condensin condenses the DNA (coils it) after replication; after replication it is unbound so this
protein packages it into chromosomes
o Phosphorylated by M-Cdk; binds DNA and condenses it
APC  degradation of certain proteins (E3 ubiquitin ligase) like M-cyclin
At the beginning of M phase, APC Is needed for the metaphase  anaphase transition
o Found an peptide inhibitor that sits in the active site of APC, cells will take it up 
spindles attach but will not be able to separate because they are stuck
o Need APC to cleave and bring cell to anaphase
To get out of M phase (and into anaphase) must get rid of M-cyclin (APC targets it for
degradation so cells can get out of mitosis and go into G1 and rest)
o Experiment: Ub always attached to Lys in proteins; identify lysine residues in proteins,
once identified, ∆ into Arg and will no longer be degraded by proteasomes
o If you introduce this ∆ into cells, cells will be stuck in the M phase because the cyclin is
still around (MTs will not get degraded, cytokinesis will not happen etc.)
o NEED PROTEOLYSIS FOR G1 RESTING phase
Separase: specific protease, cleaves cohesin molecules only; cell makes a lot of separase along
with inhibitor securin which keeps protease in the inactive state
o Activation of APC by
phosphorylation of M-Cdk
 APC Ub the securin 
securin degrades (fast
process) unleashing and
activating separase 
separase clips the cohesin
 anaphase and
chromatids pulled apart
M-cyclin: ensures replication is
blocked; allows M-cyclin dependant
activity to take place; gets
degraded so metaphase to
anaphase transition can take place
Molecular Level
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In embryonic cells, their cell cycle has no G1 or G2 just S and M
High M-cyclin levels in M phase, then have an increase in Cdc20 kinase activity (activating subunit
of APC)  APC levels go up and therefore cyclin level goes down meaning that APC activity goes
down again and cyclins level rise
o M-cyclin goes down because Cdc20-APC goes up and vice versa
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Hct1 (Cdh1): activating subunit of APC; goes up
keeping M-cyclins LOW in G1
At M phase: have active M-cdk which phosphorylates
Sic1 and Hct1 inactivating it
APC comes in together with Cdc20 (which is resistant
to M-Cdk activity)  degradation of M-cyclin making
M-Cdk inactive  the Sic1 and Hct1 become active
because nothing is keeping them phosphorylated 
Sic1 and Hsc1 accumulate and bind APC degrading all
the M-Cdk continuously
SO, in G1 phase get no M-Cdk activity 
Cells might have to go back into cell cycle though
(sensed by neighboring cell) so must go from G1 or
G0 to S phase
Returning to the Cell Cycle from G1 or G0
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G1-cyclin: insenstive to Sic & Hct (resistant); only activated upon a stimulus coming from the
outside of the cell (from neighboring cell or environment)
Initiates G1/S-Cdk activity; complex can inactivate Sic and Hct1; falls out Cdk complexes and they
have to be removed  go into S phase
DNA Damage
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How do cells sense it? Stop it? Genome damaged, DNA damage can happen through many
mechanisms (chest x-ray, suntanning, radiation etc.).
DNA damage sensed by ATM/ATR kinase sense and activate Chk1/2 kinase
Chk1/2 phoshorylates p53 (important TF)  transcription of many genes including p21
p21: in normal cells is lead to degradation by Mdm2 (Ub ligase) so not present; but when p53 is
phosphorylated, Mdm2 falls out so p21 not degraded and have lots of p53
p53: in abundance, goes to activate transcription of many genes that repairs DNA
So get inhibition of cell cycle by inhibiting G1/S cyclin complexes; cell stop in G1 so everything
can be repaired
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Cancer cells: 50% of tumors, protein p53 inactivated and cell can no longer repair DNA damage
p21: sits on and binds to G1/S-Cdk until DNA is repaired and everything is reversed
Pathway Out of G1
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mitogen: small protein, binds to small GF (mitogen receptor); comes from outside (bloodstream,
enighboring cell etc.) telling cell to proliferate
function: activates kinase which phosphorylates many proteins
route: mitogen  binds to receptor  activates Ras  MAP kinase  Myc (gene regulatory
protein, makes active G1-Cdk and allows progression to S phase)
Active G1-Cdk stimulated by mitogen, food etc.  phosphorylate Rb  Rb falls off E2F 
activates S phase genes and activates S-cyclin froming active S-Cdk  DNA synthesis
in cancer cells: this pathway is disrupted (Rb protein is inactivated) so E2F is always active (no
matter what the condition is)
o high activity of Myc  cancer
when you have too much mitogen  excessive Myc production  very strong signal  cell will
either go into cell-cycle arrest or undergo apoptosis
o Arf: binds Mdm2 inactivating it so you are left with free, stable p53 that triggers
apoptosis or cycle arrest
Growth vs. Proliferation
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Growth: cell gets bigger
Proliferation: replication
Need both to happen for cell cycle, cell needs to duplicate everything (not only DNA)
GF pathway: leads to cell growth (pre-proliferation)
Mtor Pathway: type of GF pathway; conserved from yeast to mammals; regulates proteins
synthesis and cell growth by regulation of certain translation factors
o When Mtor is active, acts on gene regulatory factors which in turn stimulate ribosome
synthesis
Mutant without the coupling of cell proliferation and growth:
o If you reduce nutrition: normally, will stop cell cycle; BUT in mutant will continue cycling
without growth and mass will decrease and will become unviable
o With nutrition cell cycle control and if you take away nutrition will take longer for cell
cycle to become complete
Cells respond to GF (for neurons, NGF)