Biochemical Control of the Cell Cycle

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Biochemical Control of the Cell Cycle

• BNS230

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Lecture programme

• Three lectures
• Aims
• Describe the cell cycle
• Discuss the importance of the cell cycle
• Discuss how the cycle is regulated

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Cell division
15 hours
M phase
G0 state
G2 phase
G1 phase
12 hours
S-phase (DNA synthesis
5 hours
16 hour cell cycle
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Cell cycle definition

• A series of distinct biochemical and
  physiological events occurring during replication
  of a cell
• Occurs in eukaryotes
• Does not occur in prokaryotes
• Time of cell cycle is variable

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Cell cycle timing

• Yeast 120 minutes (rich medium)
• Insect embryos 15-30 minutes
• Plant and mammals 15-20 hours
• Some adults dont divide
• Terminally differentiated
• e.g. Nerve cells, eye lens
• Some quiescent unless activated
• Fibroblasts in wound healing

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Components of the cell cycle

• M phase
• Cell division
• Divided into six phases
• Prophase
• Prometaphase
• Metaphase
• Anaphase
• Telophase
• Cytokinesis

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Components of the cell cycle

• G1 phase
• Cell checks everything OK for DNA replication
• Accumulates signals that activate replication
• Chloroplast and mitochondria division not linked
  to cell cycle

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Components of the cell cycle

• S-phase
• The chromosomes replicate
• Two daughter chromosomes are called chromatids
• Joined at centromere
• Number of chromosomes in diploid is four

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Components of the cell cycle

• G2-phase
• Cell checks everything is OK for cell division
• Accumulates proteins that activate cell division

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Why have a cell cycle?

• Comprises gaps and distinct phases of DNA
  replication and cell division
• If replicating DNA is forced to condense (as in
  mitosis) they fragment
• Similarly if replication before mitosis
• Unequal genetic seperation
• I.e. Important to keep DNA replication and
  mitosis separate

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Why have a cell cycle?

• Important to have divisions in mitosis
• e.g. Important metaphase complete before
  anaphase. Why?
• If not segregation of chromosomes before
  attachment of chromatids to microtubles in
  opposite poles is possible
• Down syndrome due to extra chromosome 21

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Why have a cell cycle?

• Gaps provide cell with chance to assess its
  status prior to DNA replication or cell division
• During the cell cycle there are several checks to
  monitor status
• These are called checkpoints

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Checkpoints

• Checkpoint if G1 monitors size of cell in budding
  yeast (Saccharomyces cerevisae)
• At certain size cell becomes committed to DNA
  replication
• Called start or replication site

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Evidence of size checkpoint

• Yeast cells (budding yeast) grown in rich medium
• Switch to minimal medium
• Cells recently entering G1 (buds) delayed in G1
  (longer to enter S-phase)
• Large cells above threshold size still go to
  S-phase at same time as in rich medium

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Evidence of size checkpoint

• Yeast in rich medium
• 120 minute cell cycle
• Short G1 phase
• Yeast in minimal medium
• Eight hour cell cycle primarily because of long
  G1 phase

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Checkpoints

• Checkpoint 2 in G1 monitors DNA damage
• Evidence?
• Expose cells to mutagen or irradiation
• Cell cycle arrest in either G1 phase or G2 phase
• The protein p53 involved in cell cycle arrest
• Tumour suppresser

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Checkpoints

• Checkpoint in S-phase monitors completion of DNA
  replication
• Cell does not enter M-phase until DNA synthesis
  is complete
• Checkpoint in G2
• DNA breaks cause arrest
• Otherwise when chromosomes segregate in mitosis
  DNA distal to breaak wont segregate

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Checkpoints

• Checkpoint in mitosis
• Senses when mitotic spindles have not formed
• Arrests in M-phase
• Otherwise unequal segregation of chromosomes into
  daughter cells
• Described cell cycle, now I will talk about genes
  and proteins that control this process

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Molecular control of cell cycle

• Two experimental approaches
• Biochemical
• Sea urchin fertilised eggs
• Rapid
• Synchronous division
• Analyse proteins at various stages of cycle
• Genetic analysis using
• Budding yeast Saccharomyces cerevisae
• Fission yeast Schizosaccharomyces pombe

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Using genetics to study the cell cycle

• To study the genetic basis of a biological event
• Make mutants defective in that event
• Determine which genes have been mutated
• Understand role of gene (and encoded protein) in
  the event
• Problem How do you make mutants that disrupt the
  cell cycle
• Cells will not replicate

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Using genetics to study the cell cycle

• Isolate temperature sensitive mutants that have
  defect in cell cycle
• At low temperature these mutants progress through
  cell cycle
• Arrest in cell cycle at elevated temperature
• Mutation causes gene product (protein) to be
  highly sensitive to temperature

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Using genetics to study the cell cycle

• Isolation of genes that regulate the cell cycle
• Step 1 Create strains with mutations in cell
  cycle genes

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Isolating cell cycle mutants
Yeast culture (S. pombe)
Mutagenise and plate out at high and low
temperature
30C
37C
Colonies 4 and 10 are possible cell cycle
mutants. Called cell division cycle (cdc) mutants
gt70 cdc mutants isolated
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Are the temperature sensitive mutants cdc mutants?
Grow colonies at 30C Shift temperature to
37C Look under a microscope
Colony 4 Too small enters mitosis too early
(Wee 1 mutant)
Colony 10 very long stuck in G2 (cdc25 mutant)
Wild type cells
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Using genetics to study the cell cycle

• Step 2 Insert plasmids containing fragments of
  wild type DNA
• Step 3 Look for plasmid that corrects genetic
  defects
• Step 4 Plasmid contains a cell cycle control gene

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What do we do with the mutants?
Use mutants to isolate cdc genes and then study
what the proteins do
Wild type S. pombe
Extract DNA
cdc25
Wee1
Cut with restriction enzyme and ligate into
vector
Yeast vector
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Take recombinant vectors and transform into cdc
mutants

• Wee mutant with normal gene wee1 gene in plasmid
  will grow at 37
• cdc25 mutant with normal cdc25 gene in plasmid
  will grow at 37
• I.e gene in recombinant plasmid is complementing
  the mutation

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Biochemical studies

• 1st evidence proteins regulate cell cycle
• Fuse interphase cells (G1, S or G2) withM-phase
  cells
• Cell membranes breakdown and chromosomes condense
  
• I.e Mitotic cells produce proteins that cause
  mitotic changes in other cells

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Microinjection with frog oocyte

• Oocyte stays in G2-phase
• Male gets busy and female produces progesterone
• Oocyte enters mitosis
• Purify proteins from oocyte cells treated with
  progesterone
• Inject into G2 arrested cells and see which
  protein causes mitosis (1971)

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MPF

• Protein identified that causes mitosis
• Called maturation promoting factor
• MPF in all mitotic cells from yeast to humans
• Renamed mitosis-promoting factor

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Properties of MPF

• MPF activity changes through the cell cycle

• MPF activity appears at the G2/M interphase
• and then rapidly decrease

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How does MPF cause mitosis?

• Its a protein kinase
• Phosphorylates proteins
• Phosphorylates proteins involved in mitosis
• Phosphorylates histones causing chromatin
  condensation
• Phosphorylates nuclear membrane proteins (lamins)
  causing membrane disruption

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Characterisation of MPF

• Consists of two subunits A and B
• Subunit A Protein kinase
• Subunit B Regulatory polypeptide called cyclin B
• Protein kinase present throughout cell cycle
• Cyclin B gradually increases during interphase
  (G1, S, G2)
• Cyclin B falls abruptly in anaphase
  (mid-mitosis)

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What does this profile tell you?
MPF not just due to association of subunits A and
B other factors involved
Protein kinase (subunit A)
Cyclin B levels (subunit B)
MPF activity
G1 S G2
M
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Cyclin B (subunit B)
MPF
Protein kinase (subunit A)
Metaphase
Ubiquitin
Anaphase
Prophase
Interphase (G1-S-G2)
Proteosome
Telephase
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Cyclin B

• How do Cyclin B levels decrease abruptly
• Proteolytic degradation
• Degraded in a protease complex present in
  eukaryotic cells called The Proteosome
• Specific proteins degraded by complex when tagged
  by a small peptide called ubiquitin

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Cyclin B

• Cyclin B is tagged for Proteosome degradation at
  anaphase
• Tagged at N-terminus at sequence called
• Destruction box
• DBRP binds to Destruction box
• Guides Ubiquitin ligase to add ubiquitin
  molecules to Cyclin B
• Why is Cyclin B only degraded in anaphase

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Destruction box
DBRP (inactive)
DBRP Destruction box recognition protein
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Cyclin B

• DBRP is normally inactive and is only activated
  in anaphase via phosphorylation
• Possible MPF phosphorylates DBRP causing Cyclin
  B destruction
• Binds to the destruction box
• Activates ubiquitin ligase to add ubiquitin to
  Cyclin B
• Cyclin B then targeted to the Proteosome for
  degradation

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Cyclin B

• When this causes MPF inactivation
• DBRP dephosphorylated by constitutive
  phosphorylase
• Other proteins also control MPF
• Activity doesnt increase as Cyclin B increases
• Proteins discovered in yeast by cdc mutant
  complementation

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inactive
Protein kinase (subunit A)
cdc13
cdc2
MPF
Wee1
Inactive MPF
P
CAK
Inactive MPF
P
P
cdc25
Active MPF
P
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MPF activity

• Wee mutant small Enters mitosis prematurely
• cdc 25 mutant long Stays in G2 for longer
• Wee phosphorylates Y15 and inactivates MPF
• CAK (cdc2 MPF-activating kinase) phosphorylates
  T161
• cdc25 dephosphorylates Y15 and activates MPF

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Cell cycle

• How is entry into S-phase controlled?
• Throughout cell cycle the protein kinase (cdc28
  in sc and cdc2 in sp) binds to specific cyclins
• This changes the specificity of the protein kinase

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Activity of Protein Kinase

• Cdc28-cyclins B1-4 Protein kinase activates
  proteins involved in early mitosis by
  phorphorylating them
• Cdc28-cyclins 1-3 Protein kinase activates
  proteins involved in initiation of DNA
  replication by phosphorylating them
• cdc28-cyclin 5 Phorphorylates and thus activates
  proteins that maintain DNA replication

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How many protein kinases?

• In both yeasts only one protein kinase
• In higher eukaryotes multiple protein kinases
• Active at different stages of the cell cycle
• As with yeast different cyclins