Biochemical Control of the Cell Cycle by sdfwerte


									Biochemical Control of the
       Cell Cycle
       Lecture programme
• Three lectures
• Aims
  – Describe the cell cycle
  – Discuss the importance of the cell cycle
  – Discuss how the cycle is regulated
           Cell division
           15 hours
                                                  G0 state
                G2 phase
                                   G1 phase
12 hours
                      (DNA synthesis

                                              5 hours

                   16 hour cell cycle
       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
            Cell cycle timing
•   Yeast 120 minutes (rich medium)
•   Insect embryos 15-30 minutes
•   Plant and mammals 15-20 hours
•   Some adults don’t divide
    – Terminally differentiated
    – e.g. Nerve cells, eye lens
• Some quiescent unless activated
    – Fibroblasts in wound healing
 Components of the cell cycle
• M phase
  – Cell division
  – Divided into six phases
    •   Prophase
    •   Prometaphase
    •   Metaphase
    •   Anaphase
    •   Telophase
    •   Cytokinesis
 Components of the cell cycle
• G1 phase
  – Cell checks everything OK for DNA
  – Accumulates signals that activate
  – Chloroplast and mitochondria division not
    linked to cell cycle
 Components of the cell cycle
• S-phase
  – The chromosomes replicate
  – Two daughter chromosomes are called
  – Joined at centromere
  – Number of chromosomes in diploid is four
 Components of the cell cycle
• G2-phase
  – Cell checks everything is OK for cell
  – Accumulates proteins that activate cell
     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
     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
     Why have a cell cycle?
• Gaps provide cell with chance to assess
  its status prior to DNA replication or cell
• During the cell cycle there are several
  checks to monitor status
• These are called checkpoints
• Checkpoint if G1 monitors size of cell in
  budding yeast (Saccharomyces
• At certain size cell becomes committed
  to DNA replication
• Called start or replication site
 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
 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
• Checkpoint 2 in G1 monitors DNA
• Evidence?
  – Expose cells to mutagen or irradiation
  – Cell cycle arrest in either G1 phase or G2
• The protein p53 involved in cell cycle
  – Tumour suppresser
• 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 won’t
• Checkpoint in mitosis
  – Senses when mitotic spindles have not
  – 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
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
 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
  – 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
  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
• Mutation causes gene product (protein)
  to be highly sensitive to temperature
  Using genetics to study the
          cell cycle
• Isolation of genes that regulate the cell
• Step 1: Create strains with mutations in
  cell cycle genes
             Isolating cell cycle mutants

                                           Yeast culture
                                           (S. pombe)
                                  Mutagenise and plate
                                  out at high and low temperature


Colonies 4 and 10 are possible cell cycle mutants. Called
cell division cycle (cdc) mutants >70 cdc mutants isolated
Are the temperature sensitive mutants
            cdc mutants?
        Grow colonies at 30°C
        Shift temperature to 37°C
        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
  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
       What do we do with the mutants?
      Use mutants to isolate cdc genes and
      then study what the proteins do
S. pombe
                              Extract DNA

               Wee1                          cdc25
                        Cut with restriction enzyme
                        and ligate into vector
Yeast vector
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
       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
Microinjection with frog oocyte
• Oocyte stays in G2-phase
• Male gets busy and female produces
• Oocyte enters mitosis
• Purify proteins from oocyte cells treated
  with progesterone
• Inject into G2 arrested cells and see
  which protein causes mitosis (1971)
• Protein identified that causes mitosis
• Called maturation promoting factor
• MPF in all mitotic cells from yeast to
• Renamed mitosis-promoting factor
          Properties of MPF
• MPF activity changes through the cell cycle

   • MPF activity appears at the G2/M interphase
   • and then rapidly decrease
 How does MPF cause mitosis?

• It’s a protein kinase
  – Phosphorylates proteins
• Phosphorylates proteins involved in
• Phosphorylates histones causing
  chromatin condensation
• Phosphorylates nuclear membrane
  proteins (lamins) causing membrane
     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
• Cyclin B gradually increases during
  interphase (G1, S, G2)
• Cyclin B falls abruptly in anaphase
 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
                  Cyclin B (subunit B)
                  Protein kinase (subunit A)



Prophase                                       Anaphase

 (G1-S-G2)             Telephase               Proteosome
               Cyclin B
• How do Cyclin B levels decrease
• Proteolytic degradation
• Degraded in a protease complex
  present in eukaryotic cells called “The
• Specific proteins degraded by complex
  when tagged by a small peptide called
                  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

                      P        de-phosphorylase

                    DBRP           MPF?        DBRP
           P        (active)                 (inactive)

                      Ubiquitin ligase adds ubiquitin
                      when DBRP binds to the
                      destruction box
DBRP = Destruction box recognition protein
                     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
                    Cyclin B

• When this causes MPF inactivation
  – DBRP dephosphorylated by constitutive
• Other proteins also control MPF
  – Activity doesn’t increase as Cyclin B increases
• Proteins discovered in yeast by cdc mutant
Protein kinase (subunit A)                                  Cyclin B (subunit B)
                             cdc2                  cdc13

                                    Y15 T161
                               Wee1                          Inactive MPF

                               P Y15    T161
                                                             Inactive MPF
                               P Y15    T161   P

                                                           Active MPF
                                    Y15 T161   P
            MPF activity
• Wee mutant small: Enters mitosis
• cdc 25 mutant long: Stays in G2 for
• Wee phosphorylates Y15 and
  inactivates MPF
• CAK (cdc2 [MPF]-activating kinase)
  phosphorylates T161
• cdc25 dephosphorylates Y15 and
  activates MPF
              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
     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
• cdc28-cyclin 5: Phorphorylates and thus
  activates proteins that maintain DNA
  How many protein kinases?
• In both yeasts only one protein kinase
• In higher eukaryotes multiple protein
  – Active at different stages of the cell cycle
• As with yeast different cyclins

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