Lecture 20

• G1 checkpoint


  Once cell passes G1 checkpoint it's committed to completing the cell cycle or undergoing apoptosis
• Quiescent (resting) cell


  Extracellular signals need to pull it out from G0 to G1 phase
• Passing G1 checkpoint


  • Surpassing G1 check requires mitogens
  • Once DNA replication begins (S phase) cell will complete cell cycle or die
• EGFR, cyclin D, Rb protein, and G1 checkpoint

  When mitogens bind to EGFRs, kinase cascade activates transcription factor (AP1) which increases Myc expression → Myc increases cyclin D (G1 cyclin) → activates G1-Cdk → G1-Cdk phosphorylates Rb protein
• Rb protein


  • Phosphorylation of Rb (inactivates Rb) allows E2F transcription factor to activate expression of G1/S and S-cyclins
  • When Rb is active it inhibits E2F activity
• G2 checkpoint


  To make sure all DNA has been replicated
• G1/S cyclins


  • Activate G1/S Cdks in late G1
  • Allow cell to pass G1 checkpoint and commit to cell cycle
  • G1/S cyclin levels fall at start of S phase
• S cyclins


  • Activate S-Cdks after G1 checkpoint
  • Initiate chromosome duplication in S phase
  • Levels elevated during mitosis
• 1. S cyclins are expressed near the middle of G1, but S-Cdks do not become active until S phase. This is possible because S-cdks might need even more regulatory factors to become active
• How helicases, Cdc6, ORC, and S-Cdk regulate S phase

  1. DNA replication begins at the origin of replication

  2. Helicase unwinds double helix at origins of replication to initiate DNA replication at start of S phase

  3. Inactive helicases are recruited to origins of replication by Cdc6 and ORC during G1

  4. Cdc6 and ORC must be unphosphorylated to interact and recruit helicase to origin of replication

  5. S-cyclin increases during end of G1 phase → S-cdk activated when inhibitor (CKI) removed (S phase begins)

  6. S-cdk phosphorylates proteins to activate helicases at origin of replication

  7. S-cdk phosphorylates ORC and Cdc6 so origin cannot be used → helicases cannot be recruited anymore

  8. S-cdk prevents DNA re-replication
• G0 to S phase transition

  1. Presence of mitogen activates signaling pathway: kinase cascade activates transcription factor

  2. Transcription factors induce early gene expression; cell transitions from G0 to G1

  3. Expression of myc promotes expression of cyclin D

  4. Cyclin D activates G1-cdk→ Rb is phosphorylated → E2F transcription factor is released

  5. E2F transcription factor promotes expression of G1/S cyclin → cell passes G1 checkpoint → S-cdk is activated to initiate DNA replication
• DNA damage regulation of cell cycle

  DNA damage can pause cell cycle by inhibiting cdc25 activation and promoting the expression of Cdk inhibitors
• 1. Cdk activity regulation


  1. M-cyclin-cdk complexes form during G2 phase and are held in inactive state until onset of mitosis

  2. Activation of M-cdk at G2 checkpoint: After cyclin-cdk complex forms → Cdk is phosphorylated by Wee1

  3. Inactive cyclin complex accumulates during G2 phase

  4. When concentration of M-cyclin cdk complex is high, Cdc25 removes inhibitory phosphate

  5. Active M-cdk increases activity of Cdc25

  6. Cells need a lot of M-cdk to pass G2 checkpoint and enter mitosis
• M checkpoint


  During M phase cells undergo mitosis and cytokinesis
• Mitosis


  Separates replicated chromosomes
• Cytokinesis


  Splits large cell into two daughter cells
• Key events in each phase of mitosis

  1. Prophase: centrosomes are duplicated and mitotic spindle starts to form

  2. Prometaphase: nuclear envelope broken down into vesicles and chromosomes attach to microtubules made from mitotic spindle

  3. Metaphase: organization phase; chromosomes aligned in the middle and cell begins to shut down cdk activity

  4. Anaphase: separation phase; replicated chromosomes are pulled apart by microtubules

  5. Telophase: chromosomes decondense and go to opposite poles of cell
• 1. Cdk regulation of M phase


  Nuclear lamins are dephosphorylated
• Cytokinesis


  Splits cytoplasm into two cells at end of mitosis
• Cytoskeletal filaments in cell cycle

  • Mitosis depends on microtubules
  • Cytokinesis depends on actin and myosin filaments
• After M phase


  All cyclins degraded no more active cyclin-cdk complexes → cell goes into G0 or G1