UnitE 2

• Events that take place during different phases of the cell cycle must be coordinated with one another so that they occur in the appropriate order
• This order must be preserved even if one of these events takes longer than usual
• This system switches all cell-cycle machinery on or off at different stages
• It is critically important that the cell not begin mitosis until DNA replication during S phase has been completed
• The alternative would be a catastrophic cell division, in which the daughter cells fail to inherit complete copies of daughter chromosomes
• It is crucial for cells to double in size during interphase (interphase = G1 + S + G2) before dividing in two during M phase
• Otherwise, cells would get smaller with each division
• Cell cycle progression is regulated by

  1. Checkpoints

  2. Series of protein phosphorylation followed by de-phosphorylation

  
• Phosphorylation


  Carried out by series of protein kinases called cyclin-dependent protein kinases (Cdks)
• Cdks


  Activated at different times in the cell cycle by being "switched on" by binding of cyclins
• Cyclin concentrations oscillate throughout cell cycle; cdk concentrations stay the same
• Different Cyclin–Cdk Complexes Trigger Different Steps in the Cell Cycle

  • In vertebrates
  • In yeast

  
• Cyclin Concentrations


  Regulated by transcription and by proteolysis
• Concentration of a protein is determined by rate of synthesis and degradation
• Cyclin levels gradually increase and abruptly decrease

  
• Due to continuous transcription of cyclin genes & rapid degradation of cyclin proteins

  
• Ubiquitylation


  Of S or M-cyclins by the Anaphase Promoting Complex/Cyclosome (APC/C) targets them for degradation

  
• The degradation of cyclins inactivates Cdks

  
• Anaphase Promoting Complex/Cyclosome (APC/C)


  • An E3 ubiquitin ligase that marks cell cycle proteins for degradation by the 26S proteosome
  • Composed of several subunits
  • E1 = ubiquitin-activating enzyme
  • E2 = ubiquitin-conjugating enzyme
  • Promotes the transfer of the ubiquitin residue from the E2 to the substrate protein on which the C-terminus of ubiquitin forms a covalent isopeptide bond with a lysine residue
  • Initiates the metaphase-to-anaphase transition
  • Other cell cycle proteins including G1 cyclins are regulated by Skp-Cullin F-box containing (SCF) complex

  
• Activity of Cyclin–Cdk Complexes Is Controlled by

  1. Phosphorylation

  2. Dephosphorylation

  3. Cdk inhibitor proteins

  
• Cyclin-cdk activity is regulated by phosphorylation/de-phosphorylation
• Once cyclin binds its respective Cdk, the Cdk protein becomes phosphorylated by inhibitory kinase (Wee1) and remains inactive until an activating phosphatase binds (ex: cdc25)

  
• In addition, cells use Cdk inhibitor proteins (p27) to block assembly of cyclin-Cdk complexes at specific times

  
• Protein Phosphatases Reverse the Effects of Cdks

  1. During early mitosis, M cyclin-Cdk activity is active and results in inactivation of phosphatase PP2A

  2. During later stages of mitosis, M-Cdk is inactivated by the degradation of M-cyclins by APC/C

  3. This inactivation leads to reactivation of PP2A, which then goes on to dephosphorylate M-Cdk target proteins

  
• G1 PHASE


  • Stage in which a cell decides to stay in G1, withdraw into a non-proliferative state or prepare for a new cell cycle
  • Cells must inactivate S-Cdk and M-Cdk complexes in order to allow for transition into G1 (calm state)
  • Degrades all remaining cyclins
  • Downregulates cyclin gene transcription
  • Upregulating transcription of Cdk inhibitory proteins
  • Additional mechanisms ensure shutdown of Cdk activity:
  • Release of mitogens
  • Activation of DNA damage signal mechanisms

  
• Mitogens Promote Production of Cyclins That Stimulate Cell Division

  1. Mitogen (extracellular signal) binds to its receptor on cell surface and induces a signaling cascade of events

  2. This leads to activation of G1-Cdk and G1/S-Cdks

  3. Rb (retinoblastoma) protein binds and inactivates transcription of cell cycle proliferation proteins

  4. When G1-Cdk and G1/S-Cdks are activated, they phosphorylate Rb = inactivating the protein

  5. This results in activation of transcription of S-phase specific genes = cell proliferation

  
• DNA Damage Can Temporarily Halt Progression Through G1

  1. Cell can employ several mechanisms to halt cell cycle progression in presence of DNA damage

  2. DNA damage increases p53 (transcription regulator)

  3. Active p53 in turn activates p21 (Cdk inhibitor)

  4. P21 binds to G1-Cdk and G1/S-Cdk complexes which prevents them from moving to S phase

  5. DNA repair mechanisms (non-homologous end-joining/homologous recombination) are then activated

  
• DNA helicase: Uses ATP hydrolysis to unwind double stranded DNA at front of replication machinery

  
• DNA topoisomerase: Produces small single-strand break in DNA backbone to relieve tension caused by helix unwinding. It then repairs this break.

  
• S PHASE


  • S-Cdk initiates DNA replication and blocks re-replication
  • Origin of replication (ORC) recruits Cdc6 (high in G1)
  • Orc-Cdc6 allow binding of DNA helicase à Cdc6 subsequently dissociates
  • S-cdk complex triggers the start of S phase;
  • Activates DNA helicase
  • Promotes recruitment of proteins involved in DNA replication
  • S-Cdk inactivates Cdc6 and ORC to ensure re-replication does not occur

  
• Events that take place during different phases of the cell cycle must be coordinated with one another so that they occur in the appropriate order

  
• This order must be preserved even if one of these events takes longer than usual
• This system switches all cell-cycle machinery on or off at different stages
• It is critically important that the cell not begin mitosis until DNA replication during S phase has been completed

  
• The alternative would be a catastrophic cell division, in which the daughter cells fail to inherit complete copies of daughter chromosomes
• It is crucial for cells to double in size during interphase (interphase = G1 + S + G2) before dividing in two during M phase
• Otherwise, cells would get smaller with each division
• Cell cycle progression is regulated by

  1. Checkpoints

  2. Series of protein phosphorylation followed by de-phosphorylation

  
• Phosphorylation

  Carried out by series of protein kinases called cyclin-dependent protein kinases (Cdks)

  
• Cdks

  Activated at different times in the cell cycle by being "switched on" by binding of cyclins