10. Cell Cycle Checkpoints

• cell cycles checkpoints are controlled by Cdk activity

  • Cdk is regulated by cyclin
• P21 = cyclin dependent kinase inhibitor
• G1/S checkpoint is controlled by p53 and ATM

  • DNA damage -> ATM activation -> p53 phosphorylation -> P21 activation -> binds to cyclin-Cdk complex preventing activity and entry into S
  • ATM -> CHK2 phosphorylation -> Cdc25a phosphorylation -> Cdc25a destruction -> inhibitory phosphorylations accumulate on Cdk2 complex
• p53 is normally maintained at low levels as it binds to MDM2 which targets p53 for ubiquitination and proteolysis

  • USP7 normally bound to MDM2 to prevent it ubiquitinating itself
• ATM -> MDM2 phosphorylated -> USP7 released -> MDM2 ubiquitinated and degraded -> p53 accumulates

  • p53 removed later as MDM2 is a target gene, so once enough MDM2 is produced, p53 gets degraded
• G1/S activation is monitored in a lab via:

  • western blotting with antibodies that recognise markers of p53 activation
  • FACS coupled with antibodies that recognise S phase markers
• intra s phase checkpoint:

  • ATM -> CHK2 -> decreased Cdc25a
  • ATR -> CHK1 -> decreased Cdc25a

  decreased Cdc25a leads to decreased Cdk2-cyclin E
• ATR is activated after damage at a replication fork
• S phase activation is monitored via:

  • western blotting with antibodies that recognise proteins in S
  • DNA fibre analysis to assess whether firing of new origins is suppressed after DNA damage
• G2/M checkpoint:

  • ATM/ATR activated after DNA damage, phosphorylate CHK1/2
  • phosphorylates Cdc25b/c -> gets moved out of nucleus

  inhibitory phosphorylations accumulate on Cdk2-cyclin B
• G2/M checkpoint monitored in lab via:

  • FACS coupled with antibody that recognises maker of entry into mitosis