M1L5: Cell Cycle Controls and Radiation Induced Checkpoints

Describe the structure of cyclin-Cdks that control the cell cycle

• There are many cyclins and Cdks in cells but not all regulate the cell cycle, the few that do are heterodimer kinases

• Regulatory subunit - conc varies in cyclical fashion during cell cycle

  • Can be degraded by the ubiquitin system

• Catalytic subunit (Cdk) - Ser/Thr kinase, always present but not always active

  • Cdks are active only with cyclin binding

What cyclin-Cdk complex regulates G1?

cyclin D-Cdk4/6

What cyclin-Cdk complex regulates G1/S?

cyclin E-Cdk2

What cyclin-Cdk complex regulates S?

cyclin A-Cdk2/1

What cyclin-Cdk complex regulates M?

cyclin B-Cdk1

How is cyclin-Cdk activity regulated?

• Cyclin-Cdk binding

• Activating and inactivating kinases

  • Phosphorylation at specific sites can alter the protein conformation in a way that activates or inhibits its activity

  • Cak - activates Cdk

  • Wee1 - inhibits Cdk

    • Can be used by cancer cells to avoid mitotic catastrophe, trigger senescence/repair, secrete interleukins in this time to reprogram the TME

• Activating phosphatase (Cdc25) - removes the phosphorylation from inhibitory kinase to activate the Cdk

• Cdk inhibitory proteins inhibibit cell cycle progression by binding to Cdk

  • INK4 (p16^INK4a, p15^INK4b, p18^INK4c, p19^INK4d) - inhibits Cdk4 and Cdk6

  • Cip/Kip (p21^Cip1, p27^Kip1, p57^Kip2) - inhibits Cdk2

• Mitogens and DNA damage compete to regulate cyclin:Cdk inhibitors balance and S phase entry

  

What largely regulates cell division checkpoints?

pRb

What are the cell division checkpoints?

• Start checkpoint (G1/S) /restriction point (R) - is environment favourable?

  • If conditions are favourable, and DNA is not damaged then cell commits to DNA replication, otherwise it can escape to G0 or trigger apoptosis

• G2/M checkpoint - is all DNA replicated, is environment favourable?

  • If DNA has been correctly replicated and DNA is not damaged, cell proceeds to chromosome alignment on spindle in metaphase

• Metaphase to anaphase transition/spindle assembly checkpoint - are all chromosomes attached to spindle?

  • If all chromosomes are correctly attached to spindle microtubules then cell proceeds to anaphase

Explain the control of S phase entry by pRb

• Active Rb binds to E2F family of transcription factors to inactivate it, arresting the cell cycle in G1

• G1-Cdk (cyclin D-Cdk4/6) phosphorylate Rb to inactivate it which releases E2F

• Active E2F triggers transcription of S phase genes, including G1/S cyclin (cyclin E) and S-cyclin (cyclin A), and also itself (positive feedback)

  • Cyclin E and A production activates S-Cdk (cyclin A-Cdk2) and causes DNA synthesis

  • Cyclin E–Cdk2 (G₁/S-Cdk) and cyclin A-Cdk2 (S-Cdk) further phosphorylate Rb to fully inactivate it (positive feedback) 

What largely controls DNA damage induced checkpoints?

p53

Describe the p53-p21 pathway

• p53 complexes with the E3 ligase Mdm2 which shuttles p53 into the proteasome for degradation

• p53 phosphorylation stabilises/activates p53

• p53 binds to promoter region of p21 gene - triggering transcription and translation to produce p21 protein

• p21 acts as a Cdk inhibitor (cyclin E/Cdk2 and cyclin D/Cdk4/6) - normally CycD/Cdk4/6 phosphorylates pRb to release E2F which promotes S phase gene transcription, including CycE/Cdk2 which further phosphorylates pRb (positive feedback)

Describe the Chk2 pathway

• Chk2 pathway - ATM activates Chk2 by phosphorylation which activates the checkpoint by inhibiting Cdc25 which in turn inhibits CycE/Cdk2

• ATM is activated by MRN complex at DSBs and has a faster response (replication independent) and it phosphorylates many key DDR proteins:

  • SMC1 – involved in sister chromatid cohesion

  • Nbs1 (part of MRN complex) – reinforces ATM signaling

  • MDC1 – recruits more repair factors to damage sites

  • BRCA1 – coordinates DNA repair via homologous recombination

  • Chk2 – another checkpoint kinase (parallel to Chk1)

Explain the intra S checkpoint

• Slows or shuts down replication initiation and elongation

• Fork dependent (eg. dNTP depletion, collision of fork with damaged DNA, fork stalling leading to ssDNA) - replication stress

• Fork stalling results in ssDNA which gets coated by RPA which recruits ATRIP which in turn recruits ATR

• Claspin, a mediator protein, helps ATR activate Chk1 by phosphorylation

• Activated Chk1 phosphorylates Cdc25 phosphatase

• Phosphorylated Cdc25 is targeted for ubiquitin-mediated degradation (UPS)

• Without Cdc25, Cyclin E/A–CDK2 complexes remain inactive

• CDK2 activity drops → new origin firing (via Cdc45) is prevented → DNA replication pauses

• RAD51 and BRCA1/2 help stabilise the fork and chromatin remodelers

• Stalled forks are stabilised, no new replication origins fire, and the cell buys time to repair or restart replication

What happens in the fork independent route of DNA damage induced checkpoints?

In the fork independent route (DSBs eg due to IR), structure-specific nucleases like MUS81 process the DNA ends and DNA polymerase δ subunit POLD3 can help restart replication through break-induced replication (BIR)–like mechanisms        

Between the ATR and ATM pathways which is slower?

ATR is slower and replication dependent

Describe the G2/M checkpoint

• Cancer cells strongly depend on this to prevent death by mitotic catastrophe

• ATM phosphorylates/activates Chk2 which inhibits Cdc25, preventing it from activating Cyclin B/Cdk1

• ATR phosphorylates/activates Chk1 which phosphorylates/ activates Wee1, allowing it to inhibit Cyclin B/Cdk1 via inhibitory phosphorylation

Describe the spindle assembly checkpoint

• APC/C (Anaphase-Promoting Complex/Cyclosome) is an E3 ubiquitin ligase that drives the cell from metaphase to anaphase.

  • Its co-activator, Cdc20, helps it ubiquitinate key substrates: securin (inhibitor of separase), cyclin B

• When APC/C–Cdc20 is active securin is degraded → separase cleaves cohesin → sister chromatids separate

• Cyclin B is also degraded → mitotic exit occurs

• When a kinetochore is unattached it recruits checkpoint proteins which form the mitotic checkpoint complex (MCC) - BubR1, Bub3, Cdc20, and Mad2

• MCC binds and inhibits APC/C-Cdc20 and prevents it from targeting securin and cyclin B for degradation

How can checkpoint dysregulation in cancer be used to induce synthetic lethality?

• In normal cells:

  • IR causes DNA double-strand breaks (DSBs).

  • The G₁ checkpoint, governed mainly by p53 → p21 → CDK inhibition, arrests the cell cycle before S phase.

  • This allows DNA repair mechanisms (like NHEJ and HR) to fix the damage.

  • The cell survives and resumes the cycle once repaired

• In cancer:

  • Many cancer cells have defective G₁ checkpoints because they have lost p53 function.

  • They cannot pause at G₁ after DNA damage.

  • Instead, they rely heavily on the G₂/M checkpoint, controlled by Chk1, Chk2, and Cdc25 regulation, to pause before mitosis and repair DNA damage

  • IR still induces a G₂ arrest for DNA repair, however If you treat G₁-deficient cancer cells with ionizing radiation (IR) and a G₂/M checkpoint inhibitor (e.g., an ATR, Chk1, or Wee1 inhibitor), the G₂/M checkpoint is disabled, the cell cannot repair DNA before mitosis, causing mitotic catastrophe and cell death

How is yeast genetics used to study cell cycle?

• Basic organisation of the cell cycle is essentially the same in all eukaryotes

• Many important discoveries about cell cycle control have come from systematic searches for mutations in yeast that inactivate gene encoding essential components of cell cycle control system

Describe cell free models to study cell cycle

• Gentle centrifugation can break open large batch of frog eggs and separate cytoplasm from other cell components

• Undiluted cytoplasm is collected and sperm nuclei are added with ATP

• Sperm nuclei decondense and undergo DNA replication and mitosis, showing that cell cycle control system is operating in the cell free cytoplasmic extract

What is a disadvantage of mammalian cell models vs cell free models?

Divisions are slower than cell free models

How can cell cycle progression be meaured?

• Micropscopy - cell shape, staining with DNA binding fluorescent dyes or antibodies, incorporation of nucleotide analogues

• Measure of DNA content - flow cytometry

Describe FACS analysis when staining for DNA

• Propidium iodide (PI) intercalates into DNA and fluoresces proportionally to the amount of DNA in each cell

• Cells can be pulsed with BrdU which is a synthetic analog of thymidine and an anti-BrdU antibody with fluorescent tag could be used to measure DNA replication

• During mitosis, Histone H3 becomes phosphorylated on serine 10 (Ser10), so an antibody specific for phospho-H3 (Ser10) can be used to label mitotic cells

Describe fluorescent, ubiquitination based cell cycle indicator (FUCCI)

• FUCCI uses two fluorescently tagged cell cycle–regulated proteins that are degraded at specific phases of the cell cycle, CDT1 (licensing factor that marks G₁ phase, degraded at onset of S pahse) and geminin (inhibitor of CDT1, present during S, G₂, and M phases, degraded at the end of mitosis)

• hCdt1(1/100): red fluorescence

• hGeminin(1/110): green

• G1: red (CDT1 only), G2: yellow (CDT1 ↓, Geminin ↑), G2/M: green (geminin only)

Explain replicative DNA synthesis assay

• Replicative DNA synthesis (RDS) assay - measures how much DNA replication continues after DNA damage, especially after exposure to ionizing radiation (IR) or UV light

  • Radio-resistant DNA synthesis

  • [³H]thymidine incorporation into DNA decreases inc ells that have fucntional checkpoint response

  • High [³H]thymidine incorporation indicates deficient intra-S checkpoint

What happens in DNA fibre assay?

• CldU (Chloro-deoxyuridine) → labeled with red fluorescence (e.g., anti-BrdU antibody that recognizes CldU)

• IdU (Iodo-deoxyuridine) → labeled with green fluorescence (different antibody that distinguishes it from CldU)

• Cells are first incubated with CldU (red) for a defined time (e.g., 20 min).

• Then switched to IdU (green) for another period (e.g., 20 min).

• DNA fibers are spread on glass slides (called “DNA combing”), fixed, and stained with antibodies that recognize each label.

• Under a fluorescence microscope, you see colored tracks corresponding to replicated DNA.

  

• From this we can determine fork speed, stalling frequency, new origin firing rate, fork restart efficiency, asymmetry between sister forks