23. Cell Cycle and Checkpoints

Main ideas & lessons

• Purpose of the cell division cycle: Convert one cell into two by coordinating:

  • DNA replication (so each daughter cell gets genetic material)
  • Chromosome segregation (equal distribution of chromosomes)
  • Physical cell division (the cell divides after chromosomes are separated)

• Core requirement for cell division: During the cell cycle, a cell must:

  • Duplicate organelles (so daughter cells inherit them)
  • Replicate DNA / chromosomes (so daughter cells inherit genetic information)
  • Grow in size (otherwise repeated division would make cells progressively smaller)
  • Proceed through mitosis and physically divide (chromosomes segregate, then the cell splits)

• External/internal cues determine whether a cell divides:

  • Multicellular organisms: cells decide to divide based on cell-to-cell communication, including signaling pathways (example: RTK → Ras–MAP kinase pathway), which promote entry into the cell cycle.
  • Single-celled organisms: cells respond to nutrient availability and whether the cell is the right size.

Phases of the cell division cycle (overview + what happens)

The cycle has four phases, grouped into:

• S phase (DNA synthesis)

  • DNA replication occurs
  • Machinery involved:
    • DNA polymerase
    • helicases

• M phase (mitosis)

  • Sister chromatids separate
  • Chromosome segregation occurs
  • Machinery involved:
    • mitotic spindle, built from microtubules

• G1 phase (gap 1)

  • Decision point: whether to enter the cell cycle
  • Cell checks for growth signals and appropriate environmental conditions
  • G1 → S transition is a commitment point (“start” in yeast; “restriction point” in mammals)

• G2 phase (gap 2)

  • Quality control: ensures DNA is replicated before mitosis

Ordered progression: “events must happen in the correct order”

• DNA synthesis must occur before chromosome segregation.
• This is enforced by a control system and by checkpoints.

Control system that coordinates timing (CDK–cyclin logic)

Two main components

• CDK (cyclin-dependent kinase)

  • A kinase that controls events by phosphorylating target proteins (adds phosphate groups)

• Cyclins

  • Regulatory subunits of CDK
  • Without cyclin, CDK is inactive
  • Cyclins activate CDK
  • Different cyclin variants:
    • Appear in different cell-cycle phases
    • Oscillate (rise and fall)
    • Determine which CDK is active and what substrates are phosphorylated

Functional idea emphasized

• The phase-specific cyclin present at a given time largely determines which cellular processes occur:
  • G1/S cyclin–CDK phosphorylates/activates proteins needed for DNA replication (example: helicase).
  • M cyclin–CDK phosphorylates proteins needed for mitotic spindle formation.

Link to signaling (RTK example)

• A key signaling output is increasing a G1 cyclin, helping drive G1 → S entry.

Checkpoints (quality-control mechanism)

Definition

A checkpoint is a quality control mechanism ensuring:

• an event does not proceed until the preceding event is successfully completed
• the proper order and correctness of events are maintained

Example checkpoint: DNA damage checkpoint

It verifies whether:

• DNA is damaged, or
• DNA is still under replication

If problems are detected:

• It sends a signal that inhibits cyclin–CDK
• This affects key transitions, primarily:
  • G1 → S (“beginning/start”)
  • G2 → M

Discovery and experiments supporting the system (yeast + checkpoint studies)

Yeast used: budding yeast and CDC mutants

• Budding yeast can be haploid or diploid, and its phase can be inferred from bud morphology:

  • no bud → likely G1
  • small bud → likely S
  • larger bud → likely G2

• Mutant screening strategy

  • Use haploid cells:
    • reduces the chance that a recessive mutation is masked by another allele (diploids would require multiple hits)
  • Use temperature-sensitive (ts) mutants:
    • alive at permissive temperature
    • die at restrictive temperature

Selecting specifically cell-cycle mutants (phenotype-guided reasoning)

• Temperature sensitivity alone could affect many cellular processes.
• The strategy used: look for mutants where cells accumulate in a single cell-cycle stage (a “phase arrest” phenotype).

Key mutant identified: CDC28

• At restrictive temperature, cells arrest in non-budding state (interpreted as G1 arrest).
• Confirmation:
  • DNA content measurement shows cells do not duplicate DNA
  • meaning they fail to enter S phase
• Conclusion:
  • CDC28 is necessary for G1 → S transition
  • CDC28 encodes the only cell-cycle-dependent kinase in budding yeast
• Broader significance:
  • Work described as foundational for discovery of CDKs across organisms (mentioned: Paul Nurse using fission yeast complementation to clone human CDK).

Nobel Prize attribution (as stated in video)

• 2001 Nobel Prize in Physiology or Medicine:
  • Leland Hartwell, Tim Hunt, Sir Paul Nurse
• Hartwell: CDC28 screening
• Hunt: identified cyclins using clams/sea urchins
• Nurse: cloned/linked CDK mechanism using complementation; showed conservation across species

DNA damage checkpoint experiments (Weinert + Hartwell, 1988)

• Wild-type budding yeast treated with X-rays:

  • shows a delay in G2
  • survives (interpreted as time for DNA repair)

• RAD9 mutant (radiation-sensitive):

  • removes the delay:
  • cells proceed into mitosis despite damaged DNA
  • leads to high mortality in offspring

• Conclusion:

  • RAD9 promotes checkpoint delay, preventing premature mitosis with damaged DNA

Why cyclins oscillate: regulated proteolysis (ubiquitin–proteasome)

Central mechanism

• Cyclin levels rise and fall because cyclins are degraded in a controlled way.
• The precipitous drop in mitotic cyclin (e.g., from metaphase to anaphase) is due to:
  • regulated proteolysis
  • ubiquitin-mediated targeting to the proteasome

Ubiquitin system (core steps described)

• Ubiquitin (UB) is a small protein that can attach to targets via lysines.
• Enzymatic steps (simplified):
  • E2 enzyme conjugates ubiquitin
  • ubiquitin is transferred to the target (here, cyclin)
  • E3 ubiquitin ligase builds polyubiquitin chains
• Hundreds of E3 ubiquitin ligases exist; they provide specificity (different E3s target different proteins).
• Polyubiquitinated proteins are tagged for degradation:
  • sent to the proteasome, which degrades proteins

Xenopus egg extract experiments (Andrew Murray + Mark Kirschner)

• Xenopus laevis (African clawed frog) egg extract is an in vitro system:
  • adding calcium mimics fertilization and triggers a cell-cycle-like biochemical cycle
  • cyclin (mitotic cyclin) oscillates outside of intact cells

Experiments described:

• Remove mRNA with RNases
  • oscillatory cell cycle disappears
• Add back single mRNA: mitotic cyclin
  • restores oscillations
  • shows mitotic cyclin mRNA is sufficient to drive oscillation
• Add a cyclin mutant that cannot be degraded
  • deletion in the destruction box (required for recognition by the relevant E3 ligase)
  • cyclin levels remain high
  • cell-cycle oscillation stops
  • because cyclin M stays high, cells arrest in mitosis

Additional clarification given

• Cyclin M mRNA is constant (present throughout).
• The oscillation comes from cyclin protein levels changing due to regulated degradation.

Speakers or sources mentioned (and referenced)

• Miles (student)
• Malik (student)
• Stephen (student)
• Udo (student)
• Natalie (student)
• Carmen (student)
• Diana (student)
• Professor Imperiali (instructor; referenced for prior signaling and ubiquitin/proteasome explanation)
• Andrew Murray (researcher; Xenopus extract experiments mentioned)
• Mark Kirschner (researcher; Xenopus extract experiments mentioned)
• Leland Hartwell (Nobel laureate; yeast CDC28 and checkpoint work mentioned)
• Tim Hunt (Nobel laureate; cyclin discovery mentioned)
• Sir Paul Nurse (Nobel laureate; CDK complementation/cloning mentioned)
• Weinert (researcher; checkpoint work with Hartwell mentioned)
• Tom Moresca (colleague; provided frog slides as stated)