Cell Cycle 2

Why is DNA replication essential?

It is essential for cell division and is the main cause of genetic variation

How does DNA replication initiate in different organisms?

• Yeast: From multiple origins defined by specific DNA sequences

• Higher eukaryotes: Origins specified by binding of the Origin Recognition Complex (ORC)

What are the two steps of DNA replication?

• Initiation: Activation of an origin → two replication forks form

• Elongation: Polymerisation of new DNA strands

How is DNA replication related to cancer?

Deregulated DNA replication is a feature of cancer cells

What are the three key characteristics of DNA replication?

1. Bidirectional

2. Semi-conservative

3. Semi-discontinuous

Why are multiple origins required for large genomes?

• Replication forks duplicate DNA at ~2 kb/min

• With only one origin, replication of the human genome would take ~1000 days

How is DNA replication controlled within the cell cycle?

Origins are activated only once per cell cycle; both under-activation and over-activation cause genome instability

What complex binds to origins to mark them for replication?

The Origin Recognition Complex (ORC) binds to origins to form the pre-replicative complex (pre-RC) during G1 phase

What happens during origin activation and pre-RC disassembly?

Activation leads to initiation of DNA synthesis and disassembly of the pre-RC and CDT1

How is CDT1 regulated?

• CDT1 is active during G1 (permissive state).

• CDT1 is inhibited or degraded in S phase (non-permissive state) by Geminin and CDK activity.
  This ensures only one round of replication per cell cycle

How do CDKs ensure that replication occurs only once per cycle?

By phosphorylating and inactivating replication factors (e.g., ORC, Cdc6, Cdt1), preventing re-formation of new pre-RC during S, G2, and M phases

How many DNA polymerases act at each replication fork?

Three DNA polymerases

Why do DNA polymerases require a primer?

They need a free 3’-OH group to add nucleotides; they can only extend DNA in the 5’ → 3’ direction

What are the main steps in DNA synthesis?

1. DNA is unwound by helicase

2. Primase synthesizes RNA primers

3. Replicative polymerases (Pol δ or Pol ε) extend primers (5’→3’)

4. Lagging strand synthesized discontinuously (Okazaki fragments)

5. Primers removed by nucleases

6. Okazaki fragments ligated

7. The end replication problem is solved by telomerase

Which polymerase works on each strand?

• Leading strand: DNA polymerase ε

• Lagging strand: DNA polymerase δ (after α-primase synthesis of primer)

What is DNA polymerase proofreading activity?

Exonuclease activity that corrects misincorporated nucleotides, improving replication fidelity

What is the significance of Pol δ and Pol ε proofreading mutations?

Mutations in their exonuclease domains cause replication errors and are frequently found in cancers

What is the structure of a centrosome?

• Contains two centrioles (barrels of microtubules made of tubulin and centrin)

• Surrounded by pericentriolar material (PCM), which includes γ-tubulin ring complexes that nucleate microtubules

Describe the centrosome duplication cycle

1. G1: Disorientation of centrioles

2. S: Procentriole formation

3. G2: Elongation and maturation

4. M: Separation and activation of centrosomes

What are cell cycle checkpoints?

• Control mechanisms that monitor growth, DNA replication, DNA integrity, and cellular structure to ensure proper progression

• They provide negative feedback to halt the cycle if conditions are unfavourable

Where is the G1 checkpoint located?

At the end of G1, just before the onset of S phase (DNA replication)

What options does a cell have at the G1 checkpoint?

• Divide

• Delay division

• Exit into a quiescent state (G0)

What molecular events occur when the cell passes the G1 checkpoint?

• Cyclin D–CDK4 phosphorylates Rb

• Cyclin E–CDK2 further phosphorylates Rb

• Phosphorylated Rb releases E2F transcription factor

• E2F activates transcription of S-phase genes

What is the role of p53 at the G1 checkpoint?

• Prevents cells with damaged DNA from entering S phase

• Activates transcription of p21, a cyclin kinase inhibitor (CKI), which inhibits G1 cyclin–CDK complexes

What happens when p53 is mutated?

• Loss of ability to activate p21

• Damaged cells enter S phase

• Contributes to tumor formation

• p53 mutations are found in >50% of human cancers

What happens if DNA replication is incomplete?

The cell will not proceed with mitosis

What triggers the replication stress response?

Single-stranded DNA (ssDNA) accumulation activates ATR and CHK1 kinases

Describe the steps of the DNA replication stress response

1. Recruitment of replication and signaling molecules at forks

2. Activation of ATR–ATRIP–TopBP1 complex

3. Phosphorylation cascade involving CHK1, Rad17, SMC1, and γH2AX

4. Degradation of CDC25 phosphatase

5. CDK1 inhibition → cell cycle arrest

What is the spindle-attachment checkpoint and when does it occur?

Before anaphase, to ensure all chromatids are correctly attached to the mitotic spindle

What structure is monitored at the SAC?

The kinetochore, where spindle microtubules attach to chromatids

How does the SAC prevent premature anaphase?

• Unattached kinetochores inhibit the activity of CDC20–APC (Anaphase Promoting Complex)

• This delays anaphase until all kinetochores are properly attached