12. DNA Replication and Stress

• dna replication has three stages: initiation, elongation and termination
• dna helicase = separates and unwinds the DNA double helix
• rna primase = synthesises RNA primers
• dna polymerase = synthesises DNA
• dna ligase = seals DNA ends together, sticks together Okazaki fragments
• dna topoisomerase = relaxes supercoiling. cuts dna and puts it back together
• not all origins fire at the same time
• heterochromatin is replicated later than euchromatin
• origin firing:

  1. pre-initiation complex in G1 - MCM complex recruited to origin
  2. CDK2 and CDC7 phosphorylate MCM proteins
  3. recruitment of CDC45 and GINS

  GINS, MCM and CDC45 are replicative helicases
• leading strand has one primer, lagging strand has one primer per okazaki fragment
• Pol alpha = RNA primase
• Pol epsilon = synthesises the leading strand
• Pol delta = synthesises the lagging strand
• replication stress = slowing or stalling of replication fork progression and/or DNA synthesis
• replication forks that don't move properly leads to collapse which leads to DSBs
• intracellular obstacles causing replicative stress:

  • DNA secondary structures - G-C rich regions which form G quadruplexes and promote slippase
  • RNA nucleotide incorporation
  • transcription - can collide with replication fork
  • DNA damage
• extracellular obstacles causing replicative stress:

  • UV
  • carcinogens
  • chemotherapies
• DNA fibre method of assaying replicative stress

  1. cells exposed to one nucleotide analogue for a pulse, this is incorporated into replicating strands
  2. cells exposed to another nucleotide analogue for a pulse
  3. cell lysed to release DNA
  4. antibodies to each analogue (different colours) added
• incomplete replication leads to DNA still being attached to the sister chromatid, cannot fully separate creating a lagging chromosome or an anaphase bridge
• lagging chromosome causes chromosomal instability, can have gain or loss of chromosome
• cell can detect issues with mitosis causing a mitotic catastrophe

  • necrosis, apoptosis or senescence
• cancer cells and stem cells have higher replicative stress
• replication stress activates ATR and ATM

  • gamma H2AX becomes phosphorylated
  • CHK1 and CHK2 are phosphorylated
  • DNA gets repaired via homologous recombination