12. DNA Replication and Stress

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    Evie T

    Cards (23)

    • 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
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