Repair of DNA double strand breaks by protein repair machines

DNA damage
1. DNA is composed of phosphate backbone and sugar moiety attached to a phosphate
  1. Moieties are prone to damage
2. Most damage occurs intracelluarly
  1. Replication errors
3. Double strand breaks are the most lethal form of damage
  1. Replication cannot proceed if DNA is broken
4. DNA damage can be a result of
  1. Cancer
  2. Ageing
  3. Neurological dysfunction
    1. Neuronal cells are particularly sensitive to DNA damage as they can't be replaced
Causes of DNA breaks
1. Exogenous
  1. Radiation
  2. Chemicals
2. Endogenous
  1. Oxygen: free radicals
    1. Can produce SSB and DSB
  2. DNA replication
3. Specialised
  1. V(D)J recombination
    1. Cell actively produces breaks and sticks them back together to produce antibodies
  2. Class switching
    1. Changing of one antibody to another
  3. Meiosis
4. Most of the time this damage is repaired
5. If there is lots of damage the kinase signalling pathway is activated
  1. Apoptosis
  2. If the break is incorrectly repaired this causes genome instability resulting in cancer
6. Unrepaired DNA breaks cause translocations
  1. The arm of one chromosome is transferred to another
    1. Karyotype of an advanced tumour will look very mixed up
Accumulation of broken chromosomes is an early marker of uncontrolled cell growth
1. You can grade cells on appearance of DSBs
2. As cells go from normal to cancer cells, major rearrangement occurs
3. Transition from normal to cancer cells is mediated by genome instability
Double strand break repair pathways
1. Homologous recombination
  1. 2 homologous DNA molecules aligned
    1. Despite the high degree of similarity there are small differences such as sequence variants for different alleles
    2. Formation of initial short regions of base pairing between the 2 recombining DNA molecules
      1. Strand invasion
        Single strand region of DNA from parental molecule pairs with complementary strand on homologous duplex DNA molecule
        Process regions of new duplex DNA are generated- heteroduplex DNA
        Holliday junction formation
        2 DNA molecules become connected by crossing DNA strands
        Branch migration
        Holliday junction can move along DNA by repeated melting and formation of base pairs
        Identical base pairs are formed in the recombination intermediate
        Cleavage of Holliday junction: resolution
        Cutting DNA strands in Holliday junction regenerates 2 separate duplex DNA molecules and finishes genetic exchange
        The DNA strands cut has a large impact on the extent of DNA exchange that occurs between the 2 recombining molecules
2. Non-homologous end joining (NHEJ)
  1. Sequence information is lost from broken ends
  2. Original sequence across the break is not faithfully restored during NHEJ
  3. 2 ends of broken DNA are joined to each other by misalignment between single strands protruding from both ends
  4. Misalignment occurs by pairing between tiny stretches of complementary bases
  5. Ku70 and Ku80 form a heterodimer that binds to DNA ends
    1. DNA-PKcs are recruited
      1. DNA PKcs form a complex with Artemis
        Artemis is a 5'-3' exonuclease and latent endonuclease that is activated by phosphorylation of DNA-PKcs
        These nucleolytic activities process broken ends and prepare them for ligation
        Ligation is carried out by ligase IV/XRCC4/Cernunnos-XLF
  6. Bacteria
    1. NHEJ occurs less frequently in bacteria
    2. Bacillus subtilis produces a Ku-like protein and DNA ligase when it sporulates and packages proteins into a mature spore
    3. Mutant spores lacking these proteins are susceptible to dry heat
      1. Known to cause DNA breaks
        Upon germination, heated mutant spores unable to resume growth as they are unable to rejoin heat induced breaks
    4. Spores only have 1 chromosome
      1. Cannot rely on sister chromatids
    5. Spore chromosome is tightly coiled like a doughnut
      1. Holds ends of DNA in close proximity
        Close juxtaposition could facilitate correct rejoining of ends even if chromosome sustained multiple breaks
    6. Ku is a homodimer

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Repair of DNA double strand breaks by protein repair machines

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