Repair of DNA double strand breaks by protein repair machines

Repair of DNA double strand breaks by protein repair machines

1. DNA damage

   • DNA is composed of phosphate backbone and sugar moiety attached to a phosphate
     • Moieties are prone to damage
   • Most damage occurs intracelluarly
     • Replication errors
   • Double strand breaks are the most lethal form of damage
     • Replication cannot proceed if DNA is broken
   • DNA damage can be a result of
     • Cancer
     • Ageing
     • Neurological dysfunction
       • Neuronal cells are particularly sensitive to DNA damage as they can't be replaced

2. Causes of DNA breaks

   • Exogenous
     • Radiation
     • Chemicals
   • Endogenous
     • Oxygen: free radicals
       • Can produce SSB and DSB
     • DNA replication
   • Specialised
     • V(D)J recombination
       • Cell actively produces breaks and sticks them back together to produce antibodies
     • Class switching
       • Changing of one antibody to another
     • Meiosis
   • Most of the time this damage is repaired
   • If there is lots of damage the kinase signalling pathway is activated
     • Apoptosis
     • If the break is incorrectly repaired this causes genome instability resulting in cancer
   • Unrepaired DNA breaks cause translocations
     • The arm of one chromosome is transferred to another
       • Karyotype of an advanced tumour will look very mixed up

3. Accumulation of broken chromosomes is an early marker of uncontrolled cell growth

   • You can grade cells on appearance of DSBs
   • As cells go from normal to cancer cells, major rearrangement occurs
   • Transition from normal to cancer cells is mediated by genome instability

4. Double strand break repair pathways

   • Homologous recombination
     • 2 homologous DNA molecules aligned
       • Despite the high degree of similarity there are small differences such as sequence variants for different alleles
       • Formation of initial short regions of base pairing between the 2 recombining DNA molecules
         • 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
   • Non-homologous end joining (NHEJ)
     • Sequence information is lost from broken ends
     • Original sequence across the break is not faithfully restored during NHEJ
     • 2 ends of broken DNA are joined to each other by misalignment between single strands protruding from both ends
     • Misalignment occurs by pairing between tiny stretches of complementary bases
     • Ku70 and Ku80 form a heterodimer that binds to DNA ends
       • DNA-PKcs are recruited
         • 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
     • Bacteria
       • NHEJ occurs less frequently in bacteria
       • Bacillus subtilis produces a Ku-like protein and DNA ligase when it sporulates and packages proteins into a mature spore
       • Mutant spores lacking these proteins are susceptible to dry heat
         • Known to cause DNA breaks
           • Upon germination, heated mutant spores unable to resume growth as they are unable to rejoin heat induced breaks
       • Spores only have 1 chromosome
         • Cannot rely on sister chromatids
       • Spore chromosome is tightly coiled like a doughnut
         • Holds ends of DNA in close proximity
           • Close juxtaposition could facilitate correct rejoining of ends even if chromosome sustained multiple breaks
       • Ku is a homodimer