GER lecture 4

• DNA is a chemical

  Remember that DNA is a chemical double helix.

  The DNA deoxyribose sugar-phosphate backbone is quite inert.

  • Unlike the ribose sugar in RNA, which has a reactive hydroxyl (OH) group

  DNA is double stranded – you have 2 copies of every gene.

  • If you damage 1 strand, the genetic info can be cross-checked using the other strand
• Excision repair 

  ‘Cuts’ out damaged DNA.

  There are 3 types of excision repair:

  1. Base excision repair
  2. Nucleotide excision repair
  3. Mismatch repair

  General strategy for excision repair:

  1. Find the damage

  2. Cut on both sides of the damage to ‘excise’ a piece of the DNA backbone

  3. Remove the damaged DNA – to be degraded

  4. Copy the undamaged strand to make a patch

  • High fidelity DNA polymerase starts in the 3’ end
  • Uses other strand as a template
  • DNA ligase seals nicks
• Base excision repair (BER)

  Key enzyme: DNA glycosylase (responsible for specificity of repair pathway).

  Everything else is a common abasic site repair pathway.

  Base excision repair is lesion-specific.

  Different types of glycosylase enzymes target different DNA lesions.

  But overall glycosylase enzymes tend to target intrinsic damage – except for UV damage.

  • Needs to be repaired by glycosylases since placental mammals don’t have photolyases
• DNA glycosylases recognise damage through base flipping.

  Bases are free to rotate and “flip” out from the DNA double helix and bind to enzyme active site.
• Nucleotide excision repair (NER)

  Can recognise different types of damage – less specific than BER.

  • However, DNA repair needs to be tightly regulated otherwise it could increase mutation rate

  NER mainly works to repair extrinsic DNA damage.
• Nucleotide excision repair in bacteria (simpler model)

  1. ATP-dependent damage location by UvrA2/UvrB2 damage-sensing complex

  2. UvrA and UvrB are ATPases

  3. UvrA senses if DNA can bend

  4. UvrB moves along DNA but its movement is interrupted if there's damage

  5. If the damage is confirmed, UvrA dissociates

  6. Loss of UvrA allows UvrC to bind to UvrB

  7. UvrC cleaves DNA backbone on both sides of damage to excise incorrect base

  8. UvrD helicase (motor) displaces 12-13nt oligo(nucleotide) and UvrC

  9. DNA polymerase fills gap and ligase seals nicks
• UvrA2/UvrB2 damage-sensing complex


  • 2-step damage recognition
• UvrC cleaves DNA backbone on both sides of damage to excise incorrect base, but cuts some extra nucleotides before and after the damage
• dsDNA breaks

  If not fixed properly, you could end up mismatching and joining the wrong chromosomes together…
• Accidental dsDNA breaks:

  • Ionising radiation – could also be used for ionisation cancer therapy (on purpose)
  • DNA-damaging agents
  • Inappropriate endonuclease activity
  • Replication past a DNA nick

  Consequences: extreme chromosome instability, which is potentially lethal for the cell/organism.
• Programmed dsDNA breaks:

  • Meiotic recombination
  • V(D)J recombination

  Consequences: beneficial genetic variation.
• dsDNA breaks can be double- or single-ended depending on the source.

  Double-end dsDNA breaks can be repaired in 2 ways – 1 of which is NHEJ.

  Single-end dsDNA breaks are due to ssDNA break (i.e. nick) and are harder to repair.
• Non-homologous end joining (NHEJ)

  Just glueing broken ends back together.

  1 DNA copy required

  1. End-binding: for recognition and protection (from enzymes that want to chew at DNA ends) of DNA and recruitment of other factors 
  2. End processing enzymes chew at the ends of add new nucleotides until you form regions that are compatible with one another – “microhomology” (1-4 nt) 
  3. Repair: strands anneal, DNA synthesised to fill any gaps, ligation. 

  Repaired, but usually with altered DNA sequence
• NHEJ fixes dsDNA breaks but it likely contains mutations at the point of joining due to end processing.

  • Mutations and deletions are common but you can also see insertions
• NHEJ in the human system:

  Ku70/Ku80 – recognise and protect DNA ends; and act as scaffold for later enzymes

  DNA-PKcs is a kinase that recognises and binds to the broken DNA ends to initiate repair

  Artemis is a nuclease responsible for end processing

  DNA polymerase and ligase fill out and seal the nicks to repair DNA
• NHEJ summary

  • Has no requirement for a homologous donor DNA
  • Can occur at any stage in the cell cycle
  • NHEJ products may contain errors near the break site
  • Can repair double-end DSBs only
  • Is common in eukaryotes but most bacteria lack this pathway 
  • Also plays a role in generation of antibody diversity