DSB repair by protein machines

Nucleases
1. Cutting enzymes
  1. Cleave nucleic acids
2. Exonucleases
  1. Remove terminal nucleotides from 5' and 3'
  2. DNA replication
    1. Polymerase is very accurate and has its own nuclease
      1. It can go back and immediately cut out damage
      2. 3'-5' exonuclease removes mis-incorporated nucleotides
      3. Eukaryotes
        DNA pol delta and gamma
      4. E.coli
        Pol 1
  3. DNA repair and recombination
    1. Damage is recognised and cut out
    2. DNA polymerase fills gap and DNA ligase secures
  4. NHEJ
    1. Own 3' nuclease
    2. If nuclease is left free it will cut DNA and genetic information will be lost
    3. Ku binds DNA to protect ends from nuclease
3. Endonucleases
  1. Cleave internal bonds
4. RNases
5. DNases
6. Can drive reactions forwards or backwards due to their equilibrium
DNA ligase
1. Co factors
  1. Ecoli
    1. NAD+
  2. Eukaryotes
    1. ATP
2. Need 5' P and 3' OH
3. Makes phosphodiester bond
4. Ligase binds ATP
  1. Hydrolyses ATP to AMP
    1. Attaches AMP to itself via a lysine bond
      1. Uses energy to facilitate bond formation
        Ligase transfers phosphate moiety to other side forming ADP
        Enzyme catalyses attack of OH
        AMP released back into solution
5. Ligation assay using gel to follow molecule getting larger
Types of DNA ends
1. Blunt ends
2. Sticky ends
3. Incompatible ends
Non homolgous end joining
1. Bacteria
  1. Ku binds ends of DNA break
    1. Polymerase fills DNA gap bringing ends together
      1. Phosphodiesterase cleaves nucleotides from DNA leaving 3' OH and 5'P
        Ligase repairs DNA nick
  2. If polymerases aren't controlled they will keep synthesising DNA resulting in displacement of original DNA
    1. Nuclease removes unwanted DNA
  3. Bacterial DNA ligases
    1. LigD is ATP dependent DNA ligase
      1. Multidomain protein
        Polymerase, nuclease and ligase
      2. Can add nucleotides to ssDNA or blunt dsDNA in a template independent manner
      3. PolDom can fill in ss gaps to extend 3' resected primers
      4. PolDom inserts nucleotides opposite 8-oxoG and extends base pair
      5. PolDom dislocates and realigns template strand
        Important for abasic sites
      6. Microhomology mediated DSB repair
        DSB with complementary overhangs only needs ligase from LigD as it can be aligned properly
        When ends are not complementary Ku-LigD complex searches for areas of microhomology to form a synpatic complex
        Aligns break, resection, gap filling and ligation
2. Eukaryotes
  1. Ku70/80 heterodimer binds DNA ends
    1. DNA-PKcs recruited and activated
      1. DNA-Pkcs form complex with Artemis
        Artemis is a 5'-3' exonuclease activated by DNA-PKcs
        Processes broken ends and prepares them for ligation
        Ligation carried out by XRCC4/Cernunnos XLF/DNA ligase IV
        Formation of AMP complex called adenylate complex
        AMP moiety transferred to DNA adenylate complex
        LIgase catalyses attack by 3'OH to join the polynucleotides and release AMP
3. Prokaryotes
  1. Ku binds to 3' end and recruits LigD which recognises 5'P
    1. Non-extendable 3' termini can be resected by NucDom of LigD
      1. Resynthesised by PolDom and ligation occurs

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DSB repair by protein machines

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