GER lecture 5 and 6

• NHEJ vs HR

  HR uses a template strand, whereas NHEJ does not (i.e. it’s non-homologous).

  NHEJ is not 100% accurate because we digest the broken ends to create compatibility.

  In HR, DNA is repaired accurately and is error-free.

  Both pathways are crucial for accurate DNA repair and errors in these pathways are often seen in cancer.

  Homologous recombination + CRISPR allow gene editing.
• Homologous DNA

  • Nearly identical pieces of DNA
• Recombination

  Changing the combination of the strands between the DNA duplexes
• Homologous recombination


  1. Strand is damaged with a double strand break

  2. Proteins associated with the broken DNA need to recognise equivalent bases on the other DNA strand to properly align the 2 DNA duplexes – via a "D-loop"

  3. End resection – generates long free single stranded 3' ends

  4. Homology search and strand invasion

  5. Extension of 3' end

  6. Strands anneal

  7. DNA synthesis is complete and then the strands re-align

  8. Covalently closed double Holliday Junction

  9. Seal the nicks + resolution of the Holliday Junctions
• End resection

  Nuclease degrades at the 5' end, forms long ssDNA at the 3' end that can form H-bonds with other DNA
• Homology search and strand invasion

  Formation of "D-loop" (DNA loop – analogous to 'R-loop' in CRISPR), Strand invasion by the first 3' end – looks for complementary sequence
• Extension of 3' end

  DNA polymerase extends the 3' end – replaces the DNA that was lost during end resection
• Strands anneal

  The other free 3' end anneals to the unbroken strand that was displaced by the D-loop, The 2nd free 3' end is extended by DNA polymerase
• Seal the nicks + resolution of the Holliday Junctions

  DNA ligase seals the nicks on the DNA backbone – but now the 2 duplexes are covalently joined, Nucleases cleave the junctions to separate the 2 DNA molecules, Ligase seals the nicks where nucleases cut
• Branch migration

  Requires motor protein + ATP
• Holliday Junction resolution

  Each Holliday Junction is cut independently – there’s no communication between the nucleases.

  Nucleases must cut identical strands.
• Non-crossover products

  Holliday Junction Resolution – cut both Junctions in the same direction.

  All repair activity is restricted to the middle.
• Crossover products

  Holliday Junction resolution – cuts the Junctions in different directions.

  This can happen because cutting machinery don’t know what each other are doing (i.e. independent).

  • Randomly cut vertically or horizontally

  Crossover/splice products
• Stages of a diploid cell

  G1 or G0 phase

  We have two copies of chromosome 1 

  • 1 from dad and 1 from mom

  The two copies of chromosome 1 are homologues

  • Similar but not identical

  S or G2 phase (when HR happens)

  Now we have four copies of chromosome 1

  • 2 from maternal chromosome 1
  • 2 from paternal chromosome 1

  Sister chromatid = two copies of the same chromosome 1

  Sister chromatids are identical

  • Unless there was an error in replication
• The template affects the outcome of homologous recombination repair:

  • Homologous chromosomes may have slight differences in alleles
  • When used as a template, it might shuffle the genome and recombine bits of chromosomes
  • May or may not repair the DNA back to its original form
  • Sister chromatids are always identical
  • When used as a template, the DNA is repaired back to its original form (100%) because even if there is crossover, the alleles are identical so it doesn’t matter
• HR in bacteria

  Although bacteria are usually haploid, when they are undergoing division they must replicate their genome.

  The newly made copy of the genes can be used as a template for homologous recombination repair.

  Since bacteria divide so quickly, they have 2 copies of their genome for most of their lifetime.
• HR + CRISPR for gene editing

  Normal homologous recombination repair:

  Add a new gene between the complementary genes.

  The new gene will be inserted into the chromosome during HR repair.
• RecA


  Recombinase A enzyme
• RecA

  • Catalyses the exchange of genetic material between 2 DNA molecules in an ATP-dependent manner
  • Stabilises interactions between ssDNA and dsDNA to facilitate 3' strand invasion
• RacA

  • Causes reversible distortion of dsDNA and triggers base flipping to be tested for homology
  • Extended region of sequence matching stabilises the complex allows strand exchange
• Primary binding site


  Binds ssDNA with higher affinity than dsDNA
• 1 RecA monomer

  2 binding sites
• RecA is ATP-dependent but not sequence-specific
• Step 1: RecA coats ssDNA

  1. RecA forms filaments on ssDNA

  2. Binds 3nt per RecA primary binding site

  3. 3nt ≠ 1 codon

  4. Rec A stretches DNA – we can see that the spacing between the 3nt in the binding sites is wider than the spacing between adjacent nt
• Step 2: Secondary binding site binds dsDNA repair template

  1. dsDNA is sampled randomly through base flipping and H-bonding to find a region of homology (i.e. homology search)

  2. Sampling events happen throughout the dsDNA but it keeps falling off the binding site (hence the lower affinity) until it finds the correct homologous sequence
• Step 3: If RecA samples the correct homology region

  1. The bases stay 'flipped' and remain H-bound to the single stranded 3' strand

  2. Facilitates 3' strand invasion

  3. If not, the complex is not stable enough and collapses to try again in a different region (due to the weak binding of dsDNA)
• HR key enzymes (from E. coli)

  RuvAB complex is important for Holliday Junction migration.

  • RuvA is a Junction-specific binding protein  
  • RuvB is an ATP-dependent dsDNA pump
  • i.e. rotor motor protein
  • Homoduplex DNA is drawn to the middle and comes out as heteroduplex DNA through the top and bottom
  • Also acts as a scaffolding to RuvC
• HR key enzymes (from E. coli)

  RuvC is important for Holliday Junction resolution.

  • Is an endonuclease specific to Holliday Junctions
  • Binds as a dimer on each HJ
  • Always cuts identical strands (in either direction)
  • Cleaves at 5’- A/T TT G/C -3’ (weak specificity)
• Replication fork restart

  If replication fork collapses, it needs to restart.

  • If DNA polymerase hits a nick, it makes it a big problem and strands fall apart
  • Leads to a single-end dsDNA break

  To restart, we do everything again but only once.

  Start with DNA end processing to generate compatible 3’ ends.

  RecA coats ssDNA for 3’ strand invasion.

  But there’s no 2nd strand capture.
• Homologous recombination in meiosis

  Meiosis uses programmed genome instability and HR to increase genetic diversity.

  Chromosomal crossover happens due to deliberate HR.

  • Triggered by a nuclease

  Programmed HR increases genetic diversity by generating chromosomes with new combinations of maternal and paternal alleles.

  • Must happen between homologous chromosomes
  • 1 from mother and 1 from father
  • Chromosomes cannot be identical (no new allele combinations)
• Crossover products are favoured after HR in meiotic recombination because they lead to the reassortment of large blocks of alleles.
• Programmed dsDNA breaks in meiosis

  Spo11 is the nuclease that cleaves DNA and triggers homologous recombination.

  • Functions as a dimer
  • Non-sequence specific but it’s more prone to cleave where DNA is not tightly packaged
  • Spo11 carries out a transesterification reaction
  • Forms covalent bond between Spo11 active site and 5’ phosphate group on DNA backbone
  • Spo11 cuts DNA and remains bound to it
• Generation of ssDNA 3’ ends in meiosis

  • MRX nuclease generates the 3’ ssDNA ends
  • MRX first cuts away the covalently bound Spo11 protein
  • Then, it degrades the strand that has a 5’ end at the break
  • The 3’ end involved in homology search corresponds to the Spo11 cut site
• Importance of heteroduplexes during HR in meiosis

  Leads to changes in the DNA sequence at sites adjacent to the break.

  Regions of heteroduplex DNA are not exactly the same – characterised by mismatches.

  Holliday Junction migration creates regions of heteroduplex DNA that are thousands of bp long.

  Mismatch repair leads to loss of heterozygosity (LOH) – where “a daughter cell becomes homozygous or hemizygous for one or more alleles through mitotic recombination or gene conversion.”