GER lecture 7

• How do we make more antibodies than the number of genes in the human genome?

  Because of programmed genome rearrangement in immune cells.

  • VDJ recombination and somatic hypermutation
• Heavy chain:

  1 variable N-terminal domain.

  3 constant domains (towards C-terminus).

  Light chain:

  1 N-terminal variable domain.

  1 C-terminal constant domain.
• Heavy chain DJ recombination – 1st round of DNA rearrangement.

  D and J segment are randomly combined.
• V(DJ) recombination – 2nd round of DNA rearrangement.
• Light chain variable regions only require 1 recombination event because they only have variable and joining segments
• Combinatorial diversity – combining different elements gives diversity

  Combinations of different heavy and light chains

  We have 2 types of light chain, but only 1 of them undergoes recombination and joins the heavy chain.

  • Heavy + λ light
  • Heavy + 𝜅 light

  Increases diversity by around 2-fold

  Junction diversity - trigger dsDNA break at the sequence at the point of joining the VJ or VDJ elements.

  When the break is repaired, it increases diversity.
• dsDNA break and joining requires a RSS sequence (recombination signal sequence).

  There are 2 types of RSS sequences – with 23bp spacer or with 12bp spacer.

  The RSS is recognised by Rag recombinase.
• Junction diversity is a site-specific recombination process – requires specific sequence (RSS).
• RAG1 + RAG2 = Rag endonuclease that recognises the RSS sequences and cuts the DNA.

  Rag endonuclease is only expressed in immune cells.

  Rag endonuclease simultaneously binds 2 RSS sequences following the 12/23 rule.

  • The DNA between the sequences forms a loop
  • Creates a ds break at each of the RSS sequence sites
• RAG1 and RAG2 enzymes use a water molecule as a nucleophile to cut the top and bottom strands of the sequence.

  Nick on the DNA is produced in both the top and bottom strands.

  The nuclease enzymes can use the 3’ OH as a nucleophile to cleave the opposite strands.

  Transesterification reaction covalently attaches the top and bottom strands
• After RAG1+RAG2 DNA cleavage:

  We end up with 2 dsDNA breaks but also with hairpin structures – where the DNA is joined together.

  Then, we need to join the V and J hairpins together to produce the variable domain.

  The intervening genes between the hairpins are joined into a circle and discarded.
• Why does the re-joining of V and J segment hairpins lead to diversity?

  NHEJ proteins introduce nucleotide changes during repair in the hypervariable region.

  You can’t directly ligate the hairpin ends because we don’t have a 5’ phosphate and 3’ OH.

  First we need hairpin opening and then fusion of coding elements – carried out by proteins from NHEJ.
• Somatic hypermutation

  Generates additional diversity.

  • Happens in a narrow window of B-cell development
  • After VJ and VDJ recombination
  • All 4 bases can be targeted for mutation
  • Mutation frequency: variable region > constant region
• Somatic hypermutation is induced by AID – type of cytidine deaminase.

  AID = activation induced deaminase

  AID is specific to immune cell lineage.

  AID is required for somatic hypermutation and antibody class switching (along with UNG).

  • Uracil-DNA glycosylase (UNG) activity influences the pattern of somatic hypermutationn
• Somatic hypermutation

  • AID enzyme recognises C bases
  • When DNA is ss during transcription
  • AID deaminates C to produce uracil (U)
  • C → U DNA damage
  • AID is overexpressed in these cells and produces many mutations
  • Normal repair pathways are overwhelmed
  • Leads to increased number of mutations
• Class switch recombination

  Rearrange VDJ segments of heavy chain with different isotypes of constant region genes.

  Leads to antibody isotypes with different properties – IgM, IgG, IgE, IgA, IgD.

  Variations are due to differences in constant domains of the heavy chain.
• Class switch recombination

  Genes encoding the constant region are downstream of the recombined heavy chain VDJ gene.

  Upstream of the constant region genes are switch sequences where recombination occurs.

  AID binds to the switch regions and deaminates a C residue – creates a mismatch → dsDNA break.

  Ku70/Ku80 binds the broken ends - with other proteins, the gap is sealed and the break is repaired.

  NHEJ joins switch regions quickly but it’s not error-free → gives rise to new antibody isotypes.