V(D)J Recombination Flashcards
What is the purpose of V(D)J in its simplist form?
- A process to generate antigen receptor diversity
- One of the few processes where the cell deliberately permanently alters DNA - in antigen receptor (B and T cells)
How do CD4+ T cells and CD8+ T cells differ in their response?
CD4 + T cells:
- CD4+ T helper cell binds MHC 2-antigen complex on APC (antigen presenting cell) - both APC and T cell release cytokines + T cell clones itself
- Clones T cells produce diff cytokines that activate B cells and CD8+ cells
CD8+ T cells:
- Cytotoxic T cell interacts with MHC-1 epitope complex on infected cell - it produces granzymes & perforins
- Perforins form pores in plasma membrane
- Granzymes enter cell and break down proteins - lysing the cell
Outline of the structure on an antibody
C region - heavy chain (constant & variable regions)
V region - light chain + antigen binding region (constant & variable regions)
- Have no diversity (D) region
- Hypervariable regions on both chains - generate high level of antigen specificity
How is the variable region generated?
- By joining individual gene segments in the V(D)J recombination reaction
- Any heavy chain can recombine with any light chain - required altering of antigen-binding pocket
- This generates new antibody genes - antigen diversity
What is the function of the RAG proteins?
- RAG1 & RAG2 - proteins that recognise recombination signal sequence (RSSs)
- Need 2 molecules of RAG1/RAG2 - to bind RSS
- RAG1 - active region in C-terminal core (active site DDE motif)
- RAG2 - active region in N-terminus core (allosterically) - C-terminus has PHD finger that is critical for chromatin binding
Process of V(D)J recombination
- RAGs bind the RSSs and bring segments to be recombined at synaptic complex
- RAG1 causes nick on one strand; the resulting 3’OH attacks the opposite strand - direct transesterfication reaction
- Creates hairpin structrue at coding ends; a blunt DSB at the signal ends
- Coding ends and signal ends are processed differently
How are coding joints processed?
Coding Joints: - Hypervariable region 3
- Ku70:Ku80 binds DNA ends
- DNA-Pk: Artemis asymmetrically opens hairpin (SS nick) - generating palindromic P-nucleotides (reverse in complement nucleotides)
- N-Nucleotides are added by TdT onto ends of DNA (random - increasing diversity) ~20 nucleotides
- Strands are paired and unpaired nucleotides are removed by exonuclease
- Gaps are filled by DNA synthesis; ligation - to form coding join - adds to hypervariable region 3
- Use NHEJ - but due to error prone nature - coding regions often loose or gain nucleotides
How are signal joints processed?
Signal Joints:
- Ku70:Ku80 binds DNA ends
- DNA ligase IV: XRCC4 ligates DNA ends = precise signal joint
- Use non-homologous end joining (NHEJ)
When is TdT only expressed?
- In pro B-cells and early T cells
What are the different stages where diversity can be generated?
- Combinatorial joining of gene segments - V(D)J
- Junctional diversification during gene segment joining - N and P nucleotides
- Combinatorial joining of L and H chains
- Somatic hypermutation + class switch recombination
What chain are N nucleotides added more to?
Heavy chain
How are heavy chains IgM and IgD expressed by naive B cells?
- Heavy chains IgM and IgD are expressed by naive B cells via alternative splicing
- Alternatice splicing - is RNA - moves introns in constant region
How is antigen receptor diversity obtained in a pro-B cell, and what stages does it go through to become a mature naive B cell?
- Pro-B cell - first - productive rearrangement of heavy chain - increases heavy-light chain combinations you can make = diversity (V(D)J)
- Pre-B cell
- Immature naive B cell
- Mature naive B cell - circulation through lymph organs
How do the different immunoglobulins differ?
- They differ in their interactions with complement and Fc receptors
- They differ in their locations and ability to other cells in immune system
How can you switch to IgG, IgE or IgA?
- Requires deletion of intervening DNA via class switch recombination
Briefly explain the primary and secondary responses of B cells and where Class Switch Recombination (CSR) and Somatic Hypermutation (SHM) occur?
- Stem cells in bone marrow (Ig V(D)J gene rearrangement)
- Stimulation of B cells (with antigens) via T cell signalling
Primary response - maturation process (in germinal centres of lymph nodes) - clonal expansion - CSR & SHM
- CSR - swap heavy chain constant region - doesnt alter affinity of antibody or antigen
- SHM - Iterative process - more and more mutations until highest affinity binding
- Plasma cells - secretes antibodies
Secondary Response - clonal expansion & more CSR & SHM
Class Switch Recombination (CSR) - Process
- Transcription through switch region initiated - by promoter upstream
- AID modifies DNA in switch region - allowing class switching (AID makes G-U mismatches - deamination)
- UNG (removes uracil - abasic) and APE1 (excises ribose - ss nicks) recognise G-U mismatches introduce cluster SS nicks on both DNA strands
- DNA-Pk and other repair proteins act to intiate DSBR
- DSBR machinery joins the two switch regions - and excises intervening sequences
- The selectred constant region is now located adjacent to the V(D)J region - crossing over
Where does somatic hypermutation predominantly generate antigen diversity?
In the hypervariable regions
How do SHM and CSR compare?
CSR:
- Requires DSBs
- High AID-density
- Excise large block of DNA
SHM:
- SSB is probably sufficient for mutation
- Low depedence of AID-density fusions
- Point mutation - excise small amount of DNA
What 3 different ways can SHM occur once AID has nicked?
SHM occurs in mature B cells:
1. Replication over G-U mismatch (causes G-A transition)
2. Uracil DNA-glycosylase - creates abasic site - error prone polymerase inserts random bases opposite abasic site
3. Excise small block of DNA around G-U mismatch - recuritment of MMR elements leads to insertion of mutations - preferentially at A:T
How is AID used in SHM?
- Used only to get mutation in variable exon - not constant region (directed towards variable exon via transcription factors)
- AID deaminates cytidine to uridine (without sugars attached)
Why do you get more DSBs in switch regions?
- G rich switch regions create R loops - so takes longer
- Therefore more likely to get AID activity - and get DSBs
What is the key similarities/differences between B and T cell receptors?
Similarities:
- T cell receptors have binding site similar to antibody
- Both use V(D)J recombination - T cells use (12/23 rule)
- Same RAG proteins involved
Differences:
- T cell receptors are ALWAYS membrane-bound
- T cell receptors have a single antigen binding site
- T cell receptors recognise antigen in conjunction with MHC molecules
- T cell receptors do not undergo SHM or CSR
- In T cell receptors - much more diversity lies in hypervariable region 3 - and is generated via elevated junctional diversity and the larger number of J gene segments
How is RAG cutting regulated?
RAG1/2 recognise the same RSS’s in both B and T cells
- RSS’s are only made accessible to RAGs in correct cell type
- RSS’s are only made accessible to RAGs at correct stage of lymphocyte development
- RSS’s are only made accessible to one of the two alleles
