V(D)J recombination

Question 1

what is V(D)J?

Answer 1

process of somatic DNA recombination that occurs in B and T cells to generate an extensive repertoire of antibodies and T-cell receptors.

Question 2

how does VDJ carry out its function?

Answer 2

by randomly rearranging V (variable) and J (joining) and sometimes D (diversity gene segments).
- generates antigen binding sites that recognise a multitude of diverse antigens needed for adaptive immunity

Question 3

facts for VDJ recombination

Answer 3

• rearrangement of immunoglobulin genes
• ds strands breaks
• generates variation in Ig antigen binding sites
• in SCID mice, lack of mature B and T cells ; VDJ breakage occurs but formation of funcitonal genes is severly impaired

Question 4

antibody structure

Answer 4

two identical heavy and two identical light chains each composed of variable and constant regions

Question 5

what is there at the human heavy chain Ig locus?

Answer 5

• 44 V segments, 23 D segments and 6 J segments
• k-light chain-nV segements, 5J, 1 C
• gamma-light chai-nV, 7J+C

Question 6

how does Ig heavy chain gene rearrangement proceed?

Answer 6

via two DNA recombination processes each involving NHEJ

Question 7

so what does VDJ involve?

Answer 7

• intro of DS breaks at coding signals after a randomly chosen V segment and before a J segment using the RAG recombinase, an enzyme expressed in B and T cells

Question 8

whaat happens to intervening DNA?

Answer 8

removed and DNA ends are made homologous through action of DNA polymerases and nucleases, then ligated together using NHEJ

Question 9

what happens to fused VDJ segments?

Answer 9

can be transcribed by V-segment promoter within range of powerful downstream u enhances, allowing high level expression.
-productive rearrangement rare so B-cells express only one particular heavy chain and one light chain.

Question 10

how does class switching to produce IgG, IgA, and IgE antibodies with same variable region happen?

Answer 10

recombination between switch regions upstream of gene segents that encode heavy chain constant region.
Process initiated by cytidine deaminase, involves ds break at each switch region and processing by NHEJ

Question 11

VDJ recombination mechanism

Answer 11

1. RAG binding at signals
2. synapsis of signals toether
3. nicking
4. hairpinning
5. Ku binds to DNA ends
6. stimulates DNA-PK
7. hairpin opening by artemis
8. pairing of coding ends tofether and signals together
9. nucleotide addition by TdT at all 4 ends; and alignement of terminal microhomologies of coding ends
10. nucleases remove excess DNA and polymerases fill in gaps
11. ligation by XRCC4;DNA ligase IV