Question

RAG-1,2 have bound two gene segments (V and J) that ultimately will be recombined. In the space below, briefly list all steps (be specific) required for recombination - stop at endonuclease cutting.

Answer 1

RAG-1,2 are the recombination activating genes.Their main role is to rearrange and recombine the two genes.

these two encodes for the genes which are responsible for the generation of B cells and T cells. this shuffling, cutting, recombinating of genes is known as V(D)J recombination.

V(D)J recombination is a genetic recombination occurs is lymphocytes(early developmental stages of B and T cells)

this process starts with the activity of RAG enzymes : causes the cleavage of dsDNA between the antigen receptor coding segment and the Recombinant signal segment (RSS). it is done in two steps:

1. forming a "nick" in 5' end of RSS heptamer and leaving a 3'-OH at the coding end and a 5' phosphate at RSS end.
2. binding of 0H group and PO4- group.
3. the reactivated OH group will attack the phosphodiester bond of the opposite strand
4. the formation of two DNA strands, a hairpin loop segment on the coding segment
5. a blunt end is formed at the signal segment
6. binding of blunt signals will cause the formation a the circular DNA containing all the intervening segments between the coding segments known as Signal joints.
7. further processing of the coding ends by binding of DNA-PK to each broken DNA end forming a complex that will cause its autophosphorylation, activating Artemis (protein, opens coding and hairpin)
8. alignment of DNA ends and the recruitment of TdT (deoxynucleotidyl transferase which adds non templated (N) nucleotides to the coding end is done by the proteins, XRCC4, DNA-PK, Cernunnos etc.
9. removal of bases from the coding ends by exonucleases