Question

What is the basic constraint in recombination dictated by the 12/23 rule? Why does V_kjoin to J_konly, and V_lto J_l only, and V_H to D_H to J_H only?

Answer 1

The adaptive immune response recognizes antigens through T and B lymphocytes antigen receptors. These heterodimeric molecules have a variable antigen-binding domain with variable (V), diversity (D, only present in some loci), and joining (J) gene segments.

Each gene is flanked by a canonical recombination signal sequence (RSS). RSS comprises conserved heptamer and nonamer motifs separated by non-conserved spacer of either 12 or 23 base pair. One RSS is located 3’ to each V gene segment. One RSS is located 5’ to each J gene segment while one RSS is present on both side of each D gene segment. Each Vk (kappa locus) gene is followed by a conserved sequence of one turn 12 bp spacer and preceded by a 23 two-turn spacer. The other conserved V and J sequences are inverted in orientation (12 bp at start and 23 bp at end). The V_l gene segment is preceded by a one turn 12 bp spacer and followed by a 23 bp two-turn spacer. The J_l gene segment is preceded by a one turn 12 bp spacer. V_H and J_H are flanked by 23bp RSS while D_H is flanked on both sides by a 12 bp RSS.

Recombination between 12 and 23 bp RSSs follows the 12/23 rule, which allows proper joining of the V, D, and J gene segments. The 12/23 rule states that only a 12 bp RSS can join a 23 bp RSS during a recombination event. Hence, V_l can join J_l and not another V_H. Similarly, V_H, D_H, and J_H do not join each other and are joined in proper order. Hence, V_K will join J_K, V_l will join J_l,, and V_H joins D_H that joins J_H only.

V(D)J recombination is initiated by a RAG recombinase that makes double stranded cuts in the 12 and 23 RSS sequences creating covalently sealed hairpins at the ends. Deletions and non-template N base addition are introduced before re-ligation of the ends.