Structural gymnastics of RAG-mediated DNA cleavage in V(D)J recombination

A hallmark of vertebrate immunity is the diverse repertoire of antigen-receptor genes that results from combinatorial splicing of gene coding segments by V(D)J recombination. The (RAG1-RAG2)₂ endonuclease complex (RAG) specifically recognizes and cleaves a pair of recombination signal sequences (RSSs), 12-RSS and 23-RSS, via the catalytic steps of nicking and hairpin formation. Both RSSs immediately flank the coding end segments and are composed of a conserved heptamer, a conserved nonamer, and a non-conserved spacer of either 12 base pairs (bp) or 23 bp in between. A single RAG complex only synapses a 12-RSS and a 23-RSS, which was denoted the 12/23 rule, a dogma that ensures recombination between V, D and J segments, but not within the same type of segments. This review recapitulates current structural studies to highlight the conformational transformations in both the RAG complex and the RSS during the consecutive steps of catalysis. The emerging structural mechanism emphasizes distortion of intact RSS and nicked RSS exerted by a piston-like motion in RAG1 and by dimer closure, respectively. Bipartite recognition of heptamer and nonamer, flexibly linked nonamer-binding domain dimer relatively to the heptamer recognition region dimer, and RSS plasticity and bending by HMGB1 together contribute to the molecular basis of the 12/23 rule in the RAG molecular machine.