Recombination signal sequences restrict chromosomal V(D)J recombination beyond the 12/23 rule

The genes encoding the variable regions of lymphocyte antigen receptors are assembled from variable (V), diversity (D) and joining (J) gene segments(1), V(D)J recombination is initiated by the recombinase activating gene (RAG)-1 and -2 proteins, which introduce DNA double-strand breaks between the V, D and J segments and their flanking recombination signal sequences (RSSs), Generally expressed DNA repair proteins then carry out the joining reaction(2,3), The conserved heptamer and nonamer sequences of the RSSs are separated by non-conserved spacers of 12 or 23 base pairs (forming 12-RSSs and 23-RSSs). The 12/23 rule, which is mediated at the level of RAG-1/2 recognition and cutting(4,5), specifies that V(D)J recombination occurs only between a gene segment flanked by a 12-RSS and one flanked by a 23-RSS1, V beta segments are appended to DJ beta rearrangements, with little or no direct V beta to J beta joining, despite 12/23 compatibility of V beta 23-RSSs and J beta 12-RSSs(6,7). Here we use embryonic stem cells and mice with a modified T-cell receptor (TCR)beta locus containing only one D beta (D beta 1) gene segment and one J beta (J beta 1) gene cluster to show that the 5' D beta 1 12-RSS, but not the J beta 1 12-RSSs, targets rearrangement of a diverse V beta repertoire, This targeting is precise and position-independent. This additional restriction on V(D)J recombination has important implications for the regulation of variable region gene assembly and repertoire development.