Initiation of V(D)J Recombination by Db-Associated Recombination Signal Sequences: A Critical Control Point

T cell receptor (TCR) b gene assembly by V(D)J recombination proceeds via successive Db-to-Jb and Vb-to-DJb rearrangements. This two-step process is enforced by a constraint, termed beyond (B)12/23, which prohibits direct Vb-to-Jb rearrangements. However the B12/23 restriction does not explain the order of TCRb assembly for which the regulation remains an unresolved issue. The initiation of V(D)J recombination consists of the introduction of single-strand DNA nicks at recombination signal sequences (RSSs) containing a 12 base-pairs spacer. An RSS containing a 23 base-pairs spacer is then captured to form a 12/23 RSSs synapse leading to coupled DNA cleavage. Herein, we probed RSS nicks at the TCRb locus and found that nicks were only detectable at Db-associated RSSs. This pattern implies that Db 12RSS and, unexpectedly, Db 23RSS initiate V(D)J recombination and capture their respective Vb or Jb RSS partner. Using both in vitro and in vivo assays, we further demonstrate that the Db1 23RSS impedes cleavage at the adjacent Db1 12RSS and consequently Vb-to-Db1 rearrangement first requires the Db1 23RSS excision. Altogether, our results provide the molecular explanation to the B12/ 23 constraint and also uncover a ‘Db1 23RSS-mediated ’ restriction operating beyond chromatin accessibility, which directs Db1 ordered rearrangements