V(D)J Recombination: In Vitro Coding Joint Formation

Abstract

Antigen receptor genes are assembled through a mechanism known as V(D)J recombination, which involves two different joining reactions: signal and coding joining. Formation of these joints is essential for antigen receptor assembly as well as maintaining chromosomal integrity. Here we report on a cell-free system for coding joint formation using deletion and inversion recombination substrates. In vitro coding joint formation requires RAG1, RAG2, and heat-labile factors present in the nuclear extract of nonlymphoid cells. Both inversion- and deletion-mediated coding joint reactions produce diverse coding joints, with deletions and P nucleotide addition. We also show that deletion-mediated coding joint formation follows the 12/23 rule and requires the catalytic subunit of DNA-dependent protein kinase.