V(D)J RECOMBINATION - IN-VITRO CODING JOINT FORMATION

Antigen receptor genes are assembled through a mechanism known as V(D) J recombination, which involves two different joining reactions: signa l and coding joining. Formation of these joints is essential for antig en receptor assembly as well as maintaining chromosomal integrity, Her e we report on a cell-free system for coding joint formation using del etion and inversion recombination substrates. In vitro coding joint fo rmation requires RAG1, RAG2, and heat-labile factors present in the nu clear extract of nonlymphoid cells. Both inversion- and deletion-media ted coding joint reactions produce diverse coding joints, with deletio ns and P nucleotide addition. We also show that deletion-mediated codi ng joint formation follows the 12/23 rule and requires the catalytic s ubunit of DNA-dependent protein kinase.