CODING JOINT FORMATION IN A CELL-FREE V(D)J RECOMBINATION SYSTEM

V(D)J recombination assembles the variable portion of antigen receptor genes in developing lymphocytes and is the only site-specific recombi nation reaction known in vertebrates. A cell-free system has been esta blished that performs DNA cleavage, end processing, and joining to yie ld V(D)J coding joints that exhibit structural features similar to tho se formed in vivo. The reaction has the expected substrate, metal ion, and RAG protein requirements. The efficiency of coding joint formatio n is reduced dramatically by uncoupling the cleavage and joining porti ons of the reaction, indicating that a postcleavage coding end complex facilitates joining. By varying the reaction conditions, nucleotide l oss from coding ends and heterogeneity of coding joints can be regulat ed. This cell-free system provides a novel tool for detailed mechanist ic analyses of the end processing and joining steps of V(D)J recombina tion.