INITIATION OF V(D)J RECOMBINATION IN A CELL-FREE SYSTEM

Cells performing V(D)J recombination make specific cuts in DNA at reco mbination signal sequences. Here, we show that nuclear extracts of pre -a cell lines carry out this specific cleavage. The products of cleava ge are the same as found previously in thymocytes: full-length, blunt, 5'-phosphorylated signal ends, and covalently sealed (hairpin) coding ends. A complete signal sequence is required. Recombinant RAG1 protei n greatly increases activity and complements an inactive extract from a RAG1 (-/-) pre-a cell line. When the extracts are fractionated, clea vage activity correlates with the presence of RAG2 protein. These resu lts suggest that RAG1 and RAG2 are components of the V(D)J recombinase .