(recombination activating protein RAG1yrecombination activating protein RAG2)

The first step of V(D)J recombination, specific cleavage at the recombination signal sequence (RSS), can be carried out by the recombination activating proteins RAG1 and RAG2. In vivo, the cleaved coding and signal ends must be rejoined to generate functional antigen receptors and maintain chromosomal integrity. We have investigated signal joint formation using deletion and inversion substrates in a cell free system. RAG1 and RAG2 alone or in combination were unable to generate signal joints. However, RAG1 and RAG2 complemented with nuclear extracts were able to recombine an extrachromosomal substrate and form precise signal joints. The in vitro reaction resembled authentic V(D)J recombination in being Ku-antigen-dependent. Lymphocyte antigen receptors are encoded by multiple copies of gene segments in germ-line DNA. During Band T-lymphocyte development, these gene segments are assembled into functional transcription units by the mechanism of V(D)J recombination. The DNA sequence requirements for V(D)J recombination consist of highly conserved heptamer and nonamer DNA motifs separated by a spacer of 12 or 23 base pairs (12 RSS and 23 RSS, where RSS is recombination signal sequence). During the V(D)J recombination reaction, two types of DNA joints are formed: signal joints, generally involving precise head-to-head ligation of two heptamers, and coding joints, usually containing deletions or additions of a few nucleotides (1). Several lymphoid-specific factors are known to be involved in V(D)J recombination. These include terminal deoxynucleotidyltransferase (2–4) and the recombination activating proteins RAG1 and RAG2 (5–7). RAG1 and RAG2 are sufficient for the formation of specific double-strand DNA breaks at RSSs (8, 9). Efficient recombination occurs almost exclusively between RSSs with different spacers. This restriction is known as the 12y23 rule and is evident at the initial cleavage event (10–12). The cleavage reaction involves the formation of hairpin loops at the coding ends and precise double-strand breaks at the signal ends (8, 9, 13–16). RAG1 and RAG2 can be coimmunoprecipitated from cells, suggesting that they function as part of a complex during V(D)J recombination (17, 18). Other proteins that are not restricted in their pattern of expression to lymphoid cells are also required for V(D)J recombination. These include two components of the DNAdependent protein kinase, the p86 subunit of the p70yp86 Ku antigen (product of XRCC5), and the large catalytic subunit (product of SCID), as well as the XRCC4 protein. These proteins are involved in DNA repair as well as in V(D)J recombination (19, 20). In addition other as yet unidentified factors may be required for antigen receptor assembly. Herein we report an in vitro system in which RAG1 and RAG2 complemented with a nuclear extract are able to catalyze signal joint formation. This system depends on Ku proteins and may provide the means for identification of the range of factors involved in generation of the signal joint.