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V(D)J recombination is a complex reaction that likely involves numerous activities. These include recognition of conserved heptamer-spacer-nonamer sequences (RS sequences) that flank germline V, D, or J segments, introduction of site-specific double-strand breaks between the elements to be joined and the RS sequences, potential deletion, with or without addition of nucleotides, at coding junctions, and polymerization and ligation activities [(1) and Fig. 1, A to C]. (Figure 1, A to C omitted) The differential processing of the coding and RS joins is an unusual aspect of V(D)J recombination. Nucleotides are frequently lost from the former but not the latter.
Two genes, recombination activation genes 1 and 2 (RAG-1 and RAG-2), have been identified that, when expressed simultaneously in a nonlymphoid mammalian cell, generate V(D)J recombinase activity (2). The RAG genes either encode or activate the tissue-specific activities necessary for initiation of V(D)J recombination (1). By analogy to site-specific recombination systems of yeast and bacteria, the specific components of V(D)J recombinase may recruit ubiquitously expressed cellular activities to perform certain aspects of the reaction. One such activity may be encoded by the gene affected by the mouse severe combined immune deficient (SCID) mutation (3); homozygous scid mutants are impaired in one of the terminal steps of V(D)J recombination (4) and also have a defect in double-strand DNA break repair (DSBR) in lymphoid and nonlymphoid cells (5).
To test whether or not DNA repair processes and V(D)J recombination share common factors, we assayed a number of mutant Chinese hamster ovary (CHO) cell lines defective in different pathways of DNA repair for the ability to rearrange introduced V(D)J recombination substrates. Cells were transiently transfected with RAG expression vectors driven by a long terminal repeat (LTR) promoter providing the specific V(D)J recombination functions to the nonlymphoid cells and, simultaneously, with extrachromosomal V(D)J recombination substrates that, when recovered and assayed in bacterial cells, permit evaluation of the approximate level and fidelity of V(D)J recombination (6). Two different substrates were used; one (pJH290) allows recovery of coding joins, whereas the other (pJH200) allows recovery of RS joins (7). In each case, V(D)J recombination activity was estimated by the relative amount of rearranged substrate (based on gain of choramphenicol resistance, Cam sup R ) as compared with the total amount of recovered...