DOUBLE-STRAND SIGNAL SEQUENCE BREAKS IN V(D)J RECOMBINATION ARE BLUNT, 5'-PHOSPHORYLATED, RAG-DEPENDENT, AND CELL-CYCLE-REGULATED

Immunoglobulin and T-cell receptor genes are assembled during lymphocy te development by a novel, highly regulated series of gene rearrangeme nt reactions known as V(D)J recombination. All rearranging loci are fl anked by conserved heptamer-nonamer recombination signal sequences. Ge ne rearrangement results in the imprecise fusion of coding sequences a nd the precise fusion of signal sequences. DNA molecules with double-s tranded breaks near signal sequences have been detected in cells under going V(D)J recombination of the TCRdelta locus. We have devised a lig ation-mediated PCR assay that detects broken-ended molecules in purifi ed genomic DNA. Using this assay we found that DNA breaks occurring pr ecisely at the signal sequence-coding sequence junction are a general feature of V(D)J recombination, appearing in association with each typ e of rearranging immunoglobulin gene segment. We show that a significa nt fraction of these broken ends are blunt and 5'-phosphorylated. In a ddition, detection of these broken-ended signal sequences is dependent on the activity of RAG-1 and RAG-2, and is restricted to the G0/G1 ph ase of the cell cycle. The pattern of broken-ended molecules detected in cells at various stages of development reflects the activity of the V(D)J recombinase at different loci during B- and T-cell development.