CHARACTERIZATION OF BROKEN DNA-MOLECULES ASSOCIATED WITH V(D)J RECOMBINATION

We previously demonstrated that DNA molecules with double-strand breaks at variable-(diversity)-joining [V(D)J] recombination signal sequences are relatively abundant in mouse thymocytes. This abundance strongly suggests that the mechanism of V(D)J recombination involves double-strand cleavage at recombination signals. As a first step toward understanding the mechanism of cleavage, we used a sensitive PCR assay to characterize the structure of one class of cleavage products, the signal ends, in detail. Here we demonstrate that most of these ends are blunt and terminate in 5' phosphoryl groups. Virtually all of the flush signal ends are full length. A minor subpopulation of broken ends terminates in short single-strand extensions. We have found no evidence for covalent DNA-protein linkages involving the signal ends. These data allow further refinement of the double-strand cleavage model for V(D)J recombination.