FUNCTIONAL-ANALYSIS OF COORDINATED CLEAVAGE IN V(D)J RECOMBINATION

V(D)J recombination in vivo requires a pair of signals with distinct s pacer elements of 12 and 23 bp that separate conserved heptamer and no namer motifs, Cleavage in vitro by the RAG1 and RAG2 proteins can occu r at individual signals when the reaction buffer contains Mn2+, but cl eavage is restricted to substrates containing two signals when Mg2+ is the divalent cation. By using a novel V(D)J cleavage substrate, we sh ow that while the RAG proteins alone establish a moderate preference f or a 12/23 pair versus a 12/12 pair, a much stricter dependence of cle avage on the 12/23 signal pair is produced by the inclusion of HMG1 an d competitor double-stranded DNA, The competitor DNA serves to inhibit the cleavage of substrates carrying a 12/12 or 23/23 pair, as well as the cutting at individual signals in 12/23 substrates. We show that a 23/33 pair is more efficiently recombined than a 12/33 pair, suggesti ng that the 12/23 rule can be generalized to a requirement for spacers that differ from each other by a single helical turn. Furthermore, we suggest that a fixed spatial orientation of signals is required for c leavage, In general, the same signal variants that can be cleaved sing ly can function under conditions in which a signal pair is required. H owever, a chemically modified substrate with one noncleavable signal e nables us to show that formation of a functional cleavage complex is m echanistically separable from the cleavage reaction itself and that al though cleavage requires a pair of signals, cutting does not have to o ccur simultaneously at both. The implications of these results are dis cussed with respect to the mechanism of V(D)J recombination and the ge neration of chromosomal translocations.