The nicking step in V(D)J recombination is independent of synapsis: Implications for the immune repertoire

In all of the transposition reactions that have been characterized thus far , synapsis of mo transposon ends is required before any catalytic steps (st rand nicking or strand transfer) occur. In V(D)J recombination, there have been inconclusive data concerning the role of synapsis in nicking, Synapsis between two 12-substrates or between two 23-substrates has not been ruled out in any studies thus far. Here we provide the first direct tests of this issue. We find that immobilization of signals does not affect their nickin g, even though hairpinning is affected in a manner reflecting its known syn aptic requirement, We also find that nicking is kinetically a unireactant e nzyme-catalyzed reaction. Time courses are no different between nicking see n for a 12-substrate alone and a reaction involving both a 12- and a 23-sub strate. Hence, synapsis is neither a requirement nor an effector of the rat e of nicking. These results establish V(D)J recombination as the first exam ple of a DNA transposition-type reaction in which catalytic steps begin pri or to synapsis, and the results have direct implications for the order of t he steps in V(D)J recombination, for the contribution of V(D)J recombinatio n nicks to genomic instability, and for the diversification of the immune r epertoire.