title: The nicking step in V(D)J recombination is independent of synapsis: implications for the immune repertoire.
abstract: In all of the transposition reactions that have been characterized thus far, synapsis of two transposon ends is required before any catalytic steps (strand 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 nicking, even though hairpinning is affected in a manner reflecting its known synaptic requirement. We also find that nicking is kinetically a unireactant enzyme-catalyzed reaction. Time courses are no different between nicking seen for a 12-substrate alone and a reaction involving both a 12- and a 23-substrate. Hence, synapsis is neither a requirement nor an effector of the rate of nicking. These results establish V(D)J recombination as the first example of a DNA transposition-type reaction in which catalytic steps begin prior to synapsis, and the results have direct implications for the order of the steps in V(D)J recombination, for the contribution of V(D)J recombination nicks to genomic instability, and for the diversification of the immune repertoire.
authors.affiliation: Norris Comprehensive Cancer Center and Departments of Pathology, University of Southern California School of Medicine, Los Angeles, CA 90089-9176, USA.
authors.forename: K
authors.initials: K
authors.lastname: Yu
authors.forename: M R
authors.initials: MR
authors.lastname: Lieber
mesh_terms: D000818:Animals; D002384:Catalysis; D002460:Cell Line; D004251:DNA Transposable Elements / Q000235:genetics; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000235:genetics; D004591:Electrophoresis, Polyacrylamide Gel; D016654:Genes, RAG-1 / Q000235:genetics; D005982:Glutathione Transferase / Q000378:metabolism; D006609:High Mobility Group Proteins / Q000235:genetics / Q000378:metabolism; D006801:Humans; D007700:Kinetics; D051379:Mice; D009687:Nuclear Proteins; D009841:Oligonucleotides / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D011995:Recombination, Genetic; D019809:Streptavidin / Q000378:metabolism; D013997:Time Factors; D019895:Transposases / Q000235:genetics / Q000378:metabolism
chemical_list: D004251:DNA Transposable Elements; D004268:DNA-Binding Proteins; D006609:High Mobility Group Proteins; D009687:Nuclear Proteins; D009841:Oligonucleotides; C499311:RAG2 protein, human; C499308:Rag2 protein, mouse; D011993:Recombinant Fusion Proteins; C064655:V(D)J recombination activating protein 2; D019809:Streptavidin; D005982:Glutathione Transferase; D019895:Transposases