Ordered DNA release and target capture in RAG transposition.
Following V(D)J cleavage, the newly liberated DNA signal ends can be either fused together into a signal joint or used as donor DNA in RAG-mediated transposition. We find that both V(D)J cleavage and release of flanking coding DNA occur before the target capture step of transposition can proceed; no coding DNA is ever detected in the target capture complex. Separately from its role in V(D)J cleavage, the DDE motif of the RAG1/2 active site is specifically required for target DNA capture. The requirement for cleavage and release of coding DNA prior to either physical target binding or functional target commitment suggests that the RAG1/2 transposase contains a single binding site for non-RSS DNA that can accommodate either target DNA or coding DNA, but not both together. Perhaps the presence of coding DNA may aid in preventing transpositional resolution of V(D)J recombination intermediates.
Department of Molecular Biology, Harvard Medical School, Massachusetts General Hospital, Boston, MA 02114, USA.
Adam G W
AG
Matthews
Sheryl K
SK
Elkin
Marjorie A
MA
Oettinger
D001665:Binding Sites; D004247:DNA / Q000737:chemistry / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000378:metabolism; D006367:HeLa Cells; D018398:Homeodomain Proteins / Q000378:metabolism*; D006801:Humans; D009687:Nuclear Proteins; D009690:Nucleic Acid Conformation; D011995:Recombination, Genetic
D004268:DNA-Binding Proteins; D018398:Homeodomain Proteins; D009687:Nuclear Proteins; C499311:RAG2 protein, human; C064655:V(D)J recombination activating protein 2; C064658:RAG-1 protein; D004247:DNA