In Vitro Processing of the 3′-Overhanging DNA in the Postcleavage Complex Involved in V(D)J Joining

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Abstract
The postcleavage complex involved in V(D)J joining is known to possess a transpositional strand transfer activity, whose physiological role is yet to be clarified. Here we report that RAG1 and RAG2 proteins in the signal end (SE) complex cleave the 3′-overhanging structure of the synthetic coding-end (CE) DNA in two successive steps in vitro. The 3′-overhanging structure is attacked by the SE complex imprecisely, near the double-stranded/single-stranded (ds/ss) junction, and transferred to the SE. The transferred overhang is then resolved and cleaved precisely at the ds/ss junction, generating either the linear or the circular cleavage products. Thus, the blunt-end structure is restored for the SE and variably processed ends are generated for the synthetic CE. This 3′-processing activity is observed not only with the core RAG2 but also with the full-length protein.