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

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.

We are grateful to D. G. Schatz for providing us with the expression vectors for RAG proteins. We also thank Hitomi Sakano for critical reading of the manuscript and Takeshi Imai for discussion.

This work was supported by grants from Japan Science and Technology Corporation, the Ministry of Education, Culture and Science, and the Mitsubishi Foundation.