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Molecular and Cellular Biology, May 2004, p. 3692-3702, Vol. 24, No. 9
0270-7306/04/$08.00+0     DOI: 10.1128/MCB.24.9.3692-3702.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

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

Tadashi Nishihara, Fumikiyo Nagawa, Hirofumi Nishizumi, Masami Kodama, Satoshi Hirose, Reiko Hayashi, and Hitoshi Sakano^*

Department of Biophysics and Biochemistry, Graduate School of Science, The University of Tokyo, Bunkyo-ku, Tokyo 113-0032, Japan

Received 27 October 2003/ Returned for modification 4 December 2003/ Accepted 2 February 2004

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.

Present address: Radiobiology Division, National Cancer Center Research Institute, Tsukiji, Chuo-ku, Tokyo 104-0045, Japan.

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