In vitro processing of the 3'-overhanging DNA in the postcleavage complex involved in V(D)J joining |
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Authors: | Nishihara Tadashi Nagawa Fumikiyo Nishizumi Hirofumi Kodama Masami Hirose Satoshi Hayashi Reiko Sakano Hitoshi |
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Institution: | Department of Biophysics and Biochemistry, Graduate School of Science, The University of Tokyo, Bunkyo-ku, Tokyo 113-0032, Japan. |
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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. |
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