In vitro processing of the 3'-overhanging DNA in the postcleavage complex involved in V(D)J joining.
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.
Nishihara
Tadashi
T
Department of Biophysics and Biochemistry, Graduate School of Science, The University of Tokyo, Bunkyo-ku, Tokyo 113-0032, Japan.
Nagawa
Fumikiyo
F
Nishizumi
Hirofumi
H
Kodama
Masami
M
Hirose
Satoshi
S
Hayashi
Reiko
R
Sakano
Hitoshi
H
D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D004247:DNA / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D004270:DNA, Circular; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D018398:Homeodomain Proteins / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D007133:Immunoglobulin Joining Region; D046911:Macromolecular Substances; D009687:Nuclear Proteins; D009690:Nucleic Acid Conformation; D021382:Protein Sorting Signals; D011946:Receptors, Antigen / Q000235:genetics* / Q000378:metabolism; D011995:Recombination, Genetic
D004270:DNA, Circular; D004268:DNA-Binding Proteins; D018398:Homeodomain Proteins; D007133:Immunoglobulin Joining Region; D046911:Macromolecular Substances; D009687:Nuclear Proteins; D021382:Protein Sorting Signals; C499311:RAG2 protein, human; D011946:Receptors, Antigen; C064655:V(D)J recombination activating protein 2; C064658:RAG-1 protein; D004247:DNA