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Direct binding of TReP‐132 with TdT results in reduction of TdT activity
Author(s) -
Fujisaki Seiichiro,
Sato Asami,
Toyomoto Tsuguri,
Hayano Takahide,
Sugai Maki,
Kubota Takashi,
Koiwai Osamu
Publication year - 2006
Publication title -
genes to cells
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.912
H-Index - 115
eISSN - 1365-2443
pISSN - 1356-9597
DOI - 10.1111/j.1365-2443.2005.00916.x
Subject(s) - biology , microbiology and biotechnology , primer (cosmetics) , ku70 , immunoprecipitation , dna , gene , biochemistry , dna damage , chemistry , organic chemistry
N regions at the junction of V, D and J DNA segments are synthesized with large protein complexes including terminal deoxynucleotidyltransferase (TdT) during V(D)J recombination in B‐ or T‐cells. TdT directly binds to TdIF1, TdIF2, PCNA and the Ku70/86 heterodimer. Using a yeast two‐hybrid system, we isolated a cDNA clone encoding the gene for TReP‐132, which is involved in P450scc gene expression in steroid‐hormone‐producing cells or lymphoid cells. Interaction between TReP‐132 and TdIF1 was confirmed by pull‐down assay and immunoprecipitation assay using specific antibodies against TReP‐132 both in vitro and in vivo . TdT also directly bound to TReP‐132 through its confined N‐terminal region. Furthermore, the co‐expression of TdIF1 and TReP‐132 or TdT and TReP‐132 in COS7 cells showed that these proteins are co‐localized within the nucleus. TReP‐132 reduces TdT activity to 2.5% of its maximum value in the in vitro assay system using double‐stranded DNA with a 3′ protrusion as a primer. These findings suggest that TdT synthesizes N region under a negative control of TReP‐132 during V(D)J recombination.