Direct binding of TReP-132 with TdT results in reduction of TdT activity.
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.
Fujisaki
Seiichiro
S
Faculty of Science & Technology, Department of Applied Biological Science, Tokyo University of Science, Noda, Chiba 278-8510, Japan.
Sato
Asami
A
Toyomoto
Tsuguri
T
Hayano
Takahide
T
Sugai
Maki
M
Kubota
Takashi
T
Koiwai
Osamu
O
D000818:Animals; D019556:COS Cells; D002352:Carrier Proteins / Q000235:genetics / Q000378:metabolism; D002460:Cell Line; D002522:Chlorocebus aethiops; D004247:DNA / Q000378:metabolism; D004253:DNA Nucleotidylexotransferase / Q000378:metabolism*; D004277:DNA, Single-Stranded / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000032:analysis / Q000378:metabolism*; D015536:Down-Regulation; D009687:Nuclear Proteins / Q000235:genetics / Q000378:metabolism; D013552:Swine; D013950:Thymus Gland / Q000737:chemistry / Q000166:cytology; D014157:Transcription Factors / Q000032:analysis / Q000378:metabolism*; D020798:Two-Hybrid System Techniques
D002352:Carrier Proteins; D004277:DNA, Single-Stranded; D004268:DNA-Binding Proteins; C436745:DNTTIP1 protein, human; D009687:Nuclear Proteins; C436322:TRERF1 protein, human; D014157:Transcription Factors; D004247:DNA; D004253:DNA Nucleotidylexotransferase