Direct binding of TReP-132 with TdT results in reduction of TdT activity

Abstract

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 Bor 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. © 2005 The Author(s) Journal Compilation © 2005 by the Molecular Biology Society of Japan/Blackwell Publishing Ltd.