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Terminal deoxynucleotidyltransferase is negatively regulated by direct interaction with proliferating cell nuclear antigen
Author(s) -
Ibe Shirou,
Fujita Kiyoko,
Toyomoto Tsuguri,
Shimazaki Noriko,
Kaneko Reo,
Tanabe Akiko,
Takebe Izumi,
Kuroda Sachiko,
Kobayashi Tamami,
Toji Shingo,
Tamai Katsuyuki,
Yamamoto Hiroko,
Koiwai Osamu
Publication year - 2001
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 - 10.1046/j.1365-2443.2001.00460.x
Subject(s) - proliferating cell nuclear antigen , biology , microbiology and biotechnology , immunoprecipitation , terminal deoxynucleotidyl transferase , t cell receptor , antigen , gene , dna , biochemistry , apoptosis , t cell , genetics , immune system , tunel assay
Background The repertoires of Ig and TcR are generated by a combinatorial rearrangement of variable (V), diversity (D), and joining (J) segments (V(D)J recombination) in B‐ and T‐cells. Terminal deoxynucleotidyltransferase (TdT) adds extra nucleotides (N nucleotides) at the junctions of the gene segments to enhance the Ig and TcR genes diversity. Using an anti‐TdT antibody column, TdT has been purified as a member of a megadalton protein complex from rat thymus. The N region would be synthesized with the large protein complex . Results The cDNAs for proliferating cell nuclear antigen (PCNA) were isolated by yeast two‐hybrid screening as the gene products which directly interacted with TdT. The interaction between PCNA and TdT was confirmed by co‐immunoprecipitation, both in vitro and in vivo . TdT binds directly to a PCNA trimer, as shown by gel filtration. TdT interacts with PCNA in its DNA polymerization domain (DPD), but not in its BRCA‐1 C‐terminal (BRCT) domain. TdT activity was reduced to 17% of the maximum value by TdT/PCNA complex formation . Conclusion TdT interacts directly with PCNA through its DPD. A functional consequence of this interaction is the negative regulation of TdT activity. These findings suggest that TdT catalyses the addition of N nucleotides under the negative control of PCNA during V(D)J recombination .