Terminal deoxynucleotidyltransferase is negatively regulated by direct interaction with proliferating cell nuclear antigen.
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.


CONCLUSIONS
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.
Ibe
S
S
Faculty of Science and Technology, Department of Applied Biological Science, Science University of Tokyo, Noda, Chiba 278-8510, Japan.
Fujita
K
K
Toyomoto
T
T
Shimazaki
N
N
Kaneko
R
R
Tanabe
A
A
Takebe
I
I
Kuroda
S
S
Kobayashi
T
T
Toji
S
S
Tamai
K
K
Yamamoto
H
H
Koiwai
O
O
D001665:Binding Sites; D002850:Chromatography, Gel; D004253:DNA Nucleotidylexotransferase / Q000235:genetics / Q000378:metabolism*; D015536:Down-Regulation; D017353:Gene Deletion; D005786:Gene Expression Regulation; D015723:Gene Library; D019398:Genes, BRCA1 / Q000502:physiology; D006801:Humans; D008969:Molecular Sequence Data; D011233:Precipitin Tests; D018809:Proliferating Cell Nuclear Antigen / Q000235:genetics / Q000378:metabolism*; D011995:Recombination, Genetic; D014157:Transcription Factors / Q000737:chemistry; D014407:Tumor Cells, Cultured / Q000201:enzymology; D020798:Two-Hybrid System Techniques
D018809:Proliferating Cell Nuclear Antigen; D014157:Transcription Factors; D004253:DNA Nucleotidylexotransferase