Faculty of Science and Technology, Department of Applied Biological Science, Science University of Tokyo, Noda, Chiba 278-8510, Japan.
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.