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 se gments to enhance the Ig and TcR genes diversity. Using an anti-TdT antibod y 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 isola ted by yeast two-hybrid screening as the gene products which directly inter acted with TdT. The interaction between PCNA and TdT was confirmed by co-im munoprecipitation, 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 poly merization domain (DPD), but not in its BRCA-1 C-terminal (BRCT) domain. Td T activity was reduced to 17% of the maximum value by TdT/PCNA complex form ation. 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 und er the negative control of PCNA during V(D)J recombination.