Poly(ADP‐ribosyl)ation of terminal deoxynucleotidyl transferase in vitor

The activity of purified bovine thymus terminal deoxynucleotidyl transferase was markedly inhibited when the enzyme was incubated in a poly(ADP‐ribose)‐synthesizing system containing purified bovine thymus poly(ADP‐ribose) polymerase, NAD + , Mg 2+ and DNA. All of these four components were indispensable for the inhibition. The inhibitors of poly (ADP‐ribose) polymerase counteracted the observed inhibition of the transferase. Under a Mg 2+ ‐depleted and acceptor‐dependent ADP‐ribosylating reaction condition [Tanaka, Y., Hashida, T., Yoshihara, H. and Yoshihara, K. (1979) J. Biol. Chem. 254 , 12433–12438], the addition of terminal transferase to the reaction mixture stimulated the enzyme reaction in a dose‐dependent manner, suggesting that the transferase is functioning as an acceptor for ADP‐ribose. Electrophoretic analyses of the reaction products clearly indicated that the transferase molecule itself was oligo(ADP‐ribosyl)ated. When the product was further incubated in the Mg 2+ ‐fortified reaction mixture, the activity of terminal transferase markedly decreased with increase in the apparent molecular size of the enzyme, indicating that an extensive elongation of poly(ADP‐ribose) bound to the transferase is essential for the observed inhibition. Free poly(ADP‐ribose) and the polymer bound to poly(ADP‐ribose) polymerase were ineffective on the activity of the transferase. All of these results indicate that the observed inhibition of terminal transferase is caused by the poly(ADP‐ribosyl)ation of the transferase itself.