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

Abstract

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(ADPribose) polymerase, NAD', Mg2+ 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 Mg2'-depleted and acceptor-dependent ADP-ribosylating reaction condition [Tanaka, Y., Hashida, T., Yoshihara, H. and Yoshihara, K. (1979) J. Biol. Chem. 254, 12433-124381, 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-ribosy1)ated. When the product was further incubated in the Mg2+-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(ADPribose) 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-ribosy1)ation of the transferase itself.Poly(ADP-ribose) polymerase is localized in eukaryotic cell nuclei and catalyzes the successive transfer of ADP-ribose moiety of NAD' to various acceptor proteins, forming ADPribose polymer covalently linked to these proteins. Although the biological roles of the enzyme have not yet been firmly established, several lines of evidence suggest that ADPribosylation of nuclear proteins is involved in various nuclear functions such as DNA repair, DNA synthesis, gene expression, cell differentiation and transformation (for review see [l -31). Identification of acceptor proteins of poly(ADPribose) may be important to understand the relation between poly(ADP-ribosy1)ation reaction and the nuclear functions described above. In this respect, several nuclear proteins including histones [4 -71, high-mobility-

Protein glycation by ADP-ribose: Studies of model conjugates

“…This proposal is also supported by the observation that the site of glycation of hemoglobin Aic contains an adjacent histidine residue (Koenig et al, 1977). Thus, it is possible that a similar mechanism leads to the specific glycation of mitochondrial and cytosolic proteins by ADP-ribose (Hilz et al, 1984;Tanaka et al, 1989).…”

“…Previously, Kun et al (1976) have reported glycation by ADPribose of histones or polylysine in vitro. More recently, Hilz et al (1984) and Tanaka et al (1989) have shown that glycation by ADP-ribose can occur with specific proteins in vitro. The occurrence of protein glycation by ADP-ribose has been postulated in vivo (Kreimeyer et al, 1984) although definitive evidence for its presence in vivo has not been reported.…”

Evidence for regulation of NF-κB by poly(ADP-ribose) polymerase

“…Poly(ADP-ribose) polymerase (PARP) is a nuclear enzyme, that catalyses a successive transfer of ADP-ribose moiety of NAD + to a protein, thus forming a protein, bound polymer of ADP-ribose [1]. Various nuclear proteins including histones [1], HMG proteins [2] and nuclear enzymes involved in DNA metabolism, such as Ca# + , Mg# + -dependent endonuclease [3,4], DNA topoisomerases [5,6], DNA ligase I [7,8], terminal deoxynucleotidyl transferase [9], DNA polymerases [8,10] and PARP itself (automodification ; [11,12]) have been shown to be the acceptors for poly(ADP-ribose).…”

Evidence for regulation of NF-κB by poly(ADP-ribose) polymerase

“…Poly(ADP-ribose) polymerase (PARP) is a nuclear enzyme, that catalyses a successive transfer of ADP-ribose moiety of NAD + to a protein, thus forming a protein, bound polymer of ADP-ribose [1]. Various nuclear proteins including histones [1], HMG proteins [2] and nuclear enzymes involved in DNA metabolism, such as Ca# + , Mg# + -dependent endonuclease [3,4], DNA topoisomerases [5,6], DNA ligase I [7,8], terminal deoxynucleotidyl transferase [9], DNA polymerases [8,10] and PARP itself (automodification ; [11,12]) have been shown to be the acceptors for poly(ADP-ribose).…”