Poly(ADP-Ribos)ylation of Nuclear Enzymes

Abstract

Poly(ADP-ribose) polymerase catalyzes a sequential transfer of an ADP-ribose portion of NAD⁺ to various chromatin proteins [1] and to the polymerase itself (automodification [2]), forming a polymer of ADP-ribose, which is covalently bound to protein at one end [3]. Recent studies elucidated that two chromatin enzymes, Ca²⁺, Mg²⁺-dependent endonuclease [4, 5] and DNA topoisomerase [6, 7], were markedly inhibited as a result of poly(ADP-ribos)ylation of the enzyme proteins. RNA polymerase I [8] and DNA ligase II [9] also are suggested to be poly(ADP-ribos)ylated, although the latter enzyme seems to be activated after poly(ADP-ribos)ylation in vivo. Furthermore, bull seminal RNase [10, 29] and micrococcal nuclease [29] also have been shown to be the acceptors of ADP-ribose in the enzyme reaction in vitro. These results suggest a possibility that poly(ADP-ribose) polymerase randomly modifies many kinds of chromatin enzymes rather than it selecting a few kinds of specific enzymes as its targets. Thus, in order to study whether the modification reaction is specific only for the enzymes described above, we examined six nuclear enzymes, which are involved in metabolism or function of chromatin. After several unsuccessful trials using standard and modified conditions of the reconstituted ADP-ribosylating system, we found that all of these enzymes except DNA ligase I were markedly inhibited when the enzymes were incubated in an ADP-ribosylating reaction mixture containing a limited concentration of buffer (5 mM).