The Modification of Nuclear Proteins by ADP‐ribosylation

Abstract

When incubated in vitro purified mouse nuclei incorporate NAD into poly(ADP-Rib). Analysis of the product on CsCl/urea gradients showed that a large proportion of the poly(ADP-Rib) was not attached to protein. The free poly(ADP-Rib) did not appear to arise through degradation and its chain length was significantly longer than the poly(ADP-Rib) attached to proteins. Fractionation of the proteins on hydroxyapatite revealed that tissue-specific modification of both the histones and non-histone proteins, had occurred. In the case of one protein species there was evidence for the existence of several forms with different numbers of ADP-Rib residues.It is now more than ten years since the synthesis of poly(ADP-Rib) in isolated nuclei was first demonstrated; it was shown that when nuclei were incubated in vitro with labelled NAD the radioactivity was incorporated exclusively into poly(ADP-Rib) [l]. More recently evidence has been presented to show that this polymer is also synthesized in vivo [2]. Poly(ADP-Rib) is a polynucleotide-like molecule but, in contrast to the other polynucleotides of the cell, its functional role in the cell remains unclear. It has been reported that poly(ADP-Rib) is attached to the nuclear proteins (for a review see [3]) and as such it could modify the characteristics of the proteins to which it is bound, in the same way as phosphorylation and acetylation can also modify the interaction of histones with DNA. Two problems emerge from the evidence presently available. The first concerns which proteins of the nucleus act as acceptors for the poly(ADP-Rib) molecules. So far only the histones have been identified with certainty as being modified by the polymer [4-61, but with the improved techniques now available (see [7]) it is possible to examine the role of most of the nuclear proteins in this process. The second problem relates to the large number of conflicting reports as to the rate of synthesis of poly(ADP-Rib) at different times of the cell cycle [8] and the difficulty of correlating polymer production and its modification of nuclear proteins with particular events in the cell cycle or with changes in gene activity. For some years these laboratories have been involved in developing procedures for the preparation and analysis of nuclear proteins (see [7,9,10]) and in this paper we describe the application of some of our techniques to study the modification of nuclear proteins by poly-(ADP-Rib) in a number of mouse cells.

Eukaryotic nuclear ADP-ribosylation reactions

“…are known to be modified by covalent attachment of poly(ADP-ribose) Enzymic regulation by poly(ADP-ribosyl)ation Tanaka et al, 1984; Jongstra-Bilen et al., 1983;Kawaichi et al, 1981 Rickwood et al (1977) Adamietz (1982) Adamietz ( (1984) require DNA for activity, it has been suggested that inhibition is due to an electrostatic repulsion between the natural substrate, DNA, and the ADP-ribose chains covalently bound to the enzymes Ferro & Olivera, 1982. Evidence to support this hypothesis is provided by the findings that ADP-ribosylated endonuclease and ADPRT have a reduced affinity for DNA, that free poly(ADP-ribose) is noninhibitory, and that positively charged DNAbinding proteins enhance endonuclease activity Ferro & Olivera, 1982).…”

“…2a) shows the formation of free (ADP-ribose)n chains, not linked to protein (Rickwood et al, 1977;Benjamin & Gill, 1980a). Whether these truly occur in vivo, or are artifacts produced by cleavage of ADP-ribose residues from proteins during experimental handling of tissues, is still a matter of contention.…”

In vitro ADP‐ribosylation of histones by purified calf thymus polyadenosine diphosphate ribose polymerase

“…The preferential ADP-ribosylation of histone H2B in rat liver nuclei and of histone Hl in rat brain nuclei has been demonstrated [7,9]. These findings suggest a tissue specific ADP-ribosylation of histones.…”

“…In rat liver or mouse liver nuclei, histone HZB was the major acceptor of ADPR [7]. In contrast in HeLa cell nuclei [8], rat brain nuclei or M2 cell nuclei [9], histone Hl showed the highest incorporation of ADPR. On this basis it was suggested that histone or nonhistone nuclear protein ADP-ribosylatiori may play an important role in cell differentiation or gene expression,…”

ADP-ribosylation of nuclear proteins

“…A broader study of poly(ADP-ribosy1)ation has been presented by Rickwood et al ( 1977). [32PlADP-ribosylated proteins were separated from nucleic acids by CsCI-equilibriumdensity-gradient centrifugation, fractionated on hydroxyapatite and subjected to two-dimensional gel electrophoresis.…”