Poly(adenosine diphosphate-ribose) polymerase: the distribution of a chromosome-associated enzyme within the chromatin substructure

Abstract

The distribution of a chromatin-bound, nuclear protein modifying enzyme, poly(adenosine diphosphate-ribose) polymerase, and its product, poly(ADP-ribose), among various fractions of sheared and nuclease-digested HeLa [human cervical carcinoma] cell chromatin was examined. Epichlorohydrin-tris(hydroxymethyl)aminomethane-cellulose and glycerol gradient fractionation of solubilized chromatin indicated that poly(ADP-ribose) polymerase activity was associated primarily with the template active regions (euchromatin), but the transcriptionally inert chromatin fractions contained relatively low levels of ADP-ribosylating activity. When isolated HeLa cell nuclei were digested in situ with micrococcal nuclease and the resultant chromatin was fractionated into nucleosome monomers (.nu. bodies) and oligomers by sucrose gradient centrifugation, only material sedimenting faster than the 11S monomers contained appreciable poly(ADP-ribose) polymerase activity. If isolated HeLa cell nuclei were 1st incubated with labeled NAD, the substrate for poly(ADP-ribose) polymerase, prior to the preparation and fractionation of nuclease-digested chromatin, those chromatin fractions which possess significant poly(ADP-ribose) polymerase activity (nucleosome oligomers) are relatively deficient in the labeled product of this enzyme, and a considerable portion of the homopolymeric product is ultimately associated with 11S .nu. bodies. The absence of nucleosome monomer-associated poly(ADP-ribose) polymerase activity is not due to the absence of a suitable acceptor on these structures. The activity of this enzyme within the chromatin is most probably dependent upon the physical integrity of the oligomeric structures themselves.