MULLIhS. GIRI. zyxw AND SMLLSON Poly( adenosine diphosphate-ribose) Polymerase: The Distribution of a Chromosome-Associated Enzyme within the Chromatin Substructure+ D. W. Mullins, Jr., Chandrakant P. Giri, and Mark Smulson* 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 cell chro- matin has been examined. zyxwvuts Epichlorohydrin-tris(hydroxy- methy1)aminomethane-cellulose and glycerol gradient frac- tionation of solubilized chromatin indicated that poly(ADP- ribose) polymerase activity was associated primarily with the template active regions (euchromatin), whereas the tran- scriptionally inert chromatin fractions were found to contain relatively low levels of ADP-ribosylating activity. When iso- lated HeLa cell nuclei were digested in situ with micrococcal nuclease and the resultant chromatin was fractionated into nucleosome monomers zyxwvutsr (V bodies) and oligomers by sucrose gradient centrifugation, only material sedimenting faster than the zyxwvutsrqpo 1 IS monomers was found to contain appreciable Poly(ADP-ribose)’ polymerase is a ubiquitous nuclear en- zyme which, because of its extraordinarily tight association with eukaryotic chromatin (requiring 1 M salt for dissociation) (Sugimura, 1973), provides a unique probe with which to assess both the distribution of a chromosome-associated enzyme within the chromatin substructure, as well as to aid in the elucidation of the physiological importance of the apparent highly ordered structure of proteins along the chromosomal DNA. Although Keller et al. (1975) have recently shown that the chromosome-associated enzyme, protein kinase, is localized within the transcriptionally active regions of sheared chro- matin, the present study represents the first time that the dis- tribution of such an enzyme within nuclease-treated chromatin fractions has been examined. Poly(ADP-ribose) polymerase catalyzes the formation of a homopolymer of ADP-ribose units linked by 1’-2’ glycosidic bonds. The substrate for the reaction is NAD, and in the presence of DNA, the enzyme successively adds ADP-ribose units onto an initial ADP-ribose residue which has been re- ported to be covalently attached to various nuclear proteins, + From the Department of Biochemistry, Schools of Medicine and Dentistry, Georgetown University, Washington, D.C. 20007. zyxwvutsrq Receiced Map 3, 1976. Supported by National Institutes of Health Grants CA I3195 and zyxwvutsrqpo CA 11950. Submitted by C.P.G. to the Department of Bio- chemistry in partial fulfillment of the requirements for the Ph.D. de- gree. I Abbreviations used are: (ADP-ribose), adenosine diphosphate ribose; Ado( P)-Rib-P and 4-ADP-ribose, 2’-(5”-phosphoribosy1)-5’-AMP NAD, nicotinamide adenine dinucleotide; NaDodSOd, sodium dodecyl sul- fate; CI~ACOH, trichloroacetic acid; S-MEM, spinner minimal essential medium; PhCH2SOlF, phenylmethylsulfonyl fluoride; ECTHAM, epi- chlorohydrin-tris(hydroxymethy1)aminomethane: Tris, tris(hydroxy- methy1)aminomethane; EDTA, ethylenediaminetetraacetic acid; UV. ultraviolet. poly(ADP-ribose) polymerase activity. If, on the other hand, isolated HeLa cell nuclei were first incubated with labeled NAD, the substrate for poly(ADP-ribose) polymerase, prior to the preparation and fractionation of nuclease-digested chromatin, it was found that those chromatin fractions which possess significant poly(ADP-ribose) polymerase activity (nucleosome oligomers) are relatively deficient in the labeled product of this enzyme, and that a considerable portion of the homopolymeric product is ultimately associated with the 11s Y bodies. Additional evidence is presented which indicates that the absence of nucleosome monomer-associated poly(ADP- ribose) polymerase activity is not due to the absence of a suitable acceptor on these structures, and that the activity of this enzyme within the chromatin is most probably dependent upon the physical integrity of the oligomeric structures themselves. including histones, nonhistone chromosomal proteins, and a Ca”-, Mg’+-dependent endonuclease (Nishizuka et al., 1969; Otake et al., 1969; Burzio and Koide, 1972). Although con- siderable information about this tightly bound chromosomal enzyme has been obtained in recent years, little substantive progress has been made toward an understanding of what precise function its homopolymeric product, poly(ADP-ribose), has in chromatin. Several studies have implicated a role for poly(ADP-ribose) polymerase in the regulation of eukaryotic DNA replication or repair. Results from this laboratory, and others, have shown that the synthesis of poly(ADP-ribose) causes perturbations of chromatin structure which lead either to the stimulation (Roberts et al., 1973) or inhibition (Burzio and Koide, 1970, 197 1 ; Nagao et al., 1972) of DNA synthesis, depending upon the source of tissue used. In recent years, considerable progress has also been made toward the establishment of a new and experimentally con- sistent model of chromatin structure in the eukaryotic cell. Initial evidence for this new model was provided by the neutron diffraction studies of Bradbury and his co-workers (Baldwin et al., 1975), and by electron microscopy (Olins and Olins, 1974), which revealed that the chromatin fiber has a structure resembling regularly spaced beads on a string. Spherical nu- cleoprotein particles (v bodies, nucleosomes), approximately 60-80 8, in diameter and connected by thin (1 5 A) threads, could be seen in electron micrographs of dispersed chromatin. Support for this basic model has also been obtained through biochemical studies, which have shown that a variety of nu- cleases, both endogenous and exogenous, cleave chromatin at regularly spaced intervals, and that limit digests with these enzymes produce fragments of DNA with an average length of 200 base pairs (Hewish and Burgoyne, 1973; Burgoyne et al., 1974; Van Holde et al., 1974; Finch et al., 1975). Fur- 506 BIOCHEMISTRY, VOL. 16, NO. 3, 1977