22 Pages

Fine Resolution of the Poly ADP-ribosylated Domains of Polynucleosomal Chromatin: DNA Gene and Integrity Analysis; Mechanism of Histone H1 Modification

WithMark Smulson, N. Malik, M. Wong, N. Pomato, P. Thraves

The focus of our laboratory has been to ascertain how the poly(ADP-ribosyl)ation reaction influences the structure and biological function of nucleosomal chromatin.

Antibody to poly(ADP-ribose (ADP-Rib)) was coupled to Sepharose to prepare an immunoaffinity column. The following new information concerning poly(ADP-Rib) and chromatin was obtained with this column : 1) those limiting domains of nucleosomal chromatin undergoing the modification (circa 10%) could be isolated in the “bound” fraction, 2) antibody-bound nucleosomes contained all the poly ADP-ribosylated acceptors and polymerase activity of the bulk chromatin, 3) bound nucleosomes contain significant numbers of internal DNA strand breaks, and [H]-thymidine (TdR) repair incorporation from in vivo “DNA repair label,” compared to the unbound, bulk of the chromatin, 4) the presence of actively transcribed genes in “bound” nucleosomes is being investigated, 5) the acetylation modification of histones occurs in the same domains of chromatin as does poly(ADP-ribosyl)ation, and 6) poly ADP-ribosylated histone H1 can be selectively purified by the immuno-affinity method. These same histone H1 molecules appear to be equally accessible to the histone kinase, phosphorylation modification.

In addition, new information has been obtained concerning histone H1 cross-linking by poly(ADP-ribosyl)ation, and on polymerase binding sites to chromatin. We have reconstituted histone H1-depleted chromatin with intact H1 and peptide domains of H1, and subsequently studied H1-poly(ADP-Rib)-complex synthesis. The data indicate that elongation of poly(ADP-Rib) proceeds on the amino terminal region of this histone.

By utilizing the new techniques of DNA technology, huge advances in our understanding of the programmed structure of the eukaryotic genome have been accomplished in a relatively short time. To complement this explosion of information, it is important to ascertain how these recently appreciated properties of eukaryotic DNA are packaged within extended and condensed domains of chromatin. The research to be discussed is directed, in part, at this latter topic. We have initiated a program 50aimed at furthering our comprehension of chromatin structure by studying one specific, enzymatically active chrosomal protein.

The chromatin-associated enzyme, poly(ADP-Rib) polymerase catalyzes the successive transfer of the ADP-ribose moiety of NAD to various nuclear acceptors, including core nucleosomal histones, histone H1, and a 112,000 dalton non-histone protein, shown to be poly(ADP-Rib) polymerase. With increasing concentrations of NAD, the polymer reaches chain lengths exceeding 100 units long. This reaction thus represents a considerable physical alteration of the structure of those nucleosomes undergoing this modification, since recent data suggests that histones are crosslinked upon extensive poly(ADP-ribosyl)ation. There is increasing evidence that this enzyme is activated in vivo by DNA strand breaks, and functions in some step of DNA repair. A direct correlation between NAD concentration, the level of chromatin aggregation, and the length of poly(ADP-Rib) chains in the complex was detected.

Experimentation, aimed at relating biological function to structural features of nucleosomal chromatin, has suffered from the lack of methods to isolate, selectively, large quantities of highly enriched nucleoprotein domains undergoing such reactions. In the present study, the use of anti-poly(ADP-Rib) antibody covalently attached to Sepharose has permitted the selective enrichment of chains of oligonucleosomes proximal to poly(ADP-ribosyl)ation sites in chromatin. Using this technique we have elucidated new information concerning the regions around this modification, site, related to gene activation, DNA repair/replication and alternative nuclear protein modification reactions.