Relationship between nicotinamide adenine dinucleotide concentration and in vitro synthesis of poly(adenosine diphosphate ribose) on purified nucleosomes.
When oligonucleosomes (8-10 N) were incubated under conditions favoring poly(ADP-ribosylation) with concentrations of [32P]NAD 10 microM and higher, the labeled chromatin components migrated in 3-8% gradient native chromatin polyacrylamide gels, in positions of far greater size than the starting nucleosomes. Only a small fraction of chromatin and/or oligonucleosome components was found to be involved in this aggregation. This phenomenon could be demonstrated as well by the increased sedimentation of ADP-ribosylated chromatin components in velocity sucrose gradients. In contrast, at [32P]NAD concentrations less than or equal to 1 microM, the modified nuclear proteins were found strictly associated with the original oligonucleosomes. The extent of aggregation was proportional to both substrate concentration and time of incubation with NAD. During this process, progressively longer chains of poly (ADP-Rib) (10-60 N) were generated on chromatin proteins correlating with the level of complex formation. Analysis of protein by either acid-urea or Na-DodS4O-gel electrophoresis indicated that at NAD concentrations favoring aggregation, poly(ADP-Rib) induced ADP-ribosylated complexes of histone H1, and possibly core histones were observed. Poly(ADP-Rib) polymerase requires both DNA and nuclear proteins for activity. The presence of this enzyme in the high molecular weight complexes was demonstrated on sucrose gradients and also by direct enzyme assays, in situ, in regions of 3-8% gradient chromatin gels containing the complexed chromatin components.
D004591:Electrophoresis, Polyacrylamide Gel; D006367:HeLa Cells; D006657:Histones / Q000378:metabolism; D006801:Humans; D009243:NAD / Q000378:metabolism*; D009702:Nucleoside Diphosphate Sugars / Q000096:biosynthesis*; D009707:Nucleosomes / Q000378:metabolism*; D009994:Osmolar Concentration; D011064:Poly Adenosine Diphosphate Ribose / Q000096:biosynthesis*
D006657:Histones; D009702:Nucleoside Diphosphate Sugars; D009707:Nucleosomes; D009243:NAD; D011064:Poly Adenosine Diphosphate Ribose