Some electron microscopic aspects of poly(ADPR) polymerase-DNA interactions and of auto-poly(ADP-ribosyl)ation reaction.
Interaction of calf thymus poly(ADP-ribose(ADPR] polymerase with a copurified DNA fraction (sDNA) was investigated. Electron microscopic studies of sDNA which appeared to be a powerful poly(ADPR) polymerase activator have shown that the purified poly(ADPR) polymerase-DNA complexes possess a "nucleosome like structure", with DNA wrapping around the enzyme molecule. Examination of the DNA linked poly(ADPR) polymerase preparations revealed the presence of Y-structures in sDNA. The enrichment in the sDNA fraction of the Y shape DNA suggests the existence of replication fork structures in the poly(ADPR) polymerase linked DNA and or in the vicinity of the enzyme. With increasing auto-poly(ADP-ribosyl)ation the enzyme molecule becomes much denser, increases in size and detaches from the DNA. When poly(ADPR) formed was purified and examined by electron microscopy, branched polymers of different sizes were observed. The formation of these polymers may explain the size gained by poly ADP-ribosylated enzyme molecules. When the interaction of poly(ADPR) polymerase with the plasmid pBR 322 was tested, a slight contamination of our enzyme preparation with topoisomerase I was detected. The contaminant topoisomerase I activity, however, was completely abolished by ADP-ribosylation. Further experiments with purified calf thymus topoisomerase I confirmed that this enzyme loses its activity following ADP-ribosylation with poly(ADPR) polymerase. These results may suggest that ADP-ribosylation of topoisomerase I can be one of the regulatory mechanisms of its activity. Furthermore, these results confirm that a topoisomerase I contaminant does not interfere with the ADP-ribosylation experiments of purified poly(ADPR) polymerase preparation.
D000246:Adenosine Diphosphate Ribose / Q000378:metabolism; D004247:DNA / Q000378:metabolism*; D004264:DNA Topoisomerases, Type I / Q000378:metabolism; D008854:Microscopy, Electron; D009244:NAD+ Nucleosidase / Q000378:metabolism*; D009702:Nucleoside Diphosphate Sugars / Q000378:metabolism*; D009707:Nucleosomes / Q000378:metabolism; D011064:Poly Adenosine Diphosphate Ribose / Q000378:metabolism*; D011065:Poly(ADP-ribose) Polymerases / Q000378:metabolism*
D009702:Nucleoside Diphosphate Sugars; D009707:Nucleosomes; D000246:Adenosine Diphosphate Ribose; D011064:Poly Adenosine Diphosphate Ribose; D004247:DNA; D011065:Poly(ADP-ribose) Polymerases; D009244:NAD+ Nucleosidase; D004264:DNA Topoisomerases, Type I