High resolution fractionation and characterization of ADP-ribose polymers

Abstract

Methods are described for the high resolution fractionation and characterization of ADP-ribose polymers. Polymers prepared in vitro using purified poly(ADP-ribose) polymerase were isolated free from interfering nucleic acids and salts using dihydroxyboronyl-Bio-Rex 70 chromatography and fractionated using anion exchange high-pressure liquid chromatography. The homogeneity of isolated polymer fractions was characterized by gel electrophoresis and polymer size was determined by analysis following enzymatic digestion to nucleosides. The method allows isolation of oligomers up to 50 mer as single species and larger polymers can be isolated free from oligomers according to size and branching frequency. The ability to isolate individual species of ADP-ribose polymers should prove useful for the study of the polymers and their noncovalent interactions with other components of chromatin. Microheterogeneity of individual oligomers was studied and shown to be due to differences at the protein proximal ends resulting from the chemical method of release of polymers from protein. The method also was applied to fractionate polymers generated in intact cultured mouse cells in response to treatment with the carcinogen N-methyl-N'-nitro-N-nitrosoguanidine.