Synthesis of poly(ADP-ribose)-agarose beads: an affinity resin for studying (ADP-ribose)n-protein interactions.
Polymers of ADP-ribose bind chromatosomal histones in solution and may play a role in chromatin accessibility in vivo. We have enzymatically synthesized a poly(ADP-ribose) affinity resin to further characterize binding of nuclear proteins to ADP-ribose polymers. NAD+- and (ADP-ribose)-derivatized agarose beads were recognized as polymer acceptors by the nuclear enzyme poly(ADP-ribose) polymerase. This polymerase elongated the existing ligands by successive addition of exogenously available ADP-ribose residues to form polymers covalently linked to the agarose beads. Poly(ADP-ribose) formation on the beads was dependent on incubation time and the mode of ligand attachment to the agarose. The resulting poly(ADP-ribose)-derivatized agarose beads possessed polymers which closely resembled those modifying the ADP-ribose polymerase by the automodification reaction. Fractionation of rat liver nuclear lysate over the poly(ADP-ribose) resin revealed a strong affinity of H1 for ADP-ribose polymers, thereby supporting a role for poly(ADP-ribose) in chromatin functions. Poly(ADP-ribose)-agarose beads are extremely stable and will be useful not only for affinity studies, but also for mechanistic studies involving polymer elongation and catabolism.
Panzeter
P L
PL
University of Zürich, Institute of Pharmacology and Biochemistry, Switzerland.
Zweifel
B
B
Althaus
F R
FR
D000818:Animals; D002467:Cell Nucleus / Q000737:chemistry; D002846:Chromatography, Affinity / Q000379:methods; D006657:Histones / Q000378:metabolism; D008099:Liver / Q000737:chemistry; D008297:Male; D009243:NAD / Q000737:chemistry; D009687:Nuclear Proteins / Q000737:chemistry / Q000378:metabolism*; D011064:Poly Adenosine Diphosphate Ribose / Q000737:chemistry* / Q000378:metabolism; D011065:Poly(ADP-ribose) Polymerases / Q000378:metabolism; D051381:Rats; D017207:Rats, Sprague-Dawley; D012117:Resins, Synthetic / Q000138:chemical synthesis*; D012680:Sensitivity and Specificity; D012685:Sepharose / Q000737:chemistry*
D006657:Histones; D009687:Nuclear Proteins; D012117:Resins, Synthetic; D009243:NAD; D011064:Poly Adenosine Diphosphate Ribose; D012685:Sepharose; D011065:Poly(ADP-ribose) Polymerases