Large-scale preparation and characterization of poly(ADP-ribose) and defined length polymers

Abstract

Poly(ADP-ribose) [pADPr] is a large, structurally complex polymer of repeating ADP-ribose units. It is biosynthesized from NAD+ by poly(ADP-ribose) polymerases [PARPs] and degraded to ADP-ribose by poly(ADP-ribose) glycohydrolase. PADPr is involved in many cellular processes and exerts biological function through covalent modification and non-covalent binding to specific proteins. Very little is known about molecular recognition and structure activity relationships for non-covalent interaction between pADPr and its binding proteins, in part because of lack of access to the polymer on large scale and to units of defined lengths. We prepared polydisperse pADPr from PARP1 and tankyrase 1 at hundreds of milligram scale by optimizing enzymatic synthesis and scaling up chromatographic purification methods. We developed and calibrated an anion exchange chromatography method to assign pADPr size and scaled it up to purify defined length polymers on the milligram scale. Furthermore, we present a pADPr profiling method to characterize the polydispersity of pADPr produced by PARPs under different reaction conditions and find that substrate proteins affect the pADPr size distribution. These methods will facilitate structural and biochemical studies of pADPr and its binding proteins.

A Quantitative Assay Reveals Ligand Specificity of the DNA Scaffold Repair Protein XRCC1 and Efficient Disassembly of Complexes of XRCC1 and the Poly(ADP-ribose) Polymerase 1 by Poly(ADP-ribose) Glycohydrolase

“…bose) chains were prepared by a published protocol (20). PAR polymers were synthesized in a 20-ml reaction mixture containing 50 mM Tris-HCl (pH 7.5), 10 mM MgCl 2 , 1 mM DTT, 12.5 g/ml calf thymus DNA, 2 M full-length PARP1, 4 M PARP1C, and 2 mM NAD ϩ .…”

“…The PAR chains were recovered on a dihydroxyboryl Bio-Rex 70 column and then size-fractionated on a DNA-Pac PA100 (Dionex) ionexchange column. PAR polymers were divided into pools representing different size fractions based on the HPLC elution profile (20), dried under vacuum, and then desalted into water using a PD-10 desalting column. Purified PAR chains were dried under vacuum and dissolved in water at a final concentration of 2-6 mM.…”

“…Purified PAR chains were dried under vacuum and dissolved in water at a final concentration of 2-6 mM. The molar concentration of PAR (expressed as the concentration of ADP-ribose units) was estimated as fol- (20). UV absorbance at 260 nm (A 260 ) was measured with a NanoDrop 1000 (Thermo Scientific).…”

“…We performed TR-FRET binding competition experiments to determine the minimum length of PAR oligonucleotide that binds effectively to XRCC1. Oligo(ADP-ribose) chains were prepared by enzymatic synthesis and purified with ionexchange HPLC as reported previously (20). BRCT1 binds weakly to ADP-ribose (EC 50 of 1.39 Ϯ 0.06 mM), whereas oligo(ADP-ribose) ligands with a chain length greater than 7 ADP-ribose units are bound tightly with EC 50 values of ϳ20 nM (Fig.…”

Poly(ADP-ribose): From chemical synthesis to drug design

“…PAR fragments of sufficient homogeneity can be obtained only by multiple chromatographic purification steps. 27 The development of the synthesis of regular PAR oligomers is a challenging problem for chemists. The complexity of this problem is associated with several hurdles: the synthesis of adenosine with additional ribose moiety connected by a(1 ?…”