Chain Length Analysis of ADP‐Ribose Polymers Generated by Poly(ADP‐Ribose) Polymerase (PARP) as a Function of ß‐NAD and Enzyme Concentrations

Abstract

SummaryBireactant autopoly(ADP-ribosyl)ation of poly(ADP-ribose) polymerase (PARP) (EC 2.4.2.30) was carried out by using either increasing concentrations of¯-NAD + (donor substrate) at a xed protein concentration or increasing concentrations of PARP (acceptor substrate) at a xed¯-NAD + concentration. The [ 32 P]ADPribose polymers synthesized were chemically detached from PARP by alkaline hydrolysis of the monoester bond between the carboxylate moiety of Glu and the polymer. Nucleic acid -like polymers were then analyzed by high-resolution polyacrylamide gel electrophoresis and autoradiography. The ADP-ribose chain lengths observed displayed substrate concentration -dependent elongation from 0.2 µ M to 2 mM¯-NAD + . Similar results were observed at xed concentrations of 4.5, 9, 18, 27, and 36 nM PARP. Therefore, we conclude that the concentration of the ADP-ribose donor substrate determines the average chain length of the polymer synthesized. In contrast, the polymer size was unaltered when the concentration of PARP was varied from 4.5 to 18 nM at a xed¯-NAD + concentration. However, when PARP concentrations >18 nM were used, the total amount of monomeric ADP-ribose produced was noticeably less. Therefore, we conclude that high concentrations of PARP lead to acceptor substrate inhibition at the level of the ADP-ribose chain initiation reaction.

Is There an Epigenetic Component Underlying the Resistance of Triple-Negative Breast Cancers to Parp Inhibitors?

“…Without the constraints found in vivo , ADP-ribose polymers can be extended to enormous lengths in vitro (D’Amours et al, 1999; Mendoza-Alvarez et al, 2000). It is likely that the formation of such a network of polymers could impair binding to cognate DNA sites in vitro due to simple steric hindrance.…”

Investigation of the action of poly(ADP-ribose)-synthesising enzymes on NAD+ analogues

“…1). These polymers consist of up to 200 units of ADP-ribose and may branch every 20 to 50 monomers [8–10]. To date, only four ARTD members were found to accomplish the synthesis of PAR, namely the DNA-dependent ARTD1 and ARTD2 as well as the tankyrases ARTD5 and ARTD6 [2–3].…”

Biochemical Association of Poly(ADP‐ribose) Polymerase‐1 and Its Apoptotic Peptide Fragments with DNA Polymerase β

“…We have recently reported that enzyme-bound ADP-ribose chain lengths are determined by the concentration of bNAD in the protein-poly(ADP-ribosyl)ation assay, regardless of the protein concentration in the incubation mixture [25]. Therefore, we next proceeded to determine the influence of the bNAD concentration on the electrophoretic mobility poly(ADP-ribosyl)ated-(pol b).…”

“…We accomplished this by high-resolution polyacrylamide gel electrophoresis [24]. Gel slices containing the radiolabeled bands corresponding to automodified PARP-1 as well as poly(ADP-ribosyl)ated-pol b were separately analyzed after the chemical release of ADP-ribose chains from protein in 0.1m KOH in the presence of EDTA [25]. Fig.…”