title: 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 noncovalent binding to specific proteins. Very little is known about molecular recognition and structure-activity relationships for noncovalent interaction between pADPr and its binding proteins, in part because of lack of access to the polymer on a large scale and to units of defined lengths. We prepared polydisperse pADPr from PARP1 and tankyrase 1 at the 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.
authors: Department of Systems Biology, Harvard Medical School, Boston, MA 02115, USA. edwin_tan@hms.harvard.edu
authors: Edwin S
authors: ES
authors: Tan
authors: Kristin A
authors: KA
authors: Krukenberg
authors: Timothy J
authors: TJ
authors: Mitchison
mesh_terms: D000818:Animals; D055162:Biocatalysis; D001671:Biochemistry / Q000379:methods*; D001704:Biopolymers / Q000096:biosynthesis* / Q000737:chemistry; D002138:Calibration; D002417:Cattle; D005591:Chemical Fractionation; D002851:Chromatography, High Pressure Liquid; D006026:Glycoside Hydrolases / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D006801:Humans; D011064:Poly Adenosine Diphosphate Ribose / Q000096:biosynthesis* / Q000737:chemistry; D011065:Poly(ADP-ribose) Polymerases / Q000737:chemistry / Q000378:metabolism; D058105:Polymerization; D017434:Protein Structure, Tertiary; D038501:Tankyrases / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism
chemical_list: D001704:Biopolymers; D006026:Glycoside Hydrolases; D011064:Poly Adenosine Diphosphate Ribose; D011065:Poly(ADP-ribose) Polymerases; D038501:Tankyrases; C418109:TNKS protein, human; C022407:poly ADP-ribose glycohydrolase