Immunological determination and size characterization of poly(ADP-ribose) synthesized in vitro and in vivo

• Published in: Biochimica et biophysica acta Vol. 1428; no. 2; pp. 137 - 146
• Main Authors: Affar, El Bachir, Duriez, Patrick J., Shah, Rashmi G., Winstall, Eric, Germain, Marc, Boucher, Claudia, Bourassa, Sylvie, Kirkland, James B., Poirier, Guy G.
• Format: Journal Article
• Language: English
• Published: Netherlands Elsevier B.V 05.08.1999
• Subjects: Animals; Anti-poly(ADP-ribose) antibody; Antibodies - immunology; Antibody Specificity; Cell Line; Chemical Fractionation; DNA damage; Electrophoresis, Polyacrylamide Gel; Enzyme-Linked Immunosorbent Assay; Flow Cytometry; Immunoblotting; Immunohistochemistry; Methylnitronitrosoguanidine; Mice; N-Methyl- N′-nitro- N-nitrosoguanidine; Poly Adenosine Diphosphate Ribose - biosynthesis; Poly Adenosine Diphosphate Ribose - chemistry; Poly Adenosine Diphosphate Ribose - immunology; Poly(ADP-ribose) polymerase; Poly(ADP-ribose) size; N-Methyl- N′-nitro- N-nitrosoguanidine; DNA damage; Anti-poly(ADP-ribose) antibody; DHBB, dihydroxyboronyl Bio-Rex; Poly(ADP-ribose) polymerase; ABTS, 2,2′-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid); DHQ, 1,5-dihydroxyisoquinoline; PARG, poly(ADP-ribose) glycohydrolase; MNNG, N-methyl- N′-nitro- N-nitrosoguanidine; XC, xylene cyanol; Poly(ADP-ribose) size; pADPr, poly(ADP-ribose); BPB, bromophenol blue; PARP, poly(ADP-ribose) polymerase
• ISSN: 0304-4165; 0006-3002; 1872-8006
• DOI: 10.1016/S0304-4165(99)00054-9

Poly(ADP-ribose) polymerase is a DNA break detecting enzyme playing a role in the surveillance of genome integrity. Poly(ADP-ribose) is synthesized rapidly and transiently from β-NAD in response to DNA damaging agents. In order to study the physiological significance of poly(ADP-ribose) metabolism, we have developed immunological methods which enable us to study endogenous poly(ADP-ribose) without interfering with cell metabolism and integrity. For this purpose, we produced a highly specific polyclonal anti-poly(ADP-ribose) antibody which immunoreacts with polymers and oligomers. In addition to the immunodot blot method recently described by us (Affar et al., Anal. Biochem. 259 (1998) 280–283), other applications were investigated in cells: (i) detection of poly(ADP-ribose) by ELISA; (ii) characterization of poly(ADP-ribose) size using high resolution gel electrophoresis of polymers, followed by its transfer onto a positively charged membrane and detection with anti-poly(ADP-ribose) antibody; (iii) immunocytochemistry and flow cytometry analyses allowing poly(ADP-ribose) study at the level of individual cells.