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

Poly(ADP-ribose) polymerase is a DNA break detecting enzyme playing a role in the surveillance of genome integrity. Poly(ADP-ribose) is synthesized ra pidly and transiently from P-NAD in response to DNA damaging agents. In ord er 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. F or this purpose, we produced a highly specific polyclonal anti-poly(ADP-rib ose) antibody which immunoreacts with polymers and oligomers. In addition t o the immunodot blot method recently described by us (Affar et al., Anal. B iochem. 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, fol lowed by its transfer onto a positively charged membrane and detection with anti-poly(ADP-ribose) antibody; (iii) immunocytochemistry and flow cytomet ry analyses allowing poly(ADP-ribose) study at the level of individual cell s. (C) 1999 Elsevier Science B.V. All rights reserved.