Analysis of ADP-ribose polymer sizes in intact cells.

Poly(ADP-ribose) is a polymer (pADPr) that is synthesized by poly (ADP-ribose) polymerases in response to DNA damaging agents. For instance, chemical alkylating agents such as MNNG or physical stimulation of cells by gamma-rays are well known to induce pADPr synthesis. PARPs are members of a growing family of enzymes which includes PARP-1, PARP-2, S-PARP-1, tankyrase and V-PARP. The association of PARP-1 and PARP-2 in DNA damage signaling pathways has been characterized, but tankyrase and V-PARP seem to be independent of DNA repair mechanisms. Poly(ADP-ribosyl)ation leads to heterogenous chain lengths of up to 200 units (mers) in vitro. While most of these will be covalently bound to proteins, they may be released under alkaline conditions for analysis. Previous immunological methods such as immunoblots showed that about 60-70% of the 6-8 mers pADPr were lost during fixation and that the very short pADPr (2-5 mers) were very weakly bound to the membrane. Furthermore, detection of cellular pADPr using enzyme-linked immunosorbent assay (ELISA) revealed that some molecules of pADPr are also lost during fixation and washings. This phenomenon leads to underestimation of the short pADPr population in cells. Thus, evaluating which pADPr sizes are present in cells and tissues becomes critical. We report here the development of a new highly sensitive immunological method to detect synthesized pADPr sizes distribution in intact cells.