Analysis of ADP-ribose polymer sizes in intact cells

Poly(ADP-ribose) is a polymer (pADPr) that is synthesized by poly (ADP-ribo se) polymerases in response to DNA damaging agents. For instance, chemical alkylating agents such as MNNG [1] or physical stimulation of cells by gamm a -rays [2] 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, tankyr ase and V-PARP [3]. The association of PARP-1 and PARP-2 in DNA damage sign aling 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 uni ts (mers) in vitro [3]. While most of these will be covalently bound to pro teins, they may be released under alkaline conditions for analysis. Previou s immunological methods such as immunoblots [4] 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 [5]. Furthermore, detect ion of cellular pADPr using enzyme-linked immunosorbent assay (ELISA) revea led 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 meth od to detect synthesized pADPr sizes distribution in intact cells.