High resolution size analysis of ADP-ribose polymers using modified DNA sequencing gels.

Mammalian cells respond to DNA damage by producing polymers of ADP-ribose (ADPR) on certain DNA-binding proteins (1). Synthesis of variably-sized ADPR polymers correlates with modified protein function (2). Here we present a high resolution electrophoresis technique for separating and quantifying picomole quantities of polymers up to 60 residues long. This technique allows one to study the role of specific polymer size patterns in the regulation of chromatin protein function. Intact, ^P-labeled polymers (1000-5000 cpm) were detached from proteins by incubating at 60 °C for 3 hr with 10 mM Tris, 1 mM EDTA, pH 12. Detachment using the conventional NaOH/EDTA protocol (3) resulted in significant band distortion upon electrophoresis. Samples were extracted with an equal volume of phenol/CHCyisoamyl alcohol (49:49:2) and dried in a Speed-Vac. Recovery from this procedure was >95%. Polymers were dissolved in 10 y\ of 50% urea, 25 mM NaCl, 4 mM EDTA (pH 7.5), 0.02% xylene cyanol, 0.02% bromophenol blue. A 60 cmx20cmx0.35 mm, 20% acrylamide gel (19.86:0.24 acrylamide/bisacrylamide) was polymerized in TBE buffer (0.09 M Tris, 0.09 M boric acid, 2 mM EDTA, pH 8.3) upon addition of 0.5 ml 10% ammonium persulfate and 37.5 n\ N,N,N',N'-tetramethylethylenediamine per 75 ml of gel mix. Samples were loaded immediately following preelectrophoresis for 1 hr at 55 W (constant power). Electrophoresis was carried out at 55 W until the bromophenol blue had migrated 27 cm from the origin (2 hr). The gel was transferred to Whatman 3MM paper and dried under vacuum at 30°C for 1 hr. Autoradiographs were obtained after exposure at -70°C using two fast-tungstate intensifying screens (CAWO, Zurich, Switzerland).