Isolation and Detection of DNA-Protein Crosslinks

DPCs were isolated using a modified Rapid Approach to DNA Adduct Recovery (RADAR) assay^{65 (link),25 (link)}. Briefly, 1–2 × 10⁶ cells were lysed in 1 mL of buffer containing 6 M guanidine thiocyanate (GTC); 10 mM Tris–HCl, pH 6.8; 20 mM EDTA; 4% Triton-X100; 1% Sarkosyl and 1% DTT. DNA was precipitated by adding 100% ethanol, washed three times in wash buffer (20 mM Tris–HCl, pH 6.8; 150 mM NaCl and 50% ethanol). DNA was then solubilized in 1 mL of 8 mM NaOH. The DNA concentration was quantified by treating a small aliquot of DNA with proteinase K (Invitrogen) for 3 h at 50 °C, followed by detection with PicoGreen dye (Invitrogen) according to manufacturer’s instructions. Equal dsDNA loading was confirmed by slot-blot immunodetection with an anti-dsDNA antibody (Abcam). Total DPCs after electrophoretic separation on polyacrylamide gels were visualised by silver staining using the ProteoSilver Plus Silver Stain Kit (Sigma-Aldrich) and specific proteins were detected by Western-blot.