Quantifying DNA-trapped Topoisomerase I

DNA-trapped TOP1 was determined by a modified RADAR (rapid approach to DNA adduct recovery) assay (36 (link)). After treatment of TOP1 inhibitors, DU145 cells (1x10⁶ cells/sample) were washed with 1x PBS and lysed with 600 μl of DNAzol (Invitrogen), followed by precipitation with 300 μl of 200 proof ethanol by centrifugation at 14,000 rpm. The nucleic acids were collected, washed with 75% ethanol, resuspended in 200 μl of TE buffer, and then heated at 65°C for 15 min, followed by shearing with sonication (40% output for 10 sec pulse and 10 sec rest for four times). The samples were centrifuged at 15,000 rpm for 5 min at 4°C, and the supernatant was collected. The sample (1 μl) was saved for spectrophotometric measurement of absorbance at 260 nm to quantitate DNA content (NanoDrop). Two μg of each sample was subjected to slot-blot for immunoblotting with anti-TOP1 antibody (#556597, BD Biosciences) or anti-dsDNA antibody (#3519, Abcam) as a loading control. The intensity of TOP1 and DNA was quantified by densitometric analysis using ImageJ.