DNA topoisomerase cleavage complexes were analyzed as previously described (Schellenberg et al., 2017). For the induction of cleavage complexes, serum-starved RPE-1 cells were treated as required followed by 400 μM Etoposide (Sigma, E1383), 10 μM Camptothecin (CPT, C9911) (Sigma) or DMSO vehicle (Applichem, A1584) for 5 minutes. Cells were immediately lysed using 1% (w/v) N-Lauroylsarcosine sodium salt (Sigma-Aldrich, L7414) in TE buffer supplemented with protease inhibitors. After homogenization, 0.67 g/ml CsCl (Applichem-Panreac, A1098) was added and lysated were then centrifuged at 57,000 rpm for 20 h at 25°C using 3.3 mL 13 × 33 polyallomer Optiseal tubes (Beckman Coulter) in a TLN100 rotor (Beackman Coulter).

For ICE-IP, 40 μg of ICE material was digested overnight with 0.8 U/μl PstI (NEB, R0140). Samples were incubated at 80°C for 20min to inactivate PstI and then diluted 1/10 in IP buffer (0.1% SDS, 1% TX-100, 2mM EDTA, 20 mM TrisHCl pH8, 150 mM NaCl). Samples were then incubated overnight at 4°C with 4 μg of the required primary antibody and then with 40 μL of pre-blocked (1 mg/ml BSA) Dynabeads protein A (Thermo Fisher). IPs were incubated for 2 hours at RT. Beads were then washed with Wash solution 1 (20 mM Tris HCl pH 8.1, 0.1% SDS, 1% Triton x-100, 2 mM EDTA, 150 mM NaCl), Wash solution 2 (20 mM Tris HCl pH 8.1, 0.1% SDS, 1% Triton x-100, 2 mM EDTA, 500 mM NaCl), Wash solution 3 (10 mM Tris HCl pH 8.1, 1% NP-40, 1% NaDoc, 1 mM EDTA, 250 mM LiCl), and finally with TE. DNA was then eluted with 1% SDS in TE at 65°C for 10 minutes and protein was degraded with Proteinase K for 2 hours at 37°C. Finally, DNA was purified using QIAGEN PCR purification Kit (28106, QIAGEN) and analyzed by qPCR.

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