Topoisomerase 1

Relaxed DNA was prepared in a 50 µl reaction mixture by incubating negatively supercoiled plasmid pUC19 DNA (0.5 µg) with calf thymus Topoisomerase I (1 Unit; Invitrogen) in buffer containing 50 mM Tris HCl pH 7.5, 50 mM KCl, 10 mM MgCl₂, 0.5 mM DTT, 0.1 mM EDTA, 100 µg ml⁻¹ BSA for 30 min at 37 °C. To test the unwinding activity of RAD51, RAD51 K133A, RAD51 K133R, these proteins were incubated with relaxed pUC19 (25 µM, nt) and unlabeled #160 (12.5 µM, nt) in the D-loop formation buffer supplemented with 10 mM magnesium acetate^{19 (link)} for 5 min at 37 °C. When BRC4 was used, the proteins were preincubated with BRC4 on ice for 15 min prior to addition to the reaction. Then, 1 U of calf thymus Topoisomerase I was added followed by incubation for 20 min at 37 °C. The reaction was terminated as described in D-loop formation section. The samples were analyzed by electrophoresis on 1.5% agarose gel containing TAE (40 mM Tris acetate, pH 8.0, and 1 mM EDTA) at 20 V cm⁻¹ for 1.5 h. The gels were stained with Ethidium Bromide (2 µg ml⁻¹) for 1 h and destained with nanopure water for 24 h. The DNA bands were visualized using an AlphaImager 3400 system, and auto-contrast was applied using Image Ready 7.0 (Adobe).

Histone H3-H4 (1 μM) were incubated with increasing concentrations of POLE3-POLE4 (1-8 μM) in 15 μL reaction buffer containing 10 mM Tris-HCl, pH 7.5, 150 mM NaCl, 0.5 mM EDTA, 10% glycerol and 50 μg/mL BSA at 37°C for 30 min. The circular plasmid, phix174 RF1 DNA was pre-treated with topoisomerase I (Invitrogen, 10 μ/lg DNA) for 30 min and then added (0.4 u μ g in 1 μL) into each of the chaperone-histone mix and incubated for an additional 60 min. Reactions were stopped by adding equal volume of stop buffer (glycerol 25%, 60 mM Tris-HCl, pH 8.0, 30 mM EDTA, 2.0% SDS, 2 μg/μL) followed by further 30 min incubation. Products were resolved by electrophoresis in 1.0% agarose gel followed by SYBR Gold staining.

Topoisomerase I (Thermo Fisher, cat. no. 38042024,) was used to relax the plasmid DNA to different extents depending on the ethidium bromide (Sigma-Aldrich, cat. no. E1510-10 ml) concentrations (0 to 24 µg ml⁻¹) in its 1× supplied reaction buffer (50 mM Tris–HCl, pH 7.8, 50 mM NaCl, 10 mM MgCl₂, 0.5 mM DTT, 0.1 mM EDTA and 30 µg ml⁻¹ BSA) at 37 °C for 2 h. Phenol–chloroform extraction was used to remove ethidium bromide from the plasmid DNA. Different levels of negative supercoiling counteracted the torsional stress upon removal of the intercalator. Ethanol precipitation was used to collect the DNA.