Dna topoisomerase 1

DNA supercoiling assays were performed as described previously(Osakabe et al. 2010 (link)) with the following modifications. Relaxed DNA template was prepared by combining ϕX174 RF I DNA (NEB N3021S) with excess DNA topoisomerase I (TaKaRa 2240A) in the assembly buffer (10 mM Tris-HCl at pH 7.5, 125 mM NaCl, 2 mM MgCl₂, 0.5 mM DTT, 0.1 mg/mL BSA) for 1 h at 37°C and kept at room temperature ready for use. Histone octamers (200 ng) were preincubated with 250/500 ng of yNAP1, full-length sNASP, sNASP TPR monomer, sNASP TPR dimer, or sNASP TPRΔαC (2.5-fold/fivefold to octamer), respectively, in the assembly buffer for 30 min at 37°C. To initiate the assembly reaction, 100 ng of relaxed plasmid DNA was added to the chaperone–histone mixture and incubated for 90 min at 37°C. The standard 25-μL chromatin assembly reaction was stopped by adding EDTA to 20 mM and SDS to 0.5%, with an additional 0.5 mg/mL proteinase K, and incubation for 15 min at 55°C followed by phenol-chloroform extraction. The DNA samples were analyzed on a 1% agarose gel in 1× TAE buffer (40 mM Tris-acetate, 1 mM EDTA) for 4 h at 90 V with ethidium bromide (EB) staining.