Ilustra microspin g 25 columns

The oligodeoxyribonucleotides used for the preparation of DNA substrates for the ATPase and helicase activity tests are described in Supplementary Table S1. For the helicase assays, oligonucleotides were radioactively labeled prior to annealing. 5′-ends were labeled with ³³P-γ-ATP (5000 Ci/mmol; Hartmann Analytic) and T4 PNK for 1 h at 37°C in forward reaction buffer (80 mM Tris-HCl pH 7.4, 10 mM MgCl₂ and 5 mM DTT). The reaction was stopped by heating to 72°C for 10 min. Subsequently unincorporated nucleotides were removed by size-exclusion chromatography using Ilustra MicroSpin G-25 columns (GE Healthcare). To prepare Y-shaped DNA substrates, 5′-end-labeled oligonucleotides were mixed with partially complementary unlabeled oligonucleotide at a molar ratio of 1:1.5 in binding buffer (30 mM Tris-HCl, 10 mM MgCl₂, pH 7.5), heated to 95°C for 5 min and allowed to slowly cool down to room temperature.

5΄-ends were labeled with PNK and ³³PγATP (5000 Ci/mmol) from Hartmann Analytic (Braunschweig, Germany) for 1 h at 37°C in forward reaction buffer (80 mM Tris–HCl pH 7.4, 10 mM MgCl₂, and 5 mM DTT). Reaction was stopped by heating to 72°C for 10 min. Unincorporated nucleotides were removed using Ilustra MicroSpin G-25 columns (GE Healthcare; Freiburg, Germany).