Dna sample buffer

Varying amounts of purified RuvA protein were incubated with 5′-³²P-labelled synthetic Holliday junction (HJ-Y2Ap) for 30 min at 37 °C in 5 mM EDTA, 1 mM DTT, 100 μg ml⁻¹ BSA, 30 mM Tris-HCl 8 buffer (buffer 7). DNA sample buffer (New England Biolabs) was added to the reaction and the complex formation was assayed by electrophoresis in a 6% polyacrylamide gel (1× TAE). Electrophoresis was carried out at 4 °C at 150 V for 1.5 h in a 0.5× TAE buffer. Gels were dried, and DNA bands were visualized by autoradiography.

Holliday junctions with mobile (HJ-X26)^{55 (link)} and immobile (HJ-Y2Ap, modified from Y2A^{17 (link)}) cores were prepared by annealing synthetic oligonucleotides (Sigma Aldrich) provided in Supplementary information Table 3, following a previously published protocol^{56 (link)}. In brief, the oligonucleotides were purified by native 6% PAGE (TAE buffer) and mixed in appropriate ratios in annealing buffer (buffer 4) (25 mM NaCl, 10 mM Tris-HCl pH 8). The annealing reaction was performed in a 0.2 ml tube and covered with a thin layer of mineral oil to prevent water evaporation. The mixture was heated to 95 °C for 10 min, and the temperature was subsequently decrease in 10 °C temperature steps every 10 min. To obtain homogenous four-way Holliday junction preparations, the annealing reaction was supplemented with a DNA sample buffer (New England Biolabs) and separated by native 6% PAGE (TAE buffer). Bands corresponding to four-way Holliday junctions were cut out from the gel and eluted by incubation in 5 mM Tris-HCl pH 8. For DNA-binding assays (electro mobility shift assay (EMSA)), one oligonucleotide strand was labelled with radioactive ³²P (3,000 Ci mmol⁻¹) at the 5′ end prior to annealing. For the branch migration activity assays, one oligonucleotide strand was fluorescently labelled with ATTO 647N.