In vitro DNA cleavage assay.

Linear substrate DNA used in the cleavage experiments was generated by PCR using the pBR322-based vectors as template and purified by agarose gel electrophoresis. Wild-type or mutant HzTransib (300 nM final concentration), substrate DNA (final concentration 30 nM) were incubated in reaction buffer (25 mM MOPS, pH7.0, 50 mM KCl, 2 mM DTT, 5 mM MgCl₂; 16 μl final reaction volume) at 30°C for 1 h. Reactions were stopped by adding 1.25 μl 2.5% SDS, 5 μl proteinase K (200 μg/ml) and 2 μl 0.5 M EDTA followed by incubation at 55°C for 3 h. Samples were briefly centrifuged and the supernatant mixed with 6 μl 5x high density TBE sample buffer (ThermoFisher Scientific) and loaded on a non-denaturing 1x Tris-borate-EDTA (TBE) buffered polyacrylamide gel (Bio-Rad or ThermoFisher Scientific). After 35 min electrophoresis at 160V, gels were stained with SYBR gold (ThermoFisher Scientific) in 1xTBE buffer for 1 h and imaged using a PharosFX Plus (Bio-Rad).