Figure 4.

DNA-binding properties of HpNikR to PnixA, PureA and PexbB in Ni-only and Ni/Mn conditions. The condition described in (A) corresponds to the one described by Zambelli et al. (13) with binding reactions performed in HEPES binding buffer [20 mM HEPES pH 7.85, 50 mM KCl, 0.01% Triton X-100, 0.1 mM DTT, 10% glycerol (v/v), 6 µg ml⁻¹ sonicated sperm DNA, 100 µM NiSO₄]. After a 15 min incubation with increasing HpNikR concentrations, complexes were separated on 5% acrylamide/bisacrylamide (19:1) gels, in MOPS/NaOAc running buffer (20 mM MOPS, 5 mM NaOAc pH 7.0), run at room temperature for 110 min at 170 V. EMSA conditions in (B) were Bis–Tris buffer and the binding conditions described in ‘Materials and Methods’ section of the manuscript (binding buffer with NiSO₄ and running buffer with MnSO₄).