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Electrophoretic mobility shift assays (EMSA) of GEN1-HJ complexes were carried out at different GEN1 concentrations in the pico and nano-molar ranges. In a total volume of 50 μl, GEN1 was incubated with Cy5-labeled DNA (50 pM or 2 nM) at room temperature for 30 minutes in binding buffer (40 mM Tris–HCl pH 7.5, 40 mM NaCl, 1 mM DTT, 2 mM CaCl₂, 0.1 mg/ml BSA, 5% (v/v) glycerol), and 5 ng/μl poly-dI-dC. Complexes were separated via 6% native PAGE (Invitrogen) in 1× TBE buffer at room temperature and imaged using the Typhoon Trio Imager (GE Healthcare) at 635 nm. The bands were quantified by GelQuantNET (BiochemLab Solutions). The bound substrate percentage was calculated from its contribution to the total fluorescence of the respective lane. The apparent binding constants K_{d-monomer-app-EMSA} and K_{d-dimer-app-EMSA} were calculated using the equation of the form Y = Max · [GEN1]ⁿ/(K_d-app-EMSAⁿ + [GEN1]ⁿ), where Max is the concentration at which the respective species reached its maximum (monomer or dimer), n is the Hill coefficient and K_d-app-EMSA is the apparent binding constant of the respective species, denoting the concentration of GEN1 at which half-maximum of monomer or dimer is present.

EMSA of coreTFIIH-DNA complexes were performed using different protein concentrations (0, 10, 20, 40, 60, 80, 100, 200, 300 and 400 nM). In a total volume of 12 μl, coreTFIIH and/or XPG were incubated with fluorescently labeled 5′ overhang DNA (5 nM) at 22°C for 10 min in binding buffer (40 mM Tris–HCl pH 7.5, 40 mM KCl, 1 mM DTT, 0.1 mg/ml BSA and 5% (v/v) glycerol). The complexes were separated via 6% native PAGE (Invitrogen) in 1× TBE buffer at room temperature and imaged using the Typhoon Trio Imager (GE Healthcare) at 635 nm. For band quantification, we used the GelQuantNET™ software (Biochemlab Solutions.com). The percentage of the bound substrate was calculated from its contribution to the total fluorescence of the respective lane. The binding constant K_d was calculated using the equation: Y = Max [coreTFIIH]ⁿ/(K_dⁿ + [coreTFIIH]ⁿ), where Max is the concentration at which 100% of the substrate was bound by the protein, and n is the Hill coefficient.