Mcm2-7 Helicase Loading and Regulation by DDK

Each incubation step was performed in a thermomixer (Eppendorf) with shaking at 1250 rpm at 25°C. The DNA template was the same used in CMG-formation assays as described in Champasa et al., 2019 (link). Helicase loading reactions contained 100 nM Mcm2-7, 45 nM ORC, and 45 nM Cdc6. After loading, DDK was added at varying concentrations (0 nM, 20 nM, 80 nM, 120 nM, 260 nM, and 500 nM) for 20 min. The supernatant was removed by applying the reaction to a DynaMag-2 magnet (ThermoFisher Scientific). Reactions were washed with buffer H, 300 mM KCl, and 0.01% NP-40 three times. Proteins were released from the DNA by incubation with 5 U of DNA I (Worthington) in 10 mL of 25 mM HEPES-KOH (pH 7.6), 5 mM MgOAc, 200 mM NaCl, 5% glycerol, 0.02% NP-40, and 2 mM CaCl₂ for 20 min at 25°C before running on a pre-cast 10% SuperSep Phos-tag gel at 25 mA for > 2 hr. The gels were then stained using Krypton Protein Stain (ThermoFisher Scientific).
When Mcm4 N-terminal tail fused to SNAP-tag protein was used, then 500 nM of protein was phosphorylated with varying concentrations of DDK (0 nM, 20 nM, 80 nM, 120 nM, 260 nM, and 500 nM) for 20 min. Reactions were run on a pre-cast 10% SuperSep Phos-tag gel at 25 mA for >2 hr. The gels were then stained using Krypton Protein Stain (ThermoFisher Scientific).