In gel shift assays, Mcm4/6/7 proteins were incubated at 30°C for 30 min in reaction mixtures (15 μl) containing 25 mM HEPES–NaOH (pH 7.5), 50 mM sodium acetate, 10 mM Mg(CH3COO)2, 1 mM DTT, 0.25 μg/ml BSA, 0.5 mM ATP-γ-S and labeled substrates of the amount indicated. After addition of 2 μl of 50% glycerol, the reaction mixtures were directly applied on a polyacrylamide gel containing 6 mM magnesium acetate and 5% glycerol in 0.5× TBE, and electrophoresis was conducted at room temperature. For DNA helicase assays, the reaction mixtures of the gel shift assays, as described above, were incubated at 30°C for 15 min, and then the ATP was added at 10 mM, followed by incubation at 37°C for additional 30 min. The reactions were terminated by addition of EDTA (20 mM), SDS (0.1%) and 2 μg proteinase K, and were incubated for additional 15 min. The samples were separated by electrophoresis on nondenaturing polyacrylamide gel in 1× TBE.