Unwinding DNA with MCM8/9 Complex

To prepare the substrate, the oligonucleotide (5'-(dT)₄₀GTTTTCCCAGTCACGACG-TTGTAAAACGACGGCCAGTGCC-3') containing a 40 nt region complementary to the M13mp18(+) strand and a 40 nt oligo-dT at the 5′ end was labeled at the 3′ terminus with [α-³²P] dCTP (Perkin Elmer) and annealed to the single-stranded DNA M13mp18 (Huang et al., 2020 (link)). 0.1 nM (in molecules) DNA substrates were mixed with 5 µg recombinant MCM8/9 complex as well as its mutants as indicated within each 15 µl volume reaction in the helicase buffer (25 mM HEPES, pH 7.5, 1 mM magnesium acetate, 25 mM sodium acetate, pH 5.2, 4 mM ATP, 0.1 mg/ml BSA, 1 mM DTT). 2.5 µg HROB was used as an activator. To avoid re-annealing, the reaction was supplemented with a 100-fold unlabeled oligonucleotide. The reactions were then incubated at 37 °C for 60 min and stopped by adding 1 µl of stop buffer (0.4% SDS, 30 mM EDTA, and 6% glycerol) and 1 µl of proteinase K (20 mg/ml, Sigma) into the reaction for another 10 min incubation at 37 °C. The products were separated by 15% polyacrylamide gel electrophoresis in 1x TBE buffer and analyzed by the Amersham typhoon (Cytiva).