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In order to determine the minimum requirement for double-hexamer formation, a set of synthetic bubble-like substrates (Bub-T10, Bub-T20, Bub-T30, Bub-T40 and Bub-T50) that differed in the length of the central unpaired segment were constructed, and the binding and helicase activities of Mcm4/6/7 on these substrates were examined. Mcm4/6/7 was incubated with a radiolabeled synthetic bubble substrate in the presence of 0.5 mM ATP-γ-S to allow complex formation. A half of these reaction mixtures were analyzed for DNA binding in gel shift assays (Figure 2A), and the remainder was further incubated in the presence of 10 mM ATP to measure DNA helicase activity (Figure 2B). Bub-10 generated only a low level of complexes with Mcm4/6/7, and Bub-20 generated primarily a single mobility-shifted form, which may contain a single hexamer of Mcm4/6/7. On Bub-30, significant amount of complexes containing a double hexamer were generated in addition to those containing a single hexamer (Figure 2A) (13,21). The amounts of double hexamer complexes increased on Bub-T40 and Bub-T50 with concomitant decrease of single hexamer complexes (Figure 2A). 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