Formation of double hexameric Mcm4/6/7 complexes facilitates helicase actions on bubble substrates
The above results suggest that Mcm4/6/7 generates a double hexameric complex on a bubble, each hexamer of which makes symmetric contact with the 5′-proximal central single-stranded segment. This predicts that the single-stranded segment needs to be of a sufficient length in order to accommodate the Mcm complex. In fact, we previously showed that the Mcm4/6/7 mainly forms a single hexameric complex on bubble-20 (a bubble substrate with 20 nt long central single-stranded segment) and a double hexameric complex on bubble-60 DNA (13). 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). The results indicate that a single-stranded segment of at least 40 nt long is required for efficient loading of the double-hexameric Mcm helicase on a bubble substrate.
In DNA helicase assays, Mcm4/6/7 complex did not show helicase activity on either Bub-10 or Bub-20 substrate. The efficiency of displacement slightly improved as the length of the single-stranded segment increased up to 30 nt. On Bub-T40 and Bub-T50, on the other hand, significant displacement was observed, indicating that a single-stranded segment of at least 40 nt long is required for efficient helicase actions on bubble substrates (Figure 2B). Taken together, the above results are consistent with the notion that the Mcm4/6/7 complex, loaded onto DNA as a double hexameric complex, efficiently unwinds the duplex region of the substrate DNA.