PMC:1140370 / 17348-19199 JSONTXT

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Binding and helicase actions of Mcm4/6/7 on forked and 3- or 5-extension substrates In order to examine more precisely the interaction and helicase actions with forked substrates, Y-fork substrates containing a nascent leading strand (A-fork[3]), nascent lagging strand (A-fork[5]), or both of them (A-fork[3,5]), as well as 5-extension or 3-extension structures were constructed. Mcm4/6/7 bound to A-fork[3] or A-fork[5] with affinity similar to that of a simple Y-fork (Figure 4A and data not shown). In contrast, PriA helicase, used as a control, bound to A-fork[3] more efficiently than to A-fork[5], as reported previously (23,25). Mcm4/6/7 bound also to 5-extension and 3-extension, albeit with slightly reduced affinity compared to A-fork[3] and A-fork[5] (Figure 4A). Mcm4/6/7 bound to A-fork[3,5] with much lower affinity, and the complex gave rise to a single band. These findings indicate that the Mcm4/6/7 complex is loaded onto DNA preferentially and efficiently through single-stranded region, regardless of the presence of 3- or 5-terminus. In contrast, only the substrate containing a 3 single-stranded DNA tail (A-fork[5]) was efficiently unwound and A-fork[3] or A-fork[3,5], was not unwound by Mcm4/6/7 (Figure 4B). Absence of helicase actions on A-fork[3,5] composed only of duplex regions is expected since double-stranded DNA is not able to activate the ATPase activity of Mcm4/6/7 (10). In contrast, the PriA, a structure-specific helicase used as a control, binds at the fork junction and unwinds the fork and the nascent lagging strand DNA on A-fork[5], or unwinds the nascent lagging strand on A-fork[3,5], as reported before (25). These results indicate that the Mcm4/6/7 complex binds to single-stranded DNA and that its helicase is activated only in the presence of a single-stranded 3-tail.