Abstract
The Cdc45-MCM-GINS (CMG) helicase unwinds DNA during the elongation step of eukaryotic genome duplication and this process depends on the MCM ATPase function. Whether CMG translocation occurs on single- or double-stranded DNA and how ATP hydrolysis drives DNA unwinding remain open questions. Here we use cryo-electron microscopy to describe two subnanometre resolution structures of the CMG helicase trapped on a DNA fork. In the predominant state, the ring-shaped C-terminal ATPase of MCM is compact and contacts single-stranded DNA, via a set of pre-sensor 1 hairpins that spiral around the translocation substrate. In the second state, the ATPase module is relaxed and apparently substrate free, while DNA intimately contacts the downstream amino-terminal tier of the MCM motor ring. These results, supported by single-molecule FRET measurements, lead us to suggest a replication fork unwinding mechanism whereby the N-terminal and AAA+ tiers of the MCM work in concert to translocate on single-stranded DNA.
Highlights
The Cdc45-MCM-GINS (CMG) helicase unwinds DNA during the elongation step of eukaryotic genome duplication and this process depends on the MCM ATPase function
DNA fork progression depends on the ATPase function of the MCM motor[5,14], it is unknown how the energy derived from ATP hydrolysis is converted into motion and fork unwinding[15]
To start to address these outstanding questions, we have determined two cryoelectron microscopy structures of the CMG helicase trapped on a model DNA fork
Summary
Surmount the C-terminal face of the AAA þ pore and probably correspond to DNA-free winged helix MCM appendices[4], raising the question of whether DNA occupancy in this state is lower than in the compact ATPase state. Similar results were obtained with ATP (x0 1⁄4 0.96±0.01 for naked DNA and x0 1⁄4 0.48±0.01 for CMG), binding/stretching events occurred with lower frequency (Supplementary Fig. 12) These data agree with the observation that use of a slowly hydrolysable ATP analogue is required to stabilize the interaction between the MCM AAA þ domain and single-stranded DNA6,12, and support the notion that CMG binding stabilizes and stretches single-stranded DNA. The NTD and AAA þ tiers of Mcm[2,3,4,5,6,7] rotate in opposite directions, as they transition from a relaxed to a compact ATPase form (Fig. 6c) This rotation is compatible with a nucleotide-state-controlled inter-subunit movement, previously observed using double electron–electron resonance in the archaeal MCM motor[29]. Considering that DNA interacts with the inner perimeter of NTD-MCM running 30-50 in an anticlockwise manner[30], we note that a further anticlockwise NTD rotation would result in DNA duplex underwinding, compatible with origin DNA opening
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