Cdt1 stabilizes an open MCM ring for helicase loading

Jordi Frigola,Max E Douglas,Dirk Remus,John F X Diffley,Jun He,Valerie E Pye,Ludovic Renault,Alessandro Costa,Peter Cherepanov,Kerstin Kinkelin

doi:10.1038/ncomms15720

Abstract

ORC, Cdc6 and Cdt1 act together to load hexameric MCM, the motor of the eukaryotic replicative helicase, into double hexamers at replication origins. Here we show that Cdt1 interacts with MCM subunits Mcm2, 4 and 6, which both destabilizes the Mcm2–5 interface and inhibits MCM ATPase activity. Using X-ray crystallography, we show that Cdt1 contains two winged-helix domains in the C-terminal half of the protein and a catalytically inactive dioxygenase-related N-terminal domain, which is important for MCM loading, but not for subsequent replication. We used these structures together with single-particle electron microscopy to generate three-dimensional models of MCM complexes. These show that Cdt1 stabilizes MCM in a left-handed spiral open at the Mcm2–5 gate. We propose that Cdt1 acts as a brace, holding MCM open for DNA entry and bound to ATP until ORC–Cdc6 triggers ATP hydrolysis by MCM, promoting both Cdt1 ejection and MCM ring closure.

Highlights

which would occur upon transitioning from an open lock-washer to the loaded MCM configuration
This is best visualized using the centre of mass of A
reconstituting a key active site that is essential for the ATPase function of MCM

Summary

Results

We noticed during the course of analysing Cdt[1] complexes containing different combinations of MCM subunits by gel filtration that Cdt[1] appeared to influence interactions between Mcm[2] and 5 An example of this is shown in Supplementary Fig. 1g. A stable tetrameric complex of Mcm[–] (the order of contiguous subunits in the MCM hexamer) could be identified by gel filtration, peaking in fraction 5 (Supplementary Fig. 1g(i)); upon addition of Cdt[1], this dissociated into two complexes: one containing Mcm[2, 6] and Cdt[1], peaking in fractions 3 and 4, and one containing Mcm[3] and 5, peaking in fractions 6–7 (Supplementary Fig. 1g(ii)).

PD I PD I PD I PD I PD I PD I PD I PD I PD

Cdt1 ejection

Discussion