Eukaryotic DNA replication is initiated during the G1 phase of the cell cycle, where the core of the replicative helicase, Minichromosome Maintenance complex (MCM), is loaded onto origins as an inactive double hexamer (origin licensing). During S-phase, the two MCMs are converted into two active Cdc45-MCM-GINS (CMG) holo-helicases that unwind DNA bi-directionally. This step, termed ‘origin firing’ is governed by two cell cycle regulated kinases, Dbf4-dependent kinase (DDK) and cyclin dependent kinase (CDK), which in turn mediate the recruitment of ‘firing factors’ (Cdc45, Sld3/7, Sld2, Dpb11, GINS, Polε and Mcm10). The active CMG helicase acts as the leading edge of the advancing replisome, a multi-subunit macromolecular machine that includes a primosome (Polymerase α-primase) as well as leading strand polymerase ε and lagging strand polymerase δ. CMG and Polε interact directly and GINS-Polε are simultaneously recruited during helicase assembly. Overall, several reconfigurations of the MCM ring have to occur to enable replication fork establishment, including stable recruitment of the helicase co-activators Cdc45 and GINS, double hexamer separation, DNA melting and activation of ATP hydrolysis. However, the molecular mechanism of CMG activation and replisome progression is poorly understood.

In this study, I used electron microscopy and biochemical techniques to visualise several MCM intermediates and inform the molecular basis for helicase activation. I showed that the inactive double hexamer forms a stable complex of two tilted rings and encircles bent, double-stranded DNA. Phosphorylation of N-terminal MCM tails by DDK creates a symmetrical docking platform, which may be used for downstream recruitment Cdc45 via the Sld3/7 chaperone. Double hexamer separation occurs during the GINS recruitment stage of CMG assembly prior to helicase activation, and this step is concomitant with melting of the DNA, through an MCM-mediated duplex stretching mechanism that untwists the double helix. Cdc45 and GINS bind to the side of the MCM ring at the N-terminal tier, stabilising the key Mcm2-5-3 sites that are essential for helicase activity. The active CMG helicase motor translocates on single-stranded DNA, excluding the complementary strand from the main channel. Moreover, the CMG unwinds DNA with its N-terminal MCM tier ahead of the motor domain, meaning that the two helicases cross paths upon activation to ensure that all origin DNA is unwound for genome stability maintenance. A cryo-EM structure of the CMG-Polε complex reveals that the essential, non-catalytic Polε subunits bind to the Mcm2-5-3 ATPase domains, stabilizing this region for productive fork engagement by the CMG. Polε is predominantly bound to the translocating CMG and bridges the GINS and MCM components, providing a rationale for the direct role of the leading strand polymerase in CMG assembly. Thus, regulated post-translational modifications and replisome partner protein interactions promote distinct helicase structural transitions. This flexibility is integral for MCM to fulfil its varied roles during origin licensing, helicase assembly, DNA melting and replication fork progression.