DNA unwinding mechanism of a eukaryotic replicative CMG helicase

High-resolution structures have not been reported for replicative helicases at a replication fork at atomic resolution, a prerequisite to understand the unwinding mechanism. The eukaryotic replicative CMG helicase contains a Mcm2-7 motor ring, with the N-tier ring in front and the C-tier motor ring behind. The N-tier ring is structurally divided into a zinc finger (ZF) sub-ring followed by the OB fold ring. Here we report the cryo-EM structure of CMG on forked DNA at 3.9 Å, revealing that parental DNA enters the ZF sub-ring and strand separation occurs at the bottom of the ZF sub-ring, where the lagging strand is blocked and diverted sideways by OB hairpin-loops of Mcm3, Mcm4, Mcm6, and Mcm7. Thus, instead of employing a separation pin, unwinding is achieved via a “dam-and-diversion tunnel” for steric exclusion unwinding. The C-tier motor ring contains spirally configured PS1 and H2I loops of Mcms 2, 3, 5, 6 that translocate on the spirally-configured leading strand, and thereby pull the preceding DNA segment through the diversion tunnel for strand separation.