Duplex DNA engagement and RPA oppositely regulate the DNA-unwinding rate of CMG helicase.

A ring-shaped helicase unwinds DNA during chromosome replication in all organisms. Replicative helicases generally unwind duplex DNA an order of magnitude slower compared to their in vivo replication fork rates. However, the origin of slow DNA unwinding rates by replicative helicases and the mechanism by which other replication components increase helicase speed are unclear.

Molecular Basis for ATP-Hydrolysis-Driven DNA Translocation by the CMG Helicase of the Eukaryotic Replisome.

In the eukaryotic replisome, DNA unwinding by the Cdc45-MCM-Go-Ichi-Ni-San (GINS) (CMG) helicase requires a hexameric ring-shaped ATPase named minichromosome maintenance (MCM), which spools single-stranded DNA through its central channel. Not all six ATPase sites are required for unwinding; however, the helicase mechanism is unknown. We imaged ATP-hydrolysis-driven translocation of the CMG using cryo-electron microscopy (cryo-EM) and found that the six MCM subunits engage DNA using four neighboring protomers at a time, with ATP binding promoting DNA engagement.

The mechanism of DNA unwinding by the eukaryotic replicative helicase.

Accurate DNA replication is tightly regulated in eukaryotes to ensure genome stability during cell division and is performed by the multi-protein replisome. At the core an AAA+ hetero-hexameric complex, Mcm2-7, together with GINS and Cdc45 form the active replicative helicase Cdc45/Mcm2-7/GINS (CMG). It is not clear how this replicative ring helicase translocates on, and unwinds, DNA.