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

Abstract

Summary: 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. Morphing between different helicase states leads us to suggest a non-symmetric hand-over-hand rotary mechanism, explaining the asymmetric requirements of ATPase function around the MCM ring of the CMG. By imaging of a higher-order replisome assembly, we find that the Mrc1-Csm3-Tof1 fork-stabilization complex strengthens the interaction between parental duplex DNA and the CMG at the fork, which might support the coupling between DNA translocation and fork unwinding. : Eickhoff et al. used cryo-EM to image DNA unwinding by the eukaryotic replicative helicase, Cdc45-MCM-GINS. As the hexameric MCM ring hydrolyses ATP, DNA is spooled asymmetrically around the ring pore. This asymmetry explains why selected ATPase sites are essential for DNA translocation. Understanding DNA unwinding informs on replication fork progression. Keywords: DNA replication, cryo-EM, AAA+ ATPase, helicase, molecular motor, DNA unwinding