Molecular basis for ATP-hydrolysis-driven DNA translocation by the CMG helicase of the eukaryotic replisome. Patrik Eickhoff Hazal B Kose Fabrizio Martino Tatjana Petojevic Ferdos Abid Ali Julia Locke Nele Tamberg Andrea Nans James M Berger Michael R Botchan Hasan Yardimci Alessandro Costa 10779/crick.11619507.v1 https://crick.figshare.com/articles/journal_contribution/Molecular_basis_for_ATP-hydrolysis-driven_DNA_translocation_by_the_CMG_helicase_of_the_eukaryotic_replisome_/11619507 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. 2020-01-15 16:50:22 AAA+ ATPase DNA replication DNA unwinding cryo-EM helicase molecular motor Yardimci FC001221 Costa FC001065 EM-ack SB 0601 Biochemistry and Cell Biology