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
Affiliations
Patrik Eickhoff
Macromolecular Machines Laboratory, The Francis Crick Institute, London NW1 1AT, UK
Hazal B. Kose
Single Molecule Imaging of Genome Duplication and Maintenance Laboratory, The Francis Crick Institute, London NW1 1AT, UK
Fabrizio Martino
Macromolecular Machines Laboratory, The Francis Crick Institute, London NW1 1AT, UK
Tatjana Petojevic
Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, CA 94720, USA
Ferdos Abid Ali
Macromolecular Machines Laboratory, The Francis Crick Institute, London NW1 1AT, UK
Julia Locke
Macromolecular Machines Laboratory, The Francis Crick Institute, London NW1 1AT, UK
Nele Tamberg
Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, CA 94720, USA; Institute of Technology, University of Tartu, Tartu 50411, Estonia
Andrea Nans
Structural Biology Science Technology Platform, The Francis Crick Institute, 1 Midland Road, London NW1 1AT, UK
James M. Berger
Department of Biophysics and Biophysical Chemistry, Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA
Michael R. Botchan
Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, CA 94720, USA
Hasan Yardimci
Single Molecule Imaging of Genome Duplication and Maintenance Laboratory, The Francis Crick Institute, London NW1 1AT, UK; Corresponding author
Alessandro Costa
Macromolecular Machines Laboratory, The Francis Crick Institute, London NW1 1AT, UK; Corresponding author
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
