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Cell Rep. 2019 Sep 3;28(10):2673-2688.e8. doi: 10.1016/j.celrep.2019.07.104.

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

Author information

1
Macromolecular Machines Laboratory, The Francis Crick Institute, London NW1 1AT, UK.
2
Single Molecule Imaging of Genome Duplication and Maintenance Laboratory, The Francis Crick Institute, London NW1 1AT, UK.
3
Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, CA 94720, USA.
4
Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, CA 94720, USA; Institute of Technology, University of Tartu, Tartu 50411, Estonia.
5
Structural Biology Science Technology Platform, The Francis Crick Institute, 1 Midland Road, London NW1 1AT, UK.
6
Department of Biophysics and Biophysical Chemistry, Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA.
7
Single Molecule Imaging of Genome Duplication and Maintenance Laboratory, The Francis Crick Institute, London NW1 1AT, UK. Electronic address: hasan.yardimci@crick.ac.uk.
8
Macromolecular Machines Laboratory, The Francis Crick Institute, London NW1 1AT, UK. Electronic address: alessandro.costa@crick.ac.uk.

Abstract

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.

KEYWORDS:

AAA+ ATPase; DNA replication; DNA unwinding; cryo-EM; helicase; molecular motor

PMID:
31484077
DOI:
10.1016/j.celrep.2019.07.104
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