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Molecular basis for ATP-hydrolysis-driven DNA translocation by the CMG helicase of the eukaryotic replisome.

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posted on 15.01.2020 by 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
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.

Funding

Crick (Grant ID: 10065, Grant title: Costa FC001065) Crick (Grant ID: 10221, Grant title: Yardimci FC001221) European Research Council (Grant ID: 820102 - CRYOREP, Grant title: ERC 820102 - CRYOREP)

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