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Cdt1 stabilizes an open MCM ring for helicase loading

Author

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  • Jordi Frigola

    (Chromosome Replication Laboratory, The Francis Crick Institute
    Present address: Institut d'Investigació Biomèdica de Girona Dr. Josep Trueta (IDIBGI), Parc Hospitalari Martí i Julià, 17190 Salt, Catalunya, Spain)

  • Jun He

    (Chromosome Replication Laboratory, The Francis Crick Institute
    Chromatin Structure and Mobile DNA Laboratory, The Francis Crick Institute
    Present address: UCB Celltech, 216 Bath Road, Slough SL1 3WE, UK)

  • Kerstin Kinkelin

    (Chromosome Replication Laboratory, The Francis Crick Institute)

  • Valerie E. Pye

    (Chromatin Structure and Mobile DNA Laboratory, The Francis Crick Institute)

  • Ludovic Renault

    (Macromolecular Machines Laboratory, The Francis Crick Institute
    Present address: NeCEN, Gorlaeus Laboratory, Einsteinweg 55, Leiden 2333, The Netherlands)

  • Max E. Douglas

    (Chromosome Replication Laboratory, The Francis Crick Institute)

  • Dirk Remus

    (Molecular Biology Program, Memorial Sloan Kettering Cancer Center)

  • Peter Cherepanov

    (Chromatin Structure and Mobile DNA Laboratory, The Francis Crick Institute)

  • Alessandro Costa

    (Macromolecular Machines Laboratory, The Francis Crick Institute)

  • John F. X. Diffley

    (Chromosome Replication Laboratory, The Francis Crick Institute)

Abstract

ORC, Cdc6 and Cdt1 act together to load hexameric MCM, the motor of the eukaryotic replicative helicase, into double hexamers at replication origins. Here we show that Cdt1 interacts with MCM subunits Mcm2, 4 and 6, which both destabilizes the Mcm2–5 interface and inhibits MCM ATPase activity. Using X-ray crystallography, we show that Cdt1 contains two winged-helix domains in the C-terminal half of the protein and a catalytically inactive dioxygenase-related N-terminal domain, which is important for MCM loading, but not for subsequent replication. We used these structures together with single-particle electron microscopy to generate three-dimensional models of MCM complexes. These show that Cdt1 stabilizes MCM in a left-handed spiral open at the Mcm2–5 gate. We propose that Cdt1 acts as a brace, holding MCM open for DNA entry and bound to ATP until ORC–Cdc6 triggers ATP hydrolysis by MCM, promoting both Cdt1 ejection and MCM ring closure.

Suggested Citation

  • Jordi Frigola & Jun He & Kerstin Kinkelin & Valerie E. Pye & Ludovic Renault & Max E. Douglas & Dirk Remus & Peter Cherepanov & Alessandro Costa & John F. X. Diffley, 2017. "Cdt1 stabilizes an open MCM ring for helicase loading," Nature Communications, Nature, vol. 8(1), pages 1-10, August.
    • Handle: RePEc:nat:natcom:v:8:y:2017:i:1:d:10.1038_ncomms15720
    DOI: 10.1038/ncomms15720
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