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Solving the MCM paradox by visualizing the scaffold of CMG helicase at active replisomes

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  • Hana Polasek-Sedlackova

    (University of Copenhagen
    Institute of Biophysics, Czech Academy of Sciences)

  • Thomas C. R. Miller

    (University of Copenhagen)

  • Jana Krejci

    (Institute of Biophysics, Czech Academy of Sciences)

  • Maj-Britt Rask

    (University of Copenhagen)

  • Jiri Lukas

    (University of Copenhagen)

Abstract

Genome duplication is safeguarded by constantly adjusting the activity of the replicative CMG (CDC45-MCM2-7-GINS) helicase. However, minichromosome maintenance proteins (MCMs)—the structural core of the CMG helicase—have never been visualized at sites of DNA synthesis inside a cell (the so-called MCM paradox). Here, we solve this conundrum by showing that anti-MCM antibodies primarily detect inactive MCMs. Upon conversion of inactive MCMs to CMGs, factors that are required for replisome activity bind to the MCM scaffold and block MCM antibody binding sites. Tagging of endogenous MCMs by CRISPR-Cas9 bypasses this steric hindrance and enables MCM visualization at active replisomes. Thus, by defining conditions for detecting the structural core of the replicative CMG helicase, our results explain the MCM paradox, provide visual proof that MCMs are an integral part of active replisomes in vivo, and enable the investigation of replication dynamics in living cells exposed to a constantly changing environment.

Suggested Citation

  • Hana Polasek-Sedlackova & Thomas C. R. Miller & Jana Krejci & Maj-Britt Rask & Jiri Lukas, 2022. "Solving the MCM paradox by visualizing the scaffold of CMG helicase at active replisomes," Nature Communications, Nature, vol. 13(1), pages 1-11, December.
    • Handle: RePEc:nat:natcom:v:13:y:2022:i:1:d:10.1038_s41467-022-33887-5
    DOI: 10.1038/s41467-022-33887-5
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    References listed on IDEAS

    as
    1. James M. Dewar & Magda Budzowska & Johannes C. Walter, 2015. "The mechanism of DNA replication termination in vertebrates," Nature, Nature, vol. 525(7569), pages 345-350, September.
    2. Ferdos Abid Ali & Max E. Douglas & Julia Locke & Valerie E. Pye & Andrea Nans & John F. X. Diffley & Alessandro Costa, 2017. "Cryo-EM structure of a licensed DNA replication origin," Nature Communications, Nature, vol. 8(1), pages 1-10, December.
    3. Hana Sedlackova & Maj-Britt Rask & Rajat Gupta & Chunaram Choudhary & Kumar Somyajit & Jiri Lukas, 2020. "Equilibrium between nascent and parental MCM proteins protects replicating genomes," Nature, Nature, vol. 587(7833), pages 297-302, November.
    4. Thomas C. R. Miller & Julia Locke & Julia F. Greiwe & John F. X. Diffley & Alessandro Costa, 2019. "Mechanism of head-to-head MCM double-hexamer formation revealed by cryo-EM," Nature, Nature, vol. 575(7784), pages 704-710, November.
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