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Autoregulation of the real-time kinetics of the human mitochondrial replicative helicase

Author

Listed:
  • Ismael Plaza-G A

    (IMDEA Nanociencia)

  • María Ortiz-Rodríguez

    (IMDEA Nanociencia)

  • Seth P. Buchanan

    (University of North Florida)

  • Samuel Miguez-Amil

    (Spanish National Cancer Research Centre (CNIO))

  • Kateryna M. Lemishko

    (IMDEA Nanociencia
    Quantemol Ltd)

  • Fernando Moreno-Herrero

    (Centro Nacional de Biotecnología (CSIC))

  • Rafael Fernandez-Leiro

    (Spanish National Cancer Research Centre (CNIO))

  • Grzegorz L. Ciesielski

    (University of North Florida)

  • Borja Ibarra

    (IMDEA Nanociencia
    Unidad Asociada al Centro Nacional de Biotecnología (CSIC))

Abstract

The human mitochondrial helicase Twinkle is essential for mitochondrial DNA (mtDNA) replication and integrity. Using biochemical and single-molecule techniques, we investigated Twinkle’s real-time kinetics, including DNA loading, unwinding, and rewinding, and their regulation by its N-terminal Zinc-binding domain (ZBD), C-terminal tail, and mitochondrial SSB protein (mtSSB). Our results indicate that Twinkle rapidly scans dsDNA to locate the fork, where specific interactions halt diffusion. During unwinding, ZBD-DNA interactions and C-terminal tail control of ATPase activity downregulate kinetics, slowing down the helicase. Binding of mtSSB to DNA likely outcompetes ZBD-DNA interactions, alleviating the downregulatory effects of this domain. Furthermore, we show that ZBD-DNA interactions and ATP binding also regulate rewinding kinetics following helicase stalling. Our findings reveal that ZBD and C-terminal tail play a major role in regulation of Twinkle´s real-time kinetics. Their interplay constitutes an auto-regulatory mechanism that may be relevant for coordinating the mtDNA maintenance activities of the helicase.

Suggested Citation

  • Ismael Plaza-G A & María Ortiz-Rodríguez & Seth P. Buchanan & Samuel Miguez-Amil & Kateryna M. Lemishko & Fernando Moreno-Herrero & Rafael Fernandez-Leiro & Grzegorz L. Ciesielski & Borja Ibarra, 2025. "Autoregulation of the real-time kinetics of the human mitochondrial replicative helicase," Nature Communications, Nature, vol. 16(1), pages 1-13, December.
    • Handle: RePEc:nat:natcom:v:16:y:2025:i:1:d:10.1038_s41467-025-60289-0
    DOI: 10.1038/s41467-025-60289-0
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    References listed on IDEAS

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    1. Daniel R. Burnham & Hazal B. Kose & Rebecca B. Hoyle & Hasan Yardimci, 2019. "The mechanism of DNA unwinding by the eukaryotic replicative helicase," Nature Communications, Nature, vol. 10(1), pages 1-14, December.
    2. John Jumper & Richard Evans & Alexander Pritzel & Tim Green & Michael Figurnov & Olaf Ronneberger & Kathryn Tunyasuvunakool & Russ Bates & Augustin Žídek & Anna Potapenko & Alex Bridgland & Clemens Me, 2021. "Highly accurate protein structure prediction with AlphaFold," Nature, Nature, vol. 596(7873), pages 583-589, August.
    3. Bo Sun & Daniel S. Johnson & Gayatri Patel & Benjamin Y. Smith & Manjula Pandey & Smita S. Patel & Michelle D. Wang, 2011. "ATP-induced helicase slippage reveals highly coordinated subunits," Nature, Nature, vol. 478(7367), pages 132-135, October.
    4. Zuanning Yuan & Roxana Georgescu & Lin Bai & Dan Zhang & Huilin Li & Michael E. O’Donnell, 2020. "DNA unwinding mechanism of a eukaryotic replicative CMG helicase," Nature Communications, Nature, vol. 11(1), pages 1-10, December.
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