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The nucleotide addition cycle of the SARS-CoV-2 polymerase

View ORCID ProfileSubhas Chandra Bera, View ORCID ProfileMona Seifert, View ORCID ProfileRobert N. Kirchdoerfer, Pauline van Nies, Yibulayin Wubulikasimu, Salina Quack, Flávia S. Papini, View ORCID ProfileJamie J. Arnold, View ORCID ProfileBruno Canard, Craig E. Cameron, Martin Depken, View ORCID ProfileDavid Dulin
doi: https://doi.org/10.1101/2021.03.27.437309
Subhas Chandra Bera
1Junior Research Group 2, Interdisciplinary Center for Clinical Research, Friedrich-Alexander-University Erlangen-Nürnberg (FAU), Cauerstr. 3, 91058 Erlangen, Germany
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  • ORCID record for Subhas Chandra Bera
Mona Seifert
1Junior Research Group 2, Interdisciplinary Center for Clinical Research, Friedrich-Alexander-University Erlangen-Nürnberg (FAU), Cauerstr. 3, 91058 Erlangen, Germany
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Robert N. Kirchdoerfer
2Department of Biochemistry and Institute for Molecular Virology, University of Wisconsin-Madison, Madison, WI 53706
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Pauline van Nies
1Junior Research Group 2, Interdisciplinary Center for Clinical Research, Friedrich-Alexander-University Erlangen-Nürnberg (FAU), Cauerstr. 3, 91058 Erlangen, Germany
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Yibulayin Wubulikasimu
1Junior Research Group 2, Interdisciplinary Center for Clinical Research, Friedrich-Alexander-University Erlangen-Nürnberg (FAU), Cauerstr. 3, 91058 Erlangen, Germany
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Salina Quack
1Junior Research Group 2, Interdisciplinary Center for Clinical Research, Friedrich-Alexander-University Erlangen-Nürnberg (FAU), Cauerstr. 3, 91058 Erlangen, Germany
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Flávia S. Papini
1Junior Research Group 2, Interdisciplinary Center for Clinical Research, Friedrich-Alexander-University Erlangen-Nürnberg (FAU), Cauerstr. 3, 91058 Erlangen, Germany
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Jamie J. Arnold
4Department of Microbiology and Immunology, University of North Carolina School of Medicine, Chapel Hill, NC 27599 USA
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Bruno Canard
3Architecture et Fonction des Macromolécules Biologiques, CNRS and Aix-Marseille Université, UMR 7257, Polytech Case 925, 13009 Marseille, France
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Craig E. Cameron
4Department of Microbiology and Immunology, University of North Carolina School of Medicine, Chapel Hill, NC 27599 USA
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Martin Depken
5Department of Bionanoscience, Kavli Institute of Nanoscience, Delft University of Technology, Van der Maasweg 9, 2629 HZ Delft, The Netherlands
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  • For correspondence: s.m.depken@tudelft.nl d.dulin@vu.nl
David Dulin
1Junior Research Group 2, Interdisciplinary Center for Clinical Research, Friedrich-Alexander-University Erlangen-Nürnberg (FAU), Cauerstr. 3, 91058 Erlangen, Germany
6Department of Physics and Astronomy, and LaserLaB Amsterdam, Vrije Universiteit Amsterdam, De Boelelaan 1081, 1081 HV, Amsterdam, The Netherlands
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  • For correspondence: s.m.depken@tudelft.nl d.dulin@vu.nl
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Abstract

Coronaviruses have evolved elaborate multisubunit machines to replicate and transcribe their genomes. Central to these machines are the RNA-dependent RNA polymerase subunit (nsp12) and its intimately associated cofactors (nsp7 and nsp8). We have used a high-throughput magnetic-tweezers approach to develop a mechanochemical description of this core polymerase. The core polymerase exists in at least three catalytically distinct conformations, one being kinetically consistent with incorporation of incorrect nucleotides. We provide the first evidence that an RdRp uses a thermal ratchet instead of a power stroke to transition from the pre- to post-translocated state. Ultra-stable magnetic tweezers enables the direct observation of coronavirus polymerase deep and long-lived backtrack that are strongly stimulated by secondary structure in the template. The framework presented here elucidates one of the most important structure-dynamics-function relationships in human health today, and will form the grounds for understanding the regulation of this complex.

Competing Interest Statement

The authors have declared no competing interest.

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The copyright holder for this preprint is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under a CC-BY-NC-ND 4.0 International license.
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Posted March 27, 2021.
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The nucleotide addition cycle of the SARS-CoV-2 polymerase
Subhas Chandra Bera, Mona Seifert, Robert N. Kirchdoerfer, Pauline van Nies, Yibulayin Wubulikasimu, Salina Quack, Flávia S. Papini, Jamie J. Arnold, Bruno Canard, Craig E. Cameron, Martin Depken, David Dulin
bioRxiv 2021.03.27.437309; doi: https://doi.org/10.1101/2021.03.27.437309
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The nucleotide addition cycle of the SARS-CoV-2 polymerase
Subhas Chandra Bera, Mona Seifert, Robert N. Kirchdoerfer, Pauline van Nies, Yibulayin Wubulikasimu, Salina Quack, Flávia S. Papini, Jamie J. Arnold, Bruno Canard, Craig E. Cameron, Martin Depken, David Dulin
bioRxiv 2021.03.27.437309; doi: https://doi.org/10.1101/2021.03.27.437309

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