Cryo-EM structures reveal high-resolution mechanism of a DNA polymerase sliding clamp loader

  1. Christl Gaubitz
  2. Xingchen Liu
  3. Joshua Pajak
  4. Nicholas P Stone
  5. Janelle A Hayes
  6. Gabriel Demo
  7. Brian A Kelch PhD  Is a corresponding author
  1. University of Massachusetts Medical School, United States
  2. Central European Institute of Technology - Masaryk University, Czech Republic

Abstract

Sliding clamps are ring-shaped protein complexes that are integral to the DNA replication machinery of all life. Sliding clamps are opened and installed onto DNA by clamp loader AAA+ ATPase complexes. However, how a clamp loader opens and closes the sliding clamp around DNA is still unknown. Here, we describe structures of the S. cerevisiae clamp loader Replication Factor C (RFC) bound to its cognate sliding clamp Proliferating Cell Nuclear Antigen (PCNA) en route to successful loading. RFC first binds to PCNA in a dynamic, closed conformation that blocks both ATPase activity and DNA binding. RFC then opens the PCNA ring through a large-scale 'crab-claw' expansion of both RFC and PCNA that explains how RFC prefers initial binding of PCNA over DNA. Next, the open RFC:PCNA complex binds DNA and interrogates the primer-template junction using a surprising base-flipping mechanism. Our structures indicate that initial PCNA opening and subsequent closure around DNA do not require ATP hydrolysis, but are driven by binding energy. ATP hydrolysis, which is necessary for RFC release, is triggered by interactions with both PCNA and DNA, explaining RFC’s switch-like ATPase activity. Our work reveals how a AAA+ machine undergoes dramatic conformational changes for achieving binding preference and substrate remodeling.

Data availability

All coordinates and cryoEM maps were deposited in the PDB and EMDB during revision.

The following data sets were generated

Article and author information

Author details

  1. Christl Gaubitz

    Department of Biochemistry and Molecular Pharmacology, University of Massachusetts Medical School, Worcester, United States
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-6047-9282
  2. Xingchen Liu

    Department of Biochemistry and Molecular Pharmacology, University of Massachusetts Medical School, Worcester, United States
    Competing interests
    The authors declare that no competing interests exist.
  3. Joshua Pajak

    Department of Biochemistry and Molecular Pharmacology, University of Massachusetts Medical School, Worcester, United States
    Competing interests
    The authors declare that no competing interests exist.
  4. Nicholas P Stone

    Biochemistry and Molecular Pharmacology, University of Massachusetts Medical School, Worcester, United States
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-5869-0329
  5. Janelle A Hayes

    Department of Biochemistry and Molecular Pharmacology, University of Massachusetts Medical School, Worcester, United States
    Competing interests
    The authors declare that no competing interests exist.
  6. Gabriel Demo

    Central European Institute of Technology - Masaryk University, Brno, Czech Republic
    Competing interests
    The authors declare that no competing interests exist.
  7. Brian A Kelch PhD

    Department of Biochemistry and Molecular Pharmacology, University of Massachusetts Medical School, Worcester, United States
    For correspondence
    brian.kelch@umassmed.edu
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-1369-6989

Funding

National Institute of General Medical Sciences (R01-GM127776-02)

  • Brian A Kelch PhD

Schweizerischer Nationalfonds zur Förderung der Wissenschaftlichen Forschung (177859)

  • Christl Gaubitz

Schweizerischer Nationalfonds zur Förderung der Wissenschaftlichen Forschung (168972)

  • Christl Gaubitz

MEYS CR ERC CZ (LL2008)

  • Gabriel Demo

The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.

Reviewing Editor

  1. Franziska Bleichert, Yale University, United States

Publication history

  1. Preprint posted: September 23, 2021 (view preprint)
  2. Received: September 24, 2021
  3. Accepted: February 1, 2022
  4. Accepted Manuscript published: February 18, 2022 (version 1)
  5. Version of Record published: March 3, 2022 (version 2)

Copyright

© 2022, Gaubitz et al.

This article is distributed under the terms of the Creative Commons Attribution License permitting unrestricted use and redistribution provided that the original author and source are credited.

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  1. Christl Gaubitz
  2. Xingchen Liu
  3. Joshua Pajak
  4. Nicholas P Stone
  5. Janelle A Hayes
  6. Gabriel Demo
  7. Brian A Kelch PhD
(2022)
Cryo-EM structures reveal high-resolution mechanism of a DNA polymerase sliding clamp loader
eLife 11:e74175.
https://doi.org/10.7554/eLife.74175
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