Cryo-EM structures reveal high-resolution mechanism of a DNA polymerase sliding clamp loader
- University of Massachusetts Medical School, United States
- 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.
Article and author information
Author details
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
- Franziska Bleichert, Yale University, United States
Version history
- Preprint posted: September 23, 2021 (view preprint)
- Received: September 24, 2021
- Accepted: February 1, 2022
- Accepted Manuscript published: February 18, 2022 (version 1)
- 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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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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