A second DNA binding site on RFC facilitates clamp loading at gapped or nicked DNA

  1. Xingchen Liu
  2. Christl Gaubitz  Is a corresponding author
  3. Joshua Pajak
  4. Brian A Kelch  Is a corresponding author
  1. University of Massachusetts Medical School, United States

Abstract

Clamp loaders place circular sliding clamp proteins onto DNA so that clamp-binding partner proteins can synthesize, scan, and repair the genome. DNA with nicks or small single-stranded gaps are common clamp-loading targets in DNA repair, yet these substrates would be sterically blocked given the known mechanism for binding of primer-template DNA. Here, we report the discovery of a second DNA binding site in the yeast clamp loader Replication Factor C (RFC) that aids in binding to nicked or gapped DNA. This DNA binding site is on the external surface and is only accessible in the open conformation of RFC. Initial DNA binding at this site thus provides access to the primary DNA binding site in the central chamber. Furthermore, we identify that this site can partially unwind DNA to create an extended single-stranded gap for DNA binding in RFC's central chamber and subsequent ATPase activation. Finally, we show that deletion of the BRCT domain, a major component of the external DNA binding site, results in defective yeast growth in the presence of DNA damage where nicked or gapped DNA intermediates occur. We propose that RFC’s external DNA binding site acts to enhance DNA binding and clamp loading, particularly at DNA architectures typically found in DNA repair.

Data availability

The reported cryo-EM map and atomic coordinates have been deposited in the Electron Microscopy Data Bank (entry numbers EMD-26280EMD-26298EMD-26302EMD-26297) and the Protein Data Bank (ID codes 7U1A, 7U1P, 7U19).

The following data sets were generated

Article and author information

Author details

  1. Xingchen Liu

    Department of Biochemistry and Molecular Biotechnology, University of Massachusetts Medical School, Worcester, United States
    Competing interests
    The authors declare that no competing interests exist.
  2. Christl Gaubitz

    Department of Biochemistry and Molecular Biotechnology, University of Massachusetts Medical School, Worcester, United States
    For correspondence
    cgaubitz@sund.ku.dk
    Competing interests
    The authors declare that no competing interests exist.
  3. Joshua Pajak

    Department of Biochemistry and Molecular Biotechnology, University of Massachusetts Medical School, Worcester, United States
    Competing interests
    The authors declare that no competing interests exist.
  4. Brian A Kelch

    Department of Biochemistry and Molecular Biotechnology, 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

NIGMS (1R01GM127776-01A1)

  • Brian A Kelch

Swiss National Science Foundation (177859)

  • Christl Gaubitz

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

Reviewing Editor

  1. Bruce Stillman, Cold Spring Harbor Laboratory, United States

Version history

  1. Preprint posted: January 31, 2022 (view preprint)
  2. Received: January 31, 2022
  3. Accepted: June 21, 2022
  4. Accepted Manuscript published: June 22, 2022 (version 1)
  5. Version of Record published: July 18, 2022 (version 2)

Copyright

© 2022, Liu 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. Xingchen Liu
  2. Christl Gaubitz
  3. Joshua Pajak
  4. Brian A Kelch
(2022)
A second DNA binding site on RFC facilitates clamp loading at gapped or nicked DNA
eLife 11:e77483.
https://doi.org/10.7554/eLife.77483

Share this article

https://doi.org/10.7554/eLife.77483

Further reading

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    Research Article Updated

    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 Saccharomyces 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.

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