1. Structural Biology and Molecular Biophysics

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

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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.

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

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Further reading

    1. Biochemistry and Chemical Biology

    Cryo-EM structures reveal that RFC recognizes both the 3′- and 5′-DNA ends to load PCNA onto gaps for DNA repair

    Fengwei Zheng, Roxana Georgescu ... Michael E O'Donnell
    Research Article Updated

    RFC uses ATP to assemble PCNA onto primed sites for replicative DNA polymerases δ and ε. The RFC pentamer forms a central chamber that binds 3′ ss/ds DNA junctions to load PCNA onto DNA during replication. We show here five structures that identify a second DNA binding site in RFC that binds a 5′ duplex. This 5′ DNA site is located between the N-terminal BRCT domain and AAA+ module of the large Rfc1 subunit. Our structures reveal ideal binding to a 7-nt gap, which includes 2 bp unwound by the clamp loader. Biochemical studies show enhanced binding to 5 and 10 nt gaps, consistent with the structural results. Because both 3′ and 5′ ends are present at a ssDNA gap, we propose that the 5′ site facilitates RFC’s PCNA loading activity at a DNA damage-induced gap to recruit gap-filling polymerases. These findings are consistent with genetic studies showing that base excision repair of gaps greater than 1 base requires PCNA and involves the 5′ DNA binding domain of Rfc1. We further observe that a 5′ end facilitates PCNA loading at an RPA coated 30-nt gap, suggesting a potential role of the RFC 5′-DNA site in lagging strand DNA synthesis.

    1. Structural Biology and Molecular Biophysics

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

    Christl Gaubitz, Xingchen Liu ... Brian A Kelch
    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.