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

  1. Fengwei Zheng
  2. Roxana E Georgescu
  3. Nina Y Yao
  4. Huilin Li  Is a corresponding author
  5. Michael E O'Donnell  Is a corresponding author
  1. Van Andel Institute, United States
  2. Rockefeller University, United States
  3. Howard Hughes Medical Institute, Rockefeller University, United States
https://doi.org/10.7554/eLife.77469
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  1. Fengwei Zheng
  2. Roxana E Georgescu
  3. Nina Y Yao
  4. Huilin Li
  5. Michael E O'Donnell
(2022)
Cryo-EM structures reveal that RFC recognizes both the 3′- and 5′-DNA ends to load PCNA onto gaps for DNA repair
eLife 11:e77469.
https://doi.org/10.7554/eLife.77469
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Abstract

RFC uses ATP to assemble PCNA onto primed sites for replicative DNA polymerases d and e. 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 2nd 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.

Data availability

The 3D cryo-EM maps of S. cerevisiae RFC−DNA and RFC−PCNA−DNA complexes have been deposited in the Electron Microscopy Data Bank with accession codes EMD-25872 (RFC−PCNA−DNA1−DNA2), EMD-25873 (RFC−open PCNA−DNA1), EMD-25874 (RFC−closed PCNA−DNA1), EMD-25875 (RFC−DNA1−DNA2), and EMD-25876 (RFC−DNA1). The corresponding atomic models have been deposited in the Protein Data Bank with accession codes 7TFH, 7TFI, 7TFJ, 7TFK and 7TFL.

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Author details

  1. Fengwei Zheng

    Department of Structural Biology, Van Andel Institute, Grand Rapids, United States
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-7139-4831

  2. Roxana E Georgescu

    DNA Replication Laboratory, Rockefeller University, New York, United States
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-1882-2358

  3. Nina Y Yao

    DNA Replication Laboratory, Rockefeller University, New York, United States
    Competing interests
    The authors declare that no competing interests exist.

  4. Huilin Li

    Department of Structural Biology, Van Andel Institute, Grand Rapids, United States
    For correspondence
    Huilin.Li@vai.org
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0001-8085-8928

  5. Michael E O'Donnell

    Howard Hughes Medical Institute, Rockefeller University, New York, United States
    For correspondence
    odonnel@rockefeller.edu
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0001-9002-4214

Funding

National Institute of General Medical Sciences (GM131754)

  • Huilin Li

National Institute of General Medical Sciences (GM115809)

  • Michael E O'Donnell

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. Received: January 31, 2022
  2. Preprint posted: February 5, 2022 (view preprint)
  3. Accepted: June 6, 2022
  4. Accepted Manuscript published: July 13, 2022 (version 1)
  5. Version of Record published: July 18, 2022 (version 2)

Copyright

© 2022, Zheng 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. Fengwei Zheng
  2. Roxana E Georgescu
  3. Nina Y Yao
  4. Huilin Li
  5. Michael E O'Donnell
(2022)
Cryo-EM structures reveal that RFC recognizes both the 3′- and 5′-DNA ends to load PCNA onto gaps for DNA repair
eLife 11:e77469.
https://doi.org/10.7554/eLife.77469

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