Cryo-EM structures reveal that RFC recognizes both the 3′- and 5′-DNA ends to load PCNA onto gaps for DNA repair
- Van Andel Institute, United States
- Rockefeller University, United States
- Howard Hughes Medical Institute, Rockefeller University, United States
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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3D cryo-EM map of RFC−PCNA−DNA1−DNA2Electron Microscopy Data Bank, EMD-25872.
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3D cryo-EM map of RFC−open PCNA−DNA1Electron Microscopy Data Bank, EMD-25874.
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3D cryo-EM map of RFC−closed PCNA−DNA1 2022Electron Microscopy Data Bank, EMD-25874.
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3D cryo-EM map of RFC−DNA1−DNA2 2022Electron Microscopy Data Bank, EMD-258725.
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3D cryo-EM map of RFC−DNA1Electron Microscopy Data Bank, EMD-25876.
Article and author information
Author details
Roxana E Georgescu
Michael E O'Donnell
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
- Bruce Stillman, Cold Spring Harbor Laboratory, United States
Version history
- Received: January 31, 2022
- Preprint posted: February 5, 2022 (view preprint)
- Accepted: June 6, 2022
- Accepted Manuscript published: July 13, 2022 (version 1)
- 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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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
