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.
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
Further reading
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- Biochemistry and Chemical Biology
In healthy cells, cyclin D1 is expressed during the G1 phase of the cell cycle, where it activates CDK4 and CDK6. Its dysregulation is a well-established oncogenic driver in numerous human cancers. The cancer-related function of cyclin D1 has been primarily studied by focusing on the phosphorylation of the retinoblastoma (RB) gene product. Here, using an integrative approach combining bioinformatic analyses and biochemical experiments, we show that GTSE1 (G-Two and S phases expressed protein 1), a protein positively regulating cell cycle progression, is a previously unrecognized substrate of cyclin D1–CDK4/6 in tumor cells overexpressing cyclin D1 during G1 and subsequent phases. The phosphorylation of GTSE1 mediated by cyclin D1–CDK4/6 inhibits GTSE1 degradation, leading to high levels of GTSE1 across all cell cycle phases. Functionally, the phosphorylation of GTSE1 promotes cellular proliferation and is associated with poor prognosis within a pan-cancer cohort. Our findings provide insights into cyclin D1’s role in cell cycle control and oncogenesis beyond RB phosphorylation.
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- Biochemistry and Chemical Biology
- Microbiology and Infectious Disease
Teichoic acids (TA) are linear phospho-saccharidic polymers and important constituents of the cell envelope of Gram-positive bacteria, either bound to the peptidoglycan as wall teichoic acids (WTA) or to the membrane as lipoteichoic acids (LTA). The composition of TA varies greatly but the presence of both WTA and LTA is highly conserved, hinting at an underlying fundamental function that is distinct from their specific roles in diverse organisms. We report the observation of a periplasmic space in Streptococcus pneumoniae by cryo-electron microscopy of vitreous sections. The thickness and appearance of this region change upon deletion of genes involved in the attachment of TA, supporting their role in the maintenance of a periplasmic space in Gram-positive bacteria as a possible universal function. Consequences of these mutations were further examined by super-resolved microscopy, following metabolic labeling and fluorophore coupling by click chemistry. This novel labeling method also enabled in-gel analysis of cell fractions. With this approach, we were able to titrate the actual amount of TA per cell and to determine the ratio of WTA to LTA. In addition, we followed the change of TA length during growth phases, and discovered that a mutant devoid of LTA accumulates the membrane-bound polymerized TA precursor.
