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.
-
3D cryo-EM map of RFC−PCNA−DNA1−DNA2Electron Microscopy Data Bank, EMD-25872.
-
3D cryo-EM map of RFC−open PCNA−DNA1Electron Microscopy Data Bank, EMD-25874.
-
3D cryo-EM map of RFC−closed PCNA−DNA1 2022Electron Microscopy Data Bank, EMD-25874.
-
3D cryo-EM map of RFC−DNA1−DNA2 2022Electron Microscopy Data Bank, EMD-258725.
-
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.
Metrics
-
- 2,199
- views
-
- 368
- downloads
-
- 22
- citations
Views, downloads and citations are aggregated across all versions of this paper published by eLife.
Download links
A two-part list of links to download the article, or parts of the article, in various formats.
Downloads (link to download the article as PDF)
Open citations (links to open the citations from this article in various online reference manager services)
Cite this article (links to download the citations from this article in formats compatible with various reference manager tools)
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
-
- Biochemistry and Chemical Biology
- Computational and Systems Biology
The spike protein is essential to the SARS-CoV-2 virus life cycle, facilitating virus entry and mediating viral-host membrane fusion. The spike contains a fatty acid (FA) binding site between every two neighbouring receptor-binding domains. This site is coupled to key regions in the protein, but the impact of glycans on these allosteric effects has not been investigated. Using dynamical nonequilibrium molecular dynamics (D-NEMD) simulations, we explore the allosteric effects of the FA site in the fully glycosylated spike of the SARS-CoV-2 ancestral variant. Our results identify the allosteric networks connecting the FA site to functionally important regions in the protein, including the receptor-binding motif, an antigenic supersite in the N-terminal domain, the fusion peptide region, and another allosteric site known to bind heme and biliverdin. The networks identified here highlight the complexity of the allosteric modulation in this protein and reveal a striking and unexpected link between different allosteric sites. Comparison of the FA site connections from D-NEMD in the glycosylated and non-glycosylated spike revealed that glycans do not qualitatively change the internal allosteric pathways but can facilitate the transmission of the structural changes within and between subunits.
-
- Biochemistry and Chemical Biology
- Genetics and Genomics
Deep Mutational Scanning (DMS) is an emerging method to systematically test the functional consequences of thousands of sequence changes to a protein target in a single experiment. Because of its utility in interpreting both human variant effects and protein structure-function relationships, it holds substantial promise to improve drug discovery and clinical development. However, applications in this domain require improved experimental and analytical methods. To address this need, we report novel DMS methods to precisely and quantitatively interrogate disease-relevant mechanisms, protein-ligand interactions, and assess predicted response to drug treatment. Using these methods, we performed a DMS of the melanocortin-4 receptor (MC4R), a G-protein-coupled receptor (GPCR) implicated in obesity and an active target of drug development efforts. We assessed the effects of >6600 single amino acid substitutions on MC4R’s function across 18 distinct experimental conditions, resulting in >20 million unique measurements. From this, we identified variants that have unique effects on MC4R-mediated Gαs- and Gαq-signaling pathways, which could be used to design drugs that selectively bias MC4R’s activity. We also identified pathogenic variants that are likely amenable to a corrector therapy. Finally, we functionally characterized structural relationships that distinguish the binding of peptide versus small molecule ligands, which could guide compound optimization. Collectively, these results demonstrate that DMS is a powerful method to empower drug discovery and development.
