We are upgrading the repository! A content freeze is in effect until December 6, 2024. New submissions or changes to existing items will not be allowed during this period. All content already published will remain publicly available for searching and downloading. Updates will be posted in the Website Upgrade 2024 FAQ in the sidebar Help menu. Reach out to escholarship@umassmed.edu with any questions.
Unexpected new insights into DNA clamp loaders: Eukaryotic clamp loaders contain a second DNA site for recessed 5' ends that facilitates repair and signals DNA damage: Eukaryotic clamp loaders contain a second DNA site for recessed 5' ends that facilitates repair and signals DNA damage
UMass Chan Affiliations
Biochemistry and Molecular BiotechnologyDocument Type
Journal ArticlePublication Date
2022-09-18
Metadata
Show full item recordAbstract
Clamp loaders are pentameric AAA+ assemblies that use ATP to open and close circular DNA sliding clamps around DNA. Clamp loaders show homology in all organisms, from bacteria to human. The eukaryotic PCNA clamp is loaded onto 3' primed DNA by the replication factor C (RFC) hetero-pentameric clamp loader. Eukaryotes also have three alternative RFC-like clamp loaders (RLCs) in which the Rfc1 subunit is substituted by another protein. One of these is the yeast Rad24-RFC (Rad17-RFC in human) that loads a 9-1-1 heterotrimer clamp onto a recessed 5' end of DNA. Recent structural studies of Rad24-RFC have discovered an unexpected 5' DNA binding site on the outside of the clamp loader and reveal how a 5' end can be utilized for loading the 9-1-1 clamp onto DNA. In light of these results, new studies reveal that RFC also contains a 5' DNA binding site, which functions in gap repair. These studies also reveal many new features of clamp loaders. As reviewed herein, these recent studies together have transformed our view of the clamp loader mechanism.Source
Li H, O'Donnell M, Kelch B. Unexpected new insights into DNA clamp loaders: Eukaryotic clamp loaders contain a second DNA site for recessed 5' ends that facilitates repair and signals DNA damage: Eukaryotic clamp loaders contain a second DNA site for recessed 5' ends that facilitates repair and signals DNA damage. Bioessays. 2022 Nov;44(11):e2200154. doi: 10.1002/bies.202200154. Epub 2022 Sep 18. PMID: 36116108; PMCID: PMC9927785.DOI
10.1002/bies.202200154Permanent Link to this Item
http://hdl.handle.net/20.500.14038/52969PubMed ID
36116108Rights
This is an open access article under the terms of the Creative Commons Attribution-NonCommercial License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited and is not used for commercial purposes. © 2022 The Authors. BioEssays published by Wiley Periodicals LLC; Attribution-NonCommercial 4.0 InternationalDistribution License
http://creativecommons.org/licenses/by-nc/4.0/ae974a485f413a2113503eed53cd6c53
10.1002/bies.202200154
Scopus Count
Collections
The following license files are associated with this item:
- Creative Commons
Except where otherwise noted, this item's license is described as This is an open access article under the terms of the Creative Commons Attribution-NonCommercial License, which permits use, distribution and reproduction in any
medium, provided the original work is properly cited and is not used for commercial purposes.
© 2022 The Authors. BioEssays published by Wiley Periodicals LLC
Related items
Showing items related by title, author, creator and subject.
-
Investigation of the Structural Mechanisms of a Bacterial Clamp LoaderLandeck, Jacob T (2024-04-01)The sliding clamp is an integral protein in DNA replication and repair, where it increases the speed of DNA synthesis and serves as a scaffold for repair proteins. Since the sliding clamp is a closed ring, clamp loaders must open the ring and then close it around DNA. The clamp loader and sliding clamp are the only two components of the replisome that are conserved across all three domains of life. However, differences in their structures and biochemical activities suggest that the mechanisms of opening and loading differ between prokaryotes and eukaryotes. Structures of the eukaryotic clamp loader clearly illustrate its clamp loading mechanism, but there were no comparable structures for bacterial clamp loaders. To understand how mechanisms of bacterial clamp loaders compare to their eukaryotic counterparts, I determined a series of structures of the E. coli clamp loader at distinct stages in clamp loading. To understand how ATP binding enables the clamp loader to bind to the sliding clamp, I determined a structure of the E. coli clamp loader bound to a non-hydrolyzable ATP analog. I found that the E. coli clamp loader opens its sliding clamp at a single pivot point into a planar conformation, but transitions to a helical conformation upon binding primer-template (p/t)-junctions. This behavior contrasts with eukaryotic clamp loaders, which open their sliding clamp through multiple pivot points into a helical conformation before binding p/t-junctions. My work also revealed that like the eukaryotic clamp loader, the E. coli clamp loader does not need to undergo a conformational change to close the sliding clamp on p/t-junctions. Furthermore, I explored how the E. coli clamp loader is inhibited by the bacteriophage protein gene product 8. This dissertation explores the structural mechanisms used by the bacterial clamp loader and illuminates similarities and differences between clamp loaders across the domains of life.
-
A second DNA binding site on RFC facilitates clamp loading at gapped or nicked DNALiu, Xingchen; Gaubitz, Christl; Pajak, Joshua; Kelch, Brian A (2022-06-22)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.
-
Structure of the human clamp loader bound to the sliding clamp: a further twist on AAA+ mechanism [preprint]Gaubitz, Christl; Liu, Xingchen; Magrino, Joseph; Stone, Nicholas P.; Landeck, Jacob T; Hedglin, Mark; Kelch, Brian A (2020-04-18)DNA replication requires the sliding clamp, a ring-shaped protein complex that encircles DNA, where it acts as an essential cofactor for DNA polymerases and other proteins. The sliding clamp needs to be actively opened and installed onto DNA by a clamp loader ATPase of the AAA+ family. The human clamp loader Replication Factor C (RFC) and sliding clamp PCNA are both essential and play critical roles in several diseases. Despite decades of study, no structure of human RFC has been resolved. Here, we report the structure of human RFC bound to PCNA by cryo-EM to an overall resolution of ~3.4 Å. The active sites of RFC are fully bound to ATP analogs, which is expected to induce opening of the sliding clamp. However, we observe the complex in a conformation prior to PCNA opening, with the clamp loader ATPase modules forming an over-twisted spiral that is incapable of binding DNA or hydrolyzing ATP. The autoinhibited conformation observed here has many similarities to a previous yeast RFC:PCNA crystal structure, suggesting that eukaryotic clamp loaders adopt a similar autoinhibited state early on in clamp loading. Our results point to a ‘Limited Change/Induced Fit’ mechanism in which the clamp first opens, followed by DNA binding inducing opening of the loader to release auto-inhibition. The proposed change from an over-twisted to an active conformation reveals a novel regulatory mechanism for AAA+ ATPases. Finally, our structural analysis of disease mutations leads to a mechanistic explanation for the role of RFC in human health.