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 AffiliationsBiochemistry and Molecular Biotechnology
Document TypeJournal Article
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AbstractClamp 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.
SourceLi 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.
Permanent Link to this Itemhttp://hdl.handle.net/20.500.14038/52969
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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
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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; 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.
Mechanism of Sliding Clamp Loading During DNA Replication and Repair in Atomic DetailLiu, Xingchen (2023-03-30)DNA replication is a fundamental process that is essential for all forms of life, and it is made efficient by ring-shaped sliding clamp proteins. One such protein is the eukaryotic sliding clamp, Proliferating Cellular Nuclear Antigen (PCNA), which not only facilitates replication but also coordinates multiple cellular pathways, such as DNA repair, cell cycle regulation, and apoptosis. The proper function of PCNA is critical for maintaining genome stability, making it a crucial factor in human health. The clamp loader complex is the primary regulator of sliding clamp activity. Replication Factor C (RFC), the eukaryotic clamp loader, is responsible for opening the closed PCNA and loading it onto DNA. However, the mechanism by which RFC accomplishes this task has been elusive for years. Our research has contributed to this field by revealing multiple cryo-electron microscopy (cryo-EM) structures of the RFC:PCNA complex that describe the steps involved in the clamp loading reaction. Specifically, we found that RFC opens PCNA with a 'crab-claw' motion, allowing it to preferentially bind to PCNA before DNA. Additionally, during replication, primer-template DNA, which is RFC's primary DNA substrate, directly binds to the central chamber of the complex. Our study also sheds light on the mechanism by which RFC performs its role in loading PCNA during DNA repair. When RFC binds to gapped or nicked DNA during DNA repair, it uses an additional DNA binding site, and both sites work together to melt the DNA strands with their 'separation pins.' This discovery provides the first structural insight into how RFC accomplishes its crucial functions in DNA replication and repair. Overall, our findings provide detailed atomic-level insights into how RFC efficiently loads PCNA onto different DNA substrates, advancing our understanding of this essential biological process.