Structure of the human CTF18 clamp loader bound to PCNA
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Curated by eLife
eLife Assessment
Briola and co-authors determined the structure of the human CTF18 clamp loader bound to PCNA to high resolution, analyzed the structure, and tested a new mechanism involving a human-specific Ctf18 beta-hairpin docking onto Rfc5, which represents a valuable contribution. The data are solid and complement data recently published by others.
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Abstract
Sliding clamps like PCNA are crucial processivity factors for replicative polymerases, requiring specific clamp loaders for loading onto DNA. The human alternative clamp loader CTF18 interacts with the leading strand polymerase Pol ε and loads PCNA onto primer/template DNA using its RFC pentameric module. Here, we provide a structural characterization of the human CTF18 complex and its interaction with PCNA. Our cryo-EM data support that the Ctf8 and Dcc1 subunits of CTF18, which form the regulatory module interacting with Pol ε, are flexibly tethered to the RFC module. A 2.9 Å cryo-EM structure shows the RFC module bound to PCNA in an auto-inhibited conformation similar to the canonical RFC loader, marking the initial step of the clamp-loading reaction. The unique RFC1 (Ctf18) large subunit of CTF18, which shows high relative mobility, is anchored to PCNA through an atypical low-affinity PIP box in the AAA+ domain and engages the RFC5 subunit using a novel β-hairpin at the disordered N-terminus. We show that deletion of this β-hairpin impairs the CTF18−PCNA complex stability and decreases the rate of primer synthesis by Pol ε. Our research identifies distinctive structural characteristics of the human CTF18-RFC complex, providing insights into its role in PCNA loading and the stimulation of leading strand synthesis by Pol ε.
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eLife Assessment
Briola and co-authors determined the structure of the human CTF18 clamp loader bound to PCNA to high resolution, analyzed the structure, and tested a new mechanism involving a human-specific Ctf18 beta-hairpin docking onto Rfc5, which represents a valuable contribution. The data are solid and complement data recently published by others.
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Reviewer #1 (Public review):
Summary:
The authors report the structure of the human CTF18-RFC complex bound to PCNA. Similar structures (and more) have been reported by the O'Donnell and Li labs. This study should add to our understanding of CTF18-RFC in DNA replication and clamp loaders in general. However, there are numerous major issues that I recommend the authors fix.
Strengths:
The structures reported are strong and useful for comparison with other clamp loader structures that have been reported lately.
Weaknesses:
The structures don't show how CTF18-RFC opens or loads PCNA. There are recent structures from other groups that do examine these steps in more detail, although this does not really dampen this reviewer's enthusiasm. It does mean that the authors should spend their time investigating aspects of CTF18-RFC function that …
Reviewer #1 (Public review):
Summary:
The authors report the structure of the human CTF18-RFC complex bound to PCNA. Similar structures (and more) have been reported by the O'Donnell and Li labs. This study should add to our understanding of CTF18-RFC in DNA replication and clamp loaders in general. However, there are numerous major issues that I recommend the authors fix.
Strengths:
The structures reported are strong and useful for comparison with other clamp loader structures that have been reported lately.
Weaknesses:
The structures don't show how CTF18-RFC opens or loads PCNA. There are recent structures from other groups that do examine these steps in more detail, although this does not really dampen this reviewer's enthusiasm. It does mean that the authors should spend their time investigating aspects of CTF18-RFC function that were overlooked or not explored in detail in the competing papers. The paper poorly describes the interactions of CTF18-RFC with PCNA and the ATPase active sites, which are the main interest points. The nomenclature choices made by the authors make the manuscript very difficult to read.
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Reviewer #2 (Public review):
Summary
Briola and co-authors have performed a structural analysis of the human CTF18 clamp loader bound to PCNA. The authors purified the complexes and formed a complex in solution. They used cryo-EM to determine the structure to high resolution. The complex assumed an auto-inhibited conformation, where DNA binding is blocked, which is of regulatory importance and suggests that additional factors could be required to support PCNA loading on DNA. The authors carefully analysed the structure and compared it to RFC and related structures.
Strength & Weakness
Their overall analysis is of high quality, and they identified, among other things, a human-specific beta-hairpin in Ctf18 that flexibly tethers Ctf18 to Rfc2-5. Indeed, deletion of the beta-hairpin resulted in reduced complex stability and a reduction in …
Reviewer #2 (Public review):
Summary
Briola and co-authors have performed a structural analysis of the human CTF18 clamp loader bound to PCNA. The authors purified the complexes and formed a complex in solution. They used cryo-EM to determine the structure to high resolution. The complex assumed an auto-inhibited conformation, where DNA binding is blocked, which is of regulatory importance and suggests that additional factors could be required to support PCNA loading on DNA. The authors carefully analysed the structure and compared it to RFC and related structures.
Strength & Weakness
Their overall analysis is of high quality, and they identified, among other things, a human-specific beta-hairpin in Ctf18 that flexibly tethers Ctf18 to Rfc2-5. Indeed, deletion of the beta-hairpin resulted in reduced complex stability and a reduction in a primer extension assay with Pol ε. This is potentially very interesting, although some more work is needed on the quantification. Moreover, the authors argue that the Ctf18 ATP-binding domain assumes a more flexible organisation, but their visual representation could be improved.
The data are discussed accurately and relevantly, which provides an important framework for rationalising the results.
All in all, this is a high-quality manuscript that identifies a key intermediate in CTF18-dependent clamp loading.
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Reviewer #3 (Public review):
Summary:
CTF18-RFC is an alternative eukaryotic PCNA sliding clamp loader that is thought to specialize in loading PCNA on the leading strand. Eukaryotic clamp loaders (RFC complexes) have an interchangeable large subunit that is responsible for their specialized functions. The authors show that the CTF18 large subunit has several features responsible for its weaker PCNA loading activity and that the resulting weakened stability of the complex is compensated by a novel beta hairpin backside hook. The authors show this hook is required for the optimal stability and activity of the complex.
Relevance:
The structural findings are important for understanding RFC enzymology and novel ways that the widespread class of AAA ATPases can be adapted to specialized functions. A better understanding of CTF18-RFC function …
Reviewer #3 (Public review):
Summary:
CTF18-RFC is an alternative eukaryotic PCNA sliding clamp loader that is thought to specialize in loading PCNA on the leading strand. Eukaryotic clamp loaders (RFC complexes) have an interchangeable large subunit that is responsible for their specialized functions. The authors show that the CTF18 large subunit has several features responsible for its weaker PCNA loading activity and that the resulting weakened stability of the complex is compensated by a novel beta hairpin backside hook. The authors show this hook is required for the optimal stability and activity of the complex.
Relevance:
The structural findings are important for understanding RFC enzymology and novel ways that the widespread class of AAA ATPases can be adapted to specialized functions. A better understanding of CTF18-RFC function will also provide clarity into aspects of DNA replication, cohesion establishment, and the DNA damage response.
Strengths:
The cryo-EM structures are of high quality enabling accurate modelling of the complex and providing a strong basis for analyzing differences and similarities with other RFC complexes.
Weaknesses:
The manuscript would have benefitted from more detailed biochemical analysis to tease apart the differences with the canonical RFC complex.
I'm not aware of using Mg depletion to trap active states of AAA ATPases. Perhaps the authors could provide a reference to successful examples of this and explain why they chose not to use the more standard practice in the field of using ATP analogues to increase the lifespan of reaction intermediates.
Overall appraisal:
Overall the work presented here is solid and important. The data is sufficient to support the stated conclusions and so I do not suggest any additional experiments.
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