Structure of the human CTF18 clamp loader bound to PCNA

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.

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.

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.