The molecular coupling between substrate recognition and ATP turnover in a AAA+ hexameric helicase loader

Thank you for submitting your article "The molecular coupling between substrate recognition and ATP turnover in a AAA+ hexameric helicase loader" for consideration by eLife. Your article has been reviewed favorably by 3 peer reviewers, and the evaluation has been overseen by John Kuriyan as the Reviewing Editor and Senior Editor. The reviewers have opted to remain anonymous.

The reviewers have discussed the reviews with one another and the Reviewing Editor has drafted this decision to help you prepare a revised submission.

We would like to draw your attention to changes in our revision policy that we have made in response to COVID-19 (https://elifesciences.org/articles/57162). Specifically, we are asking editors to accept without delay manuscripts, like yours, that they judge can stand as eLife papers without additional data, even if they feel that they would make the manuscript stronger. Thus the revisions requested below only address clarity and presentation.

Summary:

This manuscript advances our understanding of loading factors for assembly of ring-shaped helicases around DNA. In particular, using E. coli DnaC as a model, the authors identify several key amino acids needed for ATPase function, and a new element for which they coin the term "arginine-coupler", that enables DnaC to couple productive ATPase action to the loading of the hexameric DnaB replicative helicase onto DNA. This resolved the long standing question of how DnaC retains bound ATP when complexed with DnaB helicase to form the full DnaC hexamer, yet only hydrolyzes the ATP when the DnaB6C6 complex binds to ssDNA. Interestingly, the report also identifies, for the first time, that the ATPase of the DnaB helicase participates in the loading reaction. Furthermore, the report also shows that DnaC can couple ATP to the unloading of the DnaB helicase; this is the first time that helicase unloading has been observed in a bacterial system.

The authors address DnaC function using assays for ATPase, helicase loading, helicase unwinding, helicase unloading, and an array of 5 mutants in DnaC's: Walker A, Walker B, Arg finger, Sensor I, and Sensor II motifs. The work is systematic, employs many biochemical assays, and joins the results with amino acid homologies and previously determined structures by this group and others. The AAA+ family of proteins is widely dispersed in nature, and they function in many different ways. Typically, AAA+ proteins function as oligomers, and DnaC is no exception. Hence, the findings of this report, while focused on the AAA+ DnaC protein, will have impact on other labs that work on AAA+ factors, and especially helicase and polymerase clamp loaders.

This report represents a significant advance in our knowledge of helicase loader function, and is quite suitable for publication in eLife, once the points made below are addressed in a revised manuscript.

Essential revisions:

The reviewers have identified the following major issues to address. For Point 2, if additional experimental results are available they should be included in the revised manuscript. If such data are not available, then the revised text should clearly point out the limitations of the analysis.

1. The primary discovery by Puri et al is the Arginine Coupler residue. However, the mechanism by which the Arg coupler controls ATPase activity in response to DNA binding is unclear from the discussion in the paper. Please provide a clearer and more comprehensive discussion of how how you envisage the coupling between DNA recognition and ATP hydrolysis to be accomplished.

2. It is unclear why a double mutant of DnaB was used to ablate ATPase activity. Mutation of the glutamate of the "Walker B" alone is sufficient to kill ATPase activity, as shown by other labs, but crucially will still allow ATP binding. As the authors themselves point out, mutation of the lysine of the "Walker A" may well affect ATP binding (K→R, although K→A is used here) as well as hydrolysis. The fact that significant ATPase activity persists in the R→A mutant suggests it does still bind at some level, and this seems an unnecessary complication. Although previous work is quoted to suggest ATP binding is not required for loading, it would be reassuring to see data showing ATP binding to DnaB (or hexamer formation?) has not been impaired since hexameric helicase rings are more more stable on nucleotide binding. This would make the results much cleaner. The use of just the E262A mutation would be even better and probably sufficient.

3. The work is interesting from a DnaB/C viewpoint but also has implications for related systems, as the authors point out. However, their comparisons with related systems show some relevant omissions whose inclusions would strengthen the comparisons. For example, the polymerase clamp loader RFC is compared but it has been shown previously that one of the conserved "RFC" motifs in an archaeal RFC plays a role in coupling ATP hydrolysis to PCNA clamp loading and when mutated the complex shows elevated ATPase but cannot release the clamp. There would seem to be some relevant comparisons with the roles of the mutations described here. Furthermore, mutation of the Arg finger in RFC creates a complex that binds ATP and PCNA but cannot load onto DNA substrates. This would seem to be complementary, and consistent, evidence to the role of the Arg coupler described in regulating a step in clamp opening.

4. The authors also make pertinent comments regarding the nature of the role of this AAA+ system (DnaC) as a one-off loading "switch" rather than a processive system (like a helicase). Similar arguments have been proposed for another "switch" in AAA+ proteins, namely the "Glutamate Switch" that prevents ATP hydrolysis until the assembly is complete and ready to carry out its reaction. There is (presumably) a Glutamate Switch in DnaC so what the interplay might be between these should be addressed in the discussion.