cryo-EM structures of the E. coli replicative DNA polymerase reveal its dynamic interactions with the DNA sliding clamp, exonuclease and τ

Thank you for submitting your work entitled "cryo-EM structures of the E. coli replicative DNA polymerase reveal dynamic interactions with clamp, exonuclease and τ" for peer review at eLife. Your submission has been favorably evaluated by Michael Marletta (Senior Editor), a Reviewing Editor, and three reviewers. One of the three reviewers, Mike O’Donnell (Reviewer #3), has agreed to reveal his identity.

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

Using cryo-electron microscopy, Fernandez-Leiro et al. have determined the ~8 Å resolution structures, in a DNA-bound and DNA-free state, of the holoenzyme catalytic core comprising the E. coli DNA polymerase III α subunit, the DNA sliding clamp β, the exonuclease ε, and the C-terminal domain of the clamp loader subunit τ (τ₅₀₀). Interesting findings include: 1) showing that the rigid-body module composed of the C-terminal part of the clamp loader and the polymerase tail undergoes a large rearrangement upon DNA binding, and 2) that there is a new contact point between τ and the polymerase in the apo configuration, and different one, formed in the presence of DNA, between the OB-fold domain of the polymerase and the clamp. This work represents a highly significant advance in the science of sliding clamps, and the mechanism by which DNA polymerase functions on them. This structural study identifies an entirely new connection of the polymerase to the clamp. This new connection only occurs when the polymerase binds a primed template. The connection is in a new location, outside the two typical hydrophobic pockets on clamps that are the usual target of protein trafficking.

In general, the paper is reasonably well-written and its organization is easy to follow. The quality of the EM reconstructions is very good, especially considering the small size of the complex (~250 kDa), and the heterogeneity present in the sample. The newly identified contacts between the polymerase, clamp, and τ provide a logical and long-desired molecular picture of why holoenzyme formation leads to improved processivity and potentially of how PolIII alters its structural state in response to binding DNA (which may be important for polymerase recycling during lagging strand synthesis). Overall, the work provides valuable new insights into important DNA replication events and stands to have a long-lasting impact on the field.

Before publication, the authors need to make the following essential revisions:

1) The authors refer to the study of Georgescu et al. (EMBO, 2009) and suggest that the observed clamp-OB CTD interaction provides "an alternative explanation" for the reduced DNA synthesis seen by them. However, the Discussion section remains relatively vague and does not discuss the opposing model, although it is directly relevant for Okazaki fragment sensing. While the entire mechanism of such sensing is probably beyond the experimental scope of the present manuscript, the authors need to provide a more considered discussion of "collision" models versus "signaling". Specifically, the authors need to specifically address the conclusions in Dohrmann et al. 2011, and related work.

2) Lamers and colleagues identified recently domain-domain interactions within PolIII-clamp-exonuclease complex via cross-linking / mass spectrometry analysis (Toste Rêgo et al. EMBO 2013). In the present manuscript they frequently refer to this study in order to support their conclusions. Please show whether the crosslinking data is consistent with the molecular models derived from cryo-EM by measuring respective distances. Are there many outliers that exceed the length of the crosslinking reagent and might indicate different conformations?

3) The manuscript needs a final figure that proposes the obvious function of this new connection in the polymerase recycling process: a summary "mechanism" figure. This figure should summarize the different movements/states, and illustrate the authors' speculation as to how these changes might be involved in polymerase cycling during lagging strand synthesis. An expert in the field will connect the dots without a model figure, but for many readers, this finding deserves a figure and sufficient explanation/speculation in the text, to convey the significance of this to non-experts.

1) The authors refer to the study of Georgescu et al. (EMBO, 2009) and suggest that the observed clamp-OB CTD interaction provides "an alternative explanation" for the reduced DNA synthesis seen by them. However, the Discussion section remains relatively vague and does not discuss the opposing model, although it is directly relevant for Okazaki fragment sensing. While the entire mechanism of such sensing is probably beyond the experimental scope of the present manuscript, the authors need to provide a more considered discussion of "collision" models versus "signaling". Specifically, the authors need to specifically address the conclusions in Dohrmannet al. 2011, and related work.

We have now included a more extensive discussion regarding the "collision" vs. "signaling" model. This includes the work by Dohrmann et al. 2011 and related work.

2) Lamers and colleagues identified recently domain-domain interactions within PolIII-clamp-exonuclease complex via cross-linking / mass spectrometry analysis (Toste Rêgo et al. EMBO 2013). In the present manuscript they frequently refer to this study in order to support their conclusions. Please show whether the crosslinking data is consistent with the molecular models derived from cryo-EM by measuring respective distances. Are there many outliers that exceed the length of the crosslinking reagent and might indicate different conformations?

Indeed, our previous cross-linking results are entirely consistent with our cryo-EM models. We now refer to this in the text, and have included an additional figure (Figure 2—figure supplement 1) showing the cross-links mapped onto the old model (left) and the DNA-free cryo-EM model (right).

3) The manuscript needs a final figure that proposes the obvious function of this new connection in the polymerase recycling process: a summary "mechanism" figure. This figure should summarize the different movements/states, and illustrate the authors' speculation as to how these changes might be involved in polymerase cycling during lagging strand synthesis. An expert in the field will connect the dots without a model figure, but for many readers, this finding deserves a figure and sufficient explanation/speculation in the text, to convey the significance of this to non-experts.

We have now added a final figure that summarizes in a schematic manner the possible role of the conformational change in the polymerase. This figure is further explained in the extended discussion about the "collision" vs. "signaling" model.