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Summary:

This paper investigates the role of ssDNA binding in modulating Lon protease activity and explores its biochemical and structural consequences. It characterizes ssDNA binding and demonstrates how mutations that alter DNA binding impact biochemical activity without significantly affecting Lon oligomerization. Additionally, the study highlights alternative pathways through which Lon protease retains peptidase activity in the presence of ATP. Overall, the paper offers valuable insights into how DNA binding and electrostatic changes regulate Lon protease function. However, improvements could be made in the flow of paper, increasing sample sizes for certain experiments, and revising the title to better reflect the study's findings.

Major Concerns: 1. We believe the paper can be better communicated with a different title, emphasizing Lon’s impact with ssDNA.We suggest renaming it to “Understanding ssDNA Binding on the Activation and Regulation of Lon Protease Activity”. We emphasize removing the "allosteric modulation" portion, as it does not consistently reflect a central theme of the findings.

2. In the results section, the discussion in lines 91-97 accompanying Figure 1 reads more like a discussion. To improve clarity, we suggest removing sentences from ‘with reports’ (line 94) to ‘protein degradation (24)’ (line 96) and instead providing a concise summary of prior literature before introducing the Figure 1 results. 

3. We strongly recommend moving the structural representation from Figure 7A to Figure 2. The inclusion of this structure in Figure 2 would provide a critical foundation for understanding the binding sites and the role of oligomerization in protease activity, which is explored in the experiments that follow. 4. For presentation purposes, the gels in Figures 3 and Supplemental Figures 1 & 2 could use better contrast or rerunning, as the bands are faint. In Figure 3C, we suggest including more replicates for all conditions to address variability in the Lon4A data or conducting statistical analysis to demonstrate that the variation is not significant. 

5.  In Supplemental Figure 1B, sample sizes vary across experimental conditions despite the main text emphasizing consistency. We recommend ensuring uniform sample sizes.

6. In Figure 2B, 2C, 3D, and 4, it is unclear whether the points in panels A and B are log-transformed. If so, this should be noted on the x-axis. 

7. In  Figure 4D, the skewed fit of the lines for Lon and Lon4E raises concerns about data reliability. We recommend either reanalyzing the data or discussing how this skew impacts the interpretation of ATP hydrolysis results.

8. In Figure 5, the oligomer cartoons on the graphs appear crowded and confusing. Consider using lightly shaded background sections correlating to oligomer sizes or an alternative representation to differentiate peaks.

9. Figure 3 introduces the concept of allosteric modulation by ssDNA, but it doesn’t emphasize this idea clearly. We suggest either making this the focus of Figure 4 or move it to Figure 4, which investigates peptide hydrolysis and activity. We believe Figure 4 would fit better after fully explaining allosteric modulation.

Minor Concerns: 1. Lines 124-125 discussing the hexamer orientation do not directly relate to the experiments conducted, and we suggest they can be omitted.

2. . In the results section for Figure 1, the full protein names for the abbreviations “DnaA,” “SciP,” and “Ccrm” should be included to aid reader comprehension.

3.The terms "LMW" and "HMW," which are introduced in line 130, should be explained at that time.

4. In Figure 2A, there is an inconsistency in the molecular weight for the high molecular weight peak. It is marked as 582 kDa in the figure but listed as 588 kDa in the results text.

5. For the Figure 2 caption, specifically in line 560, there is a missing closing parenthesis.

6. We suggest including a brief introduction to Michaelis-Menten (MM) kinetics and elaborating on the measured MM parameters would also help readers better understand the enzymatic differences observed between the wildtype and mutant Lon constructs in Table 1.

7. In Supporting Information Figure 2, the sentence on line 662 that reads "contain 2 G4 DNA sequences" should be revised to "containing two G4 DNA sequences," while maintaining the numeric format (2xG4). 

8. The word "casein" has inconsistent capitalization throughout the text; please revise. 

9. In Figure 5B, the light blue shaded region is not explained in the caption or the results text; a brief explanation should be included, either in the caption or in lines 613-615, to clarify its significance. 

Competing interests

The authors declare that they have no competing interests.