This difference is due to the fact that SSB, especially at high salt concentrations is unable to remove all of the secondary structure from natural ssDNA.
This is partly because SSB does not bind with high cooperativity in the 55 or 65 modes. Therefore, the uncertainty in predicting what the expected compaction of ss lambda DNA based on site sizes measured on polydT makes the main conclusion difficult to assess. The authors need to address this before the manuscript can be published. Compaction as measured by sedimentation studies Bujalowski et al. It seems that the additional ssDNA compaction suggested in the current study should be observable also by these techniques, but none has been reported.
Reviewers agree with the 8 nm estimate, which is from the tips of the L45 loops in the crystal structure. However, the other dimension from the crystal structure can be as small of 4. When the calculation is done using 4. These two points are subject to the caveats mentioned elsewhere in the review.
How much is not clear. The difference in site sizes measured using poly dT vs.
The question is how much dissociates and is it reasonable to dismiss this? Please explain. As indicated, this result is surprising given that increasing the salt should theoretically decrease the number of available binding sites on the ssDNA. Is constant fluorescence observed when no free SSB is included in the flow Figure 2? This is the first time that this interpretation and the fact that fluorescence enhancement decreases at high [NaOAc] is mentioned — a brief discussion should be provided earlier within the Results section referring to Figure 3.
In the figure it appears as though purple and gray represent a single area separate from light purple , however the description seems to be written as if they are all separate. Both proteins have been shown to induce the 35nt binding mode of SSB in a manner dependent upon complex formation.
First, we'd like to thank the reviewers for their constructive comments, several of which seemed to focus on two major issues:. We first address these issues generally, and then we will refer back to our general comments, where applicable, as we address the more specific issues point-by-point.
The figure panel was meant to illustrate the surprising result that SSB-ssDNA compacted more than we initially anticipated based on the published results in Hamon et al. This change in length nm vs. Since lambda DNA is 6. Both of these DNA molecules are natural, mixed sequences, and we expect their propensity to form secondary structure should be similar and comparable. Though we still refer to these lengths and the typical SSB-binding modes in the text, because we now focus our comparisons to observed native DNA lengths, we have removed the figure plotting our measured site size vs. We note that both of these experiments were performed on natural ssDNA, and if anything, we expected our molecules to be longer owing to extension in flow and the absence of spermidine which they used to adsorb the molecules in the AFM experiment, and which condenses both ssDNA and dsDNA.
At lower salt concentrations, we see no net dissociation of fluorescent SSB when SSB concentrations are held constant. To clarify this important point, we have also included supplemental panels for Figure 1 , showing intensity measurements on SSB-ssDNA molecules during the length transitions at multiple salt jumps. Using a well-established standard ensemble method, we used poly dT to determine the intrinsic site size for our fluorescent SSB protein, under our conditions. These measurements allowed us to determine the dominant, intrinsic binding mode for SSB at each concentration of salt used in our single molecule experiments.
How good can cryo-EM become?
Poly dT is used as the polynucleotide of choice for SSB-binding experiments precisely for the reasons mentioned by one of the reviewers — the absence of secondary structure reduces the complexity of interpreting the site size determined using stoichiometric titrations or affinity, as the case may be. We used these experiments as verification that our SSB protein is behaving as expected under our conditions, using this method that has been extensively documented by the Lohman lab. With respect to major issue 2, SSB dissociation is something that we considered seriously, and in fact, we initially dismissed the hyper-condensation of the SSB f -ssDNA fiber based on this interpretation; however, after careful examination of both the fluorescent intensity of the SSB f -ssDNA fibers during the salt jump transitions, and even more convincingly the force-spectroscopy with wild-type, unlabeled ssDNA, we came to the conclusion that protein dissociation was negligible or non-existent in all conditions when free SSB is present, except mM NaOAc, and that dissociation could not fully account for our observations.
When natural ssDNA substrates e. In the example cited by the reviewers, at mM NaCl, a site size of 77 nts is observed instead of 65 nts. There are two ways to interpret this:. These need not be mutually exclusive, but the second interpretation is the one that is favored in the literature, and we agree with this interpretation of ensemble, equilibrium binding experiments. This assay is extremely sensitive, and allowed us to measure the condensation of the SSB-ssDNA fiber on a long, natural ssDNA substrate as a function of both force and salt perturbation.
We interpret the absence of hysteresis in the presence of SSB as strong evidence that long-range DNA secondary structure is not capable of excluding SSB and contributing to condensation until the concentration of NaOAc was increased to mM. Therefore, at least in our magnetic tweezers experiment where some force is always being exerted on the molecule owing to the bead tethering in contrast to the ensemble equilibrium binding described in the paragraph above , no measureable long-range secondary structure forms in the presence of SSB.
Based on the sum of our observations, i. We have expanded our Discussion to consider other possible explanations mentioned by the referees. That said, in the condition where free SSB is omitted from our experiment, as in Figure 2 and Figure 2—figure supplement 1 , we do observe protein dissociation during the time needed to measure condensation of the SSB-coated ssDNA fiber. We therefore conclude that the structural re-arrangements that we observe and the relatively constant amount of protein bound to the ssDNA, is normally driven or maintained through reversible mass action with free SSB.
Neither the EM or AFM studies explored a significant range of salt concentrations, and in fact, were rather limited in the range of conditions tested. Consequently, they are in fact quite perturbing of structures that are held together by weak interactions. Hence, we offer these two reasons as possible explanations for why condensation was not reported earlier.
The Hamon paper explored a rather limited range of salt concentrations, primarily focusing on developing a method to adsorb and spread SSB-ssDNA complexes onto mica for imaging, reporting contour lengths for only two conditions 20 mM TrisHCl 7. We did attempt to use AFM to image condensed complexes; however, we ran into two technical problems. We verified that conditions permitting condensation similarly resulted in poorly spread complexes that could not be used to measure contour lengths and resulted in poor imaging conditions owing to salt and aggregates on the mica.
Removal of the salt requires rinsing the samples, which obviously perturbs the intramolecular condensation of the molecules, and we know from our TIRF experiments that condensation, decondensation and remodeling are rapid. Given these technical problems — as well as the complication of requiring spermidine for surface adsorption — we reasoned that the force spectroscopy experiments would be the most accurate method for measuring contour lengths at increasing salt concentrations using wild-type unlabeled SSB. We cannot make a direct comparison to Bujalowski et al. However, upon scrutinizing the older literature, we discovered that in largely underappreciated work, Schaper et al.
When excess SSB was present as in our conditions , they observed a log-linear relationship between the sedimentation coefficient and salt concentration.
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We have extracted the values from their paper, and we have plotted those values against the length measurements from our single-molecule assays. We think that it is obvious that the log-linear trend is the same, indicating that our observations and method are in fact in good agreement with Schaper et al Author response image 1. We refer the reviewers to our general response above. Certainly several structural models could be advanced, including folding of the fiber into solenoid or fractal structures; however, our experiments are not informative as to what these structural features are.
In the Discussion section we discuss that the molecular interactions could be explained by either tetramer-ssDNA interactions for example, if tetramers do not strictly bind to ssDNA only contiguously, but rather as two or more discrete binding entities, similar to the tetramer bound to two molecules of mer in the crystal structure , or through tetramer-tetramer interactions.
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Alternatively, long-range interactions could be explained by re-annealing of the ssDNA as secondary structure forms. Because the magnetic tweezers experiments allow us to directly measure secondary structure formation, which is apparent in the hysteresis when pulling vs relaxing curves are compared, we are able to directly eliminate this latter explanation for all of our conditions except when mM NaOAc was used. A Dimerization of SSB tetramers i. The intracellular salt concentrations of E.
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As stated by Richey et al. Differences between in vitro and in vivo effects of ion concentrations on protein-DNA interactions and gene expression. Schultz Regulation of cation content. K exchange. After re-examining the SSB structure, we agree that we over-estimated the smallest possible axis.
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Nonetheless, as one reviewer pointed out in comment 2, these calculations make certain assumptions about the fiber conformation. Consequently, for reasons explained above, we have removed the figure, and instead refer to the relative length changes observed by AFM in Hamon et al. We think that this comment also relates to both of the major issues that were noted and addressed in our general response above. In addition, we now also provide intensity measurements for the molecules shown in Figure 1 see Figure 1—figure supplement 1 and also provide the intensity measurements for all molecules summarized in Figure 3E , plotted as a scatter plot as a function of salt concentration in Figure 3—figure supplement 3.
It is not our intention to dismiss the possibility of SSBf dissociation, especially in the experiment presented in Figure 2 , where free SSB is not available. We did explicitly state that there would be dissociation at mM, but we now explicitly excluded this condition from our discussions and we note that all of our conclusions remain valid even if this concentration is excluded. We have further analyzed the images in Figure 2 to quantify the dissociation in the absence of free protein. As we indicate in the text, the single-molecule experiments were performed with SSB labeled with AF, while the equilibrium titrations were performed with SSB labeled with 5,6-carboxyflourescein.
This is because AFlabeled SSB is brighter and does not photobleach as quickly as fluorescein-labeled SSB, and is less sensitive to environmental changes induced by binding. Therefore, AFlabeled SSB is most well suited for single-molecule measurements, and fluorescein-labeled SSB is more suitable for traditional, ensemble measurements.
In the absence of salt, we have observed a 2. Also, please see our response to the following question. No, the fluorescence decreases. We have included a new Supplement to Figure 2 Figure 2—figure supplement 1 , where we have analyzed and quantified the intensity of the molecules shown in Figure 2. When the salt concentration decreases back to 0 mM, we do not observe any additional intensity change, indicating that the single-molecule measurement with SSB-AF is insensitive to the molecular environmental changes coinciding with condensation and de-condensation.
Yes, this is most obvious in the force-extension traces, but it is also true of the experiments performed in Figure 1 and throughout the rest of the manuscript. The fold-increase in Figure 3B is from the amplitude from the titrations performed in Figure 3A ; however, a larger number of titrations were performed than are represented in 3A in order to prevent 3A from being overcrowded. In other words, the fold-increases in Figure 3B were determined from a full stoichiometric titration where each titration was completely and fully saturated. This data is now included as a Figure 3—figure supplement 1.
There, we articulated our caveat that the greater than expected condensation at high [NaOAc] could be due to dissociation of SSB f and formation of secondary structure. Nonetheless, we have tried to more accurately articulate this possibility throughout the manuscript. We have added these references, and have included this possibility to the Discussion. The funder had no role in study design, data collection and interpretation, or the decision to submit the work for publication.
We are grateful to members of the laboratory for their comments on this work. This article is distributed under the terms of the Creative Commons Attribution License , which permits unrestricted use and redistribution provided that the original author and source are credited. Article citation count generated by polling the highest count across the following sources: Scopus , Crossref , PubMed Central.
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