Fine-tuning of the Smc flux facilitates chromosome organization in B. subtilis

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SMC complexes are widely conserved ATP-powered loop extrusion motors indispensable for the faithful segregation of chromosomes during cell division. How SMC complexes translocate along DNA for loop extrusion and what happens when two complexes meet on the same DNA molecule is largely unknown. Revealing the origins and the consequences of SMC encounters is crucial for understanding the folding process not only of bacterial, but also of eukaryotic chromosomes. Here, we uncover several factors that influence bacterial chromosome organization by modulating the probability of such clashes. These factors include the number, the strength and the distribution of Smc loading sites, the residence time on the chromosome, the translocation rate, and the cellular abundance of Smc complexes. By studying various mutants, we show that these parameters are fine-tuned to reduce the frequency of encounters between Smc complexes, presumably as a risk mitigation strategy. Mild perturbations hamper chromosome organization by causing Smc collisions, implying that the cellular capacity to resolve them is rather limited. Altogether, we identify mechanisms that help to avoid Smc collisions and their resolution by Smc traversal or other potentially risky molecular transactions.

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  1. Reviewer #3 (Public Review):

    The manuscript of Anchimiuk and colleagues investigates the mechanism of translocation of Bacillus subtilis SMC-ScpAB, a well characterized bacterial condensin. First, the authors use several SMC constructs where the coil-coiled region has been extended and /or the hinge exchanged and test what are the effects on growth and on the organization of the chromosome. They find highly altered conformations for most of the mutants. Particularly, these altered SMCs are unable to bridge two arms in the presence of the naturally-occurring parS sequences. Interestingly, they are partially able to restore arm pairing if a single parS sequence is provided.

    Next, the authors used Chipseq to compare the binding pattern of wildtype SMC and SMC-CC425 (a mutant with an extended coil coiled region and a different hinge). They observe that the binding of wt-SMC is only midly affected by removal of most parS sequences, whilst that of the mutant is highly affected. In time-lapse experiments where ParB is depleted and then re-expressed, the authors show that in a strain with a single parS wt-SMC loads in the origin region and then redistributes over the chromosome while the mutant can only partially achieve redistribution and to a large extent remains concentrated on the origin region.

    The authors then use wt-SMC and investigate how the conformation of the chromosome changes with two different parS sites located in different positions. They observe that each parS site is able to produce arm-pairing. They observe a decrease in the strength of arm pairing when both parS sites are present.

    Finally, the authors increase the expression level of wt-SMC, and observe decreased levels of arm-pairing in the presence of all the naturally-occurring parS sites. More normal levels of arm-pairing are observed when only one parS is present, despite the higher wt-SMC levels. When two parS sites are introduced, more complex structures appear in the contact map.

    These observations are new, interesting and intriguing. However, there are multiple possible interpretations, models and mechanism that are not discerned by the data presently presented in the manuscript.

    At times, there seem to be inconsistencies in their interpretation of results, and at times the models proposed do not seem well supported by data.

    Finally, the presentation of previous models and results from the literature could be improved.

    Major issues:

    In Fig. 1 the authors make several mutant SMC constructs with larger or shorter arms and different hinges and use Hi-C to explore the changes in 3D chromosome organization. Is it not clear to me why the arc is still visible in the mutants, nor what happens to the overall organization of the chromosome in the mutants? Is chromosome choreography normal?

    In Fig. 1C the authors show that strains with parS-359 only display a secondary diagonal and conclude "chromosome arm alignment was comparable to wild-type". A quantification of the degree of pairing for each mutant normalized by the wild-type is necessary to evaluate the degree of pairing and its dependence on genomic distance to the origin.

    In Fig. 2, the authors use HiC and chip-seq to quantify the effects of changes in SMC arm length on chromosome organization and SMC genomic distributions. It would be important to verify that the expression levels of these SMC mutants are the same as wt, as as they show in Fig. 4 changes in protein levels can change also 3D chromosome organization.

    In Fig. 2C, what is the distribution of SMC at t0? Showing this result would support their claim that SMC can load in absence of ParB.

    In Fig. 2C it is claimed that SMC-CC425 moves at a slower rate than WT. Can the authors provide a quantification?

    In Fig. 2, the authors focused on one of the mutants with longer SMC arms (CC425) and performed HiC and Chip-seq in time-lapse after induction of ParB in a ParB-depleted culture. These experiments clearly establish that SMC-CC425 can redistribute from the origin and can achieve arm pairing but to a lesser extent than the WT. The authors speculate that a slower translocation rate and/or a faster dissociation rate explain the experiments. However, other possibilities exist: for instance that the mutant SMC is defective at passing through road-blocks (highly expressed genomic regions, e.g rRNA sites) or at managing collisions with RNAP/ DNAP/ other SMCs, it makes different higher-order complexes than wt-SMC, etc. This could could be due to the change in the length of the SMC, or to the use of a hinge/coiled-coil region different from that of the wt-SMC. Thus, I am not convinced that the text explores all the possible models or that the data shown discerns between any of them.

    In Fig. 3B, the authors show that use of two parS-opt sites at -304kb and -9kb lead to the formation of two secondary diagonals. They argue that these can be rationalized in terms of the diagonals formed by the strains harboring single parS-opt (either -9kb or -304kb). However, I cannot see how these can happen at the same time! If a cells makes arm pairing from -9kb then it cannot make it from -304kb right? I do not understand either how the authors can conclude from these experiments that ParS may act as unloading sites for SMC. Again, the authors are speculating over mechanisms that are not really tested.

    If parS sites triggered the unloading of SMCs, then one would assume that ~5-6 natural parS sites in the origin region are unloading the SMC complexes loaded at other parS sites? This makes little sense to me, or there is something I clearly do not understand in their explanations.

    In their text, the authors explain that "A small but noticeable fraction of SMC complexes however managed to translocate towards and beyond other parS sites apparently mostly unhindered". I am confused as to where is the evidence supporting this statement. I do not think the ensemble Hi-C experiments provided in Fig. 3 can provide conclusive evidence for this.

    The authors often hypothesize on a mechanism, but then assume this mechanism is correct. For instance, the disruption in the secondary diagonals in Fig. 3B when experiments are performed with two parS sites are initially hypothesized to be due to roadblocks (e.g with highly transcribed regions) or to collisions between SMCs loaded at different parS sites. These possibilities cannot be discerned from their data. However, the authors then assume that collisions is what is going on (e.g. paragraph in lines 274-284). I think they should provide evidence on what is producing the changes in the secondary diagonals in mutants with two ParS sites.

    Why is the ChIP-seq profile for a strain with all the natural parS sites and for a strain with only parS-9kb the same? even with the same peaks at the same locations? Does this mean that SMC peaks do not require the presence of parS? But, then SMCs do not load equally well in all naturally occurring parS sites? This is then in contradiction to their assumption that parS cannot be selectively loaded?

    Do we really know that it is a single SMC ring that is responsible for translocation? The authors assume so in their models and interpretations, but if it were not the case it could drastically modify the mechanisms proposed. For instance, SMC may be able to load on a ParS site without pairing arms (i.e. only one dsDNA strand going through the SMC ring).

    In Fig. 2C-D it is shown that a large fraction of wildtype SMC and SMC-CC425 accumulate at the origin region at early time points (Fig. 2C) however this does not seem to lead to an increased Hi-C signal in the origin region (compare early time points to the final t60). Also, despite small amounts of wt-SMC in the chromosome at the latter time points, the intensity of the secondary diagonal is very strong. Why is this? These results would be consistent with many SMCs loading at the origin region but only a fraction of them being responsible for arm-pairing. Is this not in contradiction to their assumption that SMCs pair two dsDNA arms when they load?

    The authors state that: "If SMC-CC425 indeed fails to juxtapose chromosome arms due to over-enrichment in the replication origin region, collisions may be rare in wild-type cells because of a high chromosome residence time and a limited pool of soluble SMC complexes, resulting in a small flux of SMC onto the chromosome. If so, artificially increasing the flux of SMC should lead to defects in chromosome organization with multiple parS sites but not with a single parS site (assuming that most SMC is loaded at parS sites)". However, this assumption seems inconsistent with their results in Fig. 2 that show that the peaks of SMC do not change upon removal of most parS sites.

    I am a bit confused about the interpretation of the results in Fig. 4D. The authors talk about 'loop contacts' and point to the secondary diagonal (yellow ellipses). But these are not loop contacts, but rather contacts between arms that have surpassed the two parS sequences, right? Also, it is not clear what they mean by paired-loop contacts (red ellipse). Do they mean contacts between the two loops originating at parS-359 and parS-334? If this where the case, then it means SMCs are bridging more than two dsDNA segments? Or that there are multimers of SMC linking together? Or that and SMC can circle one arm from one loop and another from the other...? But in this case, how can it load? For me it is very unclear what these experiments really mean. The explanations provided by the authors seem again highly hypothetical.

  2. Reviewer #2 (Public Review):

    In this manuscript, Anchimiuk et al reported that B. subtillis SMC can collide with each other, and that the collision is modulated by several factors including the number, strength, distribution of parS sites, the residence time of SMC on DNA, the translocation rate, and the cellular abundance of SMC. The authors suggested that these parameters are fine-tuned in the wild-type B. subtillis to minimize SMC collision. In my opinion, the finding is interesting, the experimental setup is creative, and the experiments were beautifully executed. Arguably, these experiments can only be performed in B. subtilis since parAB- and the insertion of another parS site at the mid-arm are not detrimental to cell viability (in Caulobacter crescentus, insertion of another parS mid-arm affects chromosome segregation, hence cell viability severely). Furthermore, the rare set of arm-modified SMCs from the Gruber lab also gives this manuscript a unique mechanistic angle. Given the available data, the conclusion of the manuscript is safe. I especially appreciate that the authors did not bias towards the model of SMC traversing each other by Z-loop formation.

  3. Reviewer #1 (Public Review):

    The authors investigate the role of Condensin and its loading in ensuring appropriate chromosome dynamics in the model organism Bacillus subtilis. The data are of high quality and generally support the ultimate conclusions.

    The demonstration of collisions between ectopically-loaded Condensin and their negative impact on cellular viability are important insights, particularly in light of the recent single-molecular in vitro experiments demonstrating the ability of 2 Condensins to pass one another and thereby form Z-structures on DNA.

    The main caveat is that the work lacks direct quantization of the levels of chromosome-associated Condensin—inclusion of experiments to evaluate this parameter would go a long way to validating (or refuting) the authors' conclusions.

  4. Evaluation Summary:

    This manuscript presents some intriguing data to support the notion that B. subtilis cells have tuned a variety of parameters related to SMC loading and translocation to ensure that individual complexes do not collide. This is likely an important but poorly understood aspect of condensins/SMCs, and as such represents a valuable contribution to the field and should be of interest to a broad set of readers.

    (This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. The reviewers remained anonymous to the authors.)