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

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.


Introduction
DNA may result in Smc binding in trans thus creating persistent physical linkages between 109 sister chromosomes rather than helping to resolve them.

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Here, we studied the effect of Smc, ParB and parS alterations on chromosome organization 111 to explore how Smc-ScpAB load and translocate on a chromosome with multiple loading sites.

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Based on our results, we propose that Smc complexes rarely meet on the chromosome under 113 physiological conditions. We argue that multiple parameters are fine-tuned in such a way to

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We started by generating strains in which seven parS sites were inactivated by mutations, with 156 -9kb parS (parS-359) remaining the only parS site on the chromosome (along with the weak absent) further away from the replication origin with all modified Smc constructs ( Figure 1C).

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The shortened Smc variant (CC253) also displayed more inter-arm contacts when the seven 164 parS sites were mutated. Thus, the removal of parS sites improved-rather than hindered-165 chromosome arm alignment by modified Smc proteins.

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The arc of contacts detected on the left arm of the chromosome was lost in all strains harboring 167 only the -9kb parS site ( Figure 1C) (Marbouty et al., 2015). It was also lost when only -304kb parS 168 site (parS-334) was mutated ( Figure S1C). Of note, the -304kb parS site is unique, in being 169 relatively strong as well as distantly located from other strong parS sites ( Figure 1A). DNA 170 loop extrusion starting from this site is asymmetric, presumably due to the high likelihood of a 171 clash with other Smc complexes ( Figure 1E) (see below).

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To test the impact of parS distribution in a more controlled way, we created strains with two  with an apparently reducing rate. In addition, the origin region remained highly enriched in 212 ScpB also at the later time points. These two observations suggest that the Smc-CC425 213 protein has a reduced chromosome residence time and/or a reduced translocation rate.

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Determining a meaningful translocation rate for Smc-CC425 is difficult because of a wide able to align chromosome arms in this experimental system, yet the alignment did not extend 217 all the way to the terminus region ( Figure 2D). Moreover, the onset of chromosome alignment 218 as well as the rate of progress were somewhat reduced when compared to wild-type Smc.

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These experiments demonstrate that Smc-CC425 efficiently accumulated in the replication 220 origin region, but the redistribution to the chromosome arms, particularly to distal loci, was 221 hampered.

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A simple explanation for the hyper-accumulation of Smc-CC425 in the replication origin region

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We next wondered how wild-type Smc proteins co-align chromosome arms when starting DNA 238 loop extrusion at multiple parS sites. Wild-type Smc displayed relatively low enrichment in the 239 replication origin region even when all natural parS sites were present (Figure 2A). To 240 understand how collisions between translocating Smc complexes are avoided or resolved, we 241 next aimed to increase the incidence of collisions by positioning two parSopt sequences at 242 selected sites in varying genomic distances and performed 3C-seq analysis.

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As expected, control strains with a unique parSopt sequence at positions -9 kb, at -304 kb, or

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More importantly, when two parS sites were combined on the chromosome, striking novel 252 patterns of chromosome organization by wild-type Smc arose ( Figure 3B). In all cases, parallel 253 secondary diagonals emerging from the two parS sites were detected. The pattern observed 254 with -304kb parSopt and -9kb parSopt can-to a large degree-be explained as a combination of 255 contacts observed in strains with the corresponding single parS sites, however, with clearly 256 reduced probability for contacts extending beyond the region demarcated by the parS sites.

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The latter appears to suggest that parS sequences might act as loading and unloading sites.

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To test this prediction, we first slightly increased the cellular level of all subunits of the Smc 295 complex by inserting an additional copy of the smc gene and of the scpAB operon under the 296 control of their respective endogenous promoters into the genome. The increased levels of 297 Smc-ScpAB did not noticeably affect cell growth ( Figure S4A). Immunoblotting suggested a 298 4-5-fold increase in Smc and ScpB protein levels in the SMC high strain when compared to wild 299 type ( Figure 4A). Next, we performed 3C-seq analysis. Chromosome arm co-alignment was 300 strongly hampered-rather than improved-by the presence of extra Smc complexes in the 301 cell ( Figure 4B). A prominent arc was formed at the position of the -304kb parS site and the 302 secondary diagonal originating in the origin region was weak and diffuse in the SMC high strain.

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This defect was fully restored, however, by removal of seven parS sites (with the remaining 304 strong site being either -9kb parSopt or -304kb parSopt) ( Figure 4C). Note that an additional feature 305 (a minor secondary diagonal) present on the right arm of the chromosome likely originated 306 from Smc loading at the weak +1058kb parS site. The presence of two strong parS sites ( -9kb parSopt and -304kb parSopt) led to a new pattern of chromosome folding in the SMC high strain.
Moreover, the contacts corresponding to paired loops became clearly visible ( Figure 4D).

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Finally, contacts outside the parS-demarcated region were rare and spread out, and their 311 center was shifted away from the parS sites. The latter indicated that Smc complexes that 312 were loaded at one parS site and managed to move beyond the other parS site have 313 experienced a strongly reduced translocation rate from one to the other parS site, presumably 314 due to encounters with and temporary (or partial) blockage by Smc complexes translocation 315 in opposite orientation.

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If so, then extra Smc levels may lead to artificially higher accumulation of Smc-ScpAB near 317 the replication origin when multiple parS sites are present but not with a single parS site, as

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In our study, we show that impacts from collisions are barely noticeable in wild-type cells.

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A. Spotting assay of strains with modified Smc coiled coil in wild type or sensitised background 897 (∆parB). Prepared as described in Figure 1B. Two clones for each tested mutant were spotted.

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background with or without parB. Prepared as described in Figure 1B. Two clones for each 907 tested mutant were spotted.

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A. Spotting assay comparing strains with increased amount of Smc vs wild type with respective 950 number of parSopt sites. Prepared as described in Figure 1B

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C. α-ScpB ChIP-seq read counts for SMC high strains with all parS sites present, single parSopt 955 at -9kb and two parS sites at -304kb and -9kb. Represented as in Figure S3B.