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• W2983251999 abstract "Full text Figures and data Side by side Abstract eLife digest Introduction Results Discussion Materials and methods References Decision letter Author response Article and author information Metrics Abstract Smc–ScpAB forms elongated, annular structures that promote chromosome segregation, presumably by compacting and resolving sister DNA molecules. The mechanistic basis for its action, however, is only poorly understood. Here, we have established a physical assay to determine whether the binding of condensin to native chromosomes in Bacillus subtilis involves entrapment of DNA by the Smc–ScpAB ring. To do so, we have chemically cross-linked the three ring interfaces in Smc–ScpAB and thereafter isolated intact chromosomes under protein denaturing conditions. Exclusively species of Smc–ScpA, which were previously cross-linked into covalent rings, remained associated with chromosomal DNA. DNA entrapment is abolished by mutations that interfere with the Smc ATPase cycle and strongly reduced when the recruitment factor ParB is deleted, implying that most Smc–ScpAB is loaded onto the chromosome at parS sites near the replication origin. We furthermore report a physical interaction between native Smc–ScpAB and chromosomal DNA fragments. https://doi.org/10.7554/eLife.06659.001 eLife digest The genome of any living organism holds all the genetic information that the organism needs to live and grow. This information is written in the sequence of the organism's DNA, and is often divided into sub-structures called chromosomes. Different species have different sized genomes, but even bacteria with some of the smallest genomes still contain DNA molecules that are thousand times longer than the length of their cells. DNA molecules must thus be highly compacted in order to fit inside the cells. DNA compaction is particularly important during cell division, when the DNA is being equally distributed to the newly formed cells. In plants, animals and all other eukaryotes, large protein complexes known as condensin and cohesin play a major role in compacting, and then separating, the cell's chromosomes. Many bacteria also have condensin-like complexes. At the core of all these complexes are pairs of so-called SMC proteins. However, it is not clear how these SMC proteins direct chromosomes to become highly compacted when cells are dividing. Wilhelm et al. have now developed two new approaches to investigate how SMC proteins associate with bacterial DNA. These approaches were then used to study how SMC proteins coordinate the compaction of chromosomes in a bacterium called Bacillus subtilis. The experiments revealed that SMC proteins are in direct physical contact with the bacterial chromosome, and that bacterial DNA fibers are physically captured within a ring structure formed by the SMC proteins. Wilhelm et al. suggest that these new findings, and recent technological advances, have now set the stage for future studies to gain mechanistic insight into these protein complexes that organize and segregate chromosomes. https://doi.org/10.7554/eLife.06659.002 Introduction Compaction and individualization of sister DNA molecules is a prerequisite for efficient segregation of the genetic material to daughter cells during cell division. Multi-subunit Structural Maintenance of Chromosomes (SMC) protein complexes—such as cohesin and condensin—are major determinants of chromosome structure and dynamics during the cell cycle in eukaryotes as well as in prokaryotes (Hirano, 2006; Thadani et al., 2012; Gruber, 2014). Condensin subunits were initially identified as abundant, non-histone components of mitotic chromosomes in metazoans (Hirano and Mitchison, 1994). In mitosis, condensin localizes together with topoisomerase II in punctate structures to the longitudinal core of chromatids, called the chromosome axis (Coelho et al., 2003; Maeshima and Laemmli, 2003; Ono et al., 2004). Inactivation of condensin subunits by mutation or depletion results in severe morphological aberrations and mechanical sensitivity of metaphase chromosomes, and subsequently to defects in their segregation during anaphase (Hirano and Mitchison, 1994; Ono et al., 2003; Gerlich et al., 2006). In bacteria, Smc–ScpAB is the prevalent version of SMC protein complexes. Its distant relatives MksBEF and MukBEF can be found scarcely scattered over most of the bacterial phylogenetic tree and in isolated branches of proteobacteria, respectively (Gruber, 2011). In Bacillus subtilis and Streptococcus pneumoniae, Smc–ScpAB is recruited to a region around the replication origin by ParB/Spo0J protein bound to parS sites, thereby forming a discrete focus—also called condensation center—on each nascent copy of the chromosome (Gruber and Errington, 2009; Sullivan et al., 2009; Minnen et al., 2011). Inactivation of Smc–ScpAB in B. subtilis under nutrient rich growth conditions blocks separation of sister replication origins and consequentially leads to lethal defects in chromosome partitioning (Gruber et al., 2014; Wang et al., 2014). Smc–ScpAB thus promotes the initial stages of chromosome segregation in B. subtilis, likely by condensing and individualizing the emerging copies of the chromosome in preparation for their segregation to opposite halves of the cell. The canonical SMC complex in bacteria comprises five subunits: (1) two Smc proteins, which each form a 45 nm long antiparallel coiled coil that connects an ABC-type ATPase ‘head’ domain at one end of the coiled coil with a ‘hinge’ homodimerization domain at the other end (Hirano et al., 2001), (2) a single ScpA subunit, which belongs to the kleisin family of proteins and associates via its C-terminal winged-helix domain (WHD) with the bottom ‘cap’ surface of one Smc head and via its N-terminal helical domain with the ‘neck’ coiled coil region of the other Smc protein (Bürmann et al., 2013), and (3) a dimer of ScpB protein, which binds to the central region of ScpA (Bürmann et al., 2013; Kamada et al., 2013). Overall, the pentameric Smc–ScpAB complex displays a highly extended conformation harboring a central channel, which is surrounded by a closed tripartite ring formed by the Smc dimer and the ScpAB2 sub-complex. The B. subtilis Smc coiled coils associate with one another to form rod-shaped Smc dimers (Soh et al., 2015). Furthermore, the Smc head domains can interact directly with one another—via a composite interface that includes two molecules of ATP. Binding to ATP, head engagement and ATP hydrolysis likely control and drive the biochemical action of Smc–ScpAB. Models for SMC condensation activity have been proposed based on observations made with isolated SMC dimers, SMC fragments or holo-complexes. Such protein preparations support the bridging of given DNA molecules in vitro as indicated by the re-annealing of single stranded DNA, intermolecular DNA ligation, DNA catenation and the co-purification of labeled and unlabeled DNA molecules (Sutani and Yanagida, 1997; Losada and Hirano, 2001; Cui et al., 2008). Many SMC complexes bound to different segments of DNA might thus come together and anchor DNA in condensation centers or at the chromosome axis. Oligomeric assemblies of bacterial Smc proteins have indeed been observed by Atomic Force Microscopy and Electron Microscopy (Mascarenhas et al., 2005; Fuentes-Perez et al., 2012). This model provides a straightforward explanation for the compaction activity of SMC. However, it is unclear how such apparently indiscriminate DNA aggregation would promote rather than block the individualization of sister chromosomes (Gruber, 2014). Local wrapping of DNA around the SMC complex could result in well-defined lengthwise condensation of DNA. However, too little SMC protein appears to be present in chromosomes to yield decent levels of compaction by simple wrapping. A different hypothesis is based on the finding that the structurally related cohesin complex holds sister chromatids in eukaryotes together by entrapping sister DNA fibers within its ring (Gruber et al., 2003; Gligoris et al., 2014). Accordingly, individual SMC complexes might entrap and expand loops of DNA, thereby driving lengthwise condensation of chromosomes with little limitations in the attainable levels of compaction (Nasmyth, 2001; Alipour and Marko, 2012). Here, we investigate how the prokaryotic SMC–kleisin complex binds to chromosomes in vivo using a novel whole-chromosome assay. Results A chromosome entrapment assay We initially attempted to detect topological interactions between B. subtilis Smc–ScpAB and plasmid DNA using pull-down assays as previously described (Ivanov and Nasmyth, 2005; Ghosh et al., 2009; Cuylen et al., 2011). However, several attempts failed to provide clear evidence for entrapment of small circular DNA by prokaryotic condensin. Conceivably, Smc–ScpAB does not interact with these artificial substrates in a physiological manner. To circumvent this possibility, we established an inverse assay by immobilizing whole chromosomes of B. subtilis in agarose plugs and monitoring their association with covalently closed rings of Smc–ScpA under harsh protein denaturing conditions (Figure 1A). To develop the chromosome entrapment assay we first performed experiments with the replicative sliding clamp, DnaN, in B. subtilis, which is known to entrap DNA in a topological manner. Furthermore, most of cellular DnaN protein is maintained in the vicinity of active replication forks in B. subtilis, presumably by its topological association with leading and lagging strand DNA (Su'etsugu and Errington, 2011). Figure 1 with 1 supplement see all Download asset Open asset Development of the chromosome entrapment assay using DnaN. (A) Scheme for the chromosome entrapment assay. Cells are incubated with the cysteine cross-linker BMOE, lysed in agarose plugs and subjected to an electric field in the presence of SDS buffer. Proteins stably bound to chromosomal DNA are re-isolated from nuclease treated agarose plugs, concentrated and analyzed by SDS-PAGE. (B) Crystal structure of S. pneumoniae DnaN (PDB: 3D1F) in surface representation. The monomers of DnaN are shown in dark and light blue colours, respectively. The positions of an engineered pair of cysteine residues (N114C and V313C) at the monomer–monomer interface of B. subtilis DnaN are indicated by arrows. (C) Chromosome entrapment by DnaN. Cells of strains BSG1449 (dnaN-HT) and BSG1459 (dnaN(N114C, V313C)-HT) were cross-linked with BMOE and subjected to the chromosome entrapment assay. Input and eluate fractions were analysed by in-gel detection of fluorescently labeled HT fused to DnaN (top panel). Eluate fractions of samples treated with or without nuclease during cell lysis are indicated as nuclease ‘+’ or ‘−’, respectively. Eluate fractions were further analyzed by silver staining revealing that another protein was consistently co-isolated during the chromosome entrapment assay (bottom panel). This protein—identified as flagellin by mass spectrometry—was retained independently of the integrity of the chromosome. The following figure supplement is available: Figure 1—figure supplement 1: DNA entrapment by DnaN. https://doi.org/10.7554/eLife.06659.003 Based on the crystal structure of S. pneumoniae DnaN we engineered a pair of cysteine residues (N114C, V313C) into the B. subtilis protein so that DnaN can be cross-linked into covalent rings in the presence of a cysteine-specific cross-linker such as BMOE (Figure 1B). For detection a cys-less variant of the HaloTag (‘HT’) was fused to the C-terminus of DnaN (Figure 1—figure supplement 1B) and the construct was integrated into the genome of B. subtilis via allelic replacement at the endogenous locus. The dnaN-ht genes with and without cysteine mutations supported normal growth of B. subtilis, implying that they encoded functional DnaN proteins (data not shown). In vivo cross-linking of DnaN-HT resulted in two additional, slow migrating bands in SDS-PAGE gels (Figure 1C), corresponding to single and double cross-linked species of DnaN dimers, designated as X-DnaN-HT and XX-DnaN-HT, respectively (Figure 1—figure supplement 1A). We next embedded cells in agarose plugs and disrupted their cell walls by lysozyme digestion. Agarose plugs were then subjected to an electric field in the presence of SDS to denature and remove any unattached proteins from chromosomes. Plugs were finally treated with benzonase to digest genomic DNA and to release any stably entrapped protein. DnaN-HT protein was then analysed by in-gel fluorescence. Non-crosslinkable DnaN-HT was efficiently depleted from agarose plugs during the entrapment assay (Figure 1C). In contrast, the double cross-linked, circular form of DnaN(N114C, V313C)-HT (XX-DnaN-HT) was retained in the agarose plug during electrophoresis with high efficiency (∼50% of input). A minor fraction of single cross-linked DnaN dimer (X-DnaN-HT) was also observed. This is likely generated from XX-DnaN-HT by spontaneous hydrolysis of thiol-malemide adducts during protein isolation (Kalia and Raines, 2007; Baldwin and Kiick, 2013). Importantly, the presence of benzonase during cell lysis eliminated all DnaN from the plug, indicating that circular DnaN is retained in plugs via its interaction with cellular DNA. Furthermore, in the absence of the cross-linker BMOE, no DnaN-HT was detected in the eluate fraction (Figure 1—figure supplement 1C). The chromosome entrapment assay thus specifically detects a topological association of intact chromosomes with DNA sliding clamps and confirms that a major fraction (at least 50%) of DnaN is loaded onto DNA in rapidly growing cells. Prokaryotic condensin entraps chromosomal DNA Next, we used the newly developed chromosome entrapment assay to test for an association between native chromosomes and Smc–ScpAB complexes. Cysteine pairs were introduced at the Smc–Smc and at both Smc–ScpA interfaces and a HT was fused at the C-terminus of Smc to allow in-gel fluorescence detection (Figure 2A) (Bürmann et al., 2013). Strains bearing the cysteine mutations and the Smc-HaloTag fusion supported normal growth on nutrient rich medium demonstrating the functionality of the modified Smc complex (Figure 2—figure supplement 1A). Cells were treated with BMOE and extracts were analysed by SDS-PAGE. As internal control for the chromosome entrapment assay we employed the DnaN(N114C, V313C) protein, whose double cross-linked form was detected in input and eluate samples by immunoblotting (Figure 2—figure supplement 1B). Various species of Smc–ScpAB were identified in extracts of BMOE cross-linked cells by in-gel fluorescence. These correspond to fully cross-linked Smc–ScpA–Smc rings and several intermediate cross-linking species as reported previously (Figure 2B,C) (Bürmann et al., 2013). To reveal the identity of all species, strains lacking one of six engineered cysteines were used as controls that collectively form several intermediate cross-linked species but no fully cross-linked rings of Smc–ScpAB (Figure 2—figure supplement 1C). In these control samples little or no Smc-HT protein was retained in agarose plugs under denaturing conditions as expected for any non-circular protein (Figure 2B). In the presence of all pairs of cysteine, however, a set of two closely migrating species was consistently detected at significant levels after the chromosome entrapment assay (∼10–20% of input material) (Figure 2B). We argued that the two closely migrating species might correspond to Smc–ScpAB with a single or a double cross-link at the Smc hinge. Consistent with this notion we find that only a single species of Smc2–ScpA accumulated during the chromosome entrapment assay when a single cysteine residue (R643C) was used to cross-link the Smc hinge domains (Figure 2—figure supplement 2A). These findings strongly suggest that Smc–ScpAB is bound to chromosomes via entrapment of chromosomal DNA. If this were indeed the case, then its retention in agarose plugs should depend on the integrity of Smc–ScpAB rings and of chromosomal DNA. Incubation of agarose plugs with the nuclease benzonase during cell lysis eliminated the Smc-HT and DnaN signal in the sample (Figure 2B, Figure 2—figure supplement 1B). To disrupt covalent Smc–ScpAB rings, we inserted cleavage sites for TEV protease into the linker region preceding the C-terminal WHD of ScpA and incubated cells during lysozyme treatment with recombinant TEV protease to open any circular Smc2–ScpA species. As expected, little or no Smc-HT signal was detected in agarose plugs after TEV cleavage of ScpA (Figure 2D). To exclude any artefacts due to the presence of the HT on Smc we have repeated the chromosome entrapment assay with an untagged allele of Smc using immunoblotting with anti-Smc antibodies for the detection of cross-linked species, which yielded very similar results (Figure 2—figure supplement 2C). Furthermore, we found that Smc2-ScpA rings are stably trapped in agarose plugs over extended periods of time in constant or alternating electric fields (data not shown). Thus, our chromosome entrapment assay specifically detects the association between intact chromosomal DNA and rings of Smc–ScpAB in B. subtilis, demonstrating that DNA fibers pass through the Smc ring. Figure 2 with 2 supplements see all Download asset Open asset Prokaryotic condensin entraps the chromosome. (A) Scheme for the cross-linking of Smc-HaloTag (‘HT’) and ScpA into a covalent Smc–ScpA–Smc ring. (B) Chromosome entrapment of covalent Smc2–ScpA rings. Cells of strains BSG1782, BSG1809-1813 and BSG1831 were cross-linked and subjected to the chromosome entrapment assay. Cross-linked Smc-HT species were visualized by in-gel fluorescence detection. The presence or absence of cysteine pairs at each of the three ring interfaces are indicated by ‘+’ and ‘−’, respectively. An aliquot of cells of strains BSG1782 was incubated with benzonase during cell lysis (nuclease ‘+’). The positions of uncross-linked Smc-HT and fully cross-linked, circular Smc–ScpA–Smc species are indicated by ‘Smc-HT’ and ‘Smc-HT circ.’; all species are labelled by colour-coded arrowheads (see panel C for legend). Circular species (‘h’) are labeled by a double pointed arrowhead. (C) Schematic depiction of the structure of cross-linked Smc–ScpA species (‘a’–‘i’). (D) TEV cleavage of ScpA prevents entrapment of Smc–ScpAB in agarose plugs. In-gel fluorescence detection of Smc-HT derived from strains BSG1807 and BSG1832. The presence or absence of TEV sites in ScpA and of TEV protease during cell lysis is indicated by ‘+’ and ‘−’, respectively. Cleavage of ScpA(TEVs) by TEV protease creates new species of cross-linked Smc-HT (see ‘input’ samples) and prevents entrapment of Smc-HT in agarose plugs (see ‘eluate fraction’) (top panel). ‘XX-DnaN’ serves as internal assay control visualized by immunoblotting of cross-linked species of DnaN protein (bottom panel). The following figure supplement is available: Figure 2—figure supplement 1: DNA entrapment by wild-type Smc–ScpAB (I) and Figure 2—figure supplement 2: DNA entrapment by wild-type Smc–ScpAB (II). https://doi.org/10.7554/eLife.06659.005 A full Smc ATPase cycle is required for loading of condensin onto chromosomes Next, we established the requirements for the formation of interconnections between Smc–ScpAB rings and chromosomes. The intrinsic ATPase activity of cohesin has previously been implicated in stable association with chromosomes (Arumugam et al., 2003; Weitzer et al., 2003). More specifically, ATP hydrolysis has been hypothesized to transiently open an entry gate for DNA in the cohesin ring during its loading onto chromosomes (Gruber et al., 2006; Hu et al., 2011). To test what steps of the ATP hydrolysis cycle in Smc–ScpAB are involved in the entrapment of chromosomal DNA, we made use of smc alleles harboring mutations that specifically prevent ATP binding (K37I), engagement of Smc head domains (S1090R) or ATP hydrolysis (E1118Q) (Figure 3A) (Hirano and Hirano, 2004). The three mutant proteins are expressed at normal levels in B. subtilis being indicative of proper protein folding (Figure 3—figure supplement 1A). However, they do not support growth on nutrient rich medium similar to smc null mutants, implying that all steps of the ATPase cycle are essential for Smc functionality (Figure 3—figure supplement 1B) (Gruber et al., 2014). For the chromosome entrapment assay, these Smc ATPase mutations were combined with cysteine mutations for BMOE cross-linking. To support their viability, the resulting strains as well as the wild-type controls were grown in minimal medium. The three mutant Smc proteins assembled into normal Smc–ScpAB complexes as judged by Smc–ScpA cross-linking, albeit there is a slight decrease in the fraction of ScpA proteins bridging Smc dimers and a concomitant minor increase in ScpA subunits bound to single Smc proteins (Figure 3—figure supplement 1C, species ‘e’ and ‘d’, respectively) (Bürmann et al., 2013). Intriguingly, the ATP binding and engagement mutants abolished the fraction of covalent ring species retained in the agarose plug during the chromosome entrapment assay (Figure 3B). In case of the ATP hydrolysis mutant Smc(E1118Q) only minute amounts of cross-linked rings were recovered from SDS treated plugs. This small fraction of stably bound condensin conceivably arises as a consequence of residual levels of ATP hydrolysis activity in Smc(E1118Q) (Hirano and Hirano, 2004). Thus, ATP binding and ATP dependent Smc head engagement—and most probably also ATP hydrolysis—are essential for entrapment of chromosomal DNA by condensin in bacteria, as has been supposed for cohesin in yeast. Furthermore, the strict requirement of several steps of the ATPase cycle strongly suggests that entrapment of DNA corresponds to the physiological form of association with the bacterial chromosome. Figure 3 with 1 supplement see all Download asset Open asset The Smc ATPase is required for loading of DNA into Smc–ScpAB. (A) A scheme for the ATP hydrolysis cycle of Smc. Schematic positions for Walker A, Walker B and ABC-signature motifs on the Smc head domain are shown (top row). ATP binding to the Walker A domain is blocked in Smc(K37I) ‘(1)’. ATP-dependent engagement of two Smc heads is abolished in the Smc(S1090R) mutant ‘(2)’. The E1118Q mutation strongly reduces ATP hydrolysis ‘(3)’. (B) Smc ATPase mutations abolish chromosomal loading of Smc–ScpAB. In-gel fluorescence detection of Smc-HT of input and eluate fractions from a representative chromosome entrapment assay performed with strains BSG1782 and BSG1784-6. Protein extracts (10% of input) were loaded next to samples subjected to the entrapment assay. Selected cross-linked species of Smc-HT are labeled (top panel). Detection of cross-linked species of DnaN by immunoblotting was used as internal assay control (bottom panel). The following figure supplement is available: Figure 3—figure supplement 1: ATPase mutants of Smc–ScpAB. https://doi.org/10.7554/eLife.06659.008 ScpB and ParB proteins are essential for normal loading of condensin onto chromosomes What other factors might be required for the loading of condensin onto DNA? The ScpB subunit forms homodimers that bind in an asymmetric manner to the central region of a single ScpA monomer. It thus is in close proximity of the Smc ATPase domains. Together with ScpA it putatively plays a role in the regulation of the Smc ATPase activity (Kamada et al., 2013). Its precise molecular function, however, is not clear yet. To test whether ScpB is involved in the association of Smc–ScpA rings with chromosomes we combined the cysteine mutations in Smc and ScpA with an scpB in-frame deletion (Figure 4—figure supplement 1). Ring formation was only mildly affected by the absence of ScpB as judged by BMOE cross-linking and in-gel fluorescence detection (Figure 4A) (Bürmann et al., 2013). However, Smc complexes lacking ScpB subunits failed to entrap chromosomes altogether demonstrating that ScpB is absolutely required for loading of prokaryotic condensin onto chromosomal DNA. Figure 4 with 1 supplement see all Download asset Open asset ScpB and ParB are essential for efficient DNA entrapment by Smc complexes. (A) Deletion of scpB eliminates loading of chromosomal DNA into Smc complexes. In-gel fluorescence detection of Smc-HT in input and eluate fractions is shown from chromosome entrapment assays performed with strains BSG1782 (‘WT’) and BSG1850 (‘ΔscpB’) (top panel). DnaN was used as internal control (bottom panel). (B) Several parB mutations interfere with efficient chromosomal loading of Smc–ScpAB. Input and eluate fractions from chromosome entrapment assays with strains BSG1782, BSG1783 and BSG1960-3 were analysed by in-gel fluorescence detection of Smc-HT (top panel). DnaN was used as internal control (middle panel). Immunoblotting using polyclonal rabbit anti-ParB antiserum confirms near-normal expression of mutant ParB proteins (bottom panel). The following figure supplement is available: Figure 4—figure supplement 1: Growth of smc, parB double mutants. https://doi.org/10.7554/eLife.06659.010 ParB proteins—bound to parS sites—are crucial for efficient targeting of Smc–ScpAB to a large region of the chromosome near the replication origin (Gruber and Errington, 2009; Sullivan et al., 2009; Minnen et al., 2011). ParB might act by simply increasing the local concentration of Smc–ScpAB around oriC either before or after its loading onto the chromosome. Alternatively, ParB bound to parS sites might be more directly involved in the loading reaction itself, for example, as catalytic factor, and its absence might thus affect levels of chromosomal condensin. To test this, we performed the chromosome entrapment assay with cells lacking the parB gene. Intriguingly, the levels of Smc–ScpAB entrapping chromosomal DNA were strongly reduced in the parB null mutant as judged by the limited retention of Smc–ScpA species in agarose plugs (Figure 4B). Thus, ParB protein likely promotes the entrapment of chromosomal DNA by Smc–ScpAB. This strongly suggests that most condensin is loaded onto the chromosome at parS sites, where ParB protein is bound. In all other parts of the chromosome entrapment of DNA fibers by Smc–ScpAB might be very inefficient. The cysteine bearing smc allele causes growth defects when combined with ΔparB (Figure 4—figure supplement 1). Therefore, we cannot formally exclude the possibility that the decreased loading of Smc observed in ΔparB are due to the cysteine modifications in Smc and that chromosomal loading of wild-type Smc is not or much less affected by parB deletion. Previously, two parB point mutations (N112S and R149G), which prevent the formation of Smc-GFP foci, have been isolated in B. subtilis (Gruber and Errington, 2009). We found that these mutations strongly impair loading of Smc onto the chromosome in the entrapment assay similar to ΔparB (Figure 4B). The R149G mutation is positioned on the helix-turn-helix motif of ParB and might thus directly affect binding to parS sites (Leonard et al., 2004). The N112S mutation, however, is located in another highly conserved region, which has been implicated in the ‘spreading’ of ParB protein from parS sequences into adjacent DNA (Leonard et al., 2004; Graham et al., 2014). The spreading of ParB along several kb of DNA is a feature conserved in plasmid and chromosome derived ParB proteins, however, the underlying mechanism is only poorly understood (Rodionov et al., 1999). It might possibly involve the formation of a large nucleoprotein complex (Broedersz et al., 2014). Several other mutants of ParB (including B. subtilis ParB G77S and R80A) have been reported to be defective in spreading from parS sites (Breier and Grossman, 2007; Graham et al., 2014). Intriguingly, also these mutations resulted in largely reduced levels of Smc on the chromosome in our entrapment assay, being comparable to the levels found in a parB deletion mutant (Figure 4B). This implies that ParB spreading from parS sites or formation of large nucleoprotein complexes might be essential for loading of DNA into the Smc ring by ParB. These findings are consistent with the observation that formation of Smc-GFP foci near the origin of replication are affected by the G77S mutation (Sullivan et al., 2009). In summary, these results demonstrate that several factors—including ScpB protein, a ParB/parS nucleoprotein complex and the Smc ATPase cycle—are required to promote efficient loading of condensin rings onto the chromosome. Smc–ScpAB rings physically associate with chromosomal DNA fragments Smc proteins and fragments thereof exhibit affinity for single- and double-stranded DNA in vitro (Chiu et al., 2004; Hirano and Hirano, 2006; Soh et al., 2015). The physical contacts with DNA might occur once condensin has been successfully loaded onto chromosomes and thus be a permanent feature of chromosomal Smc–ScpAB. Alternatively, the direct association with DNA might be restricted to certain intermediates in the chromosomal loading reaction. To test for interactions between Smc–ScpAB and specific chromosomal DNA fragments, we have affinity-purified endogenous Smc–ScpAB from B. subtilis cell lysates using a short Avitag peptide fused to the C-terminus of the Smc protein, which gets biotinylated when the biotin ligase gene birA is co-expressed (‘Smc-Avitag’). We then examined fractions for the co-purification of fragments of chromosomal DNA—generated by restriction digest with XbaI—using quantitative PCR with primer pairs specific for different part" @default.
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• W2983251999 title "Author response: SMC condensin entraps chromosomal DNA by an ATP hydrolysis dependent loading mechanism in Bacillus subtilis" @default.
• W2983251999 doi "https://doi.org/10.7554/elife.06659.018" @default.
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