MukB is a bacterial SMC (structural maintenance of chromosome) protein required for faithful chromosome segregation in Escherichia coli. We report here that purified MukB introduces right-handed knots into DNA in the presence of type-2 topoisomerase, indicating that the protein promotes intramolecular DNA condensation. The pattern of generated knots suggests that MukB, similar to eukaryotic condensins, stabilizes large right-handed DNA loops. In contrast to eukaryotic condensins, however, the net supercoiling stabilized by MukB was negative. Furthermore, DNA reshaping by MukB did not require ATP. These data establish that bacterial condensins alter the shape of double-stranded DNA in vitro and lend support to the notions that the right-handed knotting is the most conserved biochemical property of condensins. Finally, we found that MukB can be eluted from a heparin column in two distinct forms, one of which is inert in DNA binding or reshaping. Furthermore, we find that the activity of MukB is reversibly attenuated during chromatographic separation. Thus, MukB has a unique set of topological properties, compared with other SMC proteins, and is likely to exist in two different conformations.SMC (structural maintenance of chromosome) proteins are involved in various aspects of higher order chromatin dynamics in organisms ranging from bacteria to humans. They act as a part of multisubunit complexes that are required for such diverse cellular functions as chromosome cohesion and condensation, recombination, repair, and dosage compensation (1-6).In solution, SMC proteins form a distinctive V-shaped structure composed of two molecules joined via their hinge domains (7-9). Two long coiled-coils protruding from the hinge terminate in globular head domains of SMCs. The head domains themselves can dimerize, creating a composite ATP binding pocket at the interface of the two head domains. ATP binding and hydrolysis has been proposed to drive conformational changes in the interacting head domains, which eventually result in the formation and disruption of protein rings (10 -12) or macromolecular assemblies (13-15). Mutations within the P-loop or signature motif of SMCs inactivate the proteins in vivo (12, 16). However, the role of ATP in DNA reshaping by SMCs remains unclear.In eukaryotes, the structurally similar condensins and cohesins have very distinct roles in chromosome dynamics. In vitro, the purified SMCs act according to their intracellular functions. Condensins promote intramolecular DNA condensation, which can be detected as an increase in DNA knotting in the presence of type-2 topoisomerase (17-19). Similar reaction carried out with cohesins produces catenanes, indicating that DNA fragments from different DNA molecules are brought together (20). Rad50 protein, an SMC protein involved in double strand break repair in yeast, oligomerizes upon binding to the ends of linear DNA (15). The mechanism of DNA reshaping by SMC complexes remains unknown. It is not even clear how much of the biochemical specificity resides w...

Correct folding of the chromosome into its highly ordered structure requires the action of condensins. The multisubunit condensins are highly conserved from bacteria to humans, and at their core they contain the characteristic V-shaped dimer of structural maintenance of chromosome proteins. The mechanism of DNA rearrangements by condensins remains unclear. Using magnetic tweezers, we show that bacterial condensin MukB acts as an ATP-modulated macromolecular assemblage in DNA condensation. Condensation occurs in a highly cooperative manner, resulting in the formation of force-resilient clusters. ATP regulates nucleation but not propagation of the clusters and seems to play a structural role. MukB clusters can further interact with each other, thereby bringing distant DNA segments together. The resulting activity has not previously been described among DNA-remodeling machines and may explain how the protein can organize the global structure of the chromosome.

MukBEF is a bacterial SMC (structural maintenance of chromosome) complex required for faithful chromosome segregation in Escherichia coli. The SMC subunit of the complex, MukB, promotes DNA condensation in vitro and in vivo; however, all three subunits are required for the function of MukBEF. We report here that MukEF disrupts MukB⅐DNA complex. Preassembled MukBEF was inert in DNA binding or reshaping. Similarly, the association of MukEF with DNA-bound MukB served to displace MukB from DNA. When purified from cells, MukBEF existed as a mixture of MukEF-saturated and unsaturated complexes. The holoenzyme was unstable and could only bind DNA upon dissociation of MukEF. The DNA reshaping properties of unsaturated MukBEF were identical to those of MukB. Furthermore, the unsaturated MukBEF was stable and proficient in DNA binding. These results support the view that kleisins are not directly involved in DNA binding but rather bridge distant DNA-bound MukBs.SMCs are ubiquitous highly conserved proteins that have been implicated in virtually every aspect of higher order chromatin dynamics. Eukaryotic cells contain at least six different SMC complexes with functions in chromosome condensation and segregation, recombination, and repair (1-4). Bacteria carry two SMC complexes. In Escherichia coli, faithful chromosome segregation requires the action of MukBEF (5, 6). The second SMC complex, SbcCD nuclease, was implicated in the metabolism of double-strand DNA breaks (7).The defining feature of SMCs is their structure. They consist of two globular domains connected by a long coil-hinge-coil motif. In solution, SMCs dimerize to form an idiosyncratic V-shaped structure with two globular head domains connected at the hinge via two long coiled coils (8 -10). The Walker A and B motifs, which are found in the N-and C-terminal domains of SMCs, are located at the surface of the SMC heads. This enables further association of SMCs via nucleotide-sandwiched dimerization of the head domains, leading to the formation of protein rings (11-13) or macromolecular assemblies (14 -16).SMCs act inside the cell as a part of multisubunit complexes. Among the non-SMC subunits, a conserved family of kleisins was identified (17, 18). Kleisins bind head domains in the vicinity of the ATP binding site and apparently stabilize the dimeric form of the SMC heads (11,19,20). In several cases, a functional interaction between kleisins and ATP has been reported (11,14,21).Biochemical properties of SMCs befit their intracellular functions. In a reaction coupled to type-2 DNA topoisomerases, condensins promote formation of DNA knots of specific topology (22-24). This property is highly conserved among condensins and indicates intramolecular DNA condensation. In contrast, cohesins promote DNA catenation, indicating predominantly intermolecular DNA interactions (25). How the structurally similar condensins and cohesins distinguish between their substrates remains unclear.The mechanism of SMCs is under debate. Divergent models were proposed to explain the mecha...

Condensins play a central role in global chromatin organization. In bacteria, two families of condensins have been identified, the MukBEF and SMC-ScpAB complexes. Only one of the two complexes is usually found in a given species, giving rise to a paradigm that a single condensin organizes bacterial chromosomes. Using sequence analysis, we identified a third family of condensins, MksBEF (MukBEF-like SMC proteins), which is broadly present in diverse bacteria. The proteins appear distantly related to MukBEF, have a similar operon organization and similar predicted secondary structures albeit with notably shorter coiled coils. All three subunits of MksBEF exhibit significant sequence variation and can be divided into a series of overlapping subfamilies. MksBEF often coexists with the SMC-ScpAB, MukBEF and, sometimes, other MksBEFs. In Pseudomonas aeruginosa, both SMC and MksB contribute to faithful chromosome partitioning, with their inactivation leading to increased frequencies of anucleate cells. Moreover, MksBEF can complement anucleate cell formation in SMC-deficient cells. Purified PaMksB showed activities typical for condensins including ATP-modulated DNA binding and condensation. Notably, DNA binding by MksB is negatively regulated by ATP, which sets it apart from other known SMC proteins. Thus, several specialized condensins might be involved in organization of bacterial chromosomes.

Structural maintenance of chromosome (SMC) proteins comprise the core of several specialized complexes that stabilize the global architecture of the chromosomes by dynamically linking distant DNA fragments. This reaction however remains poorly understood giving rise to numerous proposed mechanisms of the proteins. Using two novel assays, we investigated real-time formation of DNA bridges by bacterial condensin MukBEF. We report that MukBEF can efficiently bridge two DNAs and that this reaction involves multiple steps. The reaction begins with the formation of a stable MukB-DNA complex, which can further capture another protein-free DNA fragment. The initial tether is unstable but is quickly strengthened by additional MukBs. DNA bridging is modulated but is not strictly dependent on ATP and MukEF. The reaction revealed high preference for right-handed DNA crossings indicating that bridging involves physical association of MukB with both DNAs. Our data establish a comprehensive view of DNA bridging by MukBEF, which could explain how SMCs establish both intra- and interchromosomal links inside the cell and indicate that DNA binding and bridging could be separately regulated.