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...