Abstract
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 MukB x DNA 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.
Highlights
The defining feature of SMCs is their structure
Purified MukBEF Is Unstable and Can Reshape DNA after Dissociation of MukEF—To confirm that the inhibitory effect of MukEF on DNA binding was not an artifact of the in vitro reconstitution procedure, we examined MukBEF that was purified from cells that overproduced all three subunits of the complex (“Materials and Methods”)
MukBEF plays a central role in organizing the chromosome of E. coli
Summary
The defining feature of SMCs is their structure. They consist of two globular domains connected by a long coil-hinge-coil motif. The original experiments with 13 S condensin from frogs established that the non-SMC subunits and ATP are required for DNA compaction but not for DNA binding [26] This agrees well with genetic data, which indicate similar phenotypes for mutations in SMC and non-SMC components of the complex, and the finding that the proteolytic cleavage of kleisins leads to dissolution of cohesion between sister chromosomes [20]. The yeast SMC2/4 complex and the E. coli MukB protein promote efficient DNA knotting in the absence of ATP and the cognate accessory subunits [23, 24]. Reconstitution studies with the Bacillus subtilis SMC complex indicated that the accessory subunits modulate the association of the protein with DNA [14] It remains unclear, if the assembly of the holoenzyme alters the DNA reshaping properties of the protein
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