Antagonistic Interactions of Kleisins and DNA with Bacterial Condensin MukB

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 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...

MukB-mediated Catenation of DNA Is ATP and MukEF Independent

“…These two amino acid substitutions are the equivalent of the R216E and R218E mutations made in Haemophilus ducreyi MukB that elicited DNA binding deficiency (13). These two mutations sit on the head domain of the MukB dimer.…”

“…Perhaps the head domains in a MukB dimer are also non-equivalent. (27), (ii) that the pre-formed MukBEF complex is defective in binding plasmid DNA (13), and (iii) that adding MukEF to a MukB-DNA complex appears to displace MukB from the DNA (13). Our observations are consistent with these previous observations; however, we observe distinct inhibition of the MukB-mediated catenation reaction by the addition of ATP, with less inhibition observed with AMP-PNP, and no inhibition observed with ADP.…”

“…MukB binds linear and circular DNA in vitro and can induce negative supercoils and knots in relaxed circular DNA in the presence of a topoisomerase (10). Binding of MukB to chromosomal DNA in vivo requires ATP and MukEF (11,12), although MukB DNA binding in vitro does not (10,13).…”

Escherichia coli condensin MukB stimulates topoisomerase IV activity by a direct physical interaction

“…Because full-length MukB affects the supercoiling and condensation of DNA (20,21,23,24), we also probed the ability of MukB-D, which lacks the DNA-binding head domain and much of the coiled coil (44) to stimulate Topo IV function. As with intact MukB, we observe that MukB-D is sufficient to stimulate the DNA decatenation and relaxation activities of Topo IV (Fig.…”

“…Both E. coli mukB − strains and smc − strains from Bacillus subtilis and Caulobacter crescentus show temperature-sensitive colony formation and an increase in the number of anucleate cells at the permissive temperature, suggesting a deficiency in chromosome segregation (5,7,(15)(16)(17)(18)(19). Moreover, a convincing array of experiments has demonstrated that MukB functions as a condensin in vitro and in vivo (20)(21)(22)(23)(24)(25)(26)(27)(28).…”