ATP-Induced Shrinkage of DNA with MukB Protein and the MukBEF Complex of Escherichia coli

Abstract

Fluorescence microscopic observation of individual T4 DNA molecules revealed that the MukBEF complex (bacterial condensin) and its subunit, the MukB (a member of the SMC [structural maintenance of chromosomes] superfamily) homodimer, of Escherichia coli markedly shrunk large DNA molecules in the presence of hydrolyzable ATP. In contrast, in the presence of ADP or ATP-␥S, the conformation of DNA was almost not changed. This suggests that the ATPase activity of subunit MukB is essential for shrinking large DNA molecules. Stretching experiments on the shrunken DNA molecules in the presence of ATP and MukBEF indicated a cross-bridging interaction between DNA molecules.The conformational transitions of DNA molecules have been intensively studied to understand the role of DNA compaction in various biological processes (2, 6, 23). Long DNA molecules are particularly interesting because their compaction is accompanied by an extraordinary increase in DNA molecular density on the order of 10,000-fold, and the resulting condensates can be organized into characteristic structures such as the toroid and rod. In vivo, DNA compaction is achieved by complexation with cationic proteins, while in vitro it can be realized by the addition of a variety of condensation agents, such as multivalent cations, cationic surfactants, neutral hydrophobic polymers, etc. (24).With regard to DNA compaction per se, regulation of the manner of DNA condensation-for example, the segregation between the tightly packed and swollen parts along a single genomic DNA-is considered to play an essential role in the selective activation/inactivation of desired genes.The MukB protein of Escherichia coli is known to be a structural and functional analogue of the members of the ubiquitous SMC (structural maintenance of chromosomes) family of proteins, which are responsible for maintaining the higherorder structure of DNA in chromosomes (8,12,17,19). MukB is the first bacterial SMC-like protein identified by Niki and coworkers in 1991 (13). It, along with associated proteins, forms a coiled-coiled dimer, as shown in Fig. 1A. It has been shown that MukB indeed binds and condenses DNA but is not stimulated by ATP in these reactions (12,14). In the MukB protein, the N-terminal globular domain has a nucleotide binding consensus sequence (14,21,22), and the C-terminal domain participates in DNA binding activity. It has also been shown that MukB binds to DNA even in the absence of ATP (14). While the physical origin of the driving force of the MukB protein function is unclear, a large number of reports have described the ATP-dependent activities of SMC proteins (3-5), including ATP hydrolysis, as causing a supercoiling reaction (7).The MukBEF complex is constructed through the binding of both MukF (51 kDa) and MukE (27 kDa) proteins to the globular domains of MukB (170 kDa) (22). MukE, MukF, and the MukEF complex do not demonstrate DNA binding activity (22), but the MukBEF complex, thus formed, participates in the organization of sister chromosomes and in partitionin...

MukB-mediated Catenation of DNA Is ATP and MukEF Independent

“…Biochemical and structural studies have demonstrated that the binding of two ATPs is shared by the two head domains in the dimeric protein, bringing them together in a closed conformation (23). ATPase activity is stimulated by binding of the kleisin, MukF, and its associated protein, MukE (24), suggesting that the cycle of ATP binding and hydrolysis could regulate the opening and closing of the tripartite ring of the SMC complexes. This action has been invoked as a necessary part of the ability of the eukaryotic cohesin and condensin to trap DNA and allow it to exit (3).…”

“…the ATPase activity of MukB is enhanced (24). Thus, we asked if the addition of MukEF to MukB-mediated catenation reactions that were inhibited by ATP led to a recovery of the catenation activity.…”

“…The Effect of MukEF on MukB ATPase Activity-It had been shown previously that MukEF stimulated the MukB ATPase activity (24). As communicated to us by David Sherratt, 5 only MukF is required to stimulate the MukB ATPase.…”

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).…”