MukB-mediated Catenation of DNA Is ATP and MukEF Independent

Abstract

Edited by Patrick SungProperly condensed chromosomes are necessary for accurate segregation of the sisters after DNA replication. The Escherichia coli condesin is MukB, a structural maintenance of chromosomes (SMC)-like protein, which forms a complex with MukE and the kleisin MukF. MukB is known to be able to mediate knotting of a DNA ring, an intramolecular reaction. In our investigations of how MukB condenses DNA we discovered that it can also mediate catenation of two DNA rings, an intermolecular reaction. This activity of MukB requires DNA binding by the head domains of the protein but does not require either ATP or its partner proteins MukE or MukF. The ability of MukB to mediate DNA catenation underscores its potential for bringing distal regions of a chromosome together.The Escherichia coli chromosome is condensed about 1000-fold to fit into the nucleoid of the bacterium. A number of factors contribute to this extreme DNA condensation, among them are: DNA supercoiling, the binding of various nucleoid associated proteins like HU, Fis, and H-NS, and the binding of the structural maintenance of chromosomes (SMC) 3 -like condensin MukBEF (1, 2). SMC proteins act to manage the shape and behavior of chromosomes in both prokaryotes and eukaryotes (3). These proteins dimerize at a hinge region that is flanked by long coiled-coil regions that can be 40 -50 nm in length and that end in head domains that bind and hydrolyze ATP, as well as the bridging kleisin protein (MukF for the E. coli condensin (4)). The kleisin is then itself bound by another protein (MukE for the E. coli condensin (4)). There are three versions of the eukaryotic SMC proteins: the condensin, required for packaging of the chromosomes and comprised of SMC2 and SMC4; the cohesin, required for holding sister chromosomes together during mitosis and comprised of SMC1 and SMC3; and the SMC5-SMC6 complex, required for various aspects of DNA repair (3).It is generally acknowledged that the eukaryotic condensin and cohesin trap DNA topologically in the protein triangle formed by the SMC proteins and the kleisin (5, 6), although an alternative model for DNA binding for the eukaryotic cohesin exists (7). Recent reports suggest that the Bacillus subtilis SMC protein (8) and MukB (9) also trap chromosomes topologically. 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).We (14) and the Burger and Oakley (15) labs have shown that the MukB hinge region interacts with the C-terminal ␤-propeller region of the ParC subunit of the cellular decatenase topoisomerase IV (Topo IV). We have reported (16) that this interaction stimulates the intramolecular activities of Topo IV, negative supercoil relaxation and knotting, but not the intermolecular activities of Topo IV, catenation/decatenation of DNA rings; whereas Berger, Oakley and colleag...

The MukB–topoisomerase IV interaction is required for proper chromosome compaction

“…Because the tripartite condensin also contains MukEF and MukB is an ATPase, we assessed the effect of MukEF and ATP on maximal DNA compaction. The addition of MukF alone, which does not require MukE to bind to MukB and stimulates its ATPase activity (20), had a slight stabilizing effect on DNA condensation both in the presence and absence of ATP and in the presence and absence of Topo IV (compare lanes 2 and 3, 5 and 6, 8 and 9, and 11 and 12 in Fig. 1E).…”

“…2A, lanes 7-11). To investigate this issue further, we turned to a variant MukB protein, MukB R187E/R189E (Muk-B DNA ) that is defective for binding DNA with a K D value roughly one-quarter that of the wild type (20). MukB DNA was similarly inefficient at DNA condensation, with some FMcx forming at 125 nM MukB compared with 31 nM for the wild type (compare lanes 2 and 8, Fig.…”

The bacterial condensin MukB compacts DNA by sequestering supercoils and stabilizing topologically isolated loops

“…The nicked DNA substrate was prepared as described in Bahng et al (48). Biotinylated, tailed, and nicked DNA substrate was prepared as described in Yeeles and Marians (46) using the oligonucleotide 5Ј-TTTTTTTTTTTTCCCCCTCGAGCCC-CTTTTTTTTTTTTGGGGCTCGAGGGGGTTTTTGTT-TTCCCAGTCACGAC-3Ј to prime synthesis of the complementary strand of M13mp18 viral DNA by bacteriophage T4 DNA polymerase.…”

“…Nitrocellulose filter binding-As described in Bahng et al (48), data shown are from three independent experiments.…”

Protease‐deficient SOS constitutive cells have RecN‐dependent cell division phenotypes

“…Finally, ATP hydrolysis ‘releases’ the structures. Many SMCs interact with other proteins to stabilize the DNA‐globular domain structures (e.g., MukEF with MukB [Badrinarayanan et al , ; Bahng et al , ]). RecN seems to differ from other SMCs in that no accessory proteins have yet been suggested for RecN.…”