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1.
J Biol Chem ; 298(6): 101964, 2022 06.
Article in English | MEDLINE | ID: mdl-35452680

ABSTRACT

MukBEF, a structural maintenance of chromosome-like protein complex consisting of an ATPase, MukB, and two interacting subunits, MukE and MukF, functions as the bacterial condensin. It is likely that MukBEF compacts DNA via an ATP hydrolysis-dependent DNA loop-extrusion reaction similar to that demonstrated for the yeast structural maintenance of chromosome proteins condensin and cohesin. MukB also interacts with the ParC subunit of the cellular chromosomal decatenase topoisomerase IV, an interaction that is required for proper chromosome condensation and segregation in Escherichia coli, although it suppresses the MukB ATPase activity. Other structural determinants and interactions that regulate the ATPase activity of MukBEF are not clear. Here, we have investigated the MukBEF ATPase activity, identifying intersubunit and intrasubunit interactions by protein-protein crosslinking and site-specific mutagenesis. We show that interactions between the hinge of MukB and its neck region are essential for the ATPase activity, that the ParC subunit of topoisomerase IV inhibits the MukB ATPase by preventing this interaction, that MukE interaction with DNA is likely essential for viability, and that interactions between MukF and the MukB neck region are necessary for ATPase activity and viability.


Subject(s)
Chromosomal Proteins, Non-Histone , Escherichia coli Proteins , Repressor Proteins , Adenosine Triphosphatases/metabolism , Chromosomal Proteins, Non-Histone/metabolism , Chromosomes, Bacterial/metabolism , DNA Topoisomerase IV/genetics , Escherichia coli/metabolism , Escherichia coli Proteins/metabolism , Repressor Proteins/metabolism
2.
Nucleic Acids Res ; 50(5): 2621-2634, 2022 03 21.
Article in English | MEDLINE | ID: mdl-34747485

ABSTRACT

The bacterial condensin MukB and the cellular chromosomal decatenase, topoisomerase IV interact and this interaction is required for proper condensation and topological ordering of the chromosome. Here, we show that Topo IV stimulates MukB DNA condensation by stabilizing loops in DNA: MukB alone can condense nicked plasmid DNA into a protein-DNA complex that has greater electrophoretic mobility than that of the DNA alone, but both MukB and Topo IV are required for a similar condensation of a linear DNA representing long stretches of the chromosome. Remarkably, we show that rather than MukB stimulating the decatenase activity of Topo IV, as has been argued previously, in stoichiometric complexes of the two enzymes each inhibits the activity of the other: the ParC subunit of Topo IV inhibits the MukF-stimulated ATPase activity of MukB and MukB inhibits both DNA crossover trapping and DNA cleavage by Topo IV. These observations suggest that when in complex on the DNA, Topo IV inhibits the motor function of MukB and the two proteins provide a stable scaffold for chromosomal DNA condensation.


Subject(s)
DNA Topoisomerase IV , Escherichia coli Proteins , Chromosomal Proteins, Non-Histone/metabolism , DNA/metabolism , DNA Topoisomerase IV/metabolism , Escherichia coli/genetics , Escherichia coli Proteins/metabolism
3.
Methods Mol Biol ; 2004: 169-180, 2019.
Article in English | MEDLINE | ID: mdl-31147917

ABSTRACT

Condensins in bacteria are one of the most important factors involved in the organization of long threads of DNA into compact chromosomes. The organization of DNA by condensins is vital to many DNA transactions including DNA repair and chromosome segregation. Although some of the activities of condensins are well studied, the mechanism of the overall process executed by condensins, DNA compaction, remains unclear. Here, we describe some of the methods used routinely in our laboratory to understand the mechanism of DNA compaction by Escherichia coli condensin MukB.


Subject(s)
Adenosine Triphosphatases/genetics , Chromosomal Proteins, Non-Histone/genetics , DNA, Bacterial/genetics , DNA-Binding Proteins/genetics , Escherichia coli Proteins/genetics , Multiprotein Complexes/genetics , Chromosome Segregation/genetics , Chromosomes, Bacterial/genetics , Escherichia coli/genetics
4.
J Biol Chem ; 292(41): 16921-16932, 2017 10 13.
Article in English | MEDLINE | ID: mdl-28842485

ABSTRACT

The bacterial condensin MukB and the cellular decatenating enzyme topoisomerase IV interact. This interaction stimulates intramolecular reactions catalyzed by topoisomerase IV, supercoiled DNA relaxation, and DNA knotting but not intermolecular reactions such as decatenation of linked DNAs. We have demonstrated previously that MukB condenses DNA by sequestering negative supercoils and stabilizing topologically isolated loops in the DNA. We show here that the MukB-topoisomerase IV interaction stabilizes MukB on DNA, increasing the extent of DNA condensation without increasing the amount of MukB bound to the DNA. This effect does not require the catalytic activity of topoisomerase IV. Cells carrying a mukB mutant allele that encodes a protein that does not interact with topoisomerase IV exhibit severe nucleoid decompaction leading to chromosome segregation defects. These findings suggest that the MukB-topoisomerase IV complex may provide a scaffold for DNA condensation.


Subject(s)
Chromosomal Proteins, Non-Histone/chemistry , Chromosomes, Bacterial/chemistry , DNA Topoisomerase IV/chemistry , DNA, Bacterial/chemistry , DNA, Superhelical/chemistry , Escherichia coli Proteins/chemistry , Escherichia coli/chemistry , Multiprotein Complexes/chemistry , Chromosomal Proteins, Non-Histone/genetics , Chromosomal Proteins, Non-Histone/metabolism , Chromosomes, Bacterial/genetics , Chromosomes, Bacterial/metabolism , DNA Topoisomerase IV/genetics , DNA Topoisomerase IV/metabolism , DNA, Bacterial/genetics , DNA, Bacterial/metabolism , DNA, Superhelical/genetics , DNA, Superhelical/metabolism , Escherichia coli/genetics , Escherichia coli/metabolism , Escherichia coli Proteins/genetics , Escherichia coli Proteins/metabolism , Multiprotein Complexes/genetics , Multiprotein Complexes/metabolism , Mutation
5.
J Biol Chem ; 292(41): 16904-16920, 2017 10 13.
Article in English | MEDLINE | ID: mdl-28842486

ABSTRACT

MukB is a structural maintenance of chromosome-like protein required for DNA condensation. The complete condensin is a large tripartite complex of MukB, the kleisin, MukF, and an accessory protein, MukE. As found previously, MukB DNA condensation is a stepwise process. We have defined these steps topologically. They proceed first via the formation of negative supercoils that are sequestered by the protein followed by hinge-hinge interactions between MukB dimers that stabilize topologically isolated loops in the DNA. MukB itself is sufficient to mediate both of these topological alterations; neither ATP nor MukEF is required. We show that the MukB hinge region binds DNA and that this region of the protein is involved in sequestration of supercoils. Cells carrying mutations in the MukB hinge that reduce DNA condensation in vitro exhibit nucleoid decondensation in vivo.


Subject(s)
Chromosomal Proteins, Non-Histone/chemistry , DNA, Bacterial/chemistry , DNA, Superhelical/chemistry , Escherichia coli Proteins/chemistry , Escherichia coli/chemistry , Protein Multimerization , Chromosomal Proteins, Non-Histone/genetics , Chromosomal Proteins, Non-Histone/metabolism , DNA, Bacterial/genetics , DNA, Bacterial/metabolism , DNA, Superhelical/genetics , DNA, Superhelical/metabolism , Escherichia coli/genetics , Escherichia coli/metabolism , Escherichia coli Proteins/genetics , Escherichia coli Proteins/metabolism , Mutation , Repressor Proteins/chemistry , Repressor Proteins/genetics , Repressor Proteins/metabolism
6.
J Biol Chem ; 291(46): 23999-24008, 2016 Nov 11.
Article in English | MEDLINE | ID: mdl-27697840

ABSTRACT

Properly 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.


Subject(s)
Chromosomal Proteins, Non-Histone/metabolism , DNA, Bacterial/metabolism , DNA, Catenated/metabolism , Escherichia coli Proteins/metabolism , Escherichia coli/metabolism , Repressor Proteins/metabolism , Adenosine Triphosphate/chemistry , Adenosine Triphosphate/genetics , Adenosine Triphosphate/metabolism , Chromosomal Proteins, Non-Histone/chemistry , Chromosomal Proteins, Non-Histone/genetics , DNA, Bacterial/chemistry , DNA, Bacterial/genetics , DNA, Catenated/chemistry , DNA, Catenated/genetics , Escherichia coli/chemistry , Escherichia coli/genetics , Escherichia coli Proteins/chemistry , Escherichia coli Proteins/genetics , Repressor Proteins/chemistry , Repressor Proteins/genetics
7.
J Biol Chem ; 288(11): 7653-7661, 2013 Mar 15.
Article in English | MEDLINE | ID: mdl-23349462

ABSTRACT

Proper chromosome organization is accomplished through binding of proteins such as condensins that shape the DNA and by modulation of chromosome topology by the action of topoisomerases. We found that the interaction between MukB, the bacterial condensin, and ParC, a subunit of topoisomerase IV, enhanced relaxation of negatively supercoiled DNA and knotting by topoisomerase IV, which are intramolecular DNA rearrangements but not decatenation of multiply linked DNA dimers, which is an intermolecular DNA rearrangement required for proper segregation of daughter chromosomes. MukB DNA binding and a specific chiral arrangement of the DNA was required for topoisomerase IV stimulation because relaxation of positively supercoiled DNA was unaffected. This effect could be attributed to a more effective topological reconfiguration of the negatively supercoiled compared with positively supercoiled DNA by MukB. These data suggest that the MukB-ParC interaction may play a role in chromosome organization rather than in separation of daughter chromosomes.


Subject(s)
Chromosomal Proteins, Non-Histone/metabolism , DNA Topoisomerase IV/metabolism , Escherichia coli Proteins/metabolism , Escherichia coli/enzymology , Gene Expression Regulation, Bacterial , Adenosine Triphosphatases/chemistry , Adenosine Triphosphatases/metabolism , Catalysis , Chromosomes/ultrastructure , DNA/chemistry , DNA, Superhelical/metabolism , DNA-Binding Proteins/chemistry , DNA-Binding Proteins/metabolism , Dimerization , Escherichia coli/genetics , Multiprotein Complexes/chemistry , Multiprotein Complexes/metabolism , Mutation , Nucleic Acid Conformation , Plasmids/metabolism , Protein Binding
8.
EMBO J ; 30(20): 4236-47, 2011 Aug 19.
Article in English | MEDLINE | ID: mdl-21857649

ABSTRACT

Interactions between single-stranded DNA-binding proteins (SSBs) and the DNA replication machinery are found in all organisms, but the roles of these contacts remain poorly defined. In Escherichia coli, SSB's association with the χ subunit of the DNA polymerase III holoenzyme has been proposed to confer stability to the replisome and to aid delivery of primers to the lagging-strand DNA polymerase. Here, the SSB-binding site on χ is identified crystallographically and biochemical and cellular studies delineate the consequences of destabilizing the χ/SSB interface. An essential role for the χ/SSB interaction in lagging-strand primer utilization is not supported. However, sequence changes in χ that block complex formation with SSB lead to salt-dependent uncoupling of leading- and lagging-strand DNA synthesis and to a surprising obstruction of the leading-strand DNA polymerase in vitro, pointing to roles for the χ/SSB complex in replisome establishment and maintenance. Destabilization of the χ/SSB complex in vivo produces cells with temperature-dependent cell cycle defects that appear to arise from replisome instability.


Subject(s)
Bacterial Proteins/chemistry , DNA Polymerase III/chemistry , DNA Replication , DNA, Single-Stranded/chemistry , Escherichia coli Proteins/chemistry , Escherichia coli/enzymology , Amino Acid Sequence , Bacterial Proteins/metabolism , Base Sequence , DNA Polymerase III/metabolism , DNA, Single-Stranded/metabolism , DNA-Binding Proteins/chemistry , DNA-Binding Proteins/metabolism , Escherichia coli Proteins/metabolism , Holoenzymes/chemistry , Holoenzymes/metabolism , Molecular Sequence Data
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