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1.
Seychelle M Vos Nichole K Stewart Martha G Oakley James M Berger 《The EMBO journal》2013,32(22):2950-2962
Chromosome partitioning in Escherichia coli is assisted by two interacting proteins, topoisomerase (topo) IV and MukB. MukB stimulates the relaxation of negative supercoils by topo IV; to understand the mechanism of their action and to define this functional interplay, we determined the crystal structure of a minimal MukB–topo IV complex to 2.3 Å resolution. The structure shows that the so‐called ‘hinge’ region of MukB forms a heterotetrameric assembly with a C‐terminal DNA binding domain (CTD) on topo IV's ParC subunit. Biochemical studies show that the hinge stimulates topo IV by competing for a site on the CTD that normally represses activity on negatively supercoiled DNA, while complementation tests using mutants implicated in the interaction reveal that the cellular dependency on topo IV derives from a joint need for both strand passage and MukB binding. Interestingly, the configuration of the MukB·topo IV complex sterically disfavours intradimeric interactions, indicating that the proteins may form oligomeric arrays with one another, and suggesting a framework by which MukB and topo IV may collaborate during daughter chromosome disentanglement. 相似文献
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Movement of the DNA replication machinery through the double helix induces acute positive supercoiling ahead of the fork and precatenanes behind it. Because topoisomerase I and II create transient single- and double-stranded DNA breaks, respectively, it has been assumed that type I enzymes relax the positive supercoils that precede the replication fork. Conversely, type II enzymes primarily resolve the precatenanes and untangle catenated daughter chromosomes. However, studies on yeast and bacteria suggest that type II topoisomerases may also function ahead of the replication machinery. If this is the case, then positive DNA supercoils should be the preferred relaxation substrate for topoisomerase IIalpha, the enzyme isoform involved in replicative processes in humans. Results indicate that human topoisomerase IIalpha relaxes positively supercoiled plasmids >10-fold faster than negatively supercoiled molecules. In contrast, topoisomerase IIbeta, which is not required for DNA replication, displays no such preference. In addition to its high rates of relaxation, topoisomerase IIalpha maintains lower levels of DNA cleavage complexes with positively supercoiled molecules. These properties suggest that human topoisomerase IIalpha has the potential to alleviate torsional stress ahead of replication forks in an efficient and safe manner. 相似文献
4.
We have studied the stimulation of topoisomerase IV (Topo IV) by the C-terminal AAA+ domain of FtsK. These two proteins combine to assure proper chromosome segregation in the cell. Stimulation of Topo IV activity was dependent on the chirality of the DNA substrate: FtsK stimulated decatenation of catenated DNA and relaxation of positively supercoiled [(+)ve sc] DNA, but inhibited relaxation of negatively supercoiled [(−)ve sc] DNA. The DNA translocation activity of FtsK was not required for stimulation, but was required for inhibition. DNA chirality did not affect any of the activities of FtsK, suggesting that FtsK possesses an inherent Topo IV stimulatory activity that is presumably mediated by protein–protein interactions, the stability of Topo IV on the DNA substrate dictated the effect observed. Inhibition occurs because FtsK can strip distributively acting topoisomerase off (−)ve scDNA, but not from either (+)ve scDNA or catenated DNA where the enzyme acts processively. Our analyses suggest that FtsK increases the efficiency of trapping of the transfer segment of DNA during the catalytic cycle of the topoisomerase. 相似文献
5.
Nucleoid-associated proteins play an important role in condensing chromosomal DNA and regulating gene expression. We report here the characterization of the nucleoid-associated protein YejK, which was detected in a yeast two-hybrid screen using the ParE subunit of topoisomerase IV as bait. The purified protein likely exists in a monomer-dimer equilibrium in solution and can form tetramers. Cross-linking of the protein bound to DNA suggests that the active form could be either a dimer or tetramer. YejK, which is present at about 24,000 copies of monomer per mid-log phase cell, binds double-stranded DNA with a site size of 12–14 base pairs/monomer, does not display a significant preference for either bent compared with straight DNA or supercoiled compared with relaxed DNA, and untwists DNA somewhat as it binds. YejK binds RNA, but not single-stranded DNA, with 65% of the avidity with which it binds DNA. However, cells deleted for yejK do not show defects in either RNA or protein synthesis. YejK interacts with all the subunits of both DNA gyrase and topoisomerase IV and has measurable effects on their activities. In the presence of YejK, relaxation of negatively supercoiled DNA by topoisomerase IV becomes distributive, whereas relaxation of positively supercoiled DNA is stimulated. Relaxation of negatively supercoiled DNA by DNA gyrase is inhibited, whereas the extent of supercoiling of relaxed DNA is limited. A YejK-GFP chimera is an effective marker for the nucleoid in live cell imaging. 相似文献
6.
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. 相似文献
7.
Escherichia coli topoisomerase IV (topo IV) is an essential enzyme that unlinks the daughter chromosomes for proper segregation at cell division. In vitro, topo IV readily distinguishes between the two possible chiralities of crossing segments in a DNA substrate. The enzyme relaxes positive supercoils and left-handed braids 20 times faster, and with greater processivity, than negative supercoils and right-handed braids. Here, we used chemical cross-linking of topo IV to demonstrate that enzyme bound to positively supercoiled DNA is in a different conformation from that bound to other forms of DNA. Using three different reagents, we observed novel cross-linked species of topo IV when positively supercoiled DNA was in the reaction. We show that the ParE subunits are in close enough proximity to be cross-linked only when the enzyme is bound to positively supercoiled DNA. We suggest that the altered conformation reflects efficient binding by topo IV of the two DNA segments that participate in the strand passage reaction. 相似文献
8.
High mobility group box (HMGB) proteins 1 and 2 are abundant non-histone nuclear proteins that regulate chromatin structure
because of their structure-specific binding to DNA. Here, we have investigated how the post-synthetic acetylation of HMGB1
affects its interaction with negatively supercoiled DNA by employing monoacetylated at Lys2 protein, isolated from butyrate-treated
cells. Our data reveal that this modification enhances three reaction parameters: binding affinity, supercoiling activity
and capacity to protect the supercoiled DNA from relaxation by topoisomerase I. We show that monoacetylation at Lys2 mimics
the effect of acidic tail removal but to a lesser extent thus demonstrating that in vivo acetylated HMGB1 is capable of modulating
its interaction with negatively supercoiled DNA. 相似文献
9.
Topoisomerases are essential ubiquitous enzymes, falling into two distinct classes. A number of eubacteria including Escherichia coli, typically contain four topoisomerases, two type I topoisomerases and two type II topoisomerases viz. DNA gyrase and topoisomerase IV. In contrast several other bacterial genomes including mycobacteria, encode for one type I topoisomerase and a DNA gyrase. Here we describe a new type II topoisomerase from Mycobacterium smegmatis which is different from DNA gyrase or topoisomerase IV in its characteristics and origin. The topoisomerase is distinct with respect to domain organization, properties and drug sensitivity. The enzyme catalyses relaxation of negatively supercoiled DNA in an ATP-dependent manner and also introduces positive supercoils to both relaxed and negatively supercoiled substrates. The genes for this additional topoisomerase are not found in other sequenced mycobacterial genomes and may represent a distant lineage. 相似文献
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Previous studies with human and bacterial topoisomerases suggest that the type II enzyme utilizes two distinct mechanisms to recognize the handedness of DNA supercoils. It has been proposed that the ability of some type II enzymes, such as human topoisomerase IIalpha and Escherichia coli topoisomerase IV, to distinguish supercoil geometry during DNA relaxation is mediated by elements in the variable C-terminal domain of the protein. In contrast, the ability of human topoisomerase IIalpha and topoisomerase IIbeta to discern the handedness of supercoils during DNA cleavage suggests that residues in the conserved N-terminal or central domain of the protein are involved in this process. To test this hypothesis, the ability of Paramecium bursaria chlorella virus-1 (PBCV-1) and chlorella virus Marburg-1 (CVM-1) topoisomerase II to relax and cleave negatively and positively supercoiled plasmids was assessed. These enzymes display a high degree of sequence identity with the N-terminal and central domains of eukaryotic topoisomerase II but naturally lack the C-terminal domain. While PBCV-1 and CVM-1 topoisomerase II relaxed under- and overwound substrates at similar rates, they were able to discern the handedness of supercoils during the cleavage reaction and preferentially cut negatively supercoiled DNA. Preferential cleavage was not due to a change in site specificity, DNA binding, or religation. These findings are consistent with a bimodal recognition of DNA geometry in which topoisomerase II uses elements in the C-terminal domain to sense the handedness of supercoils during DNA relaxation and elements in the conserved N-terminal or central domain during DNA cleavage. 相似文献
12.
MukB is a bacterial SMC (structural maintenance of chromosome) protein required for faithful chromosome segregation in Escherichia coli. We report here that purified MukB introduces right-handed knots into DNA in the presence of type-2 topoisomerase, indicating that the protein promotes intramolecular DNA condensation. The pattern of generated knots suggests that MukB, similar to eukaryotic condensins, stabilizes large right-handed DNA loops. In contrast to eukaryotic condensins, however, the net supercoiling stabilized by MukB was negative. Furthermore, DNA reshaping by MukB did not require ATP. These data establish that bacterial condensins alter the shape of double-stranded DNA in vitro and lend support to the notions that the right-handed knotting is the most conserved biochemical property of condensins. Finally, we found that MukB can be eluted from a heparin column in two distinct forms, one of which is inert in DNA binding or reshaping. Furthermore, we find that the activity of MukB is reversibly attenuated during chromatographic separation. Thus, MukB has a unique set of topological properties, compared with other SMC proteins, and is likely to exist in two different conformations. 相似文献
13.
Graumann PL 《Biochimie》2001,83(1):53-59
SMC proteins are a ubiquitous protein family, present in almost all organisms so far analysed except for a few bacteria. They function in chromosome condensation, segregation, cohesion, and DNA recombination repair in eukaryotes, and can introduce positive writhe into DNA in vitro. SMC proteins and the structurally homologous MukB protein are unusual ATPases that form antiparallel dimers, with long coiled coil segments separating globular ends capable of binding DNA. Recently, SMC proteins have been shown to be essential for chromosome condensation, segregation and cell cycle progression in bacteria. Identification of a suppressor mutation for MukB in topoisomerase I in Escherichia coli suggests that SMC proteins are involved in negative DNA supercoiling in vivo, and by this means organize and compact chromosomes. A model is discussed in which bacterial SMC proteins act after an initial separation of replicated chromosome origins into the future daughter cell, separating sister chromatids by condensing replicated DNA strands within both cell halves. This would be analogous to a pulling of DNA strands into opposite cell halves by a condensation mechanism exerted at two specialised subregions in the cell. 相似文献
14.
DNA topoisomerase is involved in DNA repair and replication. In this study, a novel ATP-independent 30-kDa type I DNA topoisomerase was purified and characterized from a marine methylotroph, Methylophaga sp. strain 3. The purified enzyme composed of a single polypeptide was active over a broad range of temperature and pH. The enzyme was able to relax only negatively supercoiled DNA. Mg(2+) was required for its relaxation activity, while ATP gave no effect. The enzyme was clearly inhibited by camptothecin, ethidium bromide, and single-stranded DNA, but not by nalidixic acid and etoposide. Interestingly, the purified enzyme showed Mn(2+)-activated endonuclease activity on supercoiled DNA. The N-terminal sequence of the purified enzyme showed no homology with those of other type I enzymes. These results suggest that the purified enzyme is an ATP-independent type I DNA topoisomerase that has, for the first time, been characterized from a marine methylotroph. 相似文献
15.
In order to study the double-strand DNA passage reaction of eukaryotic type II topoisomerases, a quantitative assay to monitor the enzymic conversion of supercoiled circular DNA to relaxed circular DNA was developed. Under conditions of maximal activity, relaxation catalyzed by the Drosophila melanogaster topoisomerase II was processive and the energy of activation was 14.3 kcal . mol-1. Removal of supercoils was accompanied by the hydrolysis of either ATP or dATP to inorganic phosphate and the corresponding nucleoside diphosphate. Apparent Km values were 200 microM for pBR322 plasmid DNA, 140 microM for SV40 viral DNA, 280 microM for ATP, and 630 microM for dATP. The turnover number for the Drosophila enzyme was at least 200 supercoils of DNA relaxed/min/molecule of topoisomerase II. The enzyme interacts preferentially with negatively supercoiled DNA over relaxed molecules, is capable of removing positive superhelical twists, and was found to be strongly inhibited by single-stranded DNA. Kinetic and inhibition studies indicated that the beta and gamma phosphate groups, the 2'-OH of the ribose sugar, and the C6-NH2 of the adenine ring are important for the interaction of ATP with the enzyme. While the binding of ATP to Drosophila topoisomerase II was sufficient to induce a DNA strand passage event, hydrolysis was required for enzyme turnover. The ATPase activity of the topoisomerase was stimulated 17-fold by the presence of negatively supercoiled DNA and approximately 4 molecules of ATP were hydrolyzed/supercoil removed. Finally, a kinetic model describing the switch from a processive to a distributive relaxation reaction is presented. 相似文献
16.
Tzu-Wen Huang Chin-Chen Hsu Han-Yu Yang Carton W. Chen 《Nucleic acids research》2013,41(22):10403-10413
Filamentous bacteria of the genus Streptomyces possess linear chromosomes and linear plasmids. Theoretically, linear replicons may not need a decatenase for post-replicational separation of daughter molecules. Yet, Streptomyces contain parC and parE that encode the subunits for the decatenase topoisomerase IV. The linear replicons of Streptomyces adopt a circular configuration in vivo through telomere–telomere interaction, which would require decatenation, if the circular configuration persists through replication. We investigated whether topoisomerase IV is required for separation of the linear replicons in Streptomyces. Deletion of parE from the Streptomyces coelicolor chromosome was achieved, when parE was provided on a plasmid. Subsequently, the plasmid was eliminated at high temperature, and ΔparE mutants were obtained. These results indicated that topoisomerase IV was not essential for Streptomyces. Presumably, the telomere–telomere association may be resolved during or after replication to separate the daughter chromosomes. Nevertheless, the mutants exhibited retarded growth, defective sporulation and temperature sensitivity. In the mutants, circular plasmids could not replicate, and spontaneous circularization of the chromosome was not observed, indicating that topoisomerase IV was required for decatenation of circular replicons. Moreover, site-specific integration of a plasmid is impaired in the mutants, suggesting the formation of DNA knots during integration, which must be resolved by topoisomerase IV. 相似文献
17.
Structural maintenance of chromosomes protein of Bacillus subtilis affects supercoiling in vivo
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Structural maintenance of chromosomes (SMC) proteins are found in nearly all organisms. Members of this protein family are involved in chromosome condensation and sister chromatid cohesion. Bacillus subtilis SMC protein (BsSMC) plays a role in chromosome organization and partitioning. To better understand the function of BsSMC, we studied the effects of an smc null mutation on DNA supercoiling in vivo. We found that an smc null mutant was hypersensitive to the DNA gyrase inhibitors coumermycin A1 and norfloxacin. Furthermore, depleting cells of topoisomerase I substantially suppressed the partitioning defect of an smc null mutant. Plasmid DNA isolated from an smc null mutant was more negatively supercoiled than that from wild-type cells. In vivo cross-linking experiments indicated that BsSMC was bound to the plasmid. Our results indicate that BsSMC affects supercoiling in vivo, most likely by constraining positive supercoils, an activity which contributes to chromosome compaction and organization. 相似文献
18.
The subunits of topoisomerase IV (topo IV), the ParC and ParE proteins in Escherichia coli, were purified to near homogeneity from the respective overproducers. They revealed type II topoisomerase activity only when they were combined with each other. In the presence of Mg2+ and ATP, topo IV was capable of relaxing a negatively or positively supercoiled plasmid DNA or converting the knotted P4 phage DNA, whether nicked or ligated, to a simple ring. However, supercoiling activity was not detected. The topoisomerase activity was not detectable when the purified ParC and ParE proteins were combined with the purified GyrB and GyrA proteins, respectively. This is consistent with the result that neither a parC nor a parE mutation was compensated by transformation with a plasmid carrying either the gyrA or the gyrB gene. Simultaneous introduction of both the gyrA and gyrB plasmids corrected the phenotypic defect of parC and parE mutants. The results suggest that DNA gyrase can substitute for topo IV at least in some part of the function for chromosome partitioning. Antisera were prepared against the purified ParC, ParE, GyrA, and GyrB proteins and used to investigate cellular localization of these gene products. ParC protein was found to be specifically associated with inner membranes only in the presence of DNA. This result suggests that one of the functions of topo IV might be to anchor chromosomes on membranes as previously proposed for eukaryotic topoisomerase II. 相似文献
19.
SMC (structural maintenance of chromosomes) complexes share conserved architectures and function in chromosome maintenance via an unknown mechanism. Here we have used single-molecule techniques to study MukBEF, the SMC complex in Escherichia coli. Real-time movies show MukB alone can compact DNA and ATP inhibits DNA compaction by MukB. We observed that DNA unidirectionally slides through MukB, potentially by a ratchet mechanism, and the sliding speed depends on the elastic energy stored in the DNA. MukE, MukF and ATP binding stabilize MukB and DNA interaction, and ATP hydrolysis regulates the loading/unloading of MukBEF from DNA. Our data suggests a new model for how MukBEF organizes the bacterial chromosome in vivo; and this model will be relevant for other SMC proteins. 相似文献
20.
S J Mazur K Sakaguchi E Appella X W Wang C C Harris V A Bohr 《Journal of molecular biology》1999,292(2):241-249
The C-terminal domain of the tumor suppressor protein p53 is the site of non-specific DNA binding. Purified p53 produced from baculovirus-infected insect cells binds preferentially to supercoiled DNA, forming bands with lower electrophoretic mobility. This non-covalent binding does not change the linking number of the DNA. An anti-p53 antibody targeting the C-terminal domain partially competes with supercoiled DNA in binding to p53, while antibodies targeted to the N terminus of p53 supershift the complex bands. A synthetic peptide corresponding to amino acid residues 319-393 of human p53 also displays preferential binding to supercoiled DNA, while a mutant peptide, which is unable to form tetramers, is inactive. The center of the equilibrium distribution of topoisomers formed by relaxation with topoisomerase I is not shifted in the presence of p53 although the distribution is broadened. The preferential binding of p53 is exhibited toward both positively and negatively supercoiled DNA. These observations are consistent with a model in which p53 binds to right-handed or left-handed strand crossings. 相似文献