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EC Tree
IUBMB Comments The enzyme can introduce negative superhelical turns into double-stranded circular DNA. One unit has nicking-closing activity, and another catalyses super-twisting and hydrolysis of ATP (cf. EC 5.6.2.1 DNA topoisomerase).
The taxonomic range for the selected organisms is: Saccharolobus shibatae The enzyme appears in selected viruses and cellular organisms
Reaction Schemes
ATP-dependent breakage, passage and rejoining of double-stranded DNA
Synonyms
atpase, topoisomerase ii, dna gyrase, topo ii, gyrase, top2a, dna topoisomerase ii, topoisomerase iialpha, topo iialpha, topoisomerase ii alpha,
more
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DNA topoisomerase II
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DNA topoisomerase type II
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Isomerase, deoxyribonucleate topo-, II
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Nuclear proteins 170,000-mol.wt.
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Proteins , NP170 (specific proteins and subclasses nuclear protein, 170,000-mol.-wt.)
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Topoisomerase type II
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Type II-DNA-topoisomerase
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DNA topoisomerase VI
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topo VI
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DNA topoisomerase (ATP-hydrolysing)
The enzyme can introduce negative superhelical turns into double-stranded circular DNA. One unit has nicking-closing activity, and another catalyses super-twisting and hydrolysis of ATP (cf. EC 5.6.2.1 DNA topoisomerase).
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80449-01-0
formerly not distinguished from EC 5.99.1.2
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catenated DNA + ATP + H2O
minicircular DNA + ADP + phosphate
decatenation
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DNA + ATP
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DNA topoisomerase VI relaxes both negatively and positively supercoiled DNA in the presence of ATP and has no DNA supercoiling activity
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double-stranded DNA + ATP + H2O
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the enzyme generates ATP-dependent double-strand breaks with two-nucleotide overhangs on supercoiled or linear DNA. topoVI is covalently attached to the 5'-ends of the broken DNA
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linear DNA fragments of viral SSV1 DNA + ATP + H2O
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ATP hydrolysis is necessary for DNA resealing
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network of DNA rings + ATP + H2O
monomeric DNA circles + ADP + phosphate
relaxed pBR322 plasmid + ATP + H2O
supercoiled pBR322 plasmid + ADP + phosphate
supercoiling
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supercoiled DNA + ATP + H2O
catenated DNA networks + ADP + phosphate
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catenation
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supercoiled DNA + ATP + H2O
relaxed DNA + ADP + phosphate
supercoiled pBR322 plasmid + ATP + H2O
relaxed pBR322 plasmid + ADP + phosphate
relaxation
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additional information
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changes the linking number of a closed circular molecule by an increment of two
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DNA + ATP + H2O
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the enzyme helps disentangle chromosomes to facilitate cell division
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DNA + ATP + H2O
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the enzyme catalyzes an ATP-dependent positive DNA supercoiling reaction in vitro
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DNA + ATP + H2O
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the enzyme helps disentangle chromosomes to facilitate cell division. ATP binding elicits a major structural reorganization that is propagated to the DNA-cleavage center of the enzyme, explaining how ATP is coupled to DNA capture and strand scission
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network of DNA rings + ATP + H2O
monomeric DNA circles + ADP + phosphate
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decatenation
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network of DNA rings + ATP + H2O
monomeric DNA circles + ADP + phosphate
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enzyme is more efficient in decatenation than relaxation
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supercoiled DNA + ATP + H2O
relaxed DNA + ADP + phosphate
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relaxation
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supercoiled DNA + ATP + H2O
relaxed DNA + ADP + phosphate
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ATP-dependent relaxation of negative and positive supercoils
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supercoiled DNA + ATP + H2O
relaxed DNA + ADP + phosphate
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DNA cleavage by topo VI generates two-nucleotide 5'-protruding ends
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DNA + ATP + H2O
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the enzyme helps disentangle chromosomes to facilitate cell division
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double-stranded DNA + ATP + H2O
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the enzyme generates ATP-dependent double-strand breaks with two-nucleotide overhangs on supercoiled or linear DNA. topoVI is covalently attached to the 5'-ends of the broken DNA
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ATP
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topo VI strictly requires ATP for DNA cleavage
ATP
the formation of the double-strand breaks on supercoiled or linear DNA is strictly dependent on the presence of ATP or adenosine 5'-(beta,gamma-imino)triphosphate. ATP binding is required to stabilize an enzyme conformation able to cleave the DNA backbone
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Mg2+
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required
Mg2+
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Mg2+ is effective in the concentration range 1-40 mM, optimal concentration is around 30 mM
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(1S,3S)-3-acetyl-3,5,12-trihydroxy-10-methoxy-6,11-dioxo-1,2,3,4,6,11-hexahydrotetracen-1-yl 3-amino-2,3,6-trideoxy-alpha-L-lyxo-hexopyranoside
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4'-(9-acridinylamino)methansulfon-m-anisidide
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4'-(9-acridinylamino)methansulfon-o-anisidide
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coumermycin
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no inhibition
novobiocin
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no inhibition
quinolones
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e.g. CP-115,953
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radicicol
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radicicol
inhibits the decatenation and relaxation activities
additional information
geldanamycin does not inhibit the decatenation and relaxation activities
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additional information
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geldanamycin does not inhibit the decatenation and relaxation activities
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single-strand binding-protein
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from the archaeon Sulfolobus solfataricus. Stimulates all the steps of the reverse gyrase reaction: binding to DNA, DNA cleavage, strand passage and ligation. It also stimulates reverse gyrase positive supercoiling activity on DNA templates associated with the chromatin protein Sul7d
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additional information
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7.5
assay at
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50 - 80
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50°C: no activity, 70-80°C: maximal activity
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SwissProt
brenda
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physiological function
the enzyme helps disentangle chromosomes to facilitate cell division
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RGYR_SACSH
1166
0
132080
Swiss-Prot
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210000
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derived from amino acid sequence
230000
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gel filtration, sucrose density gradient centrifugation
45055
2 * 45055 (subunit A) + 2 * 60528 (subunit B), calculated from sequence
60528
2 * 45055 (subunit A) + 2 * 60528 (subunit B), calculated from sequence
47000
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2 * 47000 + 2 * 60000, SDS-PAGE
47000
2 * 47000 + 2 * 60000
60000
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2 * 47000 + 2 * 60000, SDS-PAGE
60000
2 * 47000 + 2 * 60000
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heterotetramer
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x-ray crystallography
heterotetramer
2 * 45055 (subunit A) + 2 * 60528 (subunit B), calculated from sequence
tetramer
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2 * 47000 + 2 * 60000, SDS-PAGE
tetramer
2 * 47000 + 2 * 60000
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hanging drop vapour diffusion method, in complex with the radicicol inhibitor
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structure of the ATP-binding subunit of topoisomerase VI in two states: an unliganded monomer and a nucleotide-bound dimer
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S424C
cysteine substitution mutant capable of forming an intersubunit disulfide bond upon ATP-induced dimerization. The mutant protein has a smaller radius of gyration (Rg) than the apoprotein.
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65
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pH 7, enzyme concentration 90 ng/ml, less than 15% loss of activity after 40 min
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pH 7, enzyme concentration 90 ng/ml, half-life: about 35 min. At an enzyme concentration of 900 ng/ml less than 20% loss of activity after 40 min
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pH 7, enzyme concentration 90 ng/ml, more than 90% inactivation after 5 min
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4°C, 50 mM potassium phosphate buffer, 1 mM EDTA, 1 mM DTT, 0.0005% Triton X-100
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Ni2+ affinity column chromatography and heparin column chromatography
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purification of the two recombinant subunits of Sulfolobus shibatae Topo IV from Escherichia coli
purification procedure of a fully soluble recombinant topoVI is developed by expressing both subunits simultaneously in Escherichia coli
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expression in Escherichia coli
gene structure, transcription unit and comparative sequence analysis with two other known reverse gyrases
overexpression in Escherichia coli. The A subunit exhibits clusters of arginines encoded by rare codons in Escherichia coli. The expression of this protein requires the coexpression of the minor Escherichia coli arginyl tRNA which reads AGG and AGA codons. The A subunit expressed in Escherichia coli is obtained from inclusion bodies after denaturation and renaturation. The B subunit is overexpressed in Escherichia coli and purified in soluble form. When purified B subunit is added to the renatured A subunit, ATP-dependent relaxation and decatenation activities of the hyperthermophilic DNA topoisomerase are reconstituted. The reconstituted recombinant enzyme exhibits a specific activity similar to the enzyme purified from Sulfolobus shibatae. It catalyzes transient double-strain cleavage of DNA and becomes covalently attached to the ends of the cleaved DNA
overexpression of both subunits in Escherichia coli
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Bergerat, A.; Gadelle, D.; Forterre, P.
Purification of a DNA topoisomerase II from the hyperthermophilic archaeon Sulfolobus shibatae
J. Biol. Chem.
269
27663-27669
1994
Saccharolobus shibatae
brenda
Buhler, C.; Gadelle, D.; Forterre, P.; Wang, J.C.; Bergerat, A.
Reconstitution of DNA topoisomerase VI of the thermophilic archaeon Sulfolobus shibatae from subunits separately overexpressed in Escherichia coli
Nucleic Acids Res.
26
5157-5162
1998
Saccharolobus shibatae (O05208 and O05207), Saccharolobus shibatae
brenda
Graille, M.; Cladiere, L.; Durand, D.; Lecointe, F.; Gadelle, D.; Quevillon-Cheruel, S.; Vachette, P.; Forterre, P.; van Tilbeurgh, H.
Crystal structure of an intact type II DNA topoisomerase: insights into DNA transfer mechanisms
Structure
16
360-370
2008
Saccharolobus shibatae
brenda
Corbett, K.D.; Berger, J.M.
Structure of the topoisomerase VI-B subunit: implications for type II topoisomerase mechanism and evolution
EMBO J.
22
151-163
2003
Saccharolobus shibatae (O05207)
brenda
Jaxel, C.; Duguet, M.; Nadal, M.
Analysis of DNA cleavage by reverse gyrase from Sulfolobus shibatae B12
Eur. J. Biochem.
260
103-111
2006
Saccharolobus shibatae, Saccharolobus shibatae B12
brenda
Buhler, C.; Lebbink, J.H.; Bocs, C.; Ladenstein, R.; Forterre, P.
DNA topoisomerase VI generates ATP-dependent double-strand breaks with two-nucleotide overhangs
J. Biol. Chem.
276
37215-37222
2001
Saccharolobus shibatae (O05207 and O05208)
brenda
Corbett, K.D.; Benedetti, P.; Berger, J.M.
Holoenzyme assembly and ATP-mediated conformational dynamics of topoisomerase VI
Nat. Struct. Mol. Biol.
14
611-609
2007
Saccharolobus shibatae (O05208 and O05207), Methanosarcina mazei (Q8PUB7 and Q8PUB8), Methanosarcina mazei DSM 3647 (Q8PUB7 and Q8PUB8)
brenda
Bergerat, A.; de Massy, B.; Gadelle, D.; Varoutas, P.C.; Nicolas, A.; Forterre, P.
An atypical topoisomerase II from Archaea with implications for meiotic recombination
Nature
386
414-417
1997
Saccharolobus shibatae (O05208 and O05207), Saccharolobus shibatae
brenda
Jaxel, C.; Bouthier, de la Tour, C.; Duguet, M.; Nadal, M.
Reverse gyrase gene from Sulfolobus shibatae B12: gene structure, transcription unit and comparative sequence analysis of the two domains
Nucleic Acids Res.
24
4668-4675
1996
Saccharolobus shibatae (P74759), Saccharolobus shibatae B12 (P74759)
brenda
Gadelle, D.; Bocs, C.; Graille, M.; Forterre, P.
Inhibition of archaeal growth and DNA topoisomerase VI activities by the Hsp90 inhibitor radicicol
Nucleic Acids Res.
33
2310-2317
2005
Saccharolobus shibatae (O05207 and O05208), Saccharolobus shibatae
brenda
Napoli, A.; Valenti, A.; Salerno, V.; Nadal, M.; Garnier, F.; Rossi, M.; Ciaramella, M.
Functional interaction of reverse gyrase with single-strand binding protein of the archaeon Sulfolobus
Nucleic Acids Res.
33
564-576
2005
Saccharolobus shibatae, Saccharolobus shibatae B12
brenda