Information on EC 2.7.7.B22 - transposase

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The expected taxonomic range for this enzyme is: Bacteria, Eukaryota, Archaea

EC NUMBER
COMMENTARY hide
2.7.7.B22
preliminary BRENDA-supplied EC number
RECOMMENDED NAME
GeneOntology No.
transposase
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REACTION
REACTION DIAGRAM
COMMENTARY hide
ORGANISM
UNIPROT
LITERATURE
binds to the ends of a transposon (DNA that moves) and catalyzes the movement of the transposon to another part of the genome by a cut and paste mechanism or a replicative transposition mechanism
show the reaction diagram
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ORGANISM
COMMENTARY hide
LITERATURE
UNIPROT
SEQUENCE DB
SOURCE
Acidophilium sp. PM
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UniProt
Manually annotated by BRENDA team
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UniProt
Manually annotated by BRENDA team
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UniProt
Manually annotated by BRENDA team
GENERAL INFORMATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
evolution
malfunction
metabolism
physiological function
additional information
SUBSTRATE
PRODUCT                       
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate) hide
LITERATURE
(Substrate)
COMMENTARY
(Product) hide
LITERATURE
(Product)
Reversibility
r=reversible
ir=irreversible
?=not specified
additional information
?
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NATURAL SUBSTRATES
NATURAL PRODUCTS
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate) hide
LITERATURE
(Substrate)
COMMENTARY
(Product) hide
LITERATURE
(Product)
REVERSIBILITY
r=reversible
ir=irreversible
?=not specified
additional information
?
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METALS and IONS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
additional information
ACTIVATING COMPOUND
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
additional information
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pH OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
7.2
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assay at
7.6
P13988 AND P13989
assay at
TEMPERATURE OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
25
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assay at
30
P13988 AND P13989
assay at
30 - 37
assay at
LOCALIZATION
ORGANISM
UNIPROT
COMMENTARY hide
GeneOntology No.
LITERATURE
SOURCE
additional information
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loss of the nuclear localization signal (NLS) domain results in expression in the cytoplasm but not in the nucleus
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Manually annotated by BRENDA team
MOLECULAR WEIGHT
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
16379
2 * 16379, calculated from sequence
SUBUNITS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
dimer
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tertiary structure of SB transposase and protein core, overview
homodimer
octamer
a tetramer of dimers, the dimer is the fundamental catalytic unit
additional information
Crystallization/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
puriifed recombinant tagged transposase catalytic domain SB100X, residues 114-340, by hanging drop vapour diffusion method, mixing of 0.002 ml of 19 mg/ml protein in 20 mM HEPES, pH 7.2, 50 mM NaCl, 2% v/v glycerol, 10 mM MgCl2, and 0.2 mM TCEP, with 0.002 ml of precipitant solution containing 0.1 M Na-citrate, pH 4.8, and 3.2 M ammonium sulfate, 4C, 5 days, X-ray diffraction structure determination and analysis at 1.40 A resolution, PDB ID 3HOS
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hanging drop vapor diffusion method at 24C, crystal structure of the enzyme in both Mn2+-bound and Mn2+-free forms
hanging-drop vapour-diffusion method, crystallized at 24C using a reservoir solution consisting of 100 mM Na HEPES pH 7.5 and 20%(v/v) ethanol. X-ray diffraction data are collected to 1.78 A. The crystals belong to the monoclinic space group P2(1), with unit-cell parameters a = 65.00, b = 34.07, c = 121.58 A, alpha = 90, beta = 100.20, gamma = 90
Purification/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
recombinant N-terminally His6-tagged enzyme from Escherichia coli strain Rosetta2 (DE3) by nickel affinity chromatography
recombinant N-terminally thioredoxinA-6xHis-tagged wild-type and mutant enzymes from Escherichia coli strain RosettaII(DE3) by nickel affinity chromatography
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recombinant soluble His6-tagged enzyme from Escherichia coli strain Rossetta 1 (DE3) by nickel affinity chromatography, identification of the recombinant enzyme by MALDI-TOF-TOF tandem MS analysis
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recombinant TnsC1-85 MBP-fusion protein from Escherichia coli strain BL21Star(DE3) pET101/DTOPO-TnsD1309His, co-purification of the TnsC and TnsD fragments by nickel affinity chromatography, TnsC1-85 interacts directly with TnsD1-309
P13988 AND P13989
soluble His-tagged PAI subdomain of SB transposase from Escherichia coli strain BL21-A1 by nickel affinity chromatography
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Cloned/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
cloning of the junctions of the integrated Tgf2 cis-element and the surrounding genomic DNA using inverse PCR. Four EGFP-positive blunt snout bream are sampled from the group coinjected with donor plasmid with recombinant Tgf2 TPase. An 8-bp direct repeat of target DNA at the integration site, the signature of hAT family transposons, is created adjacent to both ends of Tgf2 at the integration sites in all fish, indicating the recombinant Tgf2 TPase insertions occur by transposition, recombinant expression of soluble His6-tagged enzyme in Escherichia coli strain Rossetta 1 (DE3)
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DNA sequence determination and analysis of several IS1341-type transposases (tnpB genes), existence of sense overlapping transcripts (sotRNAs) for all IS1341-type transposases in Halobacterium salinarum strain NRC-1, condition-dependent differential regulation between sotRNAs and their cognate genes. Identification of a UUCA tetraloop motif that is present in other archaea (ncRNA family HgcC) and in a Halobacterium salinarum intergenic ncRNA derived from a palindrome associated transposable elements (PATE). Overexpression of one sotRNA and the PATE-derived RNA harboring the tetraloop motif improves Halobacterium salinarum growth, indicating that these ncRNAs are functional. IS1341-type transposase genes harbor the right end (RE) of the IS element, which may explain the sotRNA motifs found as a retained structure formed at DNA level, also stable at RNA level, i.e. the RE-like tetraloop
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expression of wild-type and mutant enzyme Tgf2TPase and transposons in Escherichia coli strain Rosetta1 (DE3)
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full-length Hermes expressed in eukaryotic cells, such as budding yeast and Sf9 cells, similarly forms a large multimeric species consistent with an octamer as judged by gel filtration
Galileo gene structure comparisons in Drosophila species, overview
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gene APM_2825, unrooted similarity tree of IS1634 transposases and alignment of C-terminal region, overview. Insertion number determination of IS1634-21, IS1634-2, and IS1634-3, DNA sequence determination and analysis, qPCR and inverse PCR expression analysis, determination of inverted repeats and sites
Acidophilium sp. PM
gene EC990741_2310, TN10 includes genes jemA, jemB, jemC, tetR, tetA, tetC, and tetD and the left and right genes IS10L and IS10R, IS10R encodes a functional transposase protein that catalyzes the chemical steps in Tn10/IS10 transposition
genes tnsABCDE, recombinant expression in Escherichia coli strain ER2566 from an pCYB1 intein vector, recombinant expression of TnsC1-85 as MBP-fusion protein in Escherichia coli strain BL21Star(DE3) pET101/DTOPO-TnsD1-309His
P13988 AND P13989
His-tagged PAI subdomain of SB transposase (N terminal residues G1-Q53), cloned into pET 21a(+) vector, is expressed in soluble form in Escherichia coli strain BL21-A1
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insertion sequence IS1634, gene Mbar_A1742, Unrooted similarity tree of IS1634 transposases and alignment of C-terminal region, overview
IS608 encoded transposase gene tnpA, DNA sequence determination and analysis
overexpressed in intact form in Escherichia coli
recombinant expression of N-terminally His6-tagged enzyme in Escherichia coli strain Rosetta2 (DE3) in soluble form, end-point RT-PCR confirms the integrity of AcTPase mRNA during cell culture, RT-PCR expression analysis
recombinant expression of N-terminally thioredoxinA-6xHis-tagged wild-type and mutant enzymes in Escherichia coli strain RosettaII(DE3)
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recombinant expression of wild-type and mutant enzymes from plasmid pEGFP-C1, which contains EGFP driven froma CMV promoter, in HEK-293 cells
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EXPRESSION
ORGANISM
UNIPROT
LITERATURE
Hfq represses IS10/Tn10 transposase expression through both antisense RNA-dependent and independent mechanisms. Hfq binds directly to the ribosome-binding site of IS10 transposase mRNA to inhibit translation, Hfq binding to site 1 is critical for regulation of transposase expression in vivo. An interaction between the distal surface of Hfq and the ribosome-binding site of transposase mRNA (RNA-IN) is required for repressing translation initiation. Hfq represses transposase translation by facilitating antisense pairing as well as through an antisense-independent mechanism, overview. 3.2Fold decrease in transposase expression in the presence versus the absence of Hfq
impact of mutant forms of Hfq on IS10 transposase expression and transposition, Hfq binding of native RNA-IN in vivo is inhibited by site 1 mutations, overview
the small RNA ChiX activates transposase expression by titrating Hfq away from transposase mRNA, RNA-IN. Overexpression of ChiX results in a 4.5fold reduction in the amount of RNA-IN associated with Hfq. ChiX positively regulates IS10 transposase translation. ChiX competes with RNA-IN for Hfq-binding in vivo
ENGINEERING
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
D228N/E648Q
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site-directed mutagenesis
E279D
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a catalytically inactive transposase mutant
K339
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site-directed mutagenesis, the mutant shows 20% reduced activity compared to the wild-type enzyme
N280
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site-directed mutagenesis, the mutant shows 50% reduced activity compared to the wild-type enzyme
N280/K339
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site-directed mutagenesis, almost catalytically inactive mutant
additional information
APPLICATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
medicine
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Sleeping Beauty (SB) transposon is a nonviral gene transfer vector, already used in clinical trials. Full-length dysferlin transfer by the hyperactive sleeping beauty transposase restores dysferlin-deficient muscle, which can be used for nonviral gene delivery of full-length human dysferlin into muscle cells, along with a successful and efficient transplantation into skeletal muscle, important advances towards successful gene therapy of dysferlin-deficient muscular dystrophy
molecular biology