Information on EC 3.1.30.2 - Serratia marcescens nuclease

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

EC NUMBER
COMMENTARY hide
3.1.30.2
-
RECOMMENDED NAME
GeneOntology No.
Serratia marcescens nuclease
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REACTION
REACTION DIAGRAM
COMMENTARY hide
ORGANISM
UNIPROT
LITERATURE
Endonucleolytic cleavage to 5'-phosphomononucleotide and 5'-phosphooligonucleotide end-products
show the reaction diagram
REACTION TYPE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
hydrolysis of phosphoric ester
CAS REGISTRY NUMBER
COMMENTARY hide
9025-65-4
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ORGANISM
COMMENTARY hide
LITERATURE
UNIPROT
SEQUENCE DB
SOURCE
Azotobacter agilis
similar enzyme
-
-
Manually annotated by BRENDA team
tea, 2 forms: A, B
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-
Manually annotated by BRENDA team
8x = Elytrigia turcica
-
-
Manually annotated by BRENDA team
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-
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Manually annotated by BRENDA team
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-
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Manually annotated by BRENDA team
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-
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Manually annotated by BRENDA team
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-
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Manually annotated by BRENDA team
L. esculentum x L. peruvianum line 831
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-
Manually annotated by BRENDA team
wheat
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Manually annotated by BRENDA team
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-
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Manually annotated by BRENDA team
GENERAL INFORMATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
evolution
the enzyme belongs to the plant S1-like nucleases class of enzymes. Different members of this family are characterized by a surprisingly large variety of catalytic properties, nucleolytic activities of all Arabidopsis thaliana S1-like paralogues, overview. In addition to Zn2+-dependent enzymes, this family also comprises nucleases activated by Ca2+ and Mn2+, which implies that the apparently well-known S1 nuclease active site in plant nucleases is able to cooperate with different activatory ions. Particular members of this class differ in their optimum pH value and substrate specificity. Plant representatives of this family evolve toward an increase in catalytic diversity. Phylogenetic analysis, overview; the enzyme belongs to the plant S1-like nucleases class of enzymes. Different members of this family are characterized by a surprisingly large variety of catalytic properties, nucleolytic activities of all Arabidopsis thaliana S1-like paralogues, overview. In addition to Zn2+-dependent enzymes, this family also comprises nucleases activated by Ca2+ and Mn2+, which implies that the apparently well-known S1 nuclease active site in plant nucleases is able to cooperate with different activatory ions. Particular members of this class differ in their optimum pH value and substrate specificity. Plant representatives of this family evolve toward an increase in catalytic diversity. Phylogenetic analysis, overview; the enzyme belongs to the plant S1-like nucleases class of enzymes. Different members of this family are characterized by a surprisingly large variety of catalytic properties, nucleolytic activities of all Arabidopsis thaliana S1-like paralogues, overview. In addition to Zn2+-dependent enzymes, this family also comprises nucleases activated by Ca2+ and Mn2+, which implies that the apparently well-known S1 nuclease active site in plant nucleases is able to cooperate with different activatory ions. Particular members of this class differ in their optimum pH value and substrate specificity. Plant representatives of this family evolve toward an increase in catalytic diversity. Phylogenetic analysis, overview; the enzyme belongs to the plant S1-like nucleases class of enzymes. Different members of this family are characterized by a surprisingly large variety of catalytic properties, nucleolytic activities of all Arabidopsis thaliana S1-like paralogues, overview. In addition to Zn2+-dependent enzymes, this family also comprises nucleases activated by Ca2+ and Mn2+, which implies that the apparently well-known S1 nuclease active site in plant nucleases is able to cooperate with different activatory ions. Particular members of this class differ in their optimum pH value and substrate specificity. Plant representatives of this family evolve toward an increase in catalytic diversity. Phylogenetic analysis, overview
malfunction
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Deficiencies in FEN1 function or deletion of the fen1 gene have profound biological effects, including the suppression of repair of DNA damage incurred from the action of various genotoxic agents
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
3'-nucleotides + H2O
?
show the reaction diagram
5'-CCTGGCAGTT + H2O
?
show the reaction diagram
-
synthetic labeled deoxydecanucleotide, cleavage of 5'-Ap*G bond and with lower activity of Gp*T, Cp*A, and Gp*G bonds
-
-
?
B-form DNA + H2O
?
show the reaction diagram
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B form of herring testis DNA
-
-
?
B-Z-hybrid form DNA + H2O
?
show the reaction diagram
-
hybrid B-Z form of herring testis DNA
-
-
?
DNA + H2O
5'-phosphonucleotides + 5'-phosphomononucleotides
show the reaction diagram
DNA + H2O
?
show the reaction diagram
ds-oligoDNA + H2O
?
show the reaction diagram
-
-
-
-
?
dsDNA + H2O
?
show the reaction diagram
dsRNA + H2O
?
show the reaction diagram
-
-
-
-
?
flap DNA + H2O
?
show the reaction diagram
-
-
-
-
?
highly polymerized DNA + H2O
?
show the reaction diagram
-
-
-
-
?
lambda DNA + H2O
?
show the reaction diagram
-
-
size of linear DNA fragments decreases with prolonged incubation time
-
?
lambda phage DNA + H2O
?
show the reaction diagram
-
-
-
-
?
M13 flap DNA + H2O
?
show the reaction diagram
-
-
small fragment of 5-10 nucleotides
-
?
M13 mp19 (+) DNA + H2O
?
show the reaction diagram
-
-
small fragment of 5-8 nucleotides
-
?
poly(A) + H2O
?
show the reaction diagram
-
more rapidly degraded than native DNA
-
-
?
poly(C) + H2O
?
show the reaction diagram
-
more rapidly degraded than native DNA
-
-
?
poly(dA) + H2O
?
show the reaction diagram
-
more rapidly degraded than native DNA
-
-
?
poly(dA)poly(dT) + H2O
?
show the reaction diagram
-
more rapidly degraded than native DNA
-
-
?
poly(dC) + H2O
?
show the reaction diagram
-
-
-
-
?
poly(dT) + H2O
?
show the reaction diagram
-
-
-
-
?
poly(G) + H2O
?
show the reaction diagram
-
-
-
-
?
poly(I) + H2O
?
show the reaction diagram
polyA + H2O
?
show the reaction diagram
-
synthetic homopolyribonucleotide
-
-
?
polyC + H2O
?
show the reaction diagram
-
synthetic homopolyribonucleotide
-
-
?
polyG + H2O
?
show the reaction diagram
-
synthetic homopolyribonucleotide
-
-
?
polyU + H2O
?
show the reaction diagram
-
synthetic homopolyribonucleotide
-
-
?
pUC18 DNA + H2O
?
show the reaction diagram
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covalently closed circular plasmid DNA
substrate converted into relaxed circular form and than to the linear form
-
?
pUC19 DNA + H2O
?
show the reaction diagram
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relaxation of the supercoiled DNA and cutting of the open circular DNA to a linear form
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-
?
RNA + H2O
5'-phosphooligonucleotides + 5'-phosphomononucleotides
show the reaction diagram
RNA + H2O
?
show the reaction diagram
ss-oligoDNA + H2O
?
show the reaction diagram
-
-
-
-
?
ssDNA + H2O
?
show the reaction diagram
supercoiled plasmid DNA + H2O
?
show the reaction diagram
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-
-
-
?
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
DNA + H2O
?
show the reaction diagram
dsDNA + H2O
?
show the reaction diagram
flap DNA + H2O
?
show the reaction diagram
-
-
-
-
?
RNA + H2O
?
show the reaction diagram
-
hydrolysis of nucleic acids
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?
ssDNA + H2O
?
show the reaction diagram
additional information
?
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METALS and IONS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
Ca2+
activates at pH 8.0; activates at pH 8.0; activates at pH 8.0, preferred divalent cation
[Co(NH3)6]3+
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binding to the DNA substrate induces changes in the secondary structure of the enzyme, followed by a decrease of the enzyme activity
additional information
INHIBITORS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
2'-P-ADP-ribose
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-
adenine nucleotides
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CMP
uncompetitive inhibitor
dAMP
competitive inhibitor of partial type, dAMP forming a complex with Sma nuc does not completely prevent the RNA binding and reduces the enzyme affinity to RNA substrate in competition with RNA for the binding site
dithiothreitol
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Double-stranded RNA
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DTT
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slight inhibition
Fe2+
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slight
GMP
uncompetitive inhibitor
KCl
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weak inhibition up to 200 mM
Ni2+
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slight inhibition
p-chloromercuribenzoate
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Polyethylene glycol
putrescine
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RNA
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substrate inhibition above 120 µg/ml
RNA-DNA heteroduplexes
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single-stranded DNA
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Single-stranded RNA
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spermidine
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spermine
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70% inhibition at 0.1 mM
UMP
uncompetitive inhibitor
additional information
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ACTIVATING COMPOUND
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
caspase-3
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cleavage activates inactive enzyme
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Urea
Azotobacter agilis
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2 M, stimulation
additional information
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weak stimulation by addition of recombinant human PCNA
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KM VALUE [mM]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.085
DNA
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744
Mg2+
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pH 8.5, 25ºC
2898
[Co(NH3)6]3+
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pH 8.5, 25ºC
additional information
additional information
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TURNOVER NUMBER [1/s]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
737.6
B-form DNA
Serratia marcescens
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pH 8.0, 30°C, in presence of Mg2+
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0.44 - 1.2
B-Z-hybrid form DNA
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Ki VALUE [mM]
INHIBITOR
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.0765
AMP
pH 8.5, 37°C
0.0862
CMP
pH 8.5, 37°C
0.06
dAMP
pH 8.5, 37°C
0.229
GMP
pH 8.5, 37°C
0.3107
UMP
pH 8.5, 37°C
additional information
additional information
inhibition kinetics of mononucleotides, hyperbolic inhibition of the enzyme, overview
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SPECIFIC ACTIVITY [µmol/min/mg]
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
0.315
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purified enzyme
additional information
pH OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
5
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single-stranded DNA
5.2
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native DNA
5.7
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denatured DNA
6.2
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RNA, denatured DNA
7.7
Azotobacter agilis
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7.8
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3'-AMP
additional information
pH RANGE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
4 - 8
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substantially reduced activity above and below
4.2 - 9
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adenosine 3'-monophosphate, less than 20% of maximal activity above and below
4.5 - 5.7
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native DNA, less than 25% of maximal activity above and below
5 - 7
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RNA less than 10% of maximal activity above and below
5.2 - 7.2
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pH 5.2: about 15% of activity maximum, pH 7.2: about 70% of activity maximum
7.7 - 8.5
Azotobacter agilis
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sharp decrease in activity below and above
additional information
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TEMPERATURE OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
30
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assay at
60 - 70
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nuclease B: RNA, denatured DNA, 3'-AMP
TEMPERATURE RANGE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
30 - 70
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activity increases exponentially
50 - 80
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50°C: 50% nuclease B, 10% nuclease A of activity maximum, 80°C: 30% nuclease B, 60% nuclease A of activity maximum
pI VALUE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
4.7
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isoelectric focusing
SOURCE TISSUE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
SOURCE
expression of ENDO1 is associated with flower development, expression in stamen, sepal and petal
Manually annotated by BRENDA team
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at the mid-to-late uninucleate stage
Manually annotated by BRENDA team
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Manually annotated by BRENDA team
additional information
LOCALIZATION
ORGANISM
UNIPROT
COMMENTARY hide
GeneOntology No.
LITERATURE
SOURCE
-
N-terminal His-tagged protein mainly found in the culture supernatant
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Manually annotated by BRENDA team
MOLECULAR WEIGHT
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
26380
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Sm1, electrospray mass spectrometry
26590
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Sm3, electrospray mass spectrometry
26710
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Sm2, electrospray mass spectrometry
31000
-
gel filtration
34000
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1 * 34000, SDS-PAGE
36000
-
gel filtration
37000
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gel filtration
43000
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x * 43000, SDS-PAGE
49000
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gel filtration, SDS-PAGE
SUBUNITS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
?
-
x * 43000, SDS-PAGE
homodimer
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each monomer consists of 245 amino acids, they function independently of each other, monomer and dimer can function with same specific activity. Dimer form has an electrostatic advantage over the monomer to associate with DNA, inner-sphere binding in the monomer, outer-sphere in the dimer, interface of the protein and DNA is full of charged side chains, such as Arg57, Arg87, Arg125, Arg196, and Mg2+. Interfacial region is highly hydrated with an average of 27 hydration sites in the monomer (water acts more to screen the electrostatic region between the monomer and DNA) and 31 sites in the dimer (water acts more as a glue to provide structural adaptability with the protein and DNA). Dynamics of H-bonds of water in this active centre only little difference is found (water in the working region in the dimer complex has larger fluctuations than in monomer). Dimerization leads to different contacts between DNA and protein residues, especially to Mg2+
monomer
additional information
-
the subunits of the dimer function independently as monomers, molecular dynamic simulations, modelling of complex building with DNA, hydration sites of the enzyme depending on solvent density, overview
POSTTRANSLATIONAL MODIFICATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
glycoprotein
-
about 8% carbohydrate, asparagin-linked high-mannose oligosaccharide
Crystallization/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
pH STABILITY
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
6 - 8
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4°C
95082
8
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above, residual activity greater than 50%
657122
additional information
-
basic pH favors enzymatic stability
657122
TEMPERATURE STABILITY
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
0 - 60
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pre-incubation of the enzyme for 30 min, stable
65
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pre-incubation of the enzyme for 30 min, 60% of the activity
GENERAL STABILITY
ORGANISM
UNIPROT
LITERATURE
addition of 10 mM EDTA essential for storage stability
-
STORAGE STABILITY
ORGANISM
UNIPROT
LITERATURE
0°C, Tris-HCl buffer, pH 7.4, 10 mM EDTA, 10% glycerol, 15 days storage on ice, fully stable for at least 15 days
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4°C, pH 5.5, 0.04 M sodium acetate buffer, 4 months
-
Purification/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
2 forms: A, B
-
2 isoforms; Sm1 and Sm2
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affinity chromatography, 63fold purification
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ion exchange chromatography on DEAE-cellulose DE-32 and DEAE-cellulose DE-52
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native enzyme 265fold to homogeneity from seedlings by ammonium sulfate fractionation, anion exchange chromatography and heparin affinity chromatography
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nuclease Le3
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Par_DSN-t1 is insoluble and inactive after puriciation and renaturation
purified to near homogeneity
-
recombinant protein using His-tag
similar enzyme
Azotobacter agilis
-
Cloned/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
construct containing the NucA gene driven by the beta-actin promoter coupled with enhancer elements from the cytomegalovirus promoter and rabbit beta-globin gene and the blasticidin resistance gene driven by the phosphoglycerate kinase promoter are generated and electroporated into porcine fetal fibroblasts
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expression of nucA in Escherichia coli
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gene ENDO1, DNA and amino acid sequence determination and analysis, recombinant transient overexpression in Arabidosis thaliana leaf protoplasts; gene ENDO2, DNA and amino acid sequence determination and analysis, recombinant transient overexpression in Arabidosis thaliana leaf protoplasts; gene ENDO4, DNA and amino acid sequence determination and analysis, recombinant transient overexpression in Arabidosis thaliana leaf protoplasts; gene ENDO5, DNA and amino acid sequence determination and analysis, recombinant transient overexpression in Arabidosis thaliana leaf protoplasts
generation of plasmids pSS and pDS (for induction of suicide mechanism) consisting of kanamycin resistance, T7-promotor, Serratia marcescens nuclease (1 copy in pSS, 2 copies in pDS) minus its lead-peptide-coding nucleotide sequence. cotransformation with plasmid pL-EGFP-OPH (containing organophosphorus hydrolase and enhanced green fluorescent protein for degradation of organophosphates) of Escherichia coli strain BL21-AI(TM) to generate BL21AI-GOS (pDS)
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His-tag fusion protein expressed in Escherichia coli
molecular dynamics simulations of model-built monomer-DNA complexes and dimer-DNA complexes
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overexpression in Escherichia coli
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Par_DSN-t1: residues 192-379 of Par_DSN precursor (GenBank ID AAN86143) linked with a bacterial signal peptide and 6×His tag (sequence corresponds to the NUC domain). Par_DSN-t2: residues 149-406, corresponds to the NUC domain identified by multiple sequence alignment, is sensitive to proteinase K and is completely cleaved by this protease. Par_DSN-t3: residues 162-406, additionaly all unpaired Cys residues are deleted, Par_DSN-t3 is soluble, sensitive to proteinase K. Par_DSN-t4: residues 114-406, Par_DSN-t4 is degraded by proteinase K
production of catalytically active monomeric variants
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EXPRESSION
ORGANISM
UNIPROT
LITERATURE
both NucA with and without a nuclear localization signal prove to be effective in killing porcine fibroblasts compared to controls, gene expression analysis of surviving colonies indicates that survival is related to low or absent expression of the toxic genes
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ENGINEERING
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
G133V
yield after proteinase K treatment 0%
G139V
yield after proteinase K treatment 0%
H168A
yield after proteinase K treatment 0%
H171A
yield after proteinase K treatment 0%
H237A
yield after proteinase K treatment 20%
K235A
yield after proteinase K treatment 19%
K235R
yield after proteinase K treatment 21%
R121/122A
yield after proteinase K treatment 4%
R121A
for specific activity measurement
R121K
yield after proteinase K treatment 19%
R122A
for specific activity measurement
R122K
yield after proteinase K treatment 20%
R184A
yield after proteinase K treatment 22%
R184K
yield after proteinase K treatment 18%
Renatured/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
Par_DSN-t1 is insoluble and inactive after purification and renaturation. Par_DSN-t2, Par_DSN-t3, Par_DSN-t4 could be renaturated.
refolding after treatment with urea
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APPLICATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
molecular biology
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the enzyme is useful in DNA enrichment by PCR based applications for which removal of template DNA is beneficial before end repair, method overview. Mung bean nuclease treatment abolished the high molecular DNA smear in microdroplet-PCR captured DNA after end repair