Information on EC 3.1.21.8 - T4 deoxyribonuclease II

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The expected taxonomic range for this enzyme is: Enterobacteria phage T4

EC NUMBER
COMMENTARY hide
3.1.21.8
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RECOMMENDED NAME
GeneOntology No.
T4 deoxyribonuclease II
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REACTION
REACTION DIAGRAM
COMMENTARY hide
ORGANISM
UNIPROT
LITERATURE
endonucleolytic nicking and cleavage of cytosine-containing double-stranded DNA
show the reaction diagram
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ORGANISM
COMMENTARY hide
LITERATURE
UNIPROT
SEQUENCE DB
SOURCE
GENERAL INFORMATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
physiological function
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the phage T4 enzyme is involved in degradation of host DNA
SUBSTRATE
PRODUCT                       
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
DNA where cytosine is partially or completely replaced by hydroxymethylcytosine + H2O
?
show the reaction diagram
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DNA where cytosine is partially or completely replaced by hydroxymethylcytosine is degraded in vivo by T4 endonuclease II, EC 3.1.21.8, and endonuclease IV, EC 3.1.21.9
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?
plasmid pBR322 + H2O
?
show the reaction diagram
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double-stranded restriction cleavage at 12 sites in pBR322 commence before 10-min postinfection with T4 at 37°C and proceeds more slowly in the presence of competing phage DNA than in its absence, utilizing the same sites in both cases. In a 200-base pair segment of the plasmid, single-stranded nicks also are frequent. The plasmid sites are cleaved with a speed that varies with the site, yielding frequencies of cleavage at different sites varying between 10 and 90%, at 50-min postinfection. All sites contain good matches to a consensus, 5'-GRCCGCNTYGC-3', most frequently cleaved around the variable central base pair, generating fragments with blunt ends or 1-2-base 5' overhangs. A larger consensus sequence, 5'-CGRCCGCNTTGSYNGC-3', has been identified. DNA sequence elements 3' to the cut site appear important for rapid cleavage
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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
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the phage T4 enzyme is involved in degradation of host DNA
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-
?
DNA where cytosine is partially or completely replaced by hydroxymethylcytosine + H2O
?
show the reaction diagram
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DNA where cytosine is partially or completely replaced by hydroxymethylcytosine is degraded in vivo by T4 endonuclease II, EC 3.1.21.8, and endonuclease IV, EC 3.1.21.9
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?
METALS and IONS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
Mn2+
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compared to Mg2+, relative nicking activity is 0.4 with top strand, 0.8 with bottom strand
Ni2+
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compared to Mg2+, relative nicking activity is 0.1 with top strand, 0.1 with bottom strand
additional information
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ca2+ cannot substitute Mg2+
INHIBITORS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
Mg2+
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above 10 mM
NaCl
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above 50 mM
additional information
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endonuclease IV activity is almost completely inhibited in the presence of very small amounts of hydroxymethylcytosine in the DNA
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pH OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
TEMPERATURE OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
30 - 37
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activity at 30°C is somewhat lower than at 37°C
Crystallization/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
modeling based on strucuture of UvrC, PDB code 1YD0. Residues G49, R57, E118, and N130 in the putative catalytic surface are important for substrate recognition and binding. Residues G49 and R57 are essential for normal sequence recognition. and play a role in forming the DNA-binding surface and exposing the substrate scissile bond at the active site. Residues N130 and P127 likely contribute to positioning the catalytic domain correctly. Residue K76, part of a conserved NUMOD3 DNA-binding motif of homing endonucleases found to overlap the MR, affects both sequence recognition and catalysis
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mutant E118A,crystallized in space group P21 with four monomers in the asymmetric unit. EndoII forms a striking X-shaped tetrameric structure composed as a dimer of dimers, with a protruding hairpin domain providing most of the dimerization and tetramerization interfaces. A bound phosphate ion in one of the four active sites of EndoII likely mimics the scissile phosphate in a true substrate complex. A protruding loop containing a nuclease-associated modular domain 3 element is likely to be involved in substrate binding, as well as residues forming a separate nucleic acid binding surface adjacent to the active site. The substrate would bind to a primary EndoII dimer diagonally over the active sites, requiring significant distortion of the enzyme or the substrate DNA, or both, for simultaneous nicking of both DNA strands. EndoII may bind its substrate inefficiently across the two sites in the dimer, offering a plausible explanation for the catalytic preponderance of single-strand nicks
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TEMPERATURE STABILITY
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
37
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enzyme remains active for at least 120 min
Cloned/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
expression in Escherichia coli
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expression in Eschgerichia coli
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ENGINEERING
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
A83L
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similar to wild-type, cleavage activity is 20 to 70% of the nicking activity
I24A
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similar to wild-type, cleavage activity is 20 to 70% of the nicking activity
K12A
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similar to wild-type, cleavage activity is 20 to 70% of the nicking activity
K76A 
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much reduced catalytic activity
L16A
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similar to wild-type, cleavage activity is 20 to 70% of the nicking activity
L84P
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much reduced catalytic activity
N130A
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complete loss of cleavage activity
P127L
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much reduced catalytic activity
S72A
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similar to wild-type, cleavage activity is 20 to 70% of the nicking activity
V90A
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similar to wild-type, cleavage activity is 20 to 70% of the nicking activity