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Cdel5
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mutant lacking the C-terminal pentapeptide HEEDR of motif VI, 1% of wild-type DNA ligase activity
D297A
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14% of wild-type DNA ligase activity
E295A
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90% of wild-type DNA ligase activity
E296A
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52% of wild-type DNA ligase activity
F190A
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the mutant has 31% of wild type nick sealing activity
F215A
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the mutant has 70% of wild type nick sealing activity
F215L
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the mutant has 75% of wild type nick sealing activity
F276A/M278A
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the mutant has 42% of wild type nick sealing activity
F286L
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the mutant has 61% of wild type nick sealing activity
F289A
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42% of wild-type DNA ligase activity
H294A
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92% of wild-type DNA ligase activity
K274A
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the mutant has 34% of wild type nick sealing activity
K274Q
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the mutant has 72% of wild type nick sealing activity
K274R
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the mutant has 67% of wild type nick sealing activity
K27A
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the mutant cannot form the covalent ligase-adenylate intermediate and hence cannot form DNA-adenylate, but retains the ability to seal a preadenylylated nick
K281A/C283A
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the mutant has 73% of wild type nick sealing activity
K29A
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arrests ligation reaction at the substrate adenylation step
N214A
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the mutant has 51% of wild type nick sealing activity
N214D
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the mutant has 94% of wild type nick sealing activity
N214L
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the mutant has 113% of wild type nick sealing activity
N214Q
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the mutant has 98% of wild type nick sealing activity
P284A
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53% of wild-type DNA ligase activity
P287A
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59% of wild-type DNA ligase activity
R285K
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the mutant has 6% of wild type nick sealing activity
R285Q
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the mutant has 8% of wild type nick sealing activity
R293A
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30% of wild-type DNA ligase activity
R298A
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85% of wild-type DNA ligase activity
S218A/R220A
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the mutant has 49% of wild type nick sealing activity
S221A/T222A/H223A
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the mutant has 42% of wild type nick sealing activity
S235A
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the mutant has 112% of wild type nick sealing activity
S235A/K281A/C283A
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the mutant has 49% of wild type nick sealing activity
T249A
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the mutant has 92% of wild type nick sealing activity
V288A
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the mutant has 58% of wild type nick sealing activity
V288I
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the mutant has 91% of wild type nick sealing activity
V288T
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the mutant has 74% of wild type nick sealing activity
Y217A
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the mutant has 44% of wild type nick sealing activity
Y217F
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the mutant has 112% of wild type nick sealing activity
Y217L
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the mutant has 84% of wild type nick sealing activity
Y217S
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the mutant has 85% of wild type nick sealing activity
K159L
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adenylation site mutant, aboragates the ability of the ligase to covalently link to an AMP moiety
A576P
the mutation hardly affects ligation activity at pH 3.0
D162E
the mutation hardly affects ligation activity at pH 3.0
E134L
the mutation causes a 60% reduction at pH 3.0, with no net change in iron content and purple color, the mutant has an activity optimum of pH 5.0 and a 1.5fold higher turnover number as the wild type enzyme
N255G
the mutation hardly affects ligation activity at pH 3.0
T491S
the mutation hardly affects ligation activity at pH 3.0
A576P
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the mutation hardly affects ligation activity at pH 3.0
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D162E
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the mutation hardly affects ligation activity at pH 3.0
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E134L
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the mutation causes a 60% reduction at pH 3.0, with no net change in iron content and purple color, the mutant has an activity optimum of pH 5.0 and a 1.5fold higher turnover number as the wild type enzyme
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N255G
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the mutation hardly affects ligation activity at pH 3.0
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T491S
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the mutation hardly affects ligation activity at pH 3.0
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F717L
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amino acid substitution within the conserved peptide of DNA ligase IIIbeta, does not complement Escherichia coli lig mutant
G468A
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mutant enzyme shows residual adenylate complex formation at high protein concentrations, mutant complex has a lower affinity for ATP compared to wild-type complex
G468E
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mutant DNA ligase IV/Xrrc4 complex is poorly expressed, mutation completely abolishes adenylate complex formation, mutant complex has no ligation activity
G469A
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mutant enzyme shows residual adenylate complex formation
G469E
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mutant DNA ligase IV/Xrrc4 complex is poorly expressed, mutation completely abolishes adenylate complex formation, mutant complex has no ligation activity
G712A
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amino acid substitution within the conserved peptide of DNA ligase IIIbeta, complements Escherichia coli lig mutant
K323E
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the mutant has significantly reduced blunt end DNA ligation activity (87% decrease in the initial velocity of blunt end DNA ligation compared to wild type enzyme), but has very little effect on DNA nick joining activity, the mutation selectively blocks zinc finger function
K323E/E265K
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the mutations do not restore efficient blunt end DNA joining activity
K724E
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amino acid substitution within the conserved peptide of DNA ligase IIIbeta, does not complement Escherichia coli lig mutant
K727G
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amino acid substitution within the conserved peptide of DNA ligase IIIbeta, does not complement Escherichia coli lig mutant
K727R
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amino acid substitution within the conserved peptide of DNA ligase IIIbeta, does not complement Escherichia coli lig mutant
P718A
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amino acid substitution within the conserved peptide of DNA ligase IIIbeta, complements Escherichia coli lig mutant
P718T
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amino acid substitution within the conserved peptide of DNA ligase IIIbeta, complements Escherichia coli lig mutant
p719G
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amino acid substitution within the conserved peptide of DNA ligase IIIbeta, does not complement Escherichia coli lig mutant, thermolabile mutant
R278H
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the mutant form of ligase IV is severely impaired for formation of the ligase IV-adenylate and assesses DNA binding by XRCC4
R327E
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the mutant is nearly devoid of blunt end joining activity, mimicking a deletion of the zinc finger
R327E/D262R
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the mutations do not restore efficient blunt end DNA joining activity
R716G
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amino acid substitution within the conserved peptide of DNA ligase IIIbeta, does not complement Escherichia coli lig mutant
R722Q
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amino acid substitution within the conserved peptide of DNA ligase IIIbeta, complements Escherichia coli lig mutant
R722V
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amino acid substitution within the conserved peptide of DNA ligase IIIbeta, complements Escherichia coli lig mutant
R724G
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amino acid substitution within the conserved peptide of DNA ligase IIIbeta, does not complement Escherichia coli lig mutant
S714I
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amino acid substitution within the conserved peptide of DNA ligase IIIbeta, does not complement Escherichia coli lig mutant
D253A
mutant DNA ligase reacts with ATP to form the covalent intermediate, but is unable to catalyze the full ligation reaction
K251A
no DNA ligase-adenylate formation and no nick-joining
D483A
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mutant protein is inert in the ligase adenylylation reaction
E530A
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mutant protein is inert in the ligase adenylylation reaction
E613A
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mutant protein is inert in the ligase adenylylation reaction
H373A
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mutant without 3'-5' single-stranded DNA exonuclease activity
K481A
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mutant protein is inert in the ligase adenylylation reaction
K635A
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mutant is active in autoadenylylation as wild-type LigD, consistent with its retention of overall nick-joining function
K637A
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mutant enzyme forms about one-fourth the level of ligase-AMP compared to wild-type enzyme. Complete loss of function of overall nick ligation
CDELTA1
Paramecium bursaria chlorella virus
mutant with deleted C-terminal five amino acids: mutant shows 34% the specific activity of wild-type. Preformed Lig-AMP in vivo: 71% of total enzyme (wild-type: 70%), preformed Lig-AMP in vitro: 8% of total enzyme (wild-type: 21%)
CDELTA2
Paramecium bursaria chlorella virus
mutant with deleted C-terminal five amino acids: mutant shows 6% the specific activity of wild-type. Preformed Lig-AMP in vivo: 80% of total enzyme (wild-type: 70%), preformed Lig-AMP in vitro: 1.1% of total enzyme (wild-type: 21%)
CDELTA3
Paramecium bursaria chlorella virus
mutant with deleted C-terminal five amino acids: mutant shows 2% the specific activity of wild-type. Preformed Lig-AMP in vivo: 67% of total enzyme (wild-type: 70%), preformed Lig-AMP in vitro: 1.3% of total enzyme (wild-type: 21%)
CDELTA4
Paramecium bursaria chlorella virus
mutant with deleted C-terminal five amino acids: mutant shows 1% the specific activity of wild-type. Preformed Lig-AMP in vivo: 18% of total enzyme (wild-type: 70%), preformed Lig-AMP in vitro: 0.8% of total enzyme (wild-type: 21%)
CDELTA5
Paramecium bursaria chlorella virus
mutant with deleted C-terminal five amino acids: mutant shows 1% the specific activity of wild-type and is poorly responsive to ATP. Preformed Lig-AMP in vivo: 7% of total enzyme (wild-type: 70%), preformed Lig-AMP in vitro: 0.5% of total enzyme (wild-type: 21%)
D297A
Paramecium bursaria chlorella virus
preformed Lig-AMP in vivo: 76% of total enzyme (wild-type: 70%), preformed Lig-AMP in vitro: 2.9% of total enzyme (wild-type: 21%)
D297E
Paramecium bursaria chlorella virus
preformed Lig-AMP in vivo: 74% of total enzyme (wild-type: 70%), preformed Lig-AMP in vitro: 1.8% of total enzyme (wild-type: 21%)
D297N
Paramecium bursaria chlorella virus
preformed Lig-AMP in vivo: 60% of total enzyme (wild-type: 70%), preformed Lig-AMP in vitro: 2.8% of total enzyme (wild-type: 21%)
R293A
Paramecium bursaria chlorella virus
preformed Lig-AMP in vivo: 52% of total enzyme (wild-type: 70%), preformed Lig-AMP in vitro: 1.9% of total enzyme (wild-type: 21%)
R293K
Paramecium bursaria chlorella virus
preformed Lig-AMP in vivo: 70% of total enzyme (wild-type: 70%), preformed Lig-AMP in vitro: 3.4% of total enzyme (wild-type: 21%)
R293Q
Paramecium bursaria chlorella virus
preformed Lig-AMP in vivo: 48% of total enzyme (wild-type: 70%), preformed Lig-AMP in vitro: 3.8% of total enzyme (wild-type: 21%)
D15A
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3'-ribonuclease activity with D10R2 primer-template is 110% of wild-type activity, 3'-phosphomonoesterase activity with D11-p primer-template is 3.3% of wild-type activity, 3'-phosphomonoesterase activity with D9R1-p primer template is 4.9% of wild-type activity
D83A
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3'-ribonuclease activity with D10R2 primer-template is 57% of wild-type activity, 3'-phosphomonoesterase activity with D11-p primer-template is 51% of wild-type activity, 3'-phosphomonoesterase activity with D9R1-p primer template is 70% of wild-type activity
E21A
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3'-ribonuclease activity with D10R2 primer-template is 130% of wild-type activity, 3'-phosphomonoesterase activity with D11-p primer-template is less than 0.1% of wild-type activity, 3'-phosphomonoesterase activity with D9R1-p primer template is less than 0.1% of wild-type activity
H42A
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3'-ribonuclease activity with D10R2 primer-template is less than 0.1% of wild-type activity, 3'-phosphomonoesterase activity with D11-p primer-template is less than 0.1% of wild-type activity, 3'-phosphomonoesterase activity with D9R1-p primer template is 0.2% of wild-type activity
H48A
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3'-ribonuclease activity with D10R2 primer-template is less than 0.1% of wild-type activity, 3'-phosphomonoesterase activity with D11-p primer-template is 0.8% of wild-type activity, 3'-phosphomonoesterase activity with D9R1-p primer template is 0.2% of wild-type activity
K66A
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3'-ribonuclease activity with D10R2 primer-template is 2.9% of wild-type activity, 3'-phosphomonoesterase activity with D11-p primer-template is 28% of wild-type activity, 3'-phosphomonoesterase activity with D9R1-p primer template is 48% of wild-type activity
Q40A
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3'-ribonuclease activity with D10R2 primer-template is 41% of wild-type activity, 3'-phosphomonoesterase activity with D11-p primer-template is 6.7% of wild-type activity, 3'-phosphomonoesterase activity with D9R1-p primer template is 8.6% of wild-type activity
R14A
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3'-ribonuclease activity with D10R2 primer-template is 77% of wild-type activity, 3'-phosphomonoesterase activity with D11-p primer-template is 0.2% of wild-type activity, 3'-phosphomonoesterase activity with D9R1-p primer template is 0.1% of wild-type activity
R46A
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3'-ribonuclease activity with D10R2 primer-template is 16% of wild-type activity, 3'-phosphomonoesterase activity with D11-p primer-template is 19% of wild-type activity, 3'-phosphomonoesterase activity with D9R1-p primer template is 67% of wild-type activity
R76A
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3'-ribonuclease activity with D10R2 primer-template is 1.1% of wild-type activity, 3'-phosphomonoesterase activity with D11-p primer-template is 16% of wild-type activity, 3'-phosphomonoesterase activity with D9R1-p primer template is 44% of wild-type activity
Y88A
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3'-ribonuclease activity with D10R2 primer-template is 7.1% of wild-type activity, 3'-phosphomonoesterase activity with D11-p primer-template is 0.2% of wild-type activity, 3'-phosphomonoesterase activity with D9R1-p primer template is less than 0.1% of wild-type activity
D540A
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mutant exhibits notably enhanced nick-joining activity compared with that of the wild type enzyme, the mutant enzyme exhibits activity about twice as high as that of the wild type within 10 min
D540A/Q547A/K554A/K558A
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the mutant enzyme exhibits activity about twice as high as that of the wild type within 10 min. The D540A ligation is almost the same as that of the D540A/Q547A/K554A/K558A mutant enzyme, thus implying that a single substitution for Asp540 might exert a more dominant effect than the substitutions of the other three polar and ionic residues at the C terminus
D540K
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the mutant exhibits notably enhanced nick-joining activity compared with that of the wild type enzyme
D540R
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the mutant exhibits notably enhanced nick-joining activity compared with that of the wild type enzyme
D540R/delC4
DelC4 i.e. deletion of the four C-terminal residues, nick-joining activities of the mutant is enhanced as compared to that of the D540R single substitution
D540R/DELTAC4
the combination of the Asp540-replacement and the elimination of ionic residues in the helix, forming interactions with adenylylation domain, effectively enhances the activity
D540S
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the mutant exhibits notably enhanced nick-joining activity compared with that of the wild type enzyme
K249A
mutant enzyme shows no no adenylyltransferase activity
K534A
the wild-type R531A and mutant K534A enzymes exhibit almost the same DNA ligation activities both in the presence and in the absence of externally added ATP, contains AMP in the crystal
molecular biology
the enzyme is used in the ligase chain reaction
Q547A/K554A/K558A
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nick ligation activity of the mutant is slightly higher than that of the wild type enzyme
R531A
the wild-type R531A and mutant K534A enzymes exhibit almost the same DNA ligation activities both in the presence and in the absence of externally added ATP, contains AMP in the crystal
R531A/K534A
the mutations of both basic residues (R531A and K534A) severely affected the ligation activity, especially in the absence of ATP, does not contain AMP in the crystal
R544A
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mutant R544A displays a notable reduction in nick-joining activity (less than 45% of the input substrate ligated) in comparison with that of mutant R544A/Q547A/K554A/K558A
R544A/Q547A/K554A/K558A
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mutant enzyme exhibits low activity. Mutant R544A displays a notable reduction in nick-joining activity (less than 45% of the input substrate ligated) in comparison with that of mutant R544A/Q547A/K554A/K558A
K159L
Tequatrovirus T4
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the inactive His-K159L substitution mutant is unable to self-associate, but still possesses AMP-dependent DNA nicking activity, no blunt end ligation. Mutant enzyme His-N-DELTA80 catalyzes no blunt end ligation and no AMP-dependent activity. Mutant enzyme His-C-DELTA57has no blunt end ligation activity, no AMP-dependent activity, low nick joining activity and low ATP binding. Mutant enzyme His-K159L has no blunt end ligation activity, no nick joining activity, no ATP binding, no DNA binding, and no AMP-dependent activity
A287K
the mutant enzyme has a half-life time at 94°C of 20 min
A387K
the mutant enzyme has a half-life time at 94°C of 15 min
G304D
the mutant enzyme has a half-life time at 94°C of 25 min
S364I
the mutant enzyme has a half-life time at 94°C of 20 min
F115A
the ApeLig gene is originally annotated as a protein of 619 amino acids, with a calculated mass of 69196.2 Da. Later it was reannotated as a protein of 602 amino acids (67747.6 Da), in which 17 amino acids are truncated from the N-terminus of the originally annotated protein. The authors of this reference refer to the 619 amino acid protein containing the mutation at position F132. According to the UniProt numbering the position of this mutation is 115. Surface plasmon resonance analyses reveals that the F132A mutant does not interact with an immobilized subunit of the proliferating cell nuclear antigen (which is known as a DNA sliding clamp that acts as a platform for the assembly of enzymes involved in DNA replication and repair). No stimulation of the ligation activity of the F132A protein by the proliferating cell nuclear antigen can be detected in vitro. These results indicate that the phenylalanine, which is located the predicted proliferating cell nuclear antigen-binding region in the ligase, has a critical role for the physical and functional interaction with proliferating cell nuclear antigen
F115A
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the ApeLig gene is originally annotated as a protein of 619 amino acids, with a calculated mass of 69196.2 Da. Later it was reannotated as a protein of 602 amino acids (67747.6 Da), in which 17 amino acids are truncated from the N-terminus of the originally annotated protein. The authors of this reference refer to the 619 amino acid protein containing the mutation at position F132. According to the UniProt numbering the position of this mutation is 115. Surface plasmon resonance analyses reveals that the F132A mutant does not interact with an immobilized subunit of the proliferating cell nuclear antigen (which is known as a DNA sliding clamp that acts as a platform for the assembly of enzymes involved in DNA replication and repair). No stimulation of the ligation activity of the F132A protein by the proliferating cell nuclear antigen can be detected in vitro. These results indicate that the phenylalanine, which is located the predicted proliferating cell nuclear antigen-binding region in the ligase, has a critical role for the physical and functional interaction with proliferating cell nuclear antigen
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F286A
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13% of wild-type DNA ligase activity
F286A
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the mutant has 4% of wild type nick sealing activity
R285A
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26% of wild-type DNA ligase activity
R285A
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the mutant has 7% of wild type nick sealing activity
K326
catalytically inactive
K326
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catalytically inactive
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R771W
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Arg771-Trp mutation in DNA ligase I of cell line 46BR defective in DNA ligase I accounts for the malfunctioning but partly active enzyme present in 46BR cells that allows cell proliferation
R771W
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mutant enzyme used in this study shows only 3% of normal activity
D540R/K554A/K558A
the combination of the Asp540-replacement and the elimination of ionic residues in the helix, forming interactions with adenylylation domain, effectively enhances the activity
D540R/K554A/K558A
nick-joining activities of the mutant is enhanced, as compared to that of the D540R single substitution
additional information
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deletion of the zinc finger does not significantly change LigIII DNA binding affinity, nor does it abrogate the specificity of the enzyme for a nicked substrate
additional information
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loss of DNA ligase IV prevents recognition of DNA by double-strand break repair proteins XRCC4 and XLF
additional information
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the 1762delAAG and 588delK variants are associated with LIG4 syndrome, the 588delKvariant does not alter the reading frame of the DNA ligase IV protein but destabilizes the ligase IV protein
additional information
mutants (mglig4) show no defects in asexual or sexual growth, and are fully pathogenic, compared to the wild type enzyme, mglig4 exhibits weak sensitivity to a DNA-damaging agent camptothecin, non-homologous integration of DNA is frequently observed in mglig4 transformants
additional information
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mutants (mglig4) show no defects in asexual or sexual growth, and are fully pathogenic, compared to the wild type enzyme, mglig4 exhibits weak sensitivity to a DNA-damaging agent camptothecin, non-homologous integration of DNA is frequently observed in mglig4 transformants
additional information
combination of the D540R-replacement and the elimination of ionic residues in the helix, forming interactions with AdD, effectively enhances the activity
additional information
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combination of the D540R-replacement and the elimination of ionic residues in the helix, forming interactions with AdD, effectively enhances the activity