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W356F
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photobleaching of 5,10-methenyltetrahydrofolate under UV-A irradiation is strongly reduced in the mutant compared with wild-type. The amount of 5,10-methenyltetrahydrofolate after UV-A irradiation is reduced by 64% for wild-type and by 20% for the mutant. Increase in the amount of oxidized FAD under UV-A irradiation due to electron donation to 5,10-methenyltetrahydrofolate by residual FADH- but a lack of photoreduction of the flavin caused by the interrupted tryptophan triad
W432F
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photobleaching of 5,10-methenyltetrahydrofolate under UV-A irradiation is strongly reduced in the mutant compared with wild-type
R350A
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conserved arginine forms a salt bridge with phosphate+1. Among several active-site mutants this R/A mutant is found to exhibit the greatest effect by lowering the selectivity toward cyclobutane-pyrimidine dimer-containing DNA 32-fold and the quantum yield of CPD cleavage from 98% to about 60%
deltaphrA
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Rhodobacter sphaeroides mutant cells lacking the PhrA gene show a significant reduced survival rate after UV-illumination
Q336H
site-directed mutagenesis of the alpha13 conserved amino acid residue
E109D
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mutant enzyme is unable to bind the methenyltetrahydrofolate cofactor under any conditions examined
E109Q
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mutant enzyme is unable to bind the methenyltetrahydrofolate cofactor under any conditions examined
L375H
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binds methenyltetrahydrofolate more weakly than wild-type enzyme
N341A
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life-time of 2.9 ns compared to 1.3 ns for the wild-type enzyme
N378D
site-directed mutagenesis, the asparagine facing the N5 of the FAD isoalloxazine is replaced by aspartic acid, known to protonate FAD- radical (formed by electron transfer from the tryptophan chain) in plant cryptochromes (CRYs). But the mutant protein does not show this protonation. EcPL mutant protein approaches the flavin with similar kinetics to that of the aspartic acid at the corresponding position in plant CRY, but is unable to fully transfer the proton to N5 of the flavin, resulting in a FAD radical with unusual spectral properties. Possibly, the pKa values of FADH radical and/or this aspartic acid in the EcPL N378D mutant protein differ from those in native plant CRY, such that proton transfer is energetically disfavored. Absorption kinetics compared to wild-type
W277E
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the binding affinity for CPD substrate is lower for 1000fold, although the photochemical properties and the quantum yields for catalyses (under the irradiation wavelengths at 366nm and 384 nm) of the mutant is indistinguishable from the wild-type enzyme
W277R
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the binding affinity for CPD substrate is lower for 300fold, although the photochemical properties and the quantum yields for catalyses (under the irradiation wavelengths at 366nm and 384 nm) of the mutant is indistinguishable from the wild-type enzyme
N403A
replacement of asparagine N403 for either a non-polar alanine or a hydrophobic leucine causes complete loss of the catalytic FAD during purification by size exclusion chromatography
N403L
replacement of asparagine N403 for either a non-polar alanine or a hydrophobic leucine causes complete loss of the catalytic FAD during purification by size exclusion chromatography
W360F
mutation of the medial tryptophan, W360, gives a 22fold decrease of photoreduction activity relative to the wild type enzyme, no major build-up of the semiquinoid FADH radical species can be observed for W360F
W381F
the mutation causes complete loss of photoreduction activity and a loss of 70% of incorporation of the catalytic FAD compared to the wild-type enzyme
N403A
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replacement of asparagine N403 for either a non-polar alanine or a hydrophobic leucine causes complete loss of the catalytic FAD during purification by size exclusion chromatography
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N403L
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replacement of asparagine N403 for either a non-polar alanine or a hydrophobic leucine causes complete loss of the catalytic FAD during purification by size exclusion chromatography
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W360F
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mutation of the medial tryptophan, W360, gives a 22fold decrease of photoreduction activity relative to the wild type enzyme, no major build-up of the semiquinoid FADH radical species can be observed for W360F
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W381F
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the mutation causes complete loss of photoreduction activity and a loss of 70% of incorporation of the catalytic FAD compared to the wild-type enzyme
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Q126R
the mutation leads to a reduction of enzymatic activity
Q296H
the mutation leads to a reduction of enzymatic activity
S267A
site-directed mutagenesis, the mutant enzyme is phosphorylated
S494A
site-directed mutagenesis, the mutant enzyme is phosphorylated
S504A
site-directed mutagenesis, the mutant enzyme is phosphorylated
S5A
site-directed mutagenesis, the mutant enzyme is phosphorylated
S7A
site-directed mutagenesis, the mutant enzyme is not phosphorylated
S84A
site-directed mutagenesis, the mutant enzyme is phosphorylated
T115A
site-directed mutagenesis, the mutant enzyme is phosphorylated
S267A
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site-directed mutagenesis, the mutant enzyme is phosphorylated
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S504A
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site-directed mutagenesis, the mutant enzyme is phosphorylated
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S5A
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site-directed mutagenesis, the mutant enzyme is phosphorylated
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S84A
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site-directed mutagenesis, the mutant enzyme is phosphorylated
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T115A
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site-directed mutagenesis, the mutant enzyme is phosphorylated
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A385S
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is blue after purification as the wild-type. Partial formation of oxidized FAD is evident after 2 days as with the wild-type. Shows enhanced semiquinone stability
E283A
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the mutation impairs enzyme activity by diminishing the quantum yield for the repair reaction by 60%
G389N
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is yellow-green after purification, significantly less semiquinone present. Reaction time of ca. 3 days is required for complete conversion of semiquinone to oxidized FAD. Kinetic stability of the semiquinone is significantly reduced, semiquinone oxidation rates more closely resemble that in cryptochrome-DASH
M353Q
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is blue after purification as the wild-type. Partial formation of oxidized FAD is evident after 2 days as with the wild-type. Has little impact on semiquinone stability
R350A
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the mutation demonstrates a 60% decrease in quantum yield, which indicates that Arg350 plays a key role in stabilizing the dimer
W392Y
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is yellow-green after purification, significantly less semiquinone present. Reaction time of ca. 2 days is required for complete conversion of semiquinone to oxidized FAD. Kinetic stability of the semiquinone is significantly reduced, semiquinone oxidation rates more closely resemble that in cryptochrome-DASH
R46E
R46E mutant which lacks a conserved arginine in the binding site for the antenna chromophore shows no flavin-mononucleotide and discloses an eightfold lower activity at 450 nm (blue light) wheras at 370 nm (UV-A light) its activity is only three times lower than wildtype enzyme
H354A
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affinity to substrate is comparable to wild-type enzyme, no activity
H358A
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affinity to substrate is comparable to wild-type enzyme, no activity
L355A
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low affinity to the substrate
Q288A
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affinity to substrate is comparable to wild-type enzyme
W291A
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affinity to substrate is comparable to wild-type enzyme, mutant retains some activity
W398A
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affinity to substrate is comparable to wild-type enzyme, mutant retains some activity
W395R
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the mutation compromises FAD binding of wild type enzyme without significantly affecting the overall conformation of photolyase, the mutant has essentially lost its DNA binding activity, and the residual activity that remains cannot discriminate between damaged and undamaged DNA
W395R
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fully binds methenyltetrahydrofolate but not fully reduced FADH-, life-time of 2.9 ns
W395R
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the mutation compromises FAD binding of wild type enzyme without significantly affecting the overall conformation of photolyase, the mutant has essentially lost its DNA binding activity, and the residual activity that remains cannot discriminate between damaged and undamaged DNA
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E109A
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site-directed mutagenesis, inactive mutant
E109A
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non-5,10-methenyltetrahydrofolate-binding mutant
E109A
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is catalytically active but unable to bind the second cofactor methenyltetrahydrofolate
E109A
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lacks the antenna cofactor
E274A
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site-directed mutagenesis of a residue near the substrate side
E274A
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site-directed mutagenesis of an active site residue
E274A
site-directed mutagenesis of an active site residue near the substrate side, has critical effect on repair efficiency
H44F
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binds and retains methenyltetrahydrofolate under normal reconstitution conditions
H44F
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mutant enzyme retains no methenyltetrahydrofolate upon purification
M345A
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site-directed mutagenesis of an active site residue
M345A
site-directed mutagenesis of an active site residue, has a poor effect on repair efficiency
N108L
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binds and retains methenyltetrahydrofolate under normal reconstitution conditions
N108L
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mutant enzyme retains no methenyltetrahydrofolate upon purification
N378C
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site-directed mutagenesis of a residue near the flavin cofactor side
N378C
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site-directed mutagenesis of an active site residue
N378C
site-directed mutagenesis of an active site residue near the cofactor side, has critical effect on repair efficiency
N378S
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the mutation stabilizes the oxidized state of the flavin
N378S
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the recombinant mutant photolyase contains oxidized FAD (FADox) but not FADH after routine purification procedures, the mutant protein contains FADH in vivo as the wild type enzyme, the mutant photolyase is photoreducible and capable of binding cyclobutadipyrimidine dimers in DNA, but catalytically inert
N378S
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mutant shows no stable radical state of the cofactor FADH. Furthermore, catalytic activity is lost
R226A
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site-directed mutagenesis of an active site residue
R226A
site-directed mutagenesis of an active site residue, has a poor effect on repair efficiency
R342A
conserved arginine forms a salt bridge with phosphate+1. Among several active-site mutants this R/A mutant is found to exhibit the greatest effect by lowering the selectivity toward cyclobutane-pyrimidine dimer-containing DNA 32-fold and the quantum yield of CPD cleavage from 98% to about 60%
R342A
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site-directed mutagenesis of an active site residue
R342A
site-directed mutagenesis of an active site residue, has a poor effect on repair efficiency
W306F
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non-photoreducible mutant, like the wild-type enzyme the mutant enzyme carries out at least 25 rounds of photorepair at the same rate
W306F
in the W306F mutant, the terminal tryptophan W306 is replaced by an isosteric phenylalanine that is much harder to oxidize, thus leaving the electron-transfer chain cut off after the second tryptophan W359, the W359 radical formed in the W306F mutant photolyase is in its neutral form already at 10 ns after excitation
W306F
naturally occuring mutant
W306F
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lacks the ultimate intrinsic electron donor (terminal tryptophan replaced by redox inert phenylalanine), shows an important deprotonation/recombination process with a time constant of 0.85 ns
additional information
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mutation of Q411 and K414 affect the affinity toward cyclobutane-pyrimidine dimer-containing DNA
additional information
generation of deletion mutant DELTAbccry1 and overexpression strain OE::bccry1, phenotype, overview
additional information
generation of deletion mutant DELTAbccry1 and overexpression strain OE::bccry1, phenotype, overview
additional information
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generation of deletion mutant DELTAbccry1 and overexpression strain OE::bccry1, phenotype, overview
additional information
generation of deletion mutant DELTAbccry2 and overexpression strain OE::bccry2, phenotype, overview
additional information
generation of deletion mutant DELTAbccry2 and overexpression strain OE::bccry2, phenotype, overview
additional information
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generation of deletion mutant DELTAbccry2 and overexpression strain OE::bccry2, phenotype, overview
additional information
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generation of deletion mutant DELTAbccry1 and overexpression strain OE::bccry1, phenotype, overview
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additional information
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generation of deletion mutant DELTAbccry2 and overexpression strain OE::bccry2, phenotype, overview
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additional information
comparison of repair activity of the photolyase in the wild-type strain PGEX-4T-1-DsPHR2 and the mutant strain PGEX-4T-1-DsPHR2-Q336H in vitro and in vitro and under different salt concentrations, overview. The mutant shows reduced repair activity compared to wild-type, and the survival rate declines rapidly as salinity increased in the mutant Q336H, while in the wild-type strain, there is no change in the survival rate
additional information
mutation of Q403 and K406 affect the affinity toward cyclobutane-pyrimidine dimer-containing DNA
additional information
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transgenic mice are generated with a transgene for a marsupial CPD photolyase. These mice exhibit a 40% increase of repair for CPD lesions in intact skin and cultured fibroblasts that is accompanied by an improved resistance against acute UV-induced effects like erythema (sunburn), epidermal hyperplasia or apoptosis. Expression of the CPD photolyase in mice efficiently suppresses the formation of skin carcinomas
additional information
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by evolutionary sequence analysis it is shown that Met353 of the CPD photolyase derived from Anacystis nidulans is perfectly conserved throughout the putative class I CPD photolyase
additional information
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Met-353 of the CPD photolyase has been perfectly conserved throughout the putative class I CPD photolyases and plays a pivotal role in the biological function of DNA photolyase
additional information
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deletion of phr1 gene abolishes photoreactivation of UVC (200 to 280 nm)-inhibited spores
additional information
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to assess phr1 photosensory function, fluence response curves of the light-regulated promoter are tested in null mutant (deltaphr1) strains. Photoinduction of the phr1 promoter in deltaphr1 strains are more than 5fold more sensitive to light than that in the wild type, whereas in PHR1-overexpressing lines the sensitivity to light increases about 2fold
additional information
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immobilization of recombinant purified DNA photolyase using avidin-biotin-based immobilization method with neutravidin-modified BioCap chip as a sensor surface