4.1.99.13: (6-4)DNA photolyase
This is an abbreviated version!
For detailed information about (6-4)DNA photolyase, go to the full flat file.
Word Map on EC 4.1.99.13
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4.1.99.13
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photoproducts
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cyclobutane
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uv-induced
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cryptochromes
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photoreactivation
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light-dependent
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pyrimidone
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blue-light
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photorepair
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oxetane
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pyrimidine-pyrimidone
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photoreduction
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photolesion
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dash
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ostreococcus
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four-membered
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fadox
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photoreception
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cry-dashs
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dewar
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cryptochrome-dash
- 4.1.99.13
- photoproducts
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cyclobutane
-
uv-induced
-
cryptochromes
-
photoreactivation
-
light-dependent
-
pyrimidone
-
blue-light
-
photorepair
-
oxetane
-
pyrimidine-pyrimidone
-
photoreduction
-
photolesion
-
dash
- ostreococcus
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four-membered
- fadox
-
photoreception
- cry-dashs
-
dewar
- cryptochrome-dash
Reaction
Synonyms
(6-4) DNA photolyase, (6-4) photolyase, (6-4) PHR, (6-4) PL, (6-4) PP-specific PL, (6-4)-Phr, (6-4)photolyase, 6-4 DNA photolyase, 6-4 photolyase, 6-4CiPhr, 6-4PP-photolyase, animal (6-4) photolyase, At64, At64PHR, bacterial (6-4) photolyase, CmPHR1, Cry1, CryB, deoxyribodipyrimidine photolyase-related protein, Dm64, DNA photolyase, Ds64PHR, H64PRH, human (6-4) photolyase homologous protein, NF-10, OtCPF1, phr (6-4), PhrB, PL-(6-4), prokaryotic (6-4) photolyase, RSP_3077, TRIREDRAFT_77473, XELAEV_18035355mg, Xl64phr
ECTree
4.1.99.13 (6-4)DNA photolyaseAdvanced search results
results (
results (Engineering
Engineering on EC 4.1.99.13 - (6-4)DNA photolyase
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PROTEIN VARIANTS 
ORGANISM
UNIPROT
COMMENTARY 
LITERATURE
C350S
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site-directed mutagenesis, mutation of a Cys residue of the Fe-S cluster, the mutant protein is not expressed in Escherichia coli under conditions where the wild-type protein is expressed as soluble protein
C438S
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site-directed mutagenesis, mutation of a Cys residue of the Fe-S cluster, the mutant protein is not expressed in Escherichia coli under conditions where the wild-type protein is expressed as soluble protein
C441S
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site-directed mutagenesis, mutation of a Cys residue of the Fe-S cluster, the mutant protein is not expressed in Escherichia coli under conditions where the wild-type protein is expressed as soluble protein
H366A
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site-directed mutagenesis, in the mutant repair activity is lost
H366N/L370M
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site-directed mutagenesis, in the mutant repair activity is lost
I51W
site-directed mutagenesis, the mutant PhrBI51W shows loss of the DMRL chromophore (due to structural rearrangements of the residues in the DMRL binding pocket), reduced photoreduction, and reduced DNA repair capacity compared to wild-type. The mutation only affects local protein environments, whereas the overall fold remains unchanged. The crystal structure of PhrBI51W shows how the bulky Trp leads to structural rearrangements in the DMRL chromophore pocket. Structure analysis of mutant PhrBI51W
W342F
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site-directed mutagenesis, the mutant shows highly reduced light-induced spectral changes compared to wild-type
W390F
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site-directed mutagenesis, the mutant shows highly reduced light-induced spectral changes compared to wild-type
W390F/Y391F
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site-directed mutagenesis, the mutant shows highly reduced light-induced spectral changes compared to wild-type
Y391A
site-directed mutagenesis, Tyr391 replacement by alanine blocks photoreduction
Y391F
Y391W
site-directed mutagenesis, replacement of Tyr391 by Trp results in loss of FAD and DMRL chromophores, Trp might participate in the electron transfer cascade
Y399F
Y424F
Y430F
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site-directed mutagenesis, photoreduction of the mutant is indistinguishable from the wild-type, DNA binding assays are performed with single-stranded oligonucleotides with or without (-4)TT lesion, the mutant repair activity is 70% reduced compared to wild-type
Y460F
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site-directed mutagenesis, photoreduction of the mutant is indistinguishable from the wild-type, DNA binding assays are performed with single-stranded oligonucleotides with or without (6-4)TT lesion, the mutant repair activity is unaltered compared to wild-type
I51W
Agrobacterium fabrum C58 / ATCC 33970
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site-directed mutagenesis, the mutant PhrBI51W shows loss of the DMRL chromophore (due to structural rearrangements of the residues in the DMRL binding pocket), reduced photoreduction, and reduced DNA repair capacity compared to wild-type. The mutation only affects local protein environments, whereas the overall fold remains unchanged. The crystal structure of PhrBI51W shows how the bulky Trp leads to structural rearrangements in the DMRL chromophore pocket. Structure analysis of mutant PhrBI51W
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Y391A
Agrobacterium fabrum C58 / ATCC 33970
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site-directed mutagenesis, Tyr391 replacement by alanine blocks photoreduction
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Y391F
Agrobacterium fabrum C58 / ATCC 33970
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site-directed mutagenesis, Tyr391 replacement by phenylalanine does not block photoreduction
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Y391W
Agrobacterium fabrum C58 / ATCC 33970
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site-directed mutagenesis, replacement of Tyr391 by Trp results in loss of FAD and DMRL chromophores, Trp might participate in the electron transfer cascade
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Y424F
Agrobacterium fabrum C58 / ATCC 33970
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site-directed mutagenesis, the PhrBY424F mutant shows reduced binding of lesion DNA and loss of DNA repair compared to wild-type. The mutation only affects local protein environments, whereas the overall fold remains unchanged. The crystal structure of PhrBY424F reveals a water network extending to His366, which are part of the lesion-binding site. Structure analysis of mutant PhrBY424F
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H364A
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compared to wild-type mutant similar electron transfer dynamics in the range of 70-260 ps but decay to zero without any long plateaus, H364 is irreplaceable: Steady-state quantum yield measurements reveal a total lack of repair with the mutant
H364D
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compared to wild-type mutant similar electron transfer dynamics in the range of 70-260 ps but decay to zero without any long plateaus, H364 is irreplaceable: Steady-state quantum yield measurements reveal a total lack of repair with the mutant
H364K
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compared to wild-type mutant similar electron transfer dynamics in the range of 70-260 ps but decay to zero without any long plateaus, H364 is irreplaceable: Steady-state quantum yield measurements reveal a total lack of repair with the mutant
H364M
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compared to wild-type mutant similar electron transfer dynamics in the range of 70-260 ps but decay to zero without any long plateaus, H364 is irreplaceable: Steady-state quantum yield measurements reveal a total lack of repair with the mutant
H364N
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compared to wild-type mutant similar electron transfer dynamics in the range of 70-260 ps but decay to zero without any long plateaus, H364 is irreplaceable: Steady-state quantum yield measurements reveal a total lack of repair with the mutant
H364Y
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compared to wild-type mutant similar electron transfer dynamics in the range of 70-260 ps but decay to zero without any long plateaus, H364 is irreplaceable: Steady-state quantum yield measurements reveal a total lack of repair with the mutant
C434A/C437A
site-directed mutagenesis, the mutant cells show altered survival rates compaired to wild-type cells
E37F
site-directed mutagenesis, the mutant cells show altered survival rates compaired to wild-type cells
E37F/L366H
site-directed mutagenesis, the mutant cells show altered survival rates compaired to wild-type cells
E37F/W388F
site-directed mutagenesis, the mutant cells show altered survival rates compaired to wild-type cells
H362A
site-directed mutagenesis of an active site residue, the mutant cells show altered survival rates compaired to wild-type cells, in vivo and in vitro inactive mutant, no influence of an altered cofactor composition on the lack of repair. The position of H362 in the active centre next to FAD and the amino acids is involved in FAD reduction
L366A
site-directed mutagenesis, the mutant cells show altered survival rates compaired to wild-type cells, the mutant shows reduced in vitro repair activity compared to wild-type, and highly reduced FAD content
L366F
site-directed mutagenesis, the mutant cells show altered survival rates compaired to wild-type cells, the mutant shows reduced in vitro repair activity compared to wild-type, and highly reduced FAD content
L366H
site-directed mutagenesis, the mutant cells show altered survival rates compaired to wild-type cells, the mutant shows reduced in vitro repair activity compared to wild-type, and highly reduced FAD content
Q302A
site-directed mutagenesis of an active site residue, the mutant cells show altered survival rates compaired to wild-type cells
S431V
removal of the C-terminal roof-like subdomain of CryB including the [4Fe4S] cluster, two variants: variant CryBDELTAD432-V508 and CryBS431VDELTAS431-P507, both variants show highly reduced production and activity compared to wild-type
W388F
site-directed mutagenesis, the mutant cells show altered survival rates compaired to wild-type cells. The cofactor composition of CryB W338F does not differ from that of the wild-type protein. Considering the requirement of W338 for full reduction of FAD in vitro, the in vivo functionality of CryB W338F is remarkable
Y387F
site-directed mutagenesis, the mutant cells show altered survival rates compaired to wild-type cells, the mutant has a slightly reduced FAD content compared to wild-type
Y391F
site-directed mutagenesis, the mutant cells show altered survival rates compaired to wild-type cells, the mutant has a slightly reduced FAD content compared to wild-type
H362A
Cereibacter sphaeroides ATCC 17023 / 2.4.1 / NCIB 8253 / DSM 158
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site-directed mutagenesis of an active site residue, the mutant cells show altered survival rates compaired to wild-type cells, in vivo and in vitro inactive mutant, no influence of an altered cofactor composition on the lack of repair. The position of H362 in the active centre next to FAD and the amino acids is involved in FAD reduction
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S431V
Cereibacter sphaeroides ATCC 17023 / 2.4.1 / NCIB 8253 / DSM 158
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removal of the C-terminal roof-like subdomain of CryB including the [4Fe4S] cluster, two variants: variant CryBDELTAD432-V508 and CryBS431VDELTAS431-P507, both variants show highly reduced production and activity compared to wild-type
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Y387F
Cereibacter sphaeroides ATCC 17023 / 2.4.1 / NCIB 8253 / DSM 158
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site-directed mutagenesis, the mutant cells show altered survival rates compaired to wild-type cells, the mutant has a slightly reduced FAD content compared to wild-type
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Y391F
Cereibacter sphaeroides ATCC 17023 / 2.4.1 / NCIB 8253 / DSM 158
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site-directed mutagenesis, the mutant cells show altered survival rates compaired to wild-type cells, the mutant has a slightly reduced FAD content compared to wild-type
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K281G
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a single T(6-4)T photoproduct in a 10-mer oligonucleotide is photoreactivated by this mutant. Mutant shows similar capacity of photoreactivation compared to wild-type. Over the pH range 6-9 no difference to wild-type. Over the pH range 9-11 the activity of K281G mutant declines sharply and less than 30% of the activity is retained at pH 11.0
K281R
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a single T(6-4)T photoproduct in a 10-mer oligonucleotide is photoreactivated by this mutant. Mutant shows similar capacity of photoreactivation compared to wild-type. Over the pH range 6-9 no difference to wild-type. Over the pH range 9-11 the K281R mutant and the wild-type show more tolerance to the high pH (9.0-11.0), and at pH 11.0, 78.5% and 62.3% of the activity are retained, respectively
H354A
H358A
L355A
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large decrease in the affinity to the (6-4) photoproduct substrate, suggesting a hydrophobic interaction with the (6-4)photoproduct
additional information
Y391F
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site-directed mutagenesis, the mutant Y391F shows wild-type-like light-induced spectral changes
Y391F
site-directed mutagenesis, Tyr391 replacement by phenylalanine does not block photoreduction
Y399F
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site-directed mutagenesis, the mutant shows highly reduced light-induced spectral changes compared to wild-type
Y399F
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site-directed mutagenesis, the mutant Y391F shows slightly reduced light-induced spectral changes compared to wild-type
Y424F
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site-directed mutagenesis, photoreduction of the mutant is indistinguishable from the wild-type, DNA binding assays are performed with single-stranded oligonucleotides with or without (6-4)TT lesion, the mutant repair activity is lost
Y424F
site-directed mutagenesis, the PhrBY424F mutant shows reduced binding of lesion DNA and loss of DNA repair compared to wild-type. The mutation only affects local protein environments, whereas the overall fold remains unchanged. The crystal structure of PhrBY424F reveals a water network extending to His366, which are part of the lesion-binding site. Structure analysis of mutant PhrBY424F
H354A
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mutation of a conserved His residue: mutant is inactive in photorepair
H354A
site-directed mutagenesis, the mutant shows reduced repair activity compared to the wild-type enzyme
H354A
site-directed mutagenesis, unlike for the wild-type, the difference FTIR spectrum of H354A coincides with the zero line, mutant H358A to possesses limited repair activity
H358A
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almost complete loss of repair activity, suggesting that His354 and His358 are essential for catalytic activity
H358A
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mutation of a conserved His residue: mutant is inactive in photorepair
H358A
site-directed mutagenesis, unlike for the wild-type, the difference FTIR spectrum of enzyme mutant H358A is close to the zero line, mutant H358A to possesses highly limited repair activity
additional information
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mutants on cysteines that coordinate the Fe-S cluster of PhrB are either insoluble or not expressed. The same result is found for proteins with a truncated C-terminus, in which one of the Fe-S binding cysteines is mutated and for expression in minimal medium with limited Fe concentrations. Construction of two truncated versions of PhrB, designated PhrB-C and PhrB-D, which consist of amino acids 1-432 and 1-476, respectively. In PhrB-C, three of the Fe-S coordinating Cys residues aremissing, in PhrB-D, all Cys residues are present but the two C-terminal helices are missing. PhrB-C is insoluble under all tested conditions, whereas PhrB-D is partially soluble. With expression of PhrB by Escherichia coli cells growing in minimal medium without iron, PhrB is also insoluble. Thus, all conditions that might lead to a protein without Fe-S cluster result in very poor protein expression or insoluble protein
additional information
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light-dependent repair of UV-induced (6-4) photoproducts is investigated in an excision repair-deficient Arabidopsis mutant. It is demonstrated that (6-4) photoproducts are efficiently eliminated in a light-dependent manner which occurs in the presence of blue light (435 nm) but not upon exposure to light of longer wavelengths
additional information
construction of a knockout strain of cryB, termed 2.4.1DELTAcryB, which shows no detectable photoreduction of its FAD cofactor in vitro, but is able to maintain photoreactivation of Rhodobacter sphaeroides cells, in contrast, its in vitro repair activity is lost due to the inability to accumulate the repair-competent FADH- state under the assay conditions. In vivo photoreactivation is also functional when binding of FAD or the antenna chromophore is impaired, or when only low amounts of both are present. The impairment of one of the two light absorbing cofactors at a time through mutagenesis strongly influences the kinetics of photoreactivation, and the combination of both mutation completely abolishes CryB-dependent photoreactivation. The misfolding of the protein when trying to remove the iron-sulfur cluster by amino acid exchanges demonstrates its structural relevance for the protein. Survival rates of different Rhodobacter sphaeroides mutant strains after UV light treatment, and absorbance spectra of purified His-tagged CryB variants, overview
additional information
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construction of a knockout strain of cryB, termed 2.4.1DELTAcryB, which shows no detectable photoreduction of its FAD cofactor in vitro, but is able to maintain photoreactivation of Rhodobacter sphaeroides cells, in contrast, its in vitro repair activity is lost due to the inability to accumulate the repair-competent FADH- state under the assay conditions. In vivo photoreactivation is also functional when binding of FAD or the antenna chromophore is impaired, or when only low amounts of both are present. The impairment of one of the two light absorbing cofactors at a time through mutagenesis strongly influences the kinetics of photoreactivation, and the combination of both mutation completely abolishes CryB-dependent photoreactivation. The misfolding of the protein when trying to remove the iron-sulfur cluster by amino acid exchanges demonstrates its structural relevance for the protein. Survival rates of different Rhodobacter sphaeroides mutant strains after UV light treatment, and absorbance spectra of purified His-tagged CryB variants, overview
additional information
Cereibacter sphaeroides ATCC 17023 / 2.4.1 / NCIB 8253 / DSM 158
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construction of a knockout strain of cryB, termed 2.4.1DELTAcryB, which shows no detectable photoreduction of its FAD cofactor in vitro, but is able to maintain photoreactivation of Rhodobacter sphaeroides cells, in contrast, its in vitro repair activity is lost due to the inability to accumulate the repair-competent FADH- state under the assay conditions. In vivo photoreactivation is also functional when binding of FAD or the antenna chromophore is impaired, or when only low amounts of both are present. The impairment of one of the two light absorbing cofactors at a time through mutagenesis strongly influences the kinetics of photoreactivation, and the combination of both mutation completely abolishes CryB-dependent photoreactivation. The misfolding of the protein when trying to remove the iron-sulfur cluster by amino acid exchanges demonstrates its structural relevance for the protein. Survival rates of different Rhodobacter sphaeroides mutant strains after UV light treatment, and absorbance spectra of purified His-tagged CryB variants, overview
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additional information
expressing 6-4CiPhr in a photolyase-deficient Escherichia coli strain improves survival rate of the strain
additional information
construction of a theoretical 3D model shows that the protein has a FAD-binding domain. Although the amino acids identity between Dunaliella salina (6-4)photolyase and Escherichia coli CPD photolyase is just 23%, the backbone structure shows a high similarity in overall folding, suggesting a parallel photoreversal mechanism in the two enzymes
additional information
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construction of a theoretical 3D model shows that the protein has a FAD-binding domain. Although the amino acids identity between Dunaliella salina (6-4)photolyase and Escherichia coli CPD photolyase is just 23%, the backbone structure shows a high similarity in overall folding, suggesting a parallel photoreversal mechanism in the two enzymes
additional information
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expression of H64PRH does not show any photoreactivating effects on the survival of UV-irradiated Escherichia coli. Using a gel shift assay with with un-irradiated and UV-irradiated DNA probes it is shown that H64PRH protein does not possess any binding activity to either DNA probe
additional information
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the predicted amino acid sequence of the human protein has 48% identity with the Drosophila (6-4)photolyase over the entire protein
additional information
construction of a cry1 gene replacement mutants and cry1 overexpressing strains, (DELTAcry1, OEcry1, DELTAblr1 and DELTAenv1 strains). The mutant strains and the parental strain QM9414 are grown under continuous exposure to blue light or in the dark for 72 h at 28°C. The results show that neither the gene replacement mutant nor the overexpressing strain have any colony morphology alterations, and conidiated normally, the two independent gene replacement mutants and overexpressing strains show the same unaltered behavior compared to wild-type
additional information
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construction of a cry1 gene replacement mutants and cry1 overexpressing strains, (DELTAcry1, OEcry1, DELTAblr1 and DELTAenv1 strains). The mutant strains and the parental strain QM9414 are grown under continuous exposure to blue light or in the dark for 72 h at 28°C. The results show that neither the gene replacement mutant nor the overexpressing strain have any colony morphology alterations, and conidiated normally, the two independent gene replacement mutants and overexpressing strains show the same unaltered behavior compared to wild-type
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additional information
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sequencing of the cDNA clone reveals an open reading frame of encoding a protein of 526 amino acids (60600 Da) cDNA shows 58-54% amino acid identity to Drosophila (6-4)photolyase and its human homologue and 20-24% identity to the class I CPD photolyase
