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Information on EC 4.1.99.3 - deoxyribodipyrimidine photo-lyase and Organism(s) Escherichia coli and UniProt Accession P00914
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4.1.99.3 deoxyribodipyrimidine photo-lyaseIUBMB Comments
A flavoprotein (FAD), containing a second chromophore group. The enzyme catalyses the reactivation by light of irradiated DNA. A similar reactivation of irradiated RNA is probably due to a separate enzyme.
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The taxonomic range for the selected organisms is: Escherichia coli
The enzyme appears in selected viruses and cellular organisms
The enzyme appears in selected viruses and cellular organisms
Synonyms
ssdna, primase, photolyase, dna photolyase, cryptochrome 1, cpd photolyase, cry-dash, cpd-photolyase, cryptochrome dash, cyclobutane pyrimidine dimer photolyase, 
more
topSYNONYM 
ORGANISM
UNIPROT
COMMENTARY 
LITERATURE
DNA photolyase
-
CPD photolyase
-
-
deoxyribodipyrimidine photolyase
-
-
-
-
deoxyribonucleate pyrimidine dimer lyase (photosensitive)
-
-
-
-
deoxyribonucleic cyclobutane dipyrimidine photolyase
-
-
-
-
deoxyribonucleic photolyase
-
-
-
-
dipyrimidine photolyase (photosensitive)
-
-
-
-
DNA cyclobutane dipyrimidine photolyase
-
-
-
-
DNA photolyase
DNA-photoreactivating enzyme
-
-
-
-
lyase, deoxyribonucleate pyrimidine dimer
-
-
-
-
photolyase
photoreactivating enzyme
phr A photolyase
-
-
-
-
PhrB photolyase
-
-
-
-
PRE
-
-
-
-
DNA photolyase
-
-
-
-
DNA photolyase
-
-
photolyase
-
-
-
-
photoreactivating enzyme
-
-
-
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REACTION
REACTION DIAGRAM
COMMENTARY
ORGANISM
UNIPROT
LITERATURE
cyclobutadipyrimidine (in DNA) = 2 pyrimidine residues (in DNA)
results support the electron hopping mechanism by which electron transfer from W306 to the flavin is mediated in a three-step electron hopping process (W306 to W359 to W382 to FADH)
cyclobutadipyrimidine (in DNA) = 2 pyrimidine residues (in DNA)
dynamics and mechanism of CPD repair by photolyase, detailed overview. In contrast to the computational reaction model the thymine dimer splits by a sequential pathway
cyclobutadipyrimidine (in DNA) = 2 pyrimidine residues (in DNA)
photo-induced intramolecular electron transfer in photolyases and initial electron-transfer bifurcation in repair complexes. Seven electron-transfer reactions in 10 elementary steps in all classes of CPD photolyases. Unified electron-transfer pathway through a conserved structural configuration that bifurcates to favor direct tunneling in prokaryotes and a two step hopping mechanism in eukaryotes. Complete photocycles of CPD repair by class I and class II PLs, overview
cyclobutadipyrimidine (in DNA) = 2 pyrimidine residues (in DNA)
radical mechanism through a cyclic redox reaction. Photolyase binds DNA containing a CPD because the thymine dimer distorts the backbone of the DNA. Upon binding to damaged DNA, through ionic interactions between the positively charged groove on the photolyase surface and negatively charged DNA phosphodiester backbone the enzyme pulls the thymine dimer out from within the helix and into the core of the enzyme so that the thymine dimer is within Van der Waals contact with FADH-. It makes a very staple complex, and nothing happens until folate absorbs a photon and transfers the excitation energy to the flavin cofactor. The excited-state flavin, FADH- radical, repairs the thymine dimer by a cyclic redox reaction, and then the enzyme dissociates from the DNA to go on in search of other damage sites to carry out the repair reactions again
cyclobutadipyrimidine (in DNA) = 2 pyrimidine residues (in DNA)
reaction mechanisms of CPD DNA photolyase and cytochrome DASH, detailed overview. Proposed intraprotein electron transfer from W306 to FADH radical is not a part of the normal photolyase photocycle in vivo
cyclobutadipyrimidine (in DNA) = 2 pyrimidine residues (in DNA)
reduced anionic flavin adenine dinucleotide (FADH-) is the critical cofactor in DNA photolyase (PL) for the repair of cyclobutane pyrimidine dimers (CPD) in UV-damaged DNA. The initial step involves photoinduced electron transfer from FADH- radical to the CPD. The adenine (Ade) moiety is nearly stacked with the flavin ring, an unusual conformation compared to other FAD-dependent proteins
cyclobutadipyrimidine (in DNA) = 2 pyrimidine residues (in DNA)
the repair of CPD reveals seven electron-transfer (ET) reactions among ten elementary steps by a cyclic ET radical mechanism through bifurcating ET pathways, a direct tunneling route mediated by the intervening adenine and a two-step hopping path bridged by the intermediate adenine from the cofactor to damaged DNA, through the conserved folded flavin at the active site. Repair photocycle of the PLs and development of a unified repair mechanism for all CPD PLs with the critical, bifurcating electron transfer pathways through the folded flavin cofactor in the conserved active site structure, overview
cyclobutadipyrimidine (in DNA) = 2 pyrimidine residues (in DNA)
the reaction is initiated by blue light and proceeds through long-range energy transfer, single electron transfer and enzyme catalysis by a radical mechanism
-
cyclobutadipyrimidine (in DNA) = 2 pyrimidine residues (in DNA)
mechanism of photoactivation
-
cyclobutadipyrimidine (in DNA) = 2 pyrimidine residues (in DNA)
reaction mechanism, overview
-
cyclobutadipyrimidine (in DNA) = 2 pyrimidine residues (in DNA)
upon absorption of a visible photon, the photoexcited flavin radical FADH abstracts an electron from nearby W382. The tryptophanyl radical W382 thus formed abstracts an electron from the nearby middle tryptophan, W359. W359 radical abstracts an electron from W306. As the second and third steps are faster than the first one, the intermediate states are not populated in wild-type photolyase to any significant extent and escape spectroscopic detection. The described forward electron transfer steps between the tryptophans are in competition with back electron transfer from FADH- to the respective tryptophanyl cation radical, so that the quantum yield of formation of the terminal W306 radical is only 20%. W306 radical releases a proton to the aqueous phase in 200 ns. The resulting neutral radical W306 radical can be reduced by extrinsic reductants, leaving a photolyase that contains FADH as required for photorepair of DNA
-
cyclobutadipyrimidine (in DNA) = 2 pyrimidine residues (in DNA)
cyclic electron-transfer radical mechanism with two fundamental processes, electron-tunneling pathways and cyclobutane ring splitting, the cyclobutane pyrimidine dimer splits in two sequential steps within 90 ps and the electron tunnels between the cofactor and substrate through a remarkable route with an intervening adenine, dynamics and mechanism of cyclobutane pyrimidine dimer repair by DNA photolyase, overview
-
cyclobutadipyrimidine (in DNA) = 2 pyrimidine residues (in DNA)
the DNA repair enzyme recognizes a solvent-exposed cyclobutadipyrimidine as part of its damage recognition mechanism
-
REACTION TYPE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
C-C-bond cleavage
-
-
-
-
SYSTEMATIC NAME
IUBMB Comments
deoxyribocyclobutadipyrimidine pyrimidine-lyase
A flavoprotein (FAD), containing a second chromophore group. The enzyme catalyses the reactivation by light of irradiated DNA. A similar reactivation of irradiated RNA is probably due to a separate enzyme.
CAS REGISTRY NUMBER
COMMENTARY
37290-70-3
-
SUBSTRATE
PRODUCT
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate)
(Substrate)
LITERATURE
(Substrate)
(Substrate)
COMMENTARY
(Product)
(Product)
LITERATURE
(Product)
(Product)
Reversibility
r=reversible
ir=irreversible
?=not specified
r=reversible
ir=irreversible
?=not specified
cis,syn-cyclobutane pyrimidine dimer
2 pyrimidine residues
-
substrate binding and substrate conformation by isothermal titration calorimetry, overview
-
-
?
cis-syn cyclobutadipyrimidine dimer DNA
pyrimidine residues in DNA
-
-
-
-
?
cyclobutadipyrimidine in nucleosome DNA
2 pyrimidine residues in nucleosome DNA
-
folding of DNA in nucleosomes efficiently protects DNA from being repaired
-
?
cyclobutadipyrimidine (in DNA)
2 pyrimidine residues (in DNA)
-
-
-
?
cyclobutadipyrimidine (in DNA)
2 pyrimidine residues (in DNA)
-
-
-
?
cyclobutadipyrimidine (in DNA)
2 pyrimidine residues (in DNA)
the entire catalytic cycle is complete in 1.2 ns, and the enzyme repairs thymine dimer with a quantum yield of 0.9
-
-
?
cyclobutadipyrimidine (in DNA)
2 pyrimidine residues (in DNA)
the pyrimidine dimer is flipped out from the DNA helix into the central cavity, thereby coming within van der Waals contact distance of the FAD molecule. This central pocket is lined on one side with hydrophobic residues and with polar residues on the other, thus matching the asymmetric polarity of the thymidine dimer
-
-
?
cyclobutadipyrimidine (in DNA)
2 pyrimidine residues (in DNA)
-
photolyases utilize near-ultraviolet blue light to specifically repair the major photoproducts of UV-induced damaged DNA. The enzyme specifically repairs CPD lesions
-
-
?
cyclobutadipyrimidine (in DNA)
2 pyrimidine residues (in DNA)
-
repair of a single CPD lesion within a double-stranded DNA molecule
-
-
?
cyclobutadipyrimidine (in DNA)
2 pyrimidine residues (in DNA)
-
various CPD substrates, T-T, T-U, U-T, U-U dimers
-
-
?
cyclobutadipyrimidine in DNA
2 pyrimidine residues in DNA
-
enzyme uses light to repair cyclobutylpryrimidine dimers in DNA, local structure around the thymidine lesion changes dramatically upon binding to photolyase
-
?
cyclobutadipyrimidine in DNA
2 pyrimidine residues in DNA
-
upon binding of DNA, the enzyme flips the pyrimidine dimer out of the duplex into a hole that contains the catalytic cofactor. The cyclobutane ring is then split by a light-initiated electron transfer reaction
-
?
cyclobutadipyrimidine in DNA
2 pyrimidine residues in DNA
-
steady-state fluorescence measurements of single- and double-stranded oligonucleotides shows that the local region around the 5'-side of the cyclobutadipyrimidine lesion is more disrupted and destacked than the 3'-side in substrate-protein complexes
-
-
?
cyclobutadipyrimidine in DNA
?
-
the enzyme binds to DNA containing pyrimidine dimers with high affinity and then breaks the cyclobutane ring joining the two pyrimidines of the dimer in a light-dependent reaction, 300-500 nm
-
-
?
cyclobutadipyrimidine in DNA
?
-
light-dependent(300-600 nm) monomerization of cyclobutyl pyrimidine dimers, formed between adjacent pyrimidines on the same DNA strand, upon exposure to UV irradiation, 220-320 nm
-
-
?
cyclobutadipyrimidine in DNA
?
-
the enzyme converts the energy of light of near UV to visible wavelengths into chemical energy to break the cyclobutane ring of pyrimidine dimers in DNA and thus prevents the lethal and mutagenic effects of far UV, 200-300 nm
-
-
?
cyclobutadipyrimidine in DNA
?
-
about 20times more pyrimidine dimers are bound to the yeast photolyase than to the Escherichia coli photolyase. Ratio between the enzyme's binding constant for pyrimidine dimers and its binding constant for nondamaged DNA is very similar for yeast and Escherichia coli photolyases
-
-
?
cyclobutadipyrimidine in DNA
?
-
photolyase binds tighter to substrate than cryptochrome 1, binding constant is slightly sensitive to oxidation state
-
-
?
cyclobutadipyrimidine in DNA
?
-
presence of a very rigid antenna binding site, a relatively rigid active site in CPD photolyase but with large local orientation flexibility
-
-
?
cyclobutadipyrimidine in DNA
pyrimidine residues in DNA
-
-
-
-
?
cyclobutadipyrimidine in DNA
pyrimidine residues in DNA
-
the unique configuration of the phosphodiester backbone in the strand containing the pyrimidine dimer, as well as the cyclobutane ring of the dimer itself are the important structural determinants of the substrate for recognition by photolyase
-
?
cyclobutadipyrimidine in DNA
pyrimidine residues in DNA
-
binds to DNA containing pyrimidine dimers in a light-independent step and repairs the pyrimidine dimer upon absorbing a photon in the 300-600 nm range
-
?
cyclobutadipyrimidine in DNA
pyrimidine residues in DNA
-
no activity towards (6-4)pyrimidine-cytosine products in DNA
-
?
cyclobutadipyrimidine in DNA
pyrimidine residues in DNA
-
inactive on dimers in RNA
-
?
cyclobutadipyrimidine in DNA
pyrimidine residues in DNA
-
active on cis-syn-cyclobutylpyrimidine dimers in supercoiled DNA as in linear DNA
-
?
cyclobutadipyrimidine in DNA
pyrimidine residues in DNA
-
light-dependent(300-600 nm) monomerization of cyclobutyl pyrimidine dimers, formed between adjacent pyrimidines on the same DNA strand, upon exposure to ultraviolet irradiation, 220-320 nm
-
?
cyclobutadipyrimidine in DNA
pyrimidine residues in DNA
-
catalyzes the repair of cyclobutadipyrimidine dimers in DNA under near-UV or blue light irradiation
-
-
?
additional information
?
-
anaerobic repair assay in argon atmosphere
-
-
?
additional information
?
-
detailed repair dynamics of damaged DNA by photolyases and a biomimetic system through resolving all elementary steps on the ultrafast timescales, including multiple intermolecular electron- and proton-transfer reactions and bond-breaking and -making processes
-
-
?
additional information
?
-
direct measurements of photolyase binding to cyclobutane pyrimidine dimers (CPD)-containing undecamer DNA that has been labeled with a fluorophore, photolyase csCPD-DNA binding kinetics detected by fluorescence spectroscopy, overview. Preparation and purification of csCPD-containing oligonucleotides. Photolyase finds its target through a three-dimensional diffusion-controlled search. Photolyase may not recognize an intrahelical CPD but only an extrahelical CPD
-
-
?
additional information
?
-
-
direct measurements of photolyase binding to cyclobutane pyrimidine dimers (CPD)-containing undecamer DNA that has been labeled with a fluorophore, photolyase csCPD-DNA binding kinetics detected by fluorescence spectroscopy, overview. Preparation and purification of csCPD-containing oligonucleotides. Photolyase finds its target through a three-dimensional diffusion-controlled search. Photolyase may not recognize an intrahelical CPD but only an extrahelical CPD
-
-
?
additional information
?
-
enzyme in complex with CPD moiety, molecular docking study
-
-
?
additional information
?
-
photochemistry of wild-type and N378D mutant DNA photolyase with oxidized FAD cofactor studied by transient absorption spectroscopy, overview
-
-
?
additional information
?
-
-
photochemistry of wild-type and N378D mutant DNA photolyase with oxidized FAD cofactor studied by transient absorption spectroscopy, overview
-
-
?
additional information
?
-
-
major pathway to remove UV-induced DNA lesions from the genome
-
?
additional information
?
-
-
photoreduction by intraprotein electron transfer is not part of the photolyase photocycle under physiological conditions
-
-
?
additional information
?
-
-
4-amino-6-methyl-8-(2'-deoxy-beta-D-ribofuranosyl)-7(8H)-pteridone (6MAP) is a fluorescent adenine analogue that demonstrates high sensitivity to base-stacking interactions in duplex DNA. 6MAP is a sensitive probe of cyclobutylpyrimidine dimers base flipping by photolyase and does does not interfere with the repair of the substrate. It is shown that 6MAP/cyclobutylpyrimidine dimers duplexes are true substrates of photolyase
-
-
?
additional information
?
-
-
absolute dependence of catalysis by photolyase on light
-
-
?
additional information
?
-
-
a novel substrate (a modified thymidine 10-mer with a central cyclobutane pyrimidine dimer and all bases, except the one at the 3' end, replaced by 5,6-dihydrothymine) is repaired with an efficiency very similar to that of the conventional substrates (a 10-mer of unmodified thymidines containing a central cyclobutane pyrimidine dimer and an acetone-sensitized thymidine 18-mer containing in average six randomly distributed cyclobutane pyrimidine dimers per strand). Significantly lower repair quantum yield for the holoenzyme compared to its apo form due to an additional process, i.e., excitation energy transfer from the antenna cofactor to the reduced flavin
-
-
?
additional information
?
-
-
electrostatic interactions and protonation are affected by the oxidation state of the required FAD cofactor and substrate conformation
-
-
?
additional information
?
-
-
the enzyme shows light-induced reduction of FAD, and photorepair involves the transfer of an electron from the photoexcited reduced FAD to the damaged DNA for cleaving the dimers to maintain the DNA's integrity, substrate specificity, overview
-
-
?
NATURAL SUBSTRATE
NATURAL PRODUCT
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate)
(Substrate)
LITERATURE
(Substrate)
(Substrate)
COMMENTARY
(Product)
(Product)
LITERATURE
(Product)
(Product)
REVERSIBILITY
r=reversible
ir=irreversible
?=not specified
r=reversible
ir=irreversible
?=not specified
cyclobutadipyrimidine (in DNA)
2 pyrimidine residues (in DNA)
-
-
-
?
cyclobutadipyrimidine (in DNA)
2 pyrimidine residues (in DNA)
-
-
-
?
cyclobutadipyrimidine (in DNA)
2 pyrimidine residues (in DNA)
-
photolyases utilize near-ultraviolet blue light to specifically repair the major photoproducts of UV-induced damaged DNA. The enzyme specifically repairs CPD lesions
-
-
?
cyclobutadipyrimidine in DNA
?
-
the enzyme binds to DNA containing pyrimidine dimers with high affinity and then breaks the cyclobutane ring joining the two pyrimidines of the dimer in a light-dependent reaction, 300-500 nm
-
-
?
cyclobutadipyrimidine in DNA
?
-
light-dependent(300-600 nm) monomerization of cyclobutyl pyrimidine dimers, formed between adjacent pyrimidines on the same DNA strand, upon exposure to UV irradiation, 220-320 nm
-
-
?
cyclobutadipyrimidine in DNA
?
-
the enzyme converts the energy of light of near UV to visible wavelengths into chemical energy to break the cyclobutane ring of pyrimidine dimers in DNA and thus prevents the lethal and mutagenic effects of far UV, 200-300 nm
-
-
?
additional information
?
-
-
major pathway to remove UV-induced DNA lesions from the genome
-
?
additional information
?
-
-
photoreduction by intraprotein electron transfer is not part of the photolyase photocycle under physiological conditions
-
-
?
additional information
?
-
-
absolute dependence of catalysis by photolyase on light
-
-
?
COFACTOR
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
8-hydroxy-5-deazariboflavin
bound at the interface between N-terminal and C-terminal domain
5,10-methylenetetrahydrofolate
-
antenna pigment in Escherichia coli absorbs blue/near UV light and transfers the excitation energy fast and efficiently to FADH-
ATP
-
stimulates, utilization of ATP for the photorepair process of the pyrimidine dimer containing DNA, not only an allosteric effector
5,10-methenyltetrahydrofolate
bound at the interface between N-terminal and C-terminal domain
5,10-methenyltetrahydrofolate
observed in the cleft between the two domains, where it interacts with two critical amino acid residues, Cys292 and Lys293
FAD
the repair reaction involves electron transfer to the cyclobutane pyrimidine dimers from the photoexcited FAD cofactor in its fully reduced form
FAD
alpha-helical domain is harboring the FAD cofactor, essential for catalysis
FAD
critical W382 residue relative to the flavin for efficient vectorial electron transfer leading to photoreduction
FAD
bound in a C-terminal alpha-helix cavity, the C-terminal alpha-helical domain consists of 14 alpha-helices. FAD is held in a U-shaped conformation by interaction with 14 conserved amino acid residues
FAD
dependent on, adopts a uniquely folded configuration at the active site that plays a critical functional role in DNA repair, overview. Dynamics of flavin cofactor and its repair photocycles by different classes of photolyases, overview. Photolyase utilizes FADH-, not FAD- radical as the active state. Using femtosecond (fs)-resolved spectroscopy and site-directed mutagenesis, the dynamics of class I PL from Escherichia coli (EcPL) in four redox states are investigated
FAD
four redox states of FAD are relevant for the various functions of DNA photolyases: fully reduced FADH- required for DNA photorepair, and the two semireduced radical states FAD- radical and FADH radical formed in electron transfer reactions. Absorption spectra of wild-type EcPL and MTHF antenna-free mutant E109A/N378D EcPL, transient absorption kinetics on nano- and microsecond time scales at six characteristic wavelengths, spectral analysis of transient absorption kinetics, overview
FAD
reduced anionic flavin adenine dinucleotide (FADH-) is the critical cofactor in DNA photolyase (PL) for the repair of cyclobutane pyrimidine dimers (CPD) in UV-damaged DNA
FAD
steady-state spectra of flavin at various redox states and active-site solvation dynamics in photolyases, overview
FAD
the enzyme uses a fully reduced flavin, FADH-, cofactor to repair sunlight-induced DNA lesions
FADH2
results indicate that both charge recombination of the primary charge separation state FADH-W382 and electron transfer from W359 to W382 occur with time constants below 4 ps, considerably faster than the initial W382-FADH electron-transfer step.
FADH2
the isoalloxazine ring is sandwiched between a salt bridge comprising an arginine and an aspartate residue: R344/D372 and short polypeptide stretches: A377-N378/G381-W382. Another conserved interaction is a hydrogen bond formed between the N5 nitrogen of the isoalloxazine group and a conserved asparagine: N378
methenyltetrahydrofolate
is the solar panel or photoantenna of the enzyme
5,10-methenyltetrahydrofolate
-
an electron transfer pathway exists in photolyase, where the 5,10-methenyltetrahydrofolate cofactor is photoreduced to 5,10-methylenetetrahydrofolate. Reduction requires the intact tryptophan triad. Photolyase forms 5,10-methylenetetrahydrofolate when treated with UV-A. Light-driven formation of 5,10-methylenetetrahydrofolate by photolyase can be coupled with the formation of NADPH in the presence of 5,10-methylenetetrahydrofolate dehydrogenase
FAD
-
the photoexcited FAD cofactor is reduced from the semiquinone or fully oxidized state to the catalytically active FADH- state
FAD
-
the photolyase in its native state contains FAD in the two-electron reduced and deprotonated FADH- form, during purification under aerobic conditions, FADH- is oxidized to the rather stable blue neutral radical
FAD
-
the purified enzyme binds a FAD, which is in the neutral radical semiquinone form
FAD
-
photoreduction of FAD under blue light irradiation is faster in photolyase than in Arabidopsis cry3
FAD
-
photolyase's essential cofactor is a non-covalently bound flavin adenine dinucleotide in fully reduced state (FADH-)
FAD
-
the physiological form of the enzyme contains a fully reduced FAD (FADH-) that is required for its activity both in vivo and in vitro. It binds a cyclobutane pyrimidine dimer (CPD) in DNA independent of light and flips the dimer out of the double helix into the active site cavity to make a stable enzyme-substrate complex. Enzyme usually purified with FAD in the blue neutral radical form. The purified enzyme can hold its radical flavin cofactor unoxidized in aerobic conditions for several days
FAD
-
absorption spectra of FADH+, FADH radical, and FADH- of wild-type and mutant enzymes, overview. All three flavin species and decays to zero upon completion of repair
FAD
-
light-induced reduction of FAD, and transfer of an electron from the photoexcited reduced FAD to the damaged DNA for cleaving the dimers
FADH2
-
enzyme contains FADH2 and a second chromophore. Enzyme with a photodecomposed second chromophore retains full activity
FADH2
-
purified enzyme contains a stable neutral radical FAD that is not active in dimer repair. Dimer repair observed with the enzyme containing FAD in the radical form at shorter wavelength is probably photoreduction of the radical FAD followed by dimer repair by enzyme-bound FADH2
FADH2
-
electron donation by excited states of enzyme-bound FADH2 is the mechanism of flavin photosensitized dimer repair by DNA photolyase
FADH2
-
computational calculations demonstrate that the localization of the FADH-donor state on the flavin ring enhances the electronic coupling between the flavin and the dimer by permitting shorter electron-transfer pathways to the dimer that have single through-space jumps. Therefore, in photolyase, the photo-excitation itself enhances the electron transfer rate by moving the electron towards the dimer
FADH2
-
heterogeneous dynamics continuously tune local configurations to optimize photolyase's function through resonance energy transfer from the antenna to the cofactor for energy efficiency and then electron transfer between the cofactor and the substrate for repair of damaged DNA
FADH2
-
photoreduction of FADH proceeds along the conserved tryptophan triad W306-W359-W382
flavin
-
enzyme contains a stable flavin radical, the one-electron reduction potential of the excited quartet state of the flavin radical must be at least 1.23 V more positive than the ground state
flavin
-
requires fully reduced flavin for photorepair of DNA, full oxidation to FAD is not necessary for biological function, the reaction mechanism involves electron transfer to the substrate from the excited state of the flavin in its fully reduced state FADH- with subsequent electron return within a nanosecond
methenyltetrahydrofolate
-
the enzyme utilizes the the antenna cofactor to harvest light energy for the repair of thymine dimers in DNA. For this purpose, the enzyme evolved to bind the cofactor and red-shift its absorption maximum by 25 nm
methenyltetrahydrofolate
-
contains the chromophore methenyltetrahydrofolate
additional information
light-driven blue light flavophotoreceptors all operate from the excited state, whether singlet oxidized (e.g., BLUF and LOV domains) or doublet semiquinone
-
additional information
-
light-driven blue light flavophotoreceptors all operate from the excited state, whether singlet oxidized (e.g., BLUF and LOV domains) or doublet semiquinone
-
additional information
FAD analogues containing either an ethano- or etheno-bridged Ade between the AN1 and AN6 atoms (e-FAD and epsilon-FAD, respectively) are used to reconstitute apo-PL, giving e-PL and epsilon-PL, respectively. The reconstitution yield of e-PL is very poor, suggesting that the hydrophobicity of the ethano group prevents its uptake, while epsilon-PL shows 50% reconstitution yield. The substrate binding constants for epsilon-PL and rPL are identical. epsilon-PL shows a 15% higher steady-state repair yield compared to FAD-reconstituted photolyase (rPL). Evaluation of an epsilon-Ade radical intermediate versus a superexchange mechanism, preparation of apophotolyase (apo-PL) and reconstitution of apo-PL with FAD, e-FAD and epsilon-FAD, overview. Incorporation of the more hydrophobic e-FAD is so inefficient that it can not be made in sufficient quantities to study further. Ligand binding structure analysis
-
METALS and IONS
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
INHIBITOR
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
ferricyanide
-
blue light irradiation converts fully oxidized FAD to the semiquinone state, but in the presence of ferricyanide as electron acceptor further photoreduction to the fully reduced catalytically active form FADH- is inhibited
yeast DNA
-
inhibition due to inferences in the binding of photolyase with UV-irradiated DNA by yeast RNA
-
ACTIVATING COMPOUND
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
ethenoadenine
the FAD analogue accelerates UV dimer repair by DNA photolyase
5,10-methenyltetrahydrofolate
-
binds to the enzyme and acts as a light-harvesting pigment
FAD
-
enzyme contains FAD as catalytic cofactor and a second chromophore as a light harvesting antenna
FADH2
-
FADH-/FADH reduction potential of the cryptochrome1 is significantly higher than that of the photolyase at 164 mV vs normal hydrogen electrode, but it also increases upon substrate binding (to 195 mV vs normal hydrogen electrode) similar to what is observed in photolyase. FADH-/FADH reduction potential is insensitive to ATP binding
FADH2
-
in wild-type, substantial losses occur prior to formation of the ultimate (FADH- 306TrpH°+) charge pair but that there is no significant kinetic development in the 100 ps-to-10 ns time window. The quantum yield of FADH- W306°+ is at 19%, in reference to the established quantum yield of the long-lived excited state of [Ru(bpy)3]2+
additional information
-
an electric field can enhance the electron transfer from the enzyme to its substrate
-
additional information
-
reduction potential is unchanged in the presence of 120× molar excess of Mg-ATP
-
KM VALUE [mM]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
additional information
additional information
reaction kinetics, absorption spectra of wild-type EcPL and MTHF antenna-free mutant E109A/N378D EcPL, transient absorption kinetics on nano- and microsecond time scales at six characteristic wavelengths, spectral analysis of transient absorption kinetics, overview
-
additional information
additional information
-
reaction kinetics, absorption spectra of wild-type EcPL and MTHF antenna-free mutant E109A/N378D EcPL, transient absorption kinetics on nano- and microsecond time scales at six characteristic wavelengths, spectral analysis of transient absorption kinetics, overview
-
additional information
additional information
substrate binding and reaction kinetics, fluorescence stopped-flow experiments, overview
-
additional information
additional information
-
substrate binding and reaction kinetics, fluorescence stopped-flow experiments, overview
-
additional information
additional information
the entire catalytic cycle is complete in 1.2 ns, and the enzyme repairs thymine dimer with a quantum yield of 0.9
-
additional information
additional information
-
the entire catalytic cycle is complete in 1.2 ns, and the enzyme repairs thymine dimer with a quantum yield of 0.9
-
TURNOVER NUMBER [1/s]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
SPECIFIC ACTIVITY [µmol/min/mg]
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
additional information
additional information
-
activity of freshly prepared holoenzyme methenyltetrahydrofolate + FADH is considered 100%. After methenyltetrahydrofolate cofactor removal the activity of the enzyme decreases to 70%, reconstituted oxidized photolyase shows the lowest activity with 34%. Activity after dithionite reduction: 83%
additional information
-
medium-pressure UV irradiation results in a greater reduction in photolyase activity than low-pressure UV radiation. It is suggested that oxidation of the flavin adenine dinucleotide in photolyase may cause the decrease in activity
pH OPTIMUM
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
TEMPERATURE OPTIMUM
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
TEMPERATURE RANGE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
additional information
-
temperature dependence of the binding enthalpy for dsDNA and damaged DNA, overview
ORGANISM
COMMENTARY
LITERATURE
UNIPROT
SEQUENCE DB
SOURCE
GENERAL INFORMATION
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
evolution
physiological function
metabolism
-
third electron transfer pathway exists in members of the photolyase family that remained undiscovered so far
additional information
evolution
CPD photolyases are highly diversified and can be subdivided into three classes (I to III), as well as single-stranded DNA (ssDNA)-specific PLs. Unrooted phylogenetic tree of the PL-CRY protein family and representative members. The class II PL is distant from the other subfamilies, critical active-site residues that vary between the class I PLs and the other subfamilies, overview
evolution
DNA photolyases (PLs) and evolutionarily related cryptochrome (CRY) blue-light receptors form a widespread superfamily of flavoproteins involved in DNA photorepair and signaling functions. They share a flavin adenine dinucleotide (FAD) cofactor and an electron-transfer (ET) chain composed typically of three tryptophan residues that connect the flavin to the protein surface
evolution
the enzyme belongs to the enzyme superfamily of photolyase/cryptochromes. Dynamics of flavin cofactor and its repair photocycles by different classes of photolyases, overview. The unified, bifurcated electron transfer mechanism elucidates the molecular origin of various repair quantum yields of different photolyases from three life kingdoms. Classes of photolyases and structures of CPD and 6-4 photolyases, overview. The diverse subfamily of CPD photolyases consists of classes I, II and III, and ssDNA PLs
evolution
the enzyme belongs to the photolyase/cryptochrome family of proteins, phylogenetic tree of the cryptochrome/photolyase family (CPF), unrooted phylogenetic tree. The cryptochrome/photolyase family (CPF) includes photoreceptors that perform different functions in different organisms. UV is responsible for the formation of two major types of damage-associated photoproducts on DNA: cyclobutane pyrimidine dimers (CPDs) and pyrimidine-pyrimidone (6-4) photoproducts (Pyr [6-4] Pyr). Two different types of photolyases were eventually discovered: CPD and (6-4) photolyases (EC 4.1.99.13). CPD photolyases repair pyrimidine dimers, while (6-4) photolyases repair Pyr[6-4]Pyr photoproducts
physiological function
CPD photolyase is a blue-light-activated enzyme that repairs ultraviolet-induced DNA damage which occurs in the form of cyclobutane pyrimidine dimers (CPDs). The enzyme uses a fully reduced flavin (FADH-) cofactor to repair sunlight-induced DNA lesions
physiological function
CPD photolyase, a flavoenzyme containing flavin adenine dinucleotide (FAD) molecule as a catalytic cofactor, repairs UV-induced DNA damage of cyclobutane pyrimidine dimer (CPD) photoproduct using blue light. The FAD cofactor, conserved in the whole protein superfamily of photolyase/cryptochromes, adopts a unique folded configuration at the active site that plays a critical functional role in DNA repair. Class I photolyase shows electron tunneling and high repair efficiency
physiological function
photolyase, a class of flavoproteins, restores damaged DNA through absorption of blue light, CPD photolyase uses blue light to repair ultraviolet-induced DNA damage, cyclobutane pyrimidine dimers (CPDs), repair dynamics and mechanisms, cyclic electron-transfer reaction photocycle, overview
physiological function
photolyases are structure-specific DNA-repair enzymes that repair DNA lesions that have been induced by ultraviolet (UV) light. Escherichia coli DNA photolyase is a DNA-repair enzyme that repairs cyclobutane pyrimidine dimers (CPDs) which are formed on DNA upon exposure of cells to ultraviolet light. The photolyase catalyzes the CPD monomerization by a light-driven electron-transfer mechanism after the enzyme-substrate complex has formed. The enzyme requires flipping of the CPD site into an extrahelical position. The photolyase is unique in that it requires the two dimerized pyrimidine bases to flip rather than just a single damaged base
physiological function
UV irradiation converts two adjacent pyrimidines, including thymines, to a cyclobutane pyrimidine dimer (CPD), and the enzyme photolyase uses blue light energy to break the two abnormal bonds joining the thymines and thus converts the thymine dimer to two normal thymines. Photolyase therefore repairs DNA and eliminates the harmful effects of UV light. The blue light-absorbing component of photolyase are chromophores. Photolyase from Escherichia coli contains two chromophores, which are two-electron reduced flavin adenine dinucleotide (FADH-) and methenyltetrahydrofolate (folate). The folate acts like a solar panel, absorbing light and transferring the excitation energy to FADH-. The flavin is the actual catalyst, and upon excitation by energy transfer from folate (and less efficiently by direct absorption of a photon) it carries out the repair reaction on the CPD by a radical mechanism through a cyclic redox reaction
physiological function
UV is responsible for the formation of damage-associated photoproducts on DNA: cyclobutane pyrimidine dimers (CPDs) and pyrimidine-pyrimidone (6-4) photoproducts (Pyr [6-4] Pyr). CPD photolyases repair pyrimidine dimers. Photolyases repair ultraviolet-induced DNA damage by a process known as photoreactivation using photons absorbed from the blue end of the light spectrum
additional information
analysis of flavin in various redox states and the active-site solvation dynamics in photolyases, and dynamics of a similar CPD biomimetic system but with low repair efficiency, overview. High repair quantum yield by CPD photolyases. Ultrafast active-site solvation dynamics in photolyases. Dynamic solvation in binding and active sites plays a critical role in protein recognition and enzyme reaction and such local motions optimize spatial configurations and minimize energetic pathways. X-ray structures and molecular dynamics (MD) simulations show certain water molecules trapped at the active sites besides charged and polar amino acids surrounding the functional chromophore of FADH-. Thus, upon excitation the local polar environments at the active sites proceed to a series of relaxations
additional information
comparison of the photoactive center of wild-type Escherichia coli enzyme with the one from Arabidopsis thaliana CRY1 enzyme
additional information
-
comparison of the photoactive center of wild-type Escherichia coli enzyme with the one from Arabidopsis thaliana CRY1 enzyme
additional information
during purification, the flavin undergoes changes in oxidation state and as a consequence the enzyme may exhibit colors ranging from purple to orange. Three-dimensional structure and structural basis for the proposed reaction mechanism, overview
additional information
-
during purification, the flavin undergoes changes in oxidation state and as a consequence the enzyme may exhibit colors ranging from purple to orange. Three-dimensional structure and structural basis for the proposed reaction mechanism, overview
additional information
enzyme structure comparisons and molecular modeling, overview. The enzyme EcPL from Escherichia coli is mesophile. There is a significant adenine-mediated superexchange contribution to the electron transfer repair reaction when CPD is complexed with the photolyases in Anacystis nidulans (mesophile) and in the two extremophiles (Thermus thermophilus and Sulfolobus tokodaii) at their physiological temperatures. In contrast, the predominant electron transfer mechanism in the Escherichia coli photolyase at its physiological temperature (37°C) is direct electron transfer, with only about 3% of the strongest electron transfer pathways mediated by adenine. Role of adenine in the CPD repair, adenine flipping
additional information
in class I EcPL, the initial electron injection adopts dominant tunneling pathways directly from LfH- to CPD. Reaction free energy profile along the reaction coordinate for EcPL CPD repair, overview
additional information
reduced anionic flavin adenine dinucleotide (FADH-) is the critical cofactor in DNA photolyase (PL) for the repair of cyclobutane pyrimidine dimers (CPD) in UV-damaged DNA. The initial step involves photoinduced electron transfer from FADH- radical to the CPD. The adenine (Ade) moiety is nearly stacked with the flavin ring, an unusual conformation compared to other FAD-dependent proteins
additional information
-
electron-tunneling pathways and functional role of adenine moiety of wild-type and mutant enzymes, overview
additional information
-
repair mechanism and the substrate specificity that distinguish enzyme CPD-PHR from enzyme (6-4) PHR, EC 4.1.99.13, which uniquely repairs (6-4) photoproducts, using Fourier transform infrared spectroscopy, overview
PDB
SCOP
CATH
UNIPROT
ORGANISM
MOLECULAR WEIGHT
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
49000
53994
SUBUNIT
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
monomer
additional information
the N-terminal alpha/beta domain is a typical dinucleotide-binding domain with five beta-sheets and five alpha-helices. The C-terminal alpha-helical domain consists of 14 alpha-helices, with structural analysis indicating a cavity at the center where the FAD is found
POSTTRANSLATIONAL MODIFICATION
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
side-chain modification
side-chain modification
-
5 mannose, 7 glucose, 3 N-acetylglucosamine in N-glycosidic linkage to Asp
side-chain modification
-
associated with RNA, approximately 10 nucleotides per enzyme molecule, containing uracil, adenine, guanine, and cytosine with no unusual bases
side-chain modification
-
associated with a small RNA that contains ribose and the four RNA bases, uracil, adenine, guanine and cytosine. 10-15 bases per enzyme monomer
side-chain modification
-
13% carbohydrate by weight, composed of mannose, galactose, glucose and N-acetylglucosamine
CRYSTALLIZATION (Commentary)
ORGANISM
UNIPROT
LITERATURE
shows an N-terminal alpha/beta domain and a C-terminal alpha-helical domain
crystal structure of DNA photolyase from Escherichia coli is representative for the entire family. Enzyme is composed of two domains: an N-terminal alpha/beta domain (residues 1-131) and a C-terminal alpha-helical domain (residues 204-471). The two domains are connected by a long and structured interdomain loop. The photoantenna chromophore (MTHF) is located in a shallow groove between the two domains, while the FADH cofactor is deeply buried within the alpha-helical domain and is tightly bound by hydrogen bonds and ionic interactions with at least 14 amino acids. Distance between the photoantenna and the catalytic chromophore is 16.8 A center to center. A surface potential representation of the enzyme reveals the presence of a positively charged groove running nearly two-thirds of the length of the molecule and, lying in the approximate center of the groove, a hole leading to the flavin cofactor.
-
crystal structure of photolyase shows that a positively charged groove on the surface of the protein which might interact with the DNA backbone and a hydrophobic cavity locates at the center. The cavity has the right dimension to hold a cis,syn cyclobutane pyrimidine dimer, and Trp277 forms one side wall of it. Photolyase binds DNA chain containing a cyclobutane pyrimidine dimer, flips it out into the cavity, and Trp277 stacks with the 5' side of the cyclobutane pyrimidine dimer by pi-pi interaction
-
PROTEIN VARIANTS
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
E274A
site-directed mutagenesis of an active site residue near the substrate side, has critical effect on repair efficiency
M345A
site-directed mutagenesis of an active site residue, has a poor effect on repair efficiency
N378C
site-directed mutagenesis of an active site residue near the cofactor side, has critical effect on repair efficiency
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
R226A
site-directed mutagenesis of an active site residue, has a poor effect on repair efficiency
R342A
W306F
E109A
E109D
-
mutant enzyme is unable to bind the methenyltetrahydrofolate cofactor under any conditions examined
E109Q
-
mutant enzyme is unable to bind the methenyltetrahydrofolate cofactor under any conditions examined
E274A
H44F
N108L
N378C
N378S
W277E
-
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
-
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
W306F
additional information
mutation of Q403 and K406 affect the affinity toward cyclobutane-pyrimidine dimer-containing DNA
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
site-directed mutagenesis of an active site residue, has a poor effect on repair efficiency
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
E109A
-
is catalytically active but unable to bind the second cofactor methenyltetrahydrofolate
H44F
-
binds and retains methenyltetrahydrofolate under normal reconstitution conditions
N108L
-
binds and retains methenyltetrahydrofolate under normal reconstitution conditions
N378C
-
site-directed mutagenesis of a residue near the flavin cofactor side
N378S
-
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
-
mutant shows no stable radical state of the cofactor FADH. Furthermore, catalytic activity is lost
W306F
-
non-photoreducible mutant, like the wild-type enzyme the mutant enzyme carries out at least 25 rounds of photorepair at the same rate
W306F
-
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
pH STABILITY
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
GENERAL STABILITY
ORGANISM
UNIPROT
LITERATURE
STORAGE STABILITY
ORGANISM
UNIPROT
LITERATURE
-70°C, glycerol to a final concentration of 50% (v/v) is added to the photolyase
-
PURIFICATION (Commentary)
ORGANISM
UNIPROT
LITERATURE
cells are destructed by sonication in the presence of 10 mM 2-mercaptoethanol. After centrifugation photolyase is purified by chromatography on heparin-Sepharose C1-6B resin by elution with a 0.1 to 1.1 M NaCl gradient in 0.01 M potassium phosphate (pH 7.0) and 10 mM mercaptoethanol, followed by chromatography on SP-Sepharose fast flow resin eluted with a 0.04 to 0.3 M NaCl gradient
protein is purified by chromatography on heparin-Sepharose CL-6B resin by elution with a 0.1-1.1 M NaCl gradient in 0.01 M potassium phosphate pH 7.0, 10 mM 2-mercaptoethanol, followed by chromatography on SP-Sepharose Fast Flow resin eluted with a 0.04-0.3 M NaCl gradient. To minimize oxidation of the FAD chromophore during purification, solutions are flushed with and kept under nitrogen
recombinant enzyme from MS09 cells by ammonium sulfate fractionation and gel filtration, followed by an adsorption chromatography, and heparin affinity chromatography
blue Sepharose column chromatography and phenyl Sepharose column chromatography
-
by using Ni2+ chelating Sepharose Fast Flow columns and Sephadex gel filtration resins. Methenyltetrahydrofolate-free photolyase is obtained by binding the C-terminal His-tagged holoenzyme to a metal-affinity column at neutral pH and washing the column with deionized water. The defolated enzyme can be eluated with 0.5 M imidazole pH 7.2. Apo-photolyase is obtained by treating the His-tagged holoenzyme with 0.5 M imidazole pH 10.0
-
cells are purified by ammonium sulfate precipitation, blue sepharose chromatography, Bio Gel P-100 chromatography and hydroxylapatite chromatography. The described procedure typically yields 15-25 mg of above 98% pure photolyase from 5 liters of culture (850 mg total protein). Quality of the enzyme may be gauged initially by the color of the preparation. Preparations that appear deep blue are of good quality. Oxidation of the neutral blue flavin radical chromophore to FADox yields preparations displaying a green or yellow color depending on the extent of oxidation.
-
one day purification: following growth and collection of cells from 5 L of culture, cells are suspended in 25 ml (per 17.5 g of cells) of bufferA (50mM HEPES (pH 7.0), 100mM NaCl, 10% sucrose (w/v), 10 mM DTT), lysed by sonication, and centrifuged. Supernatant is applied to a Blue Sepharose CL-6B column. After washing with 510 column volumes, column is developed with a gradient of 0.12 M KCl in buffer B (50 mM HEPES (pH 7.0), 10% (v/v) glycerol, 10 mM DTT). Combined blue-colored fractions are precipitated with NH4SO4 (0.43 g/ml). Pellet is dissolved in buffer B containing 50 mM NaCl, and applied to a HiPrep 26/10 desalting column, and eluted with the same buffer. Combined blue-colored fractions are applied to a heparin Sepharose CL-6B column, washed with 5 column volumes, and eluted with a gradient of 0.1-1M KCl in buffer B. Fractions are pooled and concentrated by ultrafiltration using C-30 membranes
-
purified with neutral radical FADH and/or oxidized FAD forms and then converted to a fully reduced FADH- form under anaerobic conditions
-
rapid purification method: sufficient quality for use as repair reagents, but not sufficient purity for some photophysical experiments. 5 L of cells are grown, induced with IPTG, collected, and lysed. Following collection of the NH4SO4 precipitate, proteins are suspended in 10 ml of equilibration buffer (50 mM Tris (pH 7.5), 100 mM KCl, 1 mM EDTA, 10% glycerol, 10 mM beta-ME). Mix is dialyzed for at least 4 h against 1 L of equilibration buffer. Clear dialysate is loaded onto a 50 ml Blue Sepharose column, washed with 300 ml of equilibration buffer and 300 ml of wash buffer. Elution by washing the column with high salt elution buffer (50 mM Tris (pH 7.5), 2 M KCl, 1 mM EDTA, 10% glycerol, 10 mM beta-ME). Fractions are pooled and dialyzed against 20-50 volumes of 33 mM potassium phosphate buffer (33 mM KH2PO4 (pH 6.8), 1 mM EDTA, 10% glycerol, 10 mM beta-ME). Dialysate is applied to a 10 ml hydroxylapatite column equilibrated with 33 mM potassium phosphate buffer minus glycerol. Column is washed with 75 ml of 33 mM potassium phosphate buffer and column developed with a 75 ml gradient of 33-330 mM potassium phosphate buffer, followed by a 20 ml wash with 330 mM potassium phosphate buffer. This procedure yields up to 10 mg from peak fractions that are 90% pure.
-
recombinant His6-tagged enzyme from Escherichia coli strain BL21(DE3) by cobalt affinity chromatography
-
CLONED (Commentary)
ORGANISM
UNIPROT
LITERATURE
cloned in Escherichia coli
cloned and overexpressed in Escherichia coli by using plasmid pMS969 (TetR AmpR Phr+), a derivative of ptac12 and propagation in any RecA- strain carrying Flac iQ. Optimal levels of expression are obtained using freshly transformed cells. Single colony is inoculated into 5 ml of LB and incubated at 37°C with shaking for 8 h. 1 ml of preculture is used to prepare an overnight culture consisting of 100 ml of LB. 10 ml of the overnight culture are added to 1 liter of LB and incubated with vigorous shaking at 37°C until the A600: 0.6-0.8 (3.5-4 h), at which time 2.0 ml of 0.5 M IPTG is added. Incubation is continued for another 4 h, at which point the photolyase typically constitutes 10-15% of total cellular protein
-
holophotolyase (both flavin and antenna cofactor 5,10-methenyltetrahydrofolate present) overexpressed
-
recombinant expression of His6-tagged enzyme in Escherichia coli strain BL21(DE3)
-
APPLICATION
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
analysis
-
the novel substrate (a modified thymidine 10-mer with a central CPD and all bases, except the one at the 3' end, replaced by 5,6-dihydrothymine) is a promising tool for fast and ultrafast transient absorption studies on pyrimidine dimer splitting by CPD photolyase
REF.
AUTHORS
TITLE
JOURNAL
VOL.
PAGES
YEAR
ORGANISM (UNIPROT)
PUBMED ID
SOURCE
Weinfeld, M.; Paterson, M.C.
DNA cyclobutane pyrimidine dimers with a cleaved internal phosphodiester bond can be photoenzymatically reversed by Escherichia coli PhrB photolyase
Nucleic Acids Res.
16
5693
1988
Escherichia coli
Husain, I.; Sancar, G.B.; Holbrooks, S.R.; Sancar, A.
Mechanism of damage recognition by Escherichia coli DNA photolyase
J. Biol. Chem.
262
13188-13197
1987
Escherichia coli
Payne, G.; Heelis, P.F.; Rohrs, B.R.; Sancar, A.
The active form of Escherichia coli DNA photolyase contains a fully reduced flavin and not a flavin radical, both in vivo and in vitro
Biochemistry
26
7121-7127
1987
Escherichia coli
Sancar , G.B.; Sancar, A.
Structure and function of DNA photolyases
Trends Biochem. Sci.
12
259-261
1987
Saccharomyces cerevisiae, Escherichia coli, Streptomyces griseus
-
Sutherland, B.M.; Oliveira, O.M.; Ciarrocchi, G.; Brash, D.E.; Haseltine, W.A.; Lewis, R.J.; Hanawalt, P.C.
Substrate range of the 40,000-dalton DNA-photoreactivating enzyme from Escherichia coli
Biochemistry
25
681-687
1986
Escherichia coli
Koka, P.
Stimulation of Escherichia coli DNA photoreactivating enzyme activity by adenosine 5' triphosphate
Biochemistry
23
2914-2922
1984
Escherichia coli
Sancar, A.; Smith, F.W.; Sancar, G.B.
Purification of Escherichia coli DNA photolyase
J. Biol. Chem.
259
6028-6032
1984
Escherichia coli
Sancar, A.; Sancar, G.B.
Escherichia coli DNA photolyase is a flavoprotein
J. Mol. Biol.
172
223-227
1984
Escherichia coli
Sutherland, B.M.
Photoreactivating enzymes
The Enzymes, 3rd Ed. (Boyer, P. D. , ed. )
14
481-505
1981
Synechococcus elongatus PCC 7942 = FACHB-805, Saccharomyces cerevisiae, Caluromys derbianus, Didelphis marsupialis, Escherichia coli, Homo sapiens, Potorous tridactylus, Streptomyces griseus
-
Snapka, R.M.; Sutherland, B.M.
Escherichia coli photoreactivating enzyme: purification and properties
Biochemistry
19
4201-4208
1980
Escherichia coli
Werbin, H.
DNA photolyase
Photochem. Photobiol.
26
675-678
1977
Saccharomyces cerevisiae, Guarianthe aurantiaca, Datura stramonium, Escherichia coli, Euglena gracilis, Frog, Homo sapiens, Phaseolus vulgaris, Zea mays
Snapka, R.M.; Fuselier, C.O.
Photoreactivating enzyme from Escherichia coli
Photochem. Photobiol.
25
415-420
1977
Guarianthe aurantiaca, Escherichia coli
Boatwright, D.T.; Madden, J.J.; Denson, J.; Werbin, H.
Yeast DNA photolyase: molecular weight, subunit structure, and reconstruction of active enzyme from its subunits
Biochemistry
14
5418-5421
1975
Saccharomyces cerevisiae, Escherichia coli
Heelis, P.F.; Sancar, A.
Photochemical properties of Escherichia coli DNA photolyase: a flash photolysis study
Biochemistry
25
8163-8166
1986
Escherichia coli
Hejmadi, V.S.; Verma, N.C.
Presence of RNA from yeast inhibits the photoreactivation of UV-irradiated DNA by Phr A photolyase from Escherichia coli
Indian J. Exp. Biol.
30
756-758
1992
Escherichia coli
Park, H.W.; Kim, S.T.; Sancar, A.; Deisenhofer, J.
Crystal structure of DNA photolyase from Escherichia coli [see comments
Science
268
1866-1872
1995
Escherichia coli
Heelis, P.F.; Payne, G.; Sancar, A.
Photochemical properties of Escherichia coli DNA photolyase: selective photodecomposition of the second chromophore
Biochemistry
26
4634-4640
1987
Escherichia coli
Sancar, G.B.
DNA photolyases: physical properties, action mechanism, and roles in dark repair
Mutat. Res.
236
147-160
1990
Synechococcus elongatus PCC 7942 = FACHB-805, Saccharomyces cerevisiae, Escherichia coli, Halobacterium salinarum, Methanothermobacter thermautotrophicus, Scenedesmus acutus, Streptomyces griseus
Heelis, P.F.; Okamura, T.; Sancar, A.
Excited-state properties of Escherichia coli DNA photolyase in the picocosecond to millisecond time scale
Biochemistry
29
5684-5698
1990
Escherichia coli
-
Husain, I.; Sancar, A.
Binding of E. coli DNA photolyase to a defined substrate containing a single T mean value of T dimer
Nucleic Acids Res.
15
1109-1120
1987
Escherichia coli
Thoma, F.
Light and dark in chromatin repair: repair of UV-induced DNA lesions by photolyase and nucleotide excision repair
EMBO J.
18
6585-6598
1999
Saccharomyces cerevisiae, Escherichia coli, no activity in mammalia
Christine, K.S.; MacFarlane, A.W.t.; Yang, K.; Stanley, R.J.
Cyclobutylpyrimidine dimer base flipping by DNA photolyase
J. Biol. Chem.
277
38339-38344
2002
Escherichia coli
Aubert, C.; Mathis, P.; Eker, A.P.; Brettel, K.
Intraprotein electron transfer between tyrosine and tryptophan in DNA photolyase from Anacystis nidulans
Proc. Natl. Acad. Sci. USA
96
5423-5427
1999
Escherichia coli
Nakayama, T.; Todo, T.; Notsu, S.; Nakazono, M.; Zaitsu, K.
Assay method for Escherichia coli photolyase activity using single-strand cis-syn cyclobutane pyrimidine dimer DNA as substrate
Anal. Biochem.
329
263-268
2004
Escherichia coli
Kavakli, I.H.; Sancar, A.
Analysis of the role of intraprotein electron transfer in photoreactivation by DNA photolyase in vivo
Biochemistry
43
15103-15110
2004
Escherichia coli
Henry, A.A.; Jimenez, R.; Hanway, D.; Romesberg, F.E.
Preliminary characterization of light harvesting in E. coli DNA photolyase
ChemBioChem
5
1088-1094
2004
Escherichia coli
Kapetanaki, S.M.; Ramsey, M.; Gindt, Y.M.; Schelvis, J.P.M.
Substrate electric dipole moment exerts a pH-dependent effect on electron transfer in Escherichia coli photolyase
J. Am. Chem. Soc.
126
6214-6215
2004
Escherichia coli
Hu, J.; Quek, P.H.
Effects of UV radiation on photolyase and implications with regards to photoreactivation following low- and medium-pressure UV disinfection
Appl. Environ. Microbiol.
74
327-328
2008
Escherichia coli
Yang, K.; Stanley, R.J.
Differential distortion of substrate occurs when it binds to DNA photolyase: a 2-aminopurine study
Biochemistry
45
11239-11245
2006
Escherichia coli
Byrdin, M.; Villette, S.; Eker, A.P.; Brettel, K.
Observation of an intermediate tryptophanyl radical in W306F mutant DNA photolyase from Escherichia coli supports electron hopping along the triple tryptophan chain
Biochemistry
46
10072-10077
2007
Escherichia coli (P00914), Escherichia coli
Xu, L.; Zhang, D.; Mu, W.; van Berkel, W.J.; Luo, Z.
Reversible resolution of flavin and pterin cofactors of His-tagged Escherichia coli DNA photolyase
Biochim. Biophys. Acta
1764
1454-1461
2006
Escherichia coli
Essen, L.O.; Klar, T.
Light-driven DNA repair by photolyases
Cell. Mol. Life Sci.
63
1266-1277
2006
Arabidopsis thaliana, Aspergillus nidulans, Potorous tridactylus, Escherichia coli (P00914)
Lukacs, A.; Eker, A.P.; Byrdin, M.; Villette, S.; Pan, J.; Brettel, K.; Vos, M.H.
Role of the middle residue in the triple tryptophan electron transfer chain of DNA photolyase: ultrafast spectroscopy of a Trp-->Phe mutant
J. Phys. Chem. B
110
15654-15658
2006
Escherichia coli (P00914), Escherichia coli
Yang, K.; Matsika, S.; Stanley, R.J.
6MAP, a fluorescent adenine analogue, is a probe of base flipping by DNA photolyase
J. Phys. Chem. B
111
10615-10625
2007
Escherichia coli
Sancar, G.B.; Sancar, A.
Purification and characterization of DNA photolyases
Methods Enzymol.
408
121-156
2006
Aspergillus nidulans, Saccharomyces cerevisiae, Escherichia coli
Prytkova, T.R.; Beratan, D.N.; Skourtis, S.S.
Photoselected electron transfer pathways in DNA photolyase
Proc. Natl. Acad. Sci. USA
104
802-807
2007
Escherichia coli
Xu, L.; Mu, W.; Ding, Y.; Luo, Z.; Han, Q.; Bi, F.; Wang, Y.; Song, Q.
Active site of Escherichia coli DNA photolyase: Asn378 is crucial both for stabilizing the neutral flavin radical cofactor and for DNA repair
Biochemistry
47
8736-8743
2008
Escherichia coli
Goosen, N.; Moolenaar, G.F.
Repair of UV damage in bacteria
DNA Repair
7
353-379
2008
Synechococcus elongatus PCC 7942 = FACHB-805, Escherichia coli, Thermus thermophilus, no activity in Prochlorococcus marinus
Lukacs, A.; Eker, A.P.; Byrdin, M.; Brettel, K.; Vos, M.H.
Electron hopping through the 15 A triple tryptophan molecular wire in DNA photolyase occurs within 30 ps
J. Am. Chem. Soc.
130
14394-14395
2008
Escherichia coli
Kao, Y.T.; Tan, C.; Song, S.H.; Oztuerk, N.; Li, J.; Wang, L.; Sancar, A.; Zhong, D.
Ultrafast dynamics and anionic active states of the flavin cofactor in cryptochrome and photolyase
J. Am. Chem. Soc.
130
7695-7701
2008
Escherichia coli
Balland, V.; Byrdin, M.; Eker, A.P.; Ahmad, M.; Brettel, K.
What makes the difference between a cryptochrome and DNA photolyase? A spectroelectrochemical comparison of the flavin redox transitions
J. Am. Chem. Soc.
131
426-427
2009
Synechococcus elongatus PCC 7942 = FACHB-805, Escherichia coli
Sancar, A.
Structure and function of photolyase and in vivo enzymology: 50th Anniversary
J. Biol. Chem.
283
32153-32157
2008
Escherichia coli, Streptomyces griseus, no acitivity in Haemophilus influenzae
Murphy, A.K.; Tammaro, M.; Cortazar, F.; Gindt, Y.M.; Schelvis, J.P.
Effect of the cyclobutane cytidine dimer on the properties of Escherichia coli DNA photolyase
J. Phys. Chem. B
112
15217-15226
2008
Escherichia coli, Escherichia coli pMS969
Byrdin, M.; Villette, S.; Espagne, A.; Eker, A.P.; Brettel, K.
Polarized transient absorption to resolve electron transfer between tryptophans in DNA photolyase
J. Phys. Chem. B
112
6866-6871
2008
Escherichia coli (P00914)
Yang, K.; Stanley, R.J.
The extent of DNA deformation in DNA photolyase-substrate complexes: a solution state fluorescence study
Photochem. Photobiol.
84
741-749
2008
Escherichia coli
Henbest, K.B.; Maeda, K.; Hore, P.J.; Joshi, M.; Bacher, A.; Bittl, R.; Weber, S.; Timmel, C.R.; Schleicher, E.
Magnetic-field effect on the photoactivation reaction of Escherichia coli DNA photolyase
Proc. Natl. Acad. Sci. USA
105
14395-14399
2008
Escherichia coli
Thiagarajan, V.; Villette, S.; Espagne, A.; Eker, A.P.; Brettel, K.; Byrdin, M.
DNA repair by photolyase: a novel substrate with low background absorption around 265 nm for transient absorption studies in the UV
Biochemistry
49
297-303
2010
Synechococcus elongatus PCC 7942 = FACHB-805, Escherichia coli
Mueller, M.; Carell, T.
Structural biology of DNA photolyases and cryptochromes
Curr. Opin. Struct. Biol.
19
277-285
2009
Synechococcus elongatus PCC 7942 = FACHB-805, Arabidopsis thaliana, Homo sapiens, Sulfurisphaera tokodaii, Escherichia coli (P00914), Thermus thermophilus (P61497)
Okafuji, A.; Biskup, T.; Hitomi, K.; Getzoff, E.D.; Kaiser, G.; Batschauer, A.; Bacher, A.; Hidema, J.; Teranishi, M.; Yamamoto, K.; Schleicher, E.; Weber, S.
Light-induced activation of class II cyclobutane pyrimidine dimer photolyases
DNA Repair
9
495-505
2010
Arabidopsis thaliana, Escherichia coli, Oryza sativa
Kodali, G.; Siddiqui, S.U.; Stanley, R.J.
Charge redistribution in oxidized and semiquinone E. coli DNA photolyase upon photoexcitation: stark spectroscopy reveals a rationale for the position of Trp382
J. Am. Chem. Soc.
131
4795-4807
2009
Escherichia coli (P00914), Escherichia coli
Moldt, J.; Pokorny, R.; Orth, C.; Linne, U.; Geisselbrecht, Y.; Marahiel, M.A.; Essen, L.O.; Batschauer, A.
Photoreduction of the folate cofactor in members of the photolyase family
J. Biol. Chem.
284
21670-21683
2009
Arabidopsis thaliana, Escherichia coli
Byrdin, M.; Lukacs, A.; Thiagarajan, V.; Eker, A.P.; Brettel, K.; Vos, M.H.
Quantum yield measurements of short-lived photoactivation intermediates in DNA photolyase: toward a detailed understanding of the triple tryptophan electron transfer chain
J. Phys. Chem. A
114
3207-3214
2010
Escherichia coli
Sokolowsky, K.; Newton, M.; Lucero, C.; Wertheim, B.; Freedman, J.; Cortazar, F.; Czochor, J.; Schelvis, J.P.; Gindt, Y.M.
Spectroscopic and Thermodynamic Comparisons of Escherichia coli DNA Photolyase and Vibrio cholerae Cryptochrome 1
J. Phys. Chem. B
114
7121-7130
2010
Escherichia coli, Vibrio cholerae serotype O1
Lucas-Lledo, J.I.; Lynch, M.
Evolution of mutation rates: phylogenomic analysis of the photolyase/cryptochrome family
Mol. Biol. Evol.
26
1143-1153
2009
Cytobacillus firmus, Danio rerio, Saccharomyces cerevisiae, Escherichia coli, no activity in Caenorhabditis elegans, no activity in Candida albicans, no activity in Yarrowia lipolytica, no activity in Dictyostelium discoideum, no activity in Homo sapiens, no activity in Schizosaccharomyces pombe, Salmonella enterica subsp. enterica serovar Typhimurium, Tetraodon nigroviridis, no activity in Cryptococcus neoformans, no activity in Ashbya gossypii, no activity in Guillardia theta, no activity in Caenorhabditis briggsae
Chang, C.W.; Guo, L.; Kao, Y.T.; Li, J.; Tan, C.; Li, T.; Saxena, C.; Liu, Z.; Wang, L.; Sancar, A.; Zhong, D.
Ultrafast solvation dynamics at binding and active sites of photolyases
Proc. Natl. Acad. Sci. USA
107
2914-2919
2010
Caulobacter vibrioides, Escherichia coli
Chaves, I.; Pokorny, R.; Byrdin, M.; Hoang, N.; Ritz, T.; Brettel, K.; Essen, L.O.; van der Horst, G.T.; Batschauer, A.; Ahmad, M.
The cryptochromes: blue light photoreceptors in plants and animals
Annu. Plant Biol.
62
335-364
2011
Synechococcus elongatus PCC 7942 = FACHB-805, Escherichia coli
Brettel, K.; Byrdin, M.
Reaction mechanisms of DNA photolyase
Curr. Opin. Struct. Biol.
20
693-701
2010
Synechococcus elongatus PCC 7942 = FACHB-805, Escherichia coli
Xu, L.; Zhu, G.
The roles of several residues of Escherichia coli DNA photolyase in the highly efficient photo-repair of cyclobutane pyrimidine dimers
J. Nucleic Acids
2010
794782
2010
Escherichia coli
Wijaya, I.M.; Zhang, Y.; Iwata, T.; Yamamoto, J.; Hitomi, K.; Iwai, S.; Getzoff, E.D.; Kandori, H.
Detection of distinct alpha-helical rearrangements of cyclobutane pyrimidine dimer photolyase upon substrate binding by Fourier transform infrared spectroscopy
Biochemistry
52
1019-1027
2013
Escherichia coli
Wilson, T.J.; Crystal, M.A.; Rohrbaugh, M.C.; Sokolowsky, K.P.; Gindt, Y.M.
Evidence from thermodynamics that DNA photolyase recognizes a solvent-exposed CPD lesion
J. Phys. Chem. B
115
13746-13754
2011
Escherichia coli
Liu, Z.; Tan, C.; Guo, X.; Kao, Y.T.; Li, J.; Wang, L.; Sancar, A.; Zhong, D.
Dynamics and mechanism of cyclobutane pyrimidine dimer repair by DNA photolyase
Proc. Natl. Acad. Sci. USA
108
14831-14836
2011
Escherichia coli
Sancar, A.
Mechanisms of DNA repair by photolyase and excision nuclease (Nobel lecture)
Angew. Chem. Int. Ed. Engl.
55
8502-8527
2016
Escherichia coli (P00914), Escherichia coli
Zhang, M.; Wang, L.; Zhong, D.
Photolyase dynamics and electron-transfer mechanisms of DNA repair
Arch. Biochem. Biophys.
632
158-174
2017
Caulobacter vibrioides (A0A0H3C7H5), Escherichia coli (P00914), Synechococcus elongatus PCC 7942 = FACHB-805 (P05327), Arabidopsis thaliana (Q84KJ5), Arabidopsis thaliana (Q9SB00), Methanosarcina mazei (Q8PYK9), Synechococcus elongatus PCC 7942 = FACHB-805 ATCC 27144 / PCC 6301 / SAUG 1402/1 (P05327), Caulobacter vibrioides NA1000/CB15N (A0A0H3C7H5), Methanosarcina mazei ATCC BAA-159 (Q8PYK9)
Schelvis, J.P.; Zhu, X.; Gindt, Y.M.
Enzyme-substrate binding kinetics indicate that photolyase recognizes an extrahelical cyclobutane thymidine dimer
Biochemistry
54
6176-6185
2015
Escherichia coli (P00914), Escherichia coli
Mueller, P.; Brettel, K.; Grama, L.; Nyitrai, M.; Lukacs, A.
Photochemistry of wild-type and N378D mutant E. coli DNA photolyase with oxidized FAD cofactor studied by transient absorption spectroscopy
Chemphyschem
17
1329-1340
2016
Escherichia coli (P00914), Escherichia coli
Rousseau, B.J.G.; Shafei, S.; Migliore, A.; Stanley, R.J.; Beratan, D.N.
Determinants of photolyases DNA repair mechanism in Mesophiles and Extremophiles
J. Am. Chem. Soc.
140
2853-2861
2018
Sulfurisphaera tokodaii (F9VNB1), Escherichia coli (P00914), Synechococcus elongatus PCC 7942 = FACHB-805 (P05327), Thermus thermophilus (P61497), Synechococcus elongatus PCC 7942 = FACHB-805 ATCC 27144 / PCC 6301 / SAUG 1402/1 (P05327), Thermus thermophilus HB8 / ATCC 27634 / DSM 579 (P61497), Sulfurisphaera tokodaii DSM 16993 / JCM 10545 / NBRC 100140 / 7 (F9VNB1)
Narayanan, M.; Singh, V.R.; Kodali, G.; Moravcevic, K.; Stanley, R.J.
An ethenoadenine FAD analog accelerates UV dimer repair by DNA photolyase
Photochem. Photobiol.
93
343-354
2017
Escherichia coli (P00914)
Kavakli, I.H.; Baris, I.; Tardu, M.; Guel, S.; Oener, H.; Cal, S.; Bulut, S.; Yarparvar, D.; Berkel, C.; Ustaoglu, P.; Aydin, C.
The photolyase/cryptochrome family of proteins as DNA repair enzymes and transcriptional repressors
Photochem. Photobiol.
93
93-103
2017
Cyanidioschyzon merolae (M1V3I5), Escherichia coli (P00914), Vibrio cholerae serotype O1 (Q9KNA8), Vibrio cholerae serotype O1 (Q9KR33), Vibrio cholerae serotype O1 ATCC 39315 / El Tor Inaba N16961 (Q9KNA8), Vibrio cholerae serotype O1 ATCC 39315 / El Tor Inaba N16961 (Q9KR33), Cyanidioschyzon merolae 10D (M1V3I5)
Liu, Z.; Wang, L.; Zhong, D.
Dynamics and mechanisms of DNA repair by photolyase
Phys. Chem. Chem. Phys.
17
11933-11949
2015
Escherichia coli (P00914)
Zhang, M.; Wang, L.; Shu, S.; Sancar, A.; Zhong, D.
Bifurcating electron-transfer pathways in DNA photolyases determine the repair quantum yield
Science
354
209-213
2016
Caulobacter vibrioides (A0A0H3C7H5), Escherichia coli (P00914), Synechococcus elongatus PCC 7942 = FACHB-805 (P05327), Drosophila melanogaster (Q24443), Arabidopsis thaliana (Q84KJ5), Arabidopsis thaliana (Q9SB00), Caulobacter vibrioides NA1000 / CB15N (A0A0H3C7H5), Synechococcus elongatus PCC 7942 = FACHB-805 ATCC 27144 / PCC 6301 / SAUG 1402/1 (P05327)
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