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Aziz Sancar - One of the best experts on this subject based on the ideXlab platform.
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bifurcating electron transfer pathways in dna Photolyases determine the repair quantum yield
Science, 2016Co-Authors: Meng Zhang, Lijuan Wang, Aziz Sancar, Dongping ZhongAbstract:Photolyase is a blue-light–activated enzyme that repairs ultraviolet-induced DNA damage that occurs in the form of cyclobutane pyrimidine dimers (CPDs) and pyrimidine-pyrimidone (6-4) photoproducts. Previous studies on microbial Photolyases have revealed an electron-tunneling pathway that is critical for the repair mechanism. In this study, we used femtosecond spectroscopy to deconvolute seven electron-transfer reactions in 10 elementary steps in all classes of CPD Photolyases. We report a 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. Both bifurcation routes are operative, but their relative contributions, dictated by the reduction potentials of the flavin cofactor and the substrate, determine the overall quantum yield of repair.
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the second chromophore in drosophila Photolyase cryptochrome family photoreceptors
Biochemistry, 2012Co-Authors: Christopher P. Selby, Aziz SancarAbstract:The Photolyase/cryptochrome family of proteins are FAD-containing flavoproteins which carry out blue-light-dependent functions including DNA repair, plant growth and development, and regulation of the circadian clock. In addition to FAD, many members of the family contain a second chromophore which functions as a photoantenna, harvesting light and transferring the excitation energy to FAD and thus increasing the efficiency of the system. The second chromophore is methenyltetrahydrofolate (MTHF) in most Photolyases characterized to date and FAD, FMN, or 5-deazariboflavin in others. To date, no second chromophore has been identified in cryptochromes. Drosophila contains three members of the cryptochrome/Photolyase family: cyclobutane pyrimidine dimer (CPD) Photolyase, (6–4) photoproduct Photolyase, and cryptochrome. We developed an expression system capable of incorporating all known second chromophores into the cognate cryptochrome/Photolyase family members. Using this system, we demonstrate that Drosophil...
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purification and characterization of a type iii Photolyase from caulobacter crescentus
Biochemistry, 2008Co-Authors: Nuri Ozturk, Dongping Zhong, Carrie L Partch, Christopher P. Selby, Ya Ting Kao, Halil I Kavakli, Aziz SancarAbstract:The Photolyase/cryptochrome family is a large family of flavoproteins that encompasses DNA repair proteins, Photolyases, and cryptochromes that regulate blue-light-dependent growth and development in plants, and light-dependent and light-independent circadian clock setting in animals. Phylogenetic analysis has revealed a new class of the family, named type III Photolyase, which cosegregates with plant cryptochromes. Here we describe the isolation and characterization of a type III Photolyase from Caulobacter crescentus. Spectroscopic analysis shows that the enzyme contains both the methenyl tetrahydrofolate photoantenna and the FAD catalytic cofactor. Biochemical analysis shows that it is a bona fide Photolyase that repairs cyclobutane pyrimidine dimers. Mutation of an active site Trp to Arg disrupts FAD binding with no measurable effect on MTHF binding. Using enzyme preparations that contain either both chromophores or only folate, we were able to determine the efficiency and rate of transfer of energy from MTHF to FAD.
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ultrafast dynamics and anionic active states of the flavin cofactor in cryptochrome and Photolyase
Journal of the American Chemical Society, 2008Co-Authors: Ya Ting Kao, Nuri Ozturk, Sang-hun Song, Aziz Sancar, Lijuan Wang, Chuang Tan, Dongping ZhongAbstract:We report here our systematic studies of the dynamics of four redox states of the flavin cofactor in both Photolyases and insect type 1 cryptochromes. With femtosecond resolution, we observed ultrafast photoreduction of oxidized state flavin adenine dinucleotide (FAD) in subpicosecond and of neutral radical semiquinone (FADH(*)) in tens of picoseconds through intraprotein electron transfer mainly with a neighboring conserved tryptophan triad. Such ultrafast dynamics make these forms of flavin unlikely to be the functional states of the Photolyase/cryptochrome family. In contrast, we find that upon excitation the anionic semiquinone (FAD(*-)) and hydroquinone (FADH(-)) have longer lifetimes that are compatible with high-efficiency intermolecular electron transfer reactions. In Photolyases, the excited active state (FADH(-)*) has a long (nanosecond) lifetime optimal for DNA-repair function. In insect type 1 cryptochromes known to be blue-light photoreceptors the excited active form (FAD(*-)*) has complex deactivation dynamics on the time scale from a few to hundreds of picoseconds, which is believed to occur through conical intersection(s) with a flexible bending motion to modulate the functional channel. These unique properties of anionic flavins suggest a universal mechanism of electron transfer for the initial functional steps of the Photolyase/cryptochrome blue-light photoreceptor family.
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Structure and Function of Photolyase and in Vivo Enzymology: 50th Anniversary
Journal of Biological Chemistry, 2008Co-Authors: Aziz SancarAbstract:Photoreactivation is the reversal of the harmful effects of far-UV radiation (200-300 nm) on organisms, such as growth delay, mutation, cell death, and cancer, by concomitant or subsequent exposure of the organism to near-UV/blue light (300-500 nm). The two major lesions induced in DNA by UV light are cyclobutane pyrimidine dimers (Pyr Pyr(2) or CPD), which constitute similar to 80-90% of the photoproducts, and pyrimidine-pyrimidone (6-4) photoproducts (Pyr[6-4] Pyr), which account for the 10-20% of the UV lesions. Photoreactivation results from the repair of these lesions in situ by flavoproteins called photoreactivating enzymes (Photolyase) that use a blue-light photon as a co-substrate. Photolyases that repair these two photoproducts are evolutionarily related but functionally distinct. Enzymes that repair CPDs are referred to as CPD Photolyase, and enzymes that repair (6-4) photoproducts are called (6-4) Photolyase. For historical reasons and as a matter of common practice, the term "Photolyase" without further qualification means CPD Photolyase, and it will be used as such in this review, which celebrates the 50th anniversary of the discovery of Photolyase.
Takeshi Todo - One of the best experts on this subject based on the ideXlab platform.
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light induced conformational change and product release in dna repair by 6 4 Photolyase
Journal of the American Chemical Society, 2011Co-Authors: Masato Kondoh, Takeshi Todo, Junpei Yamamoto, Kenichi Hitomi, Shigenori Iwai, Elizabeth D. Getzoff, Masahide TerazimaAbstract:Proteins of the cryptochrome/Photolyase family share high sequence similarities, common folds, and the flavin adenine dinucleotide (FAD) cofactor, but exhibit diverse physiological functions. Mammalian cryptochromes are essential regulatory components of the 24 h circadian clock, whereas (6-4) Photolyases recognize and repair UV-induced DNA damage by using light energy absorbed by FAD. Despite increasing knowledge about physiological functions from genetic analyses, the molecular mechanisms and conformational dynamics involved in clock signaling and DNA repair remain poorly understood. The (6-4) Photolyase, which has strikingly high similarity to human clock cryptochromes, is a prototypic biological system to study conformational dynamics of cryptochrome/Photolyase family proteins. The entire light-dependent DNA repair process for (6-4) Photolyase can be reproduced in a simple in vitro system. To decipher pivotal reactions of the common FAD cofactor, we accomplished time-resolved measurements of radical formation, diffusion, and protein conformational changes during light-dependent repair by full-length (6-4) Photolyase on DNA carrying a single UV-induced damage. The (6-4) Photolyase by itself showed significant volume changes after blue-light activation, indicating protein conformational changes distant from the flavin cofactor. A drastic diffusion change was observed only in the presence of both (6-4) Photolyase and damaged DNA, and not for (6-4) Photolyase alone or with undamaged DNA. Thus, we propose that this diffusion change reflects the rapid (50 μs time constant) dissociation of the protein from the repaired DNA product. Conformational changes with such fast turnover would likely enable DNA repair Photolyases to access the entire genome in cells.
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diatom ptcpf1 is a new cryptochrome Photolyase family member with dna repair and transcription regulation activity
EMBO Reports, 2009Co-Authors: Sacha Coesel, Manuela Mangogna, Tomoko Ishikawa, Marc Heijde, Alessandra Rogato, Takeshi Todo, Giovanni Finazzi, Chris Bowler, Angela FalciatoreAbstract:Members of the cryptochrome/Photolyase family (CPF) are widely distributed throughout all kingdoms, and encode photosensitive proteins that typically show either photoreceptor or DNA repair activity. Animal and plant cryptochromes have lost DNA repair activity and now perform specialized photoperceptory functions, for example, plant cryptochromes regulate growth and circadian rhythms, whereas mammalian and insect cryptochromes act as transcriptional repressors that control the circadian clock. However, the functional differentiation between Photolyases and cryptochromes is now being questioned. Here, we show that the PtCPF1 protein from the marine diatom Phaeodactylum tricornutum shows 6-4 photoproduct repair activity and can act as a transcriptional repressor of the circadian clock in a heterologous mammalian cell system. Conversely, it seems to have a wide role in blue-light-regulated gene expression in diatoms. The protein might therefore represent a missing link in the evolution of CPFs, and act as a novel ultraviolet/blue light sensor in marine environments.
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similarities and differences between cyclobutane pyrimidine dimer Photolyase and 6 4 Photolyase as revealed by resonance raman spectroscopy electron transfer from the fad cofactor to ultraviolet damaged dna
Journal of Biological Chemistry, 2006Co-Authors: Takeshi Uchida, Takeshi Todo, Teizo KitagawaAbstract:Abstract The cyclobutane pyrimidine dimer (CPD) and (6-4) photoproduct, two major types of DNA damage caused by UV light, are repaired under illumination with near UV-visible light by CPD and (6-4) Photolyases, respectively. To understand the mechanism of DNA repair, we examined the resonance Raman spectra of complexes between damaged DNA and the neutral semiquinoid and oxidized forms of (6-4) and CPD Photolyases. The marker band for a neutral semiquinoid flavin and band I of the oxidized flavin, which are derived from the vibrations of the benzene ring of FAD, were shifted to lower frequencies upon binding of damaged DNA by CPD Photolyase but not by (6-4) Photolyase, indicating that CPD interacts with the benzene ring of FAD directly but that the (6-4) photoproduct does not. Bands II and VII of the oxidized flavin and the 1398/1391 cm-1 bands of the neutral semiquinoid flavin, which may reflect the bending of U-shaped FAD, were altered upon substrate binding, suggesting that CPD and the (6-4) photoproduct interact with the adenine ring of FAD. When substrate was bound, there was an upshifted 1528 cm-1 band of the neutral semiquinoid flavin in CPD Photolyase, indicating a weakened hydrogen bond at N(5)-H of FAD, and band X seemed to be downshifted in (6-4) Photolyase, indicating a weakened hydrogen bond at N(3)-H of FAD. These Raman spectra led us to conclude that the two Photolyases have different electron transfer mechanisms as well as different hydrogen bonding environments, which account for the higher redox potential of CPD Photolyase.
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investigation of the cyclobutane pyrimidine dimer cpd Photolyase dna recognition mechanism by nmr analyses
Journal of Biological Chemistry, 2004Co-Authors: Takuya Torizawa, Kosuke Morikawa, Takeshi Todo, Kenichi Hitomi, Shigenori Iwai, Seiki Kuramitsu, Takumi Ueda, Ichio ShimadaAbstract:Abstract The cyclobutane pyrimidine dimer (CPD) is one of the major forms of DNA damage caused by irradiation with ultraviolet (UV) light. CPD Photolyases recognize and repair UV-damaged DNA. The DNA recognition mechanism of the CPD Photolyase has remained obscure because of a lack of structural information about DNA-CPD Photolyase complexes. In order to elucidate the CPD Photolyase DNA binding mode, we performed NMR analyses of the DNA-CPD Photolyase complex. Based upon results from 31P NMR measurements, in combination with site-directed mutagenesis, we have demonstrated the orientation of CPD-containing single-stranded DNA (ssDNA) on the CPD Photolyase. In addition, chemical shift perturbation analyses, using stable isotope-labeled DNA, revealed that the CPD is buried in a cavity within CPD Photolyase. Finally, NMR analyses of a double-stranded DNA (dsDNA)-CPD Photolyase complex indicated that the CPD is flipped out of the dsDNA by the enzyme, to gain access to the active site.
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role of two histidines in the 6 4 Photolyase reaction
Journal of Biological Chemistry, 2001Co-Authors: Kenichi Hitomi, Tomoko Ishikawa, Shigenori Iwai, Haruki Nakamura, Sang Tae Kim, Toshimi Mizukoshi, Takeshi TodoAbstract:The reaction mechanism of Xenopus(6-4) Photolyase was investigated using several mutant enzymes. In the active site, which is homologous between thecis,syn-cyclobutane pyrimidine dimer and (6-4) Photolyases, four amino acid residues that are specific to (6-4) Photolyase, Gln288, His354, Leu355, and His358, and two conserved tryptophans, Trp291and Trp398, were substituted with alanine. Only the L355A mutant had a lower affinity for the substrate, which suggested a hydrophobic interaction with the (6-4) photoproduct. Both the H354A and H358A mutations resulted in an almost complete loss of the repair activity, although the Trp291 and Trp398mutants retained some activity. Taking the pH profile of the (6-4) Photolyase reaction into consideration with this observation, we propose a mechanism in which these histidines catalyze the formation of the four-membered ring intermediate in the repair process of this enzyme. When deuterium oxide was used as a solvent, the repair activity was decreased. The proton transfer shown by this isotope effect supports the proposed mechanism. The substrate binding and the reaction mechanism are discussed in detail using a molecular model.
Dongping Zhong - One of the best experts on this subject based on the ideXlab platform.
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a compass at weak magnetic fields using thymine dimer repair
ACS central science, 2018Co-Authors: Theodore J. Zwang, Dongping Zhong, Jacqueline K. BartonAbstract:How birds sense the variations in Earth’s magnetic field for navigation is poorly understood, although cryptochromes, proteins homologous to Photolyases, have been proposed to participate in this magnetic sensing. Here, in electrochemical studies with an applied magnetic field, we monitor the repair of cyclobutane pyrimidine dimer lesions in duplex DNA by Photolyase, mutants of Photolyase, and a modified cryptochrome. We find that the yield of dimer repair is dependent on the strength and angle of the applied magnetic field even when using magnetic fields weaker than 1 gauss. This high sensitivity to weak magnetic fields depends upon a fast radical pair reaction on the thymines leading to repair. These data illustrate chemically how cyclobutane pyrimidine dimer repair may be used in a biological compass informed by variations in Earth’s magnetic field.
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A Compass at Weak Magnetic Fields Using Thymine Dimer Repair
2018Co-Authors: Theodore J. Zwang, Dongping Zhong, Edmund C. M. Tse, Jacqueline K. BartonAbstract:How birds sense the variations in Earth’s magnetic field for navigation is poorly understood, although cryptochromes, proteins homologous to Photolyases, have been proposed to participate in this magnetic sensing. Here, in electrochemical studies with an applied magnetic field, we monitor the repair of cyclobutane pyrimidine dimer lesions in duplex DNA by Photolyase, mutants of Photolyase, and a modified cryptochrome. We find that the yield of dimer repair is dependent on the strength and angle of the applied magnetic field even when using magnetic fields weaker than 1 gauss. This high sensitivity to weak magnetic fields depends upon a fast radical pair reaction on the thymines leading to repair. These data illustrate chemically how cyclobutane pyrimidine dimer repair may be used in a biological compass informed by variations in Earth’s magnetic field
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Photolyase dynamics and electron transfer mechanisms of dna repair
Archives of Biochemistry and Biophysics, 2017Co-Authors: Meng Zhang, Lijuan Wang, Dongping ZhongAbstract:Photolyase, a flavoenzyme containing flavin adenine dinucleotide (FAD) molecule as a catalytic cofactor, repairs UV-induced DNA damage of cyclobutane pyrimidine dimer (CPD) and pyrimidine-pyrimidone (6-4) 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. Here, we review our comprehensive characterization of the dynamics of flavin cofactor and its repair photocycles by different classes of Photolyases on the most fundamental level. Using femtosecond spectroscopy and molecular biology, significant advances have recently been made to map out the entire dynamical evolution and determine actual timescales of all the catalytic processes in Photolyases. 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. The unified, bifurcated ET mechanism elucidates the molecular origin of various repair quantum yields of different Photolyases from three life kingdoms. For 6-4 photoproduct repair, a similar cyclic ET mechanism operates and a new cyclic proton transfer with a conserved histidine residue at the active site of (6-4) Photolyases is revealed.
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Photolyase: Dynamics and Mechanisms of Repair of Sun-Induced DNA Damage.
Photochemistry and Photobiology, 2017Co-Authors: Meng Zhang, Lijuan Wang, Dongping ZhongAbstract:Photolyase, a photomachine discovered half a century ago for repair of sun-induced DNA damage of cyclobutane pyrimidine dimers (CPDs) and pyrimidine (6-4) pyrimidone photoproducts (6-4PPs), has been characterized extensively in biochemistry (function), structure and dynamics since 1980s. The molecular mechanism and repair photocycle have been revealed at the most fundamental level. Using femtosecond spectroscopy, we have mapped out the entire dynamical evolution and determined all actual timescales of the catalytic processes. Here, we review our recent efforts in studies of the dynamics of DNA repair by Photolyases. The repair of CPDs in three life kingdoms includes seven electron transfer (ET) reactions among 10 elementary steps through initial bifurcating ET pathways, a direct tunneling route and a two-step hopping path both through an intervening adenine from the cofactor to CPD, with a conserved folded structure at the active site. The repair of 6-4PPs is challenging and requires similar ET reactions and a new cyclic proton transfer with a conserved histidine residue at the active site of (6-4) Photolyases. Finally, we also summarize our efforts on multiple intraprotein ET of Photolyases in different redox states and such mechanistic studies are critical to the functional mechanism of homologous cryptochromes of blue-light photoreceptors.
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bifurcating electron transfer pathways in dna Photolyases determine the repair quantum yield
Science, 2016Co-Authors: Meng Zhang, Lijuan Wang, Aziz Sancar, Dongping ZhongAbstract:Photolyase is a blue-light–activated enzyme that repairs ultraviolet-induced DNA damage that occurs in the form of cyclobutane pyrimidine dimers (CPDs) and pyrimidine-pyrimidone (6-4) photoproducts. Previous studies on microbial Photolyases have revealed an electron-tunneling pathway that is critical for the repair mechanism. In this study, we used femtosecond spectroscopy to deconvolute seven electron-transfer reactions in 10 elementary steps in all classes of CPD Photolyases. We report a 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. Both bifurcation routes are operative, but their relative contributions, dictated by the reduction potentials of the flavin cofactor and the substrate, determine the overall quantum yield of repair.
Kenichi Hitomi - One of the best experts on this subject based on the ideXlab platform.
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detection of distinct α helical rearrangements of cyclobutane pyrimidine dimer Photolyase upon substrate binding by fourier transform infrared spectroscopy
Biochemistry, 2013Co-Authors: Made Mahaputra I Wijaya, Junpei Yamamoto, Tatsuya Iwata, Kenichi Hitomi, Shigenori Iwai, Elizabeth D. Getzoff, Yu Zhang, Hideki KandoriAbstract:Photolyases (PHRs) utilize near-ultraviolet (UV)–blue light to specifically repair the major photoproducts (PPs) of UV-induced damaged DNA. The cyclobutane pyrimidine dimer PHR (CPD-PHR) from Escherichia coli binds flavin adenine dinucleotide (FAD) as a cofactor and 5,10-methenyltetrahydrofolate as a light-harvesting pigment and specifically repairs CPD lesions. By comparison, a second Photolyase known as (6–4) PHR, present in a range of higher organisms, uniquely repairs (6–4) PPs. To understand the repair mechanism and the substrate specificity that distinguish CPD-PHR from (6–4) PHR, we applied Fourier transform infrared (FTIR) spectroscopy to bacterial CPD-PHR in the presence or absence of a well-defined DNA substrate, as we have studied previously for vertebrate (6–4) PHR. PHRs show light-induced reduction of FAD, and photorepair by CPD-PHR 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. Here, we measur...
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light induced conformational change and product release in dna repair by 6 4 Photolyase
Journal of the American Chemical Society, 2011Co-Authors: Masato Kondoh, Takeshi Todo, Junpei Yamamoto, Kenichi Hitomi, Shigenori Iwai, Elizabeth D. Getzoff, Masahide TerazimaAbstract:Proteins of the cryptochrome/Photolyase family share high sequence similarities, common folds, and the flavin adenine dinucleotide (FAD) cofactor, but exhibit diverse physiological functions. Mammalian cryptochromes are essential regulatory components of the 24 h circadian clock, whereas (6-4) Photolyases recognize and repair UV-induced DNA damage by using light energy absorbed by FAD. Despite increasing knowledge about physiological functions from genetic analyses, the molecular mechanisms and conformational dynamics involved in clock signaling and DNA repair remain poorly understood. The (6-4) Photolyase, which has strikingly high similarity to human clock cryptochromes, is a prototypic biological system to study conformational dynamics of cryptochrome/Photolyase family proteins. The entire light-dependent DNA repair process for (6-4) Photolyase can be reproduced in a simple in vitro system. To decipher pivotal reactions of the common FAD cofactor, we accomplished time-resolved measurements of radical formation, diffusion, and protein conformational changes during light-dependent repair by full-length (6-4) Photolyase on DNA carrying a single UV-induced damage. The (6-4) Photolyase by itself showed significant volume changes after blue-light activation, indicating protein conformational changes distant from the flavin cofactor. A drastic diffusion change was observed only in the presence of both (6-4) Photolyase and damaged DNA, and not for (6-4) Photolyase alone or with undamaged DNA. Thus, we propose that this diffusion change reflects the rapid (50 μs time constant) dissociation of the protein from the repaired DNA product. Conformational changes with such fast turnover would likely enable DNA repair Photolyases to access the entire genome in cells.
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light induced activation of class ii cyclobutane pyrimidine dimer Photolyases
DNA Repair, 2010Co-Authors: Asako Okafuji, Kenichi Hitomi, Elizabeth D. Getzoff, Till Biskup, Gebhard Kaiser, Alfred Batschauer, Adelbert Bacher, Jun HidemaAbstract:Abstract Light-induced activation of class II cyclobutane pyrimidine dimer (CPD) Photolyases of Arabidopsis thaliana and Oryza sativa has been examined by UV/Vis and pulsed Davies-type electron-nuclear double resonance (ENDOR) spectroscopy, and the results compared with structure-known class I enzymes, CPD Photolyase and (6–4) Photolyase. By ENDOR spectroscopy, the local environment of the flavin adenine dinucleotide (FAD) cofactor is probed by virtue of proton hyperfine couplings that report on the electron-spin density at the positions of magnetic nuclei. Despite the amino-acid sequence dissimilarity as compared to class I enzymes, the results indicate similar binding motifs for FAD in the class II Photolyases. Furthermore, the photoreduction kinetics starting from the FAD cofactor in the fully oxidized redox state, FAD ox , have been probed by UV/Vis spectroscopy. In Escherichia coli (class I) CPD Photolyase, light-induced generation of FADH from FAD ox , and subsequently FADH − from FADH , proceeds in a step-wise fashion via a chain of tryptophan residues. These tryptophans are well conserved among the sequences and within all known structures of class I Photolyases, but completely lacking from the equivalent positions of class II Photolyase sequences. Nevertheless, class II Photolyases show photoreduction kinetics similar to those of the class I enzymes. We propose that a different, but also effective, electron-transfer cascade is conserved among the class II Photolyases. The existence of such electron transfer pathways is supported by the observation that the catalytically active fully reduced flavin state obtained by photoreduction is maintained even under oxidative conditions in all three classes of enzymes studied in this contribution.
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investigation of the cyclobutane pyrimidine dimer cpd Photolyase dna recognition mechanism by nmr analyses
Journal of Biological Chemistry, 2004Co-Authors: Takuya Torizawa, Kosuke Morikawa, Takeshi Todo, Kenichi Hitomi, Shigenori Iwai, Seiki Kuramitsu, Takumi Ueda, Ichio ShimadaAbstract:Abstract The cyclobutane pyrimidine dimer (CPD) is one of the major forms of DNA damage caused by irradiation with ultraviolet (UV) light. CPD Photolyases recognize and repair UV-damaged DNA. The DNA recognition mechanism of the CPD Photolyase has remained obscure because of a lack of structural information about DNA-CPD Photolyase complexes. In order to elucidate the CPD Photolyase DNA binding mode, we performed NMR analyses of the DNA-CPD Photolyase complex. Based upon results from 31P NMR measurements, in combination with site-directed mutagenesis, we have demonstrated the orientation of CPD-containing single-stranded DNA (ssDNA) on the CPD Photolyase. In addition, chemical shift perturbation analyses, using stable isotope-labeled DNA, revealed that the CPD is buried in a cavity within CPD Photolyase. Finally, NMR analyses of a double-stranded DNA (dsDNA)-CPD Photolyase complex indicated that the CPD is flipped out of the dsDNA by the enzyme, to gain access to the active site.
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role of two histidines in the 6 4 Photolyase reaction
Journal of Biological Chemistry, 2001Co-Authors: Kenichi Hitomi, Tomoko Ishikawa, Shigenori Iwai, Haruki Nakamura, Sang Tae Kim, Toshimi Mizukoshi, Takeshi TodoAbstract:The reaction mechanism of Xenopus(6-4) Photolyase was investigated using several mutant enzymes. In the active site, which is homologous between thecis,syn-cyclobutane pyrimidine dimer and (6-4) Photolyases, four amino acid residues that are specific to (6-4) Photolyase, Gln288, His354, Leu355, and His358, and two conserved tryptophans, Trp291and Trp398, were substituted with alanine. Only the L355A mutant had a lower affinity for the substrate, which suggested a hydrophobic interaction with the (6-4) photoproduct. Both the H354A and H358A mutations resulted in an almost complete loss of the repair activity, although the Trp291 and Trp398mutants retained some activity. Taking the pH profile of the (6-4) Photolyase reaction into consideration with this observation, we propose a mechanism in which these histidines catalyze the formation of the four-membered ring intermediate in the repair process of this enzyme. When deuterium oxide was used as a solvent, the repair activity was decreased. The proton transfer shown by this isotope effect supports the proposed mechanism. The substrate binding and the reaction mechanism are discussed in detail using a molecular model.
Junpei Yamamoto - One of the best experts on this subject based on the ideXlab platform.
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key interactions with deazariboflavin cofactor for light driven energy transfer in xenopus 6 4 Photolyase
Photochemical and Photobiological Sciences, 2021Co-Authors: Ayaka Morimoto, Shigenori Iwai, Yuhei Hosokawa, Hiromu Miyamoto, Rajiv Kumar Verma, Ryuma Sato, Junpei YamamotoAbstract:Photolyases are flavoenzymes responsible for light-driven repair of carcinogenic crosslinks formed in DNA by UV exposure. They possess two non-covalently bound chromophores: flavin adenine dinucleotide (FAD) as a catalytic center and an auxiliary antenna chromophore that harvests photons and transfers solar energy to the catalytic center. Although the energy transfer reaction has been characterized by time-resolved spectroscopy, it is strikingly important to understand how well natural biological systems organize the chromophores for the efficient energy transfer. Here, we comprehensively characterized the binding of 8-hydroxy-7,8-didemethyl-5-deazariboflavin (8-HDF) to Xenopus (6-4) Photolyase. In silico simulations indicated that a hydrophobic amino acid residue located at the entrance of the binding site dominates translocation of a loop upon binding of 8-HDF, and a mutation of this residue caused dysfunction of the efficient energy transfer in the DNA repair reaction. Mutational analyses of the protein combined with modification of the chromophore suggested that Coulombic interactions between positively charged residues in the protein and the phenoxide moiety in 8-HDF play a key role in accommodation of 8-HDF in the proper direction. This study provides a clear evidence that Xenopus (6-4) Photolyase can utilize 8-HDF as the light-harvesting chromophore. The obtained new insights into binding of the natural antenna molecule will be helpful for the development of artificial light-harvesting chromophores and future characterization of the energy transfer in (6-4) Photolyase by spectroscopic studies.
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Twist and turn: a revised structural view on the unpaired bubble of class II CPD Photolyase in complex with damaged DNA
International Union of Crystallography, 2018Co-Authors: Manuel Maestre-reyna, Lars-oliver Essen, Junpei Yamamoto, Wei-cheng Huang, Ming-daw Tsai, Yoshitaka BesshoAbstract:Cyclobutane pyrimidine dimer (CPD) Photolyases harness the energy of blue light to repair UV-induced DNA CPDs. Upon binding, CPD Photolyases cause the photodamage to flip out of the duplex DNA and into the catalytic site of the enzyme. This process, called base-flipping, induces a kink in the DNA, as well as an unpaired bubble, which are stabilized by a network of protein–nucleic acid interactions. Previously, several co-crystal structures have been reported in which the binding mode of CPD Photolyases has been studied in detail. However, in all cases the internucleoside linkage of the photodamage site was a chemically synthesized formacetal analogue and not the natural phosphodiester. Here, the first crystal structure and conformational analysis via molecular-dynamics simulations of a class II CPD Photolyase in complex with photodamaged DNA that contains a natural cyclobutane pyrimidine dimer with an intra-lesion phosphodiester linkage are presented. It is concluded that a highly conserved bubble-intruding region (BIR) mediates stabilization of the open form of CPD DNA when complexed with class II CPD Photolyases
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ultrafast flavin photoreduction in an oxidized animal 6 4 Photolyase through an unconventional tryptophan tetrad
Physical Chemistry Chemical Physics, 2017Co-Authors: Ryan Martin, Klaus Brettel, Junpei Yamamoto, Pascal Plaza, Agathe Espagne, Pavel Muller, Fabien Lacombat, Nadia Dozova, Aurelien De La LandeAbstract:Photolyases are flavoenzymes repairing UV-induced lesions in DNA, which may be activated by a photoreduction of their FAD cofactor. In most Photolyases, this photoreduction proceeds by electron transfer along a chain of three tryptophan (Trp) residues, connecting the flavin to the protein surface. Much less studied, animal (6-4) Photolyases (repairing pyrimidine-pyrimidone (6-4) photoproducts) are particularly interesting as they were recently shown to have a longer electron transfer chain, counting four Trp residues. Using femtosecond polarized transient absorption spectroscopy, we performed a detailed analysis of the photoactivation reaction in the (6-4) Photolyase of Xenopus laevis with oxidized FAD. We showed that the excited flavin is very quickly reduced (∼0.5 ps) by a nearby tryptophan residue, yielding FAD˙- and WH˙+ radicals. Subsequent kinetic steps in the picosecond regime were assigned to the migration of the positive charge along the Trp tetrad, in competition with charge recombination. We propose that the positive charge is actually delocalized over various Trp residues during most of the dynamics and that charge recombination essentially occurs through the proximal tryptophanyl radical. Oxidation of the fourth tryptophan is thought to be reached about as fast as that of the third one (∼40 ps), based on a comparison with a mutant protein lacking the distal Trp, implying ultrafast electron transfer between these two residues. This unusual mechanism sheds light on the rich diversity of electron transfer pathways found in various Photolyases, and evolution-related cryptochromes alike.
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Repair of (6-4) Lesions in DNA by (6-4) Photolyase: 20 Years of Quest for the Photoreaction Mechanism.
Photochemistry and Photobiology, 2016Co-Authors: Junpei Yamamoto, Pascal Plaza, Klaus BrettelAbstract:Exposure of DNA to ultraviolet (UV) light from the Sun or from other sources causes formation of harmful and carcinogenic crosslinks between adjacent pyrimidine nucleobases, namely cyclobutane pyrimidine dimers and pyrimidine(6-4)pyrimidone photoproducts. Nature has developed unique flavoenzymes, called DNA Photolyases, that utilize blue light, i.e. photons of lower energy than those of the damaging light, to repair these lesions. In this review, we focus on the chemically challenging repair of the (6-4) photoproducts by (6-4) Photolyase and describe the major events along the quest for the reaction mechanisms, over the 20 years since the discovery of (6-4) Photolyase.
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detection of distinct α helical rearrangements of cyclobutane pyrimidine dimer Photolyase upon substrate binding by fourier transform infrared spectroscopy
Biochemistry, 2013Co-Authors: Made Mahaputra I Wijaya, Junpei Yamamoto, Tatsuya Iwata, Kenichi Hitomi, Shigenori Iwai, Elizabeth D. Getzoff, Yu Zhang, Hideki KandoriAbstract:Photolyases (PHRs) utilize near-ultraviolet (UV)–blue light to specifically repair the major photoproducts (PPs) of UV-induced damaged DNA. The cyclobutane pyrimidine dimer PHR (CPD-PHR) from Escherichia coli binds flavin adenine dinucleotide (FAD) as a cofactor and 5,10-methenyltetrahydrofolate as a light-harvesting pigment and specifically repairs CPD lesions. By comparison, a second Photolyase known as (6–4) PHR, present in a range of higher organisms, uniquely repairs (6–4) PPs. To understand the repair mechanism and the substrate specificity that distinguish CPD-PHR from (6–4) PHR, we applied Fourier transform infrared (FTIR) spectroscopy to bacterial CPD-PHR in the presence or absence of a well-defined DNA substrate, as we have studied previously for vertebrate (6–4) PHR. PHRs show light-induced reduction of FAD, and photorepair by CPD-PHR 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. Here, we measur...