DNA Photolyase Research Articles

Cryptochrome 3 from Arabidopsis thaliana: Structural and Functional Analysis of its Complex with a Folate Light Antenna

Cryptochromes are almost ubiquitous blue-light receptors and act in several species as central components of the circadian clock. Despite being evolutionary and structurally related with DNA photolyases, a class of light-driven DNA-repair enzymes, and having similar cofactor compositions, cryptochromes lack DNA-repair activity. Cryptochrome 3 from the plant Arabidopsis thaliana belongs to the DASH-type subfamily. Its crystal structure determined at 1.9 Å resolution shows cryptochrome 3 in a dimeric state with the antenna cofactor 5,10-methenyltetrahydrofolate (MTHF) bound in a distance of 15.2 Å to the U-shaped FAD chromophore. Spectroscopic studies on a mutant where a residue crucial for MTHF-binding, E149, was replaced by site-directed mutagenesis demonstrate that MTHF acts in cryptochrome 3 as a functional antenna for the photoreduction of FAD.

Computational analysis of DNA photolyases using digital signal processing methods

Sun light energy is used by plants to trigger their growth and development. However, an increase of UV-B light may lead to DNA damage. DNA photolyases are enzymes that repair the cyclobutane pyridine dimer (CPD) and 6–4 photoproduct lesions formed through UV irradiation of DNA. Many aspects of the repair process are under intense scientific investigation but still poorly understood. Here we have computationally analysed DNA-photolyases using the resonant recognition model (RRM), a physico-mathematical approach based on digital signal processing methods. The RRM proposes that protein interactions represent the transfer of resonant electromagnetic energy between interacting molecules at the particular frequency. Within this study we have determined photolyases characteristic frequency, “hot spots” amino acids corresponding to the functional mutations and functional active/binding sites, and designed photolyase peptide analogous. A mutual relationship between photolyase and p53 tumour suppressor protein has also been investigated. The results obtained provide new insights into the structure–function relationships of photolyase protein family.

Efficient cycloreversion of cis,syn-thymine photodimer by a Zn2+–1,4,7,10-tetraazacyclododecane complex bearing a lumiflavin and tryptophan by chemical reduction and photoreduction of a lumiflavin unit

DNA photolyases (EC 4.1.99.3) are enzymes that catalyze photoreversion of cis,syn-thymine photodimer (T[c,s]T), which is one of major photolesion products in DNA, by utilizing UV light. In this work, we have designed and synthesized Zn2+ -1,4,7,10-tetraazacyclododecane complexes bearing a lumiflavin and L: -tryptophan (ZnL3) or L: -phenylalanine (ZnL4) as artificial DNA photolyases. We have found that (ZnL3)red, whose flavin unit was reduced in situ by Na2S2O4, accelerates the photoreversion of T[c,s]T utilizing near-UV light in aqueous solution at pH 7.6 and 11. Interestingly, more efficient photoreversion of T[c,s]T was achieved by UV irradiation of an oxidized form of ZnL3 [(ZnL3)ox] in the presence of an excess amount of Et3N at pH 11. UV-vis and fluorescence measurements and action spectra showed that an oxidized form of flavin of (ZnL3)ox was photoreduced by Et3N into its reduced form (ZnL3)red, which promoted the photoreduction of T[c,s]T. Comparison of the photochemical properties of ZnL3 with those of ZnL4 suggested that a tryptophan unit in ZnL3 contributed to the stabilities of the flavin through intramolecular photoinduced electron transfer.

Natural and Non‐natural Antenna Chromophores in the DNA Photolyase from Thermus Thermophilus

X-ray crystallographic and functional analysis of the class I DNA photolyase from Thermus thermophilus revealed the binding of flavin mononucleotide (FMN) as an antenna chromophore. The binding mode of FMN closely coincides with the binding of a deazaflavin-like chromophore in the related class I DNA photolyase from Anacystis nidulans. Compared to the R46E mutant, which lacks a conserved arginine in the binding site for the antenna chromophore, the FMN-comprising holophotolyase exhibits an eightfold higher activity at 450 nm. The facile incorporation of the flavin cofactors 8-hydroxy-deazariboflavin and 8-iodo-8-demethyl-riboflavin into the binding site for the antenna chromophore paves the way for wavelength-tuning of the activity spectra of DNA photolyases by using synthetic flavins.

Differential Distortion of Substrate Occurs When It Binds to DNA Photolyase: A 2-Aminopurine Study

Cyclobutylpyrimidine dimers (CPDs) are formed between adjacent pyrimidines in DNA when it is exposed to ultraviolet light. CPDs can be directly repaired by DNA photolyase (PL) upon absorption of blue-green light. We have used the fluorescent adenine analogue 2-aminopurine (2Ap) to probe the local double-helical structure of the DNA substrate when it binds to the protein. Duplex melting temperatures and van't Hoff enthalpies were obtained by both UV-vis absorption and fluorescence spectroscopies to ascertain the effect of the probe and CPD on DNA stability. Steady-state fluorescence measurements of the single- and double-stranded oligos showed that the local region around the 5'-side of the CPD lesion was more disrupted and destacked than the 3'-side in substrate-protein complexes. These results were compared with those of a protein-substrate crystal structure, demonstrating that the crystal structure and solution-state studies are in agreement with regard to the differential distortions of the target DNA at the active site of the protein.

Investigation of the Pathways of Excess Electron Transfer in DNA with Flavin‐Donor and Oxetane‐Acceptor Modified DNA Hairpins

Oxetane is a potential intermediate that is enzymatically formed during the repair of (6-4) DNA lesions by special repair enzymes (6-4 DNA photolyases). These enzymes use a reduced and deprotonated flavin to cleave the oxetane by single electron donation. Herein we report synthesis of DNA hairpin model compounds containing a flavin as the hairpin head and two different oxetanes in the stem structure of the hairpin. The data show that the electron moves through the duplex even over distances of 17 A. Attempts to trap the moving electron with N2O showed no reduction of the cleavage efficiency showing that the electron moves through the duplex and not through solution. The electron transfer is sequence dependent. The efficiency is reduced by a factor of 2 in GC rich DNA hairpins.

Reversible resolution of flavin and pterin cofactors of His-tagged Escherichia coli DNA photolyase

Escherichia coli photolyase catalyzes the repair of cyclobutane pyrimidine dimers (CPD) in DNA under near UV/blue-light irradiation. The enzyme contains flavin adenine dinucleotide (FAD) and methenyltetrahydrofolate (MTHF) as noncovalently bound light sensing cofactors. To study the apoprotein–chromophore interactions we developed a new procedure to prepare apo-photolyase. MTHF-free photolyase was obtained by binding the C-terminal His-tagged holoenzyme to a metal-affinity column at neutral pH and washing the column with deionized water. Under these conditions the flavin remains bound and the defolated enzyme can be released from the column with 0.5 M imidazole pH 7.2. The MTHF-free protein was still capable of DNA repair, showing 70% activity of native enzyme. Fluorescence polarization experiments confirmed that MTHF binding is weakened at low ionic strength. Apo-photolyase was obtained by treating the His-tagged holoenzyme with 0.5 M imidazole pH 10.0. The apo-photolyase thus obtained was highly reconstitutable and bound nearly stoichiometric amounts of FAD ox. Photolyase reconstituted with FAD ox had about 34% activity of native enzyme, which increased to 83% when FAD ox was reduced to FADH −. Reconstitution kinetics performed at 20 °C showed that apo-photolyase associates with FADH − much faster ( k obs ∼ 3000 M − 1 s − 1 ) than with FAD ox ( k obs = 34 M − 1 s − 1 ). The dissociation constant of the photolyase–FAD ox complex is about 2.3 μM and that of E-FADH − is not higher than 20 nM (pH 7.2).

Role of the Middle Residue in the Triple Tryptophan Electron Transfer Chain of DNA Photolyase: Ultrafast Spectroscopy of a Trp→Phe Mutant

Photoreduction of the semi-reduced flavin adenine dinucleotide cofactor FADH* in DNA photolyase from Escherichia coli into FADH- involves three tryptophan (W) residues that form a closely spaced electron-transfer chain FADH*-W382-W359-W306. To investigate this process, we have constructed a mutant photolyase in which W359 is replaced by phenylalanine (F). Monitoring its photoproducts by femtosecond spectroscopy, the excited-state FADH* was found to decay in approximately 30 ps, similar as in wild type (WT) photolyase. In contrast to WT, however, in W359F mutant photolyase the ground-state FADH* fully recovered virtually concomitantly with the decay of its excited state and, despite the presence of the primary electron donor W382, no measurable flavin reduction was observed at any time. Thus, W359F photolyase appears to behave like many other flavoproteins, where flavin excited states are quenched by very short-lived oxidation of aromatic residues. Our analysis indicates that both charge recombination of the primary charge separation state FADH-W382*+ and (in WT) electron transfer from W359 to W382*+ occur with time constants <4 ps, considerably faster than the initial W382-->FADH* electron-transfer step. Our results provide a first experimental indication that electron transfer between aromatic residues can take place on the time scale of approximately 10(-12) s.

Light-driven DNA repair by photolyases

DNA photolyases are highly efficient light-driven DNA repair enzymes which revert the genome-damaging effects caused by ultraviolet (UV) radiation. These enzymes occur in almost all living organisms exposed to sunlight, the only exception being placental mammals like humans and mice. Their catalytic mechanism employs the light-driven injection of an electron onto the DNA lesion to trigger the cleavage of cyclobutane- pyrimidine dimers or 6-4 photoproducts inside duplex DNA. Spectroscopic and structural analysis has recently yielded a concise view of how photolyases recognize these DNA lesions involving two neighboring bases, catalyze the repair reaction within a nanosecond and still achieve quantum efficiencies of close to one. Apart from these mechanistic aspects, the potential of DNA photolyases for the generation of highly UV-resistant organisms, or for skin cancer prevention by ectopical application is increasingly recognized.

Explicit and implicit solvation of radical ions: the cycloreversion of CPD dimers

Abstract The electron transfer catalyzed cycloreversion of cyclobutane pyrimidine dimers is the key step in repair of light-induced DNA lesions catalyzed by the enzyme CPD photolyase. The formation of the CPD radical anion was found to be strongly solvent dependent due to a specific hydrogen bond that stabilizes the valence bound state over the dipole bound state of the additional electron. The effect of solvation on the vertical and adiabatic electron affinity of uracil and uracil dimers as well as on the mechanism of the cycloreversion of the uracil dimer radical anion is explored for three model systems that include explicit solvent molecules at the B3LYP/6-311++G ∗∗ /B3LYP/6-31+G ∗ level of theory. The second solvation shell is described using the implicit C-PCM solvation model. These calculations indicate an effectively barrierless mechanism. These results are in agreement with the available experimental data for the reaction energies and isotope effects. It is also shown that a single hydrogen bond donor is a sufficient minimal model for the first solvation shell by adequately describing the stabilization of the valence bound state of the radical anion through hydrogen bonding. The relationship of these model systems with the enzymatic reaction catalyzed by DNA photolyase is also discussed.

Production of cis–syn thymine–thymine cyclobutane dimer oligonucleotide in the presence of acetone photosensitizer

cis–syn Cyclobutane pyrimidine dimer (CPD) oligonucleotide was produced by UV irradiation in the presence of acetone photosensitizer. Acetone could enhance the productivity but evidently induced the photocleavage of oligonucleotide under a long time irradiation. A statistical approach of orthogonal design was applied to optimize the preparation condition for the production of the modified oligonucleotide. Optimal conditions for maximal cis–syn CPD oligonucleotide productivity were determined based on three factors: acetone concentration, initial oligonucleotide concentration, and irradiation time at several different levels. The optimal modified oligonucleotide that this optimization could produce was 32.7%. Through analysis of 20% polyacrylamide gel electrophoresis, it was found that modified oligonucleotide migrated slightly more slowly than the parent oligonucleotide. The photoreactivation of cis–syn thymine–thymine dimer oligonucleotide displayed the selectivity of the substrate specificity of DNA photolyase with high-performance liquid chromatography (HPLC) analysis.

Cloning and Sequence Analysis of the Gene Encoding (6-4)photolyase from Dunaliella salina

DNA photolyase can repair UV-induced DNA damage in a light-dependent manner. A cDNA of (6-4)photolyase from Dunaliella salina (GenBank accession number: AY845324) was cloned, sequenced and its amino acid sequence was deduced. The derived amino acid sequence showed high homology with other (6-4)photolyases and a predicted 3D model was constructed by homology modeling.

Blue-Light-Induced Changes in Arabidopsis Cryptochrome 1 Probed by FTIR Difference Spectroscopy

Cryptochromes are blue-light photoreceptors that regulate a variety of responses in animals and plants, including circadian entrainment in Drosophila and photomorphogenesis in Arabidopsis. They comprise a photolyase homology region (PHR) of about 500 amino acids and a C-terminal extension of varying length. In the PHR domain, flavin adenine dinucleotide (FAD) is noncovalently bound. The presence of a second chromophore, such as methenyltetrahydrofolate, in animal and plant cryptochromes is still under debate. Arabidopsis cryptochrome 1 (CRY1) has been intensively studied with regard to function and interaction of the protein in vivo and in vitro. However, little is known about the pathway from light absorption to signal transduction on the molecular level. We investigated the full-length CRY1 protein by Fourier transform infrared (FTIR) and UV/vis difference spectroscopy. Starting from the fully oxidized state of the chromophore FAD, a neutral flavoprotein radical is formed upon illumination in the absence of any exogenous electron donor. A preliminary assignment of the chromophore bands is presented. The FTIR difference spectrum reveals only moderate changes in secondary structure of the apoprotein in response to the photoreduction of the chromophore. Deprotonation of an aspartic or glutamic acid, probably D396, accompanies radical formation, as is deduced from the negative band at 1734 cm(-)(1) in D(2)O. The main positive band at 1524 cm(-)(1) in the FTIR spectrum shows a strong shift to lower frequencies as compared to other flavoproteins. Together with the unusual blue-shift of the absorption in the visible range to 595 nm, this clearly distinguishes the radical form of CRY1 from those of structurally highly homologous DNA photolyases. As a consequence, the direct comparison of cryptochrome to photolyase in terms of photoreactivity and mechanism has to be made with caution.

Base Flipping in Nucleotide Excision Repair

UvrB, the ultimate damage-binding protein in bacterial nucleotide excision repair is capable of binding a vast array of structurally unrelated lesions. A beta-hairpin structure in the protein plays an important role in damage-specific binding. In this paper we have monitored DNA conformational alterations in the UvrB-DNA complex, using the fluorescent adenine analogue 2-aminopurine. We show that binding of UvrB to a DNA fragment with cholesterol damage moves the base adjacent to the lesion at the 3' side into an extrahelical position. This extrahelical base is not accessible for acrylamide quenching, suggesting that it inserts into a pocket of the UvrB protein. Also the base opposite this flipped base is extruded from the DNA helix. The degree of solvent exposure of both residues varies with the type of cofactor (ADP/ATP) bound by UvrB. Fluorescence of the base adjacent to the damage is higher when UvrB is in the ADP-bound configuration, but concomitantly this UvrB-DNA complex is less stable. In the ATP-bound form the UvrB-DNA complex is very stable and in this configuration the base in the non-damaged strand is more exposed. Hairpin residue Tyr-95 is specifically involved in base flipping in the non-damaged strand. We present evidence that this conformational change in the non-damaged strand is important for 3' incision by UvrC.

Enzymatic Reversal of Polymeric Thymine Photocrosslinking with E. coli DNA Photolyase

Polymers containing pendant thymine groups undergo a 2π+2π cycloaddition crosslinking reaction. The enzyme DNA photolyase has been demonstrated to reverse this photocrosslinking in thymine containing styrene derivatives.

Identification and Characterization of a Second Chromophore of DNA Photolyase from Thermus thermophilus HB27

Cyclobutane pyrimidine dimer (CPD) photolyases use light to repair CPDs. For efficient light absorption, CPD photolyases use a second chromophore. We purified Thermus thermophilus CPD photolyase with its second chromophore. UV-visible absorption spectra, reverse-phase HPLC, and NMR analyses of the chromophores revealed that the second chromophore of the enzyme is flavin mononucleotide (FMN). To clarify the role of FMN in the CPD repair reaction, the enzyme without FMN (Enz-FMN(-) and that with a stoichiometric amount of FMN (Enz-FMN(+)) were both successfully obtained. The CPD repair activity of Enz-FMN(+) was higher than that of Enz-FMN(-), and the CPD repair activity ratio of Enz-FMN(+) and Enz-FMN(-) was dependent on the wavelength of light. These results suggest that FMN increases the light absorption efficiency of the enzyme. NMR analyses of Enz-FMN(+) and Enz-FMN(-) revealed that the binding mode of FMN is similar to that of 7,8-didemethyl-8-hydroxy-5-deazariboflavin in Anacystis nidulans CPD photolyase, and thus a direct electron transfer between FMN and CPD is not likely to occur. Based on these results, we concluded that FMN acts as a highly efficient light harvester that gathers light and transfers the energy to FAD.

Crystallization and preliminary X-ray analysis of cryptochrome 3 from Arabidopsis thaliana.

Cryptochromes are flavoproteins which serve as blue-light receptors in plants, animals, fungi and prokaryotes and belong to the same protein family as the catalytically active DNA photolyases. Cryptochrome 3 from the plant Arabidopsis thaliana (cry3; 525 amino acids, 60.7 kDa) is a representative of the novel cryDASH subfamily of UV-A/blue-light receptors and has been expressed as a mature FAD-containing protein in Escherichia coli without the signal sequence that directs the protein into plant organelles. The purified cryptochrome was found to be complexed to methenyltetrahydrofolate as an antenna pigment. Crystals of the cryptochrome-antenna pigment complex were obtained by vapour diffusion and display orthorhombic symmetry, with unit-cell parameters a = 76.298, b = 116.782, c = 135.024 A. X-ray diffraction data were collected to 1.9 A resolution using synchrotron radiation. The asymmetric unit comprises a cry3 dimer, the physiological role of which remains to be elucidated.

Computational Studies of DNA Photolyase

The electron transfer catalyzed (ETC) repair of the DNA photolesion cyclobutane pyrimidine dimer (CPD) is mediated by the enzyme DNA photolyase. Due to its importance as part of the cancer prevention mechanism in many organisms, but also due to its unique mechanism, this DNA photoreactivation is a topic of intense study. The progress in the application of computational methods to three aspects of the ETC repair of CPD is reviewed: (i) electronic structure calculations of the cycloreversion of the CPD radical cation and radical anion, (ii) MD simulations of the DNA photolyase and its complex to photodamaged DNA, and (iii) the structure and dynamics of photodamaged DNA. The contributions of this work to the overall understanding of the reaction and its relationship to the available experimental work are highlighted.

Substrate Binding Modulates the Reduction Potential of DNA Photolyase

The reduction potential of the (FADH-/FADH*) couple in DNA photolyase was measured, and the value was found to be significantly higher than the values estimated in the literature. In the absence of substrate, the enzyme has a reduction potential of 16 +/- 6 mV vs NHE. In the presence of excess substrate the reduction potential increases to 81 +/- 8 mV vs NHE. The increase in reduction potential has physiological relevance since it gives the catalytic state greater resistance to oxidation. This is the first measurement of a reduction potential for this class of DNA-repair enzymes and the larger family of blue-light photoreceptors.

Genome sequence of Chrysodeixis chalcites nucleopolyhedrovirus, a baculovirus with two DNA photolyase genes

The complete genome sequence of a single nucleocapsid nucleopolyhedrovirus recently isolated from Chrysodeixis chalcites (ChchNPV) was determined. The viral genome has a size of 149 622 bp and an overall G+C content of 39.1 mol%. The sequence contains 151 predicted open reading frames (ORFs) with a minimal size of 50 codons. The similarity of these ORFs with those of other completely sequenced baculoviruses was calculated using a newly developed database, named GECCO. Phylogenetic analysis of the whole genome confirmed the evolutionary relationship of ChchNPV with group II NPVs, as did the absence of the NPV group I-specific gp64 gene. It is the first group II NPV to encode proliferating cell nuclear antigen. Most noteworthy is the presence of two ORFs encoding a class II cyclobutane pyrimidine dimer DNA photolyase. These two ORFs share only 45 % amino acid identity and have different promoter motifs. Twenty-two additional unique baculovirus genes were identified, including a gene encoding a novel putative RING finger protein with a possible homologue in poxviruses.