A Review of Spectroscopic and Biophysical‐Chemical Studies of the Complex of Cyclobutane Pyrimidine Dimer Photolyase and Cryptochrome DASH with Substrate DNA

Cyclobutane pyrimidine dimer (CPD) photolyase (PL) is a structure‐specific DNA repair enzyme that uses blue light to repair CPD on DNA. Cryptochrome (CRY) DASH enzymes use blue light for the repair of CPD lesions on single‐stranded (ss) DNA, although some may also repair these lesions on double‐stranded (ds) DNA. In addition, CRY DASH may be involved in blue light signaling, similar to cryptochromes. The focus of this review is on spectroscopic and biophysical‐chemical experiments of the enzyme–substrate complex that have contributed to a more detailed understanding of all the aspects of the CPD repair mechanism of CPD photolyase and CRY DASH. This will be performed in the backdrop of the available X‐ray crystal structures of these enzymes bound to a CPD‐like lesion. These structures helped to confirm conclusions that were drawn earlier from spectroscopic and biophysical‐chemical experiments, and they have a critical function as a framework to design new experiments and to interpret new experimental data. This review will show the important synergy between X‐ray crystallography and spectroscopic/biophysical‐chemical investigations that is essential to obtain a sufficiently detailed picture of the overall mechanism of CPD photolyases and CRY DASH proteins.     

The Effect of Chronic Treatment of Mice with Urocanic Acid Isomers

Abstract— Trans-urocanic acid (trans-UCA) accumulates in the upper layers of the epidermis and can be isomerized to cis-UCA by UV light irradiation. Cis-urocanic acid possesses immunosuppressive properties that have led to its consideration as one of the initiators of UV-induced immunosuppression. High quantities of cis-UCA persist in human skin for prolonged periods in the summer months. In the present study, mice were injected intradermaUy with trans-UCA and cis-UCA three times a week for 4 weeks in order to ascertain the long-term effects of the presence of these compounds in the skin. The weight of mice and of their spleens were unaffected by the cis- or trans-UCA treatment. A decrease in thymus weight, accompanied by an increase in lymph node weight, was detected in the cis-UCA-treated mice compared with trans-UCA-treated mice and untreated controls. A net accumulation of lymphocytes and dendritic cells (DC) in lymph nodes was evident following cis-UCA treatment but the percentage of both CD4+and CD8+lymphocytes as well as Ia+DC remained constant among the different treatment groups, indicating that there was no specific migration or proliferation of a particular subset of cells. The in vitro lymphoproliferative response of lymph node cells to the mitogen concanavalin A was significantly sup pressed by cis-UCA treatment. The density of Langerhans cells in the epidermis of the ears was not altered by the chronic cis-UCA treatment. However, chronic cis-UCA treatment did suppress the mixed skin lymphocyte reaction response utilizing epidermal cells from the ears (an uninjected area of skin), indicating a systemic suppression. Compared with trans-UCA treatment, chronic cis-UCA treatment did not cause a significant reduction in the contact hypersensitivity response to oxazolone or the delayed hypersensitivity response to herpes simplex virus. Thus, chronic treatment with cis-UCA led to the suppression of some, but not all, of the immune parameters that are affected by UVB irradiation.     

Determination of the Dissociation Constants of Urocanic Acid Isomers in Aqueous Solutions

Summary. (E)- and (Z)-Urocanic acids are endogenous chemicals in the normal mammalian skin. The first and the second thermodynamic dissociation constants (pK _a1 and pK _a2) of urocanic acid isomers were determined using UV spectrophotometry in aqueous solutions. The values with standard deviation (pK _a1 = 3.43 ± 0.12 and pK _a2 = 5.80 ± 0.04) and (pK _a1 = 2.7 ± 0.3 and pK _a2 = 6.65 ± 0.04) were obtained to (E)- and (Z)-urocanic acids, respectively. The second dissociations were studied also by potentiometric titration in aqueous sodium chloride solutions up to the isotonic salt concentration (0.154 mol dm⁻³), and the second thermodynamic dissociation constants as well as activity parameters for both isomers were determined at temperature 25°C and for (E)-urocanic acid also at 37°C. The obtained pK _a2 values were close to those found by UV spectrophotometry. The equations for the calculation of the second stoichiometric dissociation constants of urocanic acid isomers on molality and molarity scale in aqueous sodium chloride solutions were derived. The obtained pK _a1 and pK _a2 values for (Z)-urocanic acid appear to be essentially lower than some previously reported values in literature. An erratum to this article is available at .     

Determination of the Dissociation Constants of Urocanic Acid Isomers in Aqueous Solutions

(E)- and (Z)-Urocanic acids are endogenous chemicals in the normal mammalian skin. The first and the second thermodynamic dissociation constants (pK _a1 and pK _a2) of urocanic acid isomers were determined using UV spectrophotometry in aqueous solutions. The values with standard deviation (pK _a1 = 3.43 ± 0.12 and pK _a2 = 5.80 ± 0.04) and (pK _a1 = 2.7 ± 0.3 and pK _a2 = 6.65 ± 0.04) were obtained to (E)- and (Z)-urocanic acids, respectively. The second dissociations were studied also by potentiometric titration in aqueous sodium chloride solutions up to the isotonic salt concentration (0.154 mol dm⁻³), and the second thermodynamic dissociation constants as well as activity parameters for both isomers were determined at temperature 25°C and for (E)-urocanic acid also at 37°C. The obtained pK _a2 values were close to those found by UV spectrophotometry. The equations for the calculation of the second stoichiometric dissociation constants of urocanic acid isomers on molality and molarity scale in aqueous sodium chloride solutions were derived. The obtained pK _a1 and pK _a2 values for (Z)-urocanic acid appear to be essentially lower than some previously reported values in literature.     

The Structure of Adducts of Phosphoryl Compounds with Nitric Acid

Abstract

The sctructure of adducts of phosphoryl compounds with HNO₃ has been studied by means of IR spectra, thermometric titration and X-ray diffraction.     

Concerning the Photodiastereomerization and Protic Equilibria of Urocanic Acid and its Complex with Human Serum Albumin

Summary. Photodiastereomerization of urocanic acid and its human serum albumin complex (its binding constant was estimated to amount 4.1·10^–4dm³·mol^–1) was investigated. It was found that although the photodiastereomerization rates were similar, the photoequilibrium positions differed significantly ((E):(Z)=33:77 for free urocanic acid, and 50:50 for the complex). This is thought to be due to a different stabilization of the corresponding orthogonal excited states. The thermal barrier of diastereomerization was estimated to amount to more than 250kJ·mol^–1 making it a very unlikely process under physiological and photodiastereomerization conditions. The various prototropic species of the two diastereomers at various pH values were analyzed by means of a mathematical model and from these results a novel photoinduced pH-jump methodology allowing for fast, persistent, diffusion controlled, and bidirectional jumps is proposed.     

Photolyase: Dynamics and Mechanisms of Repair of Sun‐Induced DNA Damage

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.     

Investigating the Red Shift between in vitro and in vivo Urocanic Acid Photoisomerization Action Spectra

Abstract— Trans-urocanic acid (UCA) is found in the upper layer of the skin and UV irradiation induces its photoisomerization to cis -UCA. Cis -UCA mimics some of the immunosuppressive properties of UV exposure. The wavelength dependence for in vitro photoisomerization of trans-UCA (15 μM) over the spectral range 250 nm-340 nm (10 nm intervals) was determined. The action spectrum revealed that maximal cis-UCA production occurred at 280 nm, which is red-shifted by 10-12 nm from its absorption peak at 268 nm and differs markedly from the reported action spectra for cis-UCA production in mouse skin in vivo , which peaks at 300-310 nm. The reasons for the red shift between the in vitro and in vivo action spectra are not clear. There is limited evidence suggesting that the UV absorption maximum of trans- UCA red shifts from 268 nm in vitro to 310 nm on interaction with stratum corneum proteins in vivo. This phenomenon was investigated by applying trans-UCA (2.5 mg/cm²) in an oil emulsion to isolated human stratum corneum. After incubation at 37°C for 1 h, the absorption spectra of stratum corneum with UCA and with oil only were compared using a Xe arc source and a spectrora-diometer. A moderate red shift in trans-UCA absorption from ∼268 nm to 280 nm was observed. In summary, we suggest that the 10-12 nm red shift between the UCA absorption spectrum peak and the action spectrum peak in vitro may be accounted for by the wavelength dependence of quantum yields reported over the 254-313 nm range. The red shift between the in vitro and in vivo photoisomerization action spectra may result from the 10 to 12 nm red shift in the absorption of UCA in association with stratum corneum proteins, combined with increasing quantum yields over the 254-313 nm range.     

Targeted Inactivation of DNA Photolyase Genes in Medaka Fish (Oryzias latipes)

Proteins of the cryptochrome/photolyase family (CPF) exhibit sequence and structural conservation, but their functions are divergent. Photolyase is a DNA repair enzyme that catalyzes the light‐dependent repair of ultraviolet (UV)‐induced photoproducts, whereas cryptochrome acts as a photoreceptor or circadian clock protein. Two types of DNA photolyase exist: CPD photolyase, which repairs cyclobutane pyrimidine dimers (CPDs), and 6‐4 photolyase, which repairs 6‐4 pyrimidine–pyrimidone photoproducts (6‐4PPs). Although the Cry‐DASH protein is classified as a cryptochrome, it also has light‐dependent DNA repair activity. To determine the significance of the three light‐dependent repair enzymes in recovering from solar UV‐induced DNA damage at the organismal level, we generated mutants in each gene in medaka using the CRISPR genome editing technique. The light‐dependent repair activity of the mutants was examined in vitro in cultured cells and in vivo in skin tissue. Light‐dependent repair of CPD was lost in the CPD photolyase‐deficient mutant, whereas weak repair activity against 6‐4PPs persisted in the 6‐4 photolyase‐deficient mutant. These results suggest the existence of a heretofore unknown 6‐4PP repair pathway and thus improve our understanding of the mechanisms of defense against solar UV in vertebrates.     

Attempted Oxidative Deamination of N-Deacetylcolchicinoids with 3,5-Di(tert-butyl)-1,2-benzoquinone: Synthesis of 2H-1,4-Benzoxazine-Type Adducts

In an attempt to use 3,5-di(tert-butyl)-1,2-benzoquinone for the oxidative deamination of N-deacetylcolchicine ( 4 ) and N-deacetylthiocolchicine (=N-deacetyl-10-demethoxy-10-(methylthio)colchicine; 5 ) to give the corresponding ketones 2 and 3 , the 2H-1,4-benzoxazine-type adducts 8 / 9 and 11 / 12 , respectively, were formed instead, showing a new and unexpected behavior of Corey's reagent. The adducts were separated and spectroscopically characterized, and a plausible scheme of formation is reported.     

Coexistence of Different Electron‐Transfer Mechanisms in the DNA Repair Process by Photolyase

DNA photolyase has been the topic of extensive studies due to its important role of repairing photodamaged DNA, and its unique feature of using light as an energy source. A crucial step in the repair by DNA photolyase is the forward electron transfer from its cofactor (FADH^?) to the damaged DNA, and the detailed mechanism of this process has been controversial. In the present study, we examine the forward electron transfer in DNA photolyase by carrying out high‐level ab initio calculations in combination with a quantum mechanical/molecular mechanical (QM/MM) approach, and by measuring fluorescence emission spectra at low temperature. On the basis of these computational and experimental results, we demonstrate that multiple decay pathways exist in DNA photolyase depending on the wavelength at excitation and the subsequent transition. This implies that the forward electron transfer in DNA photolyase occurs not only by superexchange mechanism but also by sequential electron transfer.     

Quantum-Chemical Study of the Adducts of Silicon Halides with Nitrogen-containing Donors: I. Adducts with Ammonia

Ab initio and DFT methods (RHF and B₃LYP) were used to calculate the structural and thermodynamic characteristics of adducts SiX₄·NH₃ and SiX₄·2NH₃ (X = H, F, Cl, Br). With the resulting data, the enthalpies and entropies of sublimation of SiF₄·2NH₃ were estimated for the first time. The effect of the nature of heteroatom X on the Si-N and Si-X bond lengths and atomic charges in the adducts and their dissociation enthalpies was analyzed. The hydride system was shown to differ from the halide systems, and the adducts with X = F have some peculiar features compared with those with X = Cl, Br. The Si-X bond is found to be sensitive to the charge redistribution produced by complex formation.     

UVB-lnduced DNA Damage and Its Photorepair in Nuclei and Chloroplasts of Spinacia oleracea L.

Ultraviolet-B-induced lesions and their photorepair in nuclear and chloroplast DNA of spinach (Spinacia oleracea L.) leaves were examined with two photoproducts, cyclobutane pyrimidine dimers (CPD) and pyrimidine (6-4) pyrimidinone photoproducts (6-4PP). These photoproducts were induced both in nuclear and chloroplast DNA by UVB irradiation and could be detected by enzyme-linked immunosorbent assay using their respective monoclonal antibodies. Formation of CPD was greater in nuclear DNA than in chloroplast DNA (about 10 to 7), whereas 6-4PP formation was comparable in both DNA. On subsequent exposure of leaves to blue/UVA after UVB irradiation, photorepair of CPD and 6-4PP occurred in nuclear DNA but not in chloroplast DNA. When isolated chloroplasts were irradiated with UVB, CPD was also induced in their DNA. But photorepair of CPD did not occur in them by subsequent exposure to blue/UVA, suggesting that no photorepair system operates in chloroplasts.     

Model Studies of DNA Photorepair: Enthalpy of Cleavage of a Pyrimidine Dimer Measured by Photothermal Beam Deflection Calorimetry

Abstract— The enzyme DNA photolyase mediates the repair of pyrimidine dimers. This repair step, a net retro [²+2] reaction, proceeds through either the cation or anion radical of the pyrimidine dimer. In order to understand how electron transfer makes the repair process possible, its energetics have been examined by photothermal beam deflection calorimetry, fluorescence quenching and quantum yield studies. The enthalpy for the cleavage reaction of cis-syn 1,3-dimethylthymine dimer itself was found to be -19 kcal/mol. In addition, from the redox potentials, the enthalpies for the cleavage reactions of the dimer cation radical and the anion radical were determined to be -19 kcal/mol and -28 kcal/mol, respectively.     

Borane Adducts of Hydrazoic Acid and Organic Azides: Intermediates for the Formation of Aminoboranes

The reaction of HN₃ with the strong Lewis acid B(C₆F₅)₃ led to the formation of a very labile HN₃?B(C₆F₅)₃ adduct, which decomposed to an aminoborane, H(C₆F₅)NB(C₆F₅)₂, above ?20 °C with release of molecular nitrogen and simultaneous migration of a C₆F₅ group from boron to the nitrogen atom. The intermediary formation of azide–borane adducts with B(C₆F₅)₃ was also demonstrated for a series of organic azides, RN₃ (R=Me₃Si, Ph, 3,5‐(CF₃)₂C₆H₃), which also underwent Staudinger‐like decomposition along with C₆F₅ group migration. In accord with experiment, computations revealed rather small barriers towards nitrogen release for these highly labile azide adducts for all organic substituents except R=Me₃Si (m.p. 120 °C, T_dec=189 °C). Hydrolysis of the aminoboranes provided C₆F₅‐substituted amines, HN(R)(C₆F₅), in good yields.     

Quantum-Chemical Study of Adducts of Silicon Halides with Nitrogen-containing Donors: IV. Adducts with Pyridine

The structural and thermodynamic characteristics of SiX₄·Py and SiX₄·2Py adducts (X = H, F, Cl, Br) were calculated by ab initio and DFT methods (RHF and B3LYP). The resulting data were used to estimate for the first time the enthalpies of sublimation of trans-SiX₄·2Py complexes. The distortion energies of the donor and acceptor fragments and the energies of the Si-N bonds in the 1:1 and 1:2 halide complexes were calculated. The high distortion energy makes thermodynamically unfavorable equatorial monopyridine adducts with Si-N bond energies of 150-200 kJ/mol. In trans 1:2 complexes, pyridine acts as a weaker donor than ammonia with respect to silicon tetrahalides.     

Quantitative Determination of Cyclobutane Thymine Dimers in DNA by Stable Isotope-Dilution Mass Spectrometry

In order to understand the role of UV-induced DNA lesions in biological processes such as mutagenesis and carcinogenesis, it is essential to detect and quantify DNA damage in cells. In this paper we present a novel and both highly selective and sensitive assay using capillary gas chromatography (GC) combined with mass spectrometry (MS) for the detection and accurate quantitation of a major product of UV-induced DNA damage (cissyn cyclobutadithymine). Quantitation of the cyclobutane thymine dimer was achieved by the use of an internal standard in the form of a stable ²H-labeled analogue. Both isotopically labeled and nonlabeled dimers were prepared directly from their corresponding monomers. Each was identified as their trimethylsilyl ether derivative by GC-MS. Calibration plots were obtained for known quantities of both nonlabeled analyte and internal standard. Quantitation of cis-syn cyclobutadithymine was demonstrated in DNA exposed to UVC radiation over a dose range of 0 to 3500 J m^?2. Under the conditions used, the limit of detection was found to be 20–50 fmol on column (equivalent to 0.02–0.05 nmol dimer per mg DNA). The results of the present study indicate that capillary GC-MS is an ideally suited technique for selective and sensitive quantification of cis-syn cyclobutadithymine in DNA and hence UV-induced DNA damage.