Reaction of thymidine and ascorbic acid induced by UV in the presence of salicylic acid.

When a neutral solution of thymidine and ascorbic acid was irradiated with UV light of wavelength longer than 300 nm in the presence of salicylic acid as a photosensitizer, six product peaks appeared in an HPLC chromatogram in addition to small amounts of thymidine dimers. The six products were identified as three pairs of diastereomers of 5-(2-deoxy-2-l-ascorbyl)-5,6-dihydrothymidine, 5-(2-l-ascorbyl)-5,6-dihydrothymidine, and 5,6-dihydrothymidine. These results suggest that novel DNA damage may be generated by ascorbic acid with salicylic acid induced by sunlight.

Thymidine decomposition induced by low-energy electrons and soft X rays under N2 and O2 atmospheres.

A novel technique has been employed to investigate the simultaneous damage to DNA components induced by soft X rays (1.5 keV) and low-energy electrons (0-30 eV) in thin films of thymidine deposited on glass and tantalum substrates and irradiated under atmospheric pressure and temperature. The films were surrounded by either an N2 or O2 environment. The formation of four radiation-induced products is reported in this article: base release, 5-hydroxymethyl-2'-deoxyuridine (5-HMdUrd), 5-formyl-2'-deoxyuridine (5-FordUrd) and 5,6-dihydrothymidine (5,6-DHThd). Analysis with LC-MS/MS shows larger damage yields in the samples deposited on tantalum than in those deposited on glass, which is attributed to the interaction of the additional low-energy electrons that are photoemitted from the metal surface. From a comparison of the results obtained from N2 and O2 environment, we report a dramatic effect from 6 O2: an approximately threefold increase in the yield of products, attributed to the reaction of O2 with initial carbon-centered thymidine radicals generated in the film during irradiation.

Atomic hydrogen surrounded by water molecules, H(H2O)m, modulates basal and UV-induced gene expressions in human skin in vivo.

Recently, there has been much effort to find effective ingredients which can prevent or retard cutaneous skin aging after topical or systemic use. Here, we investigated the effects of the atomic hydrogen surrounded by water molecules, H(H2O)m, on acute UV-induced responses and as well as skin aging. Interestingly, we observed that H(H2O)m application to human skin prevented UV-induced erythema and DNA damage. And H(H2O)m significantly prevented UV-induced MMP-1, COX-2, IL-6 and IL-1ß mRNA expressions in human skin in vivo. We found that H(H2O)m prevented UV-induced ROS generation and inhibited UV-induced MMP-1, COX-2 and IL-6 expressions, and UV-induced JNK and c-Jun phosphorylation in HaCaT cells. Next, we investigated the effects of H(H2O)m on intrinsically aged or photoaged skin of elderly subjects. In intrinsically aged skin, H(H2O)m application significantly reduced constitutive expressions of MMP-1, IL-6, and IL-1ß mRNA. Additionally, H(H2O)m significantly increased procollagen mRNA and also decreased MMP-1 and IL-6 mRNA expressions in photoaged facial skin. These results demonstrated that local application of H(H2O)m may prevent UV-induced skin inflammation and can modulate intrinsic skin aging and photoaging processes. Therefore, we suggest that modifying the atmospheric gas environment within a room may be a new way to regulate skin functions or skin aging.

Structure of the DNA (6-4) photoproduct dTT(6-4)TT in complex with the 64M-2 antibody Fab fragment implies increased antibody-binding affinity by the flanking nucleotides.

Pyrimidine (6-4) pyrimidone DNA photoproducts produced by ultraviolet light are highly mutagenic and carcinogenic. The crystal structure of the dTT(6-4)TT photoproduct in complex with the Fab fragment of the antibody 64M-2 that is specific for (6-4) photoproducts was determined at 2.4 Šresolution. The dT(6-4)T segment is fully accommodated in the concave binding pocket of the Fab, as observed in the complex of dT(6-4)T with the Fab. The pyrimidine and pyrimidone bases of the dT(6-4)T segment are positioned nearly perpendicularly to each other. The thymidine segments flanking both ends extend away from the dT(6-4)T segment. The 5'-side thymine base is parallel to the side chain of Tyr100iH of the antibody heavy chain and is also involved in electrostatic interactions with Asn30L, Tyr32L and Lys50L of the antibody light chain. The 5'-side and 3'-side phosphate groups exhibit electrostatic interactions with Asn28L and Ser58H, respectively. These interactions with the flanking nucleotides explain why longer oligonucleotides containing dT(6-4)T segments in the centre show higher antibody-binding affinities than the dT(6-4)T ligand.

Identification of potentially cytotoxic lesions induced by UVA photoactivation of DNA 4-thiothymidine in human cells.

Photochemotherapy-in which a photosensitizing drug is combined with ultraviolet or visible radiation-has proven therapeutic effectiveness. Existing approaches have drawbacks, however, and there is a clinical need to develop alternatives offering improved target cell selectivity. DNA substitution by 4-thiothymidine (S(4)TdR) sensitizes cells to killing by ultraviolet A (UVA) radiation. Here, we demonstrate that UVA photoactivation of DNA S(4)TdR does not generate reactive oxygen or cause direct DNA breakage and is only minimally mutagenic. In an organotypic human skin model, UVA penetration is sufficiently robust to kill S(4)TdR-photosensitized epidermal cells. We have investigated the DNA lesions responsible for toxicity. Although thymidine is the predominant UVA photoproduct of S(4)TdR in dilute solution, more complex lesions are formed when S(4)TdR-containing oligonucleotides are irradiated. One of these, a thietane/S(5)-(6-4)T:T, is structurally related to the (6-4) pyrimidine:pyrimidone [(6-4) Py:Py] photoproducts induced by UVB/C radiation. These lesions are detectable in DNA from S(4)TdR/UVA-treated cells and are excised from DNA more efficiently by keratinocytes than by leukaemia cells. UVA irradiation also induces DNA interstrand crosslinking of S(4)TdR-containing duplex oligonucleotides. Cells defective in repairing (6-4) Py:Py DNA adducts or processing DNA crosslinks are extremely sensitive to S(4)TdR/UVA indicating that these lesions contribute significantly to S(4)TdR/UVA cytotoxicity.

Evidence for direct contact between the RPA3 subunit of the human replication protein A and single-stranded DNA.

Replication Protein A is a single-stranded (ss) DNA-binding protein that is highly conserved in eukaryotes and plays essential roles in many aspects of nucleic acid metabolism, including replication, recombination, DNA repair and telomere maintenance. It is a heterotrimeric complex consisting of three subunits: RPA1, RPA2 and RPA3. It possesses four DNA-binding domains (DBD), DBD-A, DBD-B and DBD-C in RPA1 and DBD-D in RPA2, and it binds ssDNA via a multistep pathway. Unlike the RPA1 and RPA2 subunits, no ssDNA-RPA3 interaction has as yet been observed although RPA3 contains a structural motif found in the other DBDs. We show here using 4-thiothymine residues as photoaffinity probe that RPA3 interacts directly with ssDNA on the 3'-side on a 31 nt ssDNA.

Flavin-sensitized photoreduction of thymidine glycol.

Photochemical reactivity of flavin adenine dinucleotide (FAD) and flavin mononucleotide (FMN) toward thymidine glycol (dTg) has been investigated. Fluorescence intensity of FAD was enhanced as increasing the concentration of dTg, suggesting that adenosine moiety of FAD interacts with dTg. However, photoreduction of dTg using reduced form of FAD gave repaired thymidine in almost the same yield as when reduced FMN was used alternatively, and thus such interaction seems to have no effect on the reduction. Oligodeoxynucleotides containing dTg were also photochemically repaired by reduced form of flavins in different yields depending on the sequence, which could be related to electron affinity of the nucleobases in DNA.

Ring opening of the cyclobutane in a thymine dimer radical anion.

The reactions of hydrated electrons (e(aq) (-)) with thymine dimer 2 and thymidine have been investigated by radiolytic methods coupled with product studies, and addressed computationally by means of BB1K-HMDFT calculations. Pulse radiolysis revealed that one-electron reduction of the thymine dimer 2 affords the radical anion of thymidine (5) with t(1/2)<35 ns. Indeed, the theoretical study suggests that radical anion 3, in which the spin density and charge distribution are located in both thymine rings, undergoes a fast partially ionic splitting of the cyclobutane with a half-life of a few ps. This model fits with the in vivo observation of thymine dimer repair in DNA by photolyase. gamma-Radiolysis of thymine dimer 2 demonstrates that the one-electron reduction and the subsequent cleavage of the cyclobutane ring does not proceed by means of a radical chain mechanism, that is, in this model reaction the T(-)* is unable to transfer an electron to the thymine dimer 2.

A new photoactive building block for investigation of DNA backbone interactions: photoaffinity labeling of human DNA polymerase beta.

The cross-linking of target proteins or nucleic acids to light-activatable ligands is an important tool for elucidating molecular interactions. Through the use of photoaffinity-labeling reagents, several new insights into nucleic acid interactions have been obtained, for example in DNA replication and repair. In most known photoprobes, the applied light-sensitive functionalities are placed directly at the nucleobase or are attached via linkers to either the nucleobase or the phosphate backbone. Here we describe the first photoprobe that bears a light-sensitive aryl(trifluoromethyl)diazirine at the sugar moiety of a DNA oligonucleotide. We devised a route for the synthesis of the modified nucleoside and its incorporation into an oligonucleotide. The photoactive species was proven to be stable under the conditions employed in routine automated DNA synthesis. The modified oligonucleotide was shown by subsequent photolabeling studies of human DNA polymerase beta to form a covalent complex to the enzyme upon irradiation with near-UV light.

DNA-binding properties of the antibody specific for the Dewar photoproduct of thymidylyl-(3-5')-thymidine.

A monoclonal antibody (DEM-1) specific for the Dewar photoproduct is used for detection and quantification of photolesions in DNA. To help understand the molecular recognition of damaged DNA by the antibody protein, we have cloned and sequenced the variable region genes of DEM-1. We have also prepared Fab fragments of DEM-1 (DEM1Fab), and synthesized two kinds of 3'-biotinylated oligonucleotides of different lengths containing a central Dewar photoproduct of TpT to analyze the effects of the antigen size on the binding rates by means of surface plasmon resonance (SPR). Results obtained from SPR analyses suggest that DEM1Fab may recognize tetranucleotide unit as the epitope.

Intramolecular interactions in the triplet excited states of benzophenone-thymine dyads.

Time-resolved and product studies on the synthesized dyads 1 and 2 have provided evidence that the benzophenone-to-thymine orientation strongly influences intramolecular photophysical and photochemical processes. The prevailing reaction mechanism has been established as a Paterno-Büchi cycloaddition to give oxetanes 3-6; however, the ability of benzophenone to achieve a formal hydrogen abstraction from the methyl group of thymidine has also been evidenced by the formation of photoproducts 7 and 8. These processes have been observed only in the case of the cisoid dyad 1. Adiabatic photochemical cycloreversion of the oxetane ring is achieved upon direct photolysis to give the starting dyad 1 in its excited triplet state. The photobiological implications of the above results are discussed with respect to benzophenone-photosensitized damage of thymidine.

Photosensitization of DNA by dipicolinic acid, a major component of spores of Bacillus species.

The DNA in spores of Bacillus species exhibits a relatively novel photochemistry, as 5-thyminyl-5,6-dihydrothymine (spore photoproduct (SP)) is by far the major UV photoproduct whereas cyclobutane dimers (CPDs) and (6-4) photoproducts (6-4PPs) are the major photoproducts in growing cells. Dehydration and more importantly complexation of DNA by alpha/beta-type small, acid-soluble spore proteins (SASP) have been shown to partly explain the photochemistry of spore DNA. The large amount ( approximately 10% of dry weight) of the spore's dipicolinic acid (DPA) also has been shown to play a role in spore DNA photochemistry. In the present work we showed by exposing spores of various strains of B. subtilis to UVC radiation that DPA photosensitizes spore DNA to damage and favors the formation of SP. The same result was obtained in either the presence or absence of the alpha/beta-type SASP that saturate the spore chromosome. Addition of DPA to dry films of isolated DNA or to frozen solutions of thymidine also led to a higher yield of SP and increased ratio of CPDs to 6-4PPs; DPA also significantly increased the yield of CPDs in thymidine exposed to UVC in liquid solution. These observations strongly support a triplet energy transfer between excited DPA and thymine residues. We further conclude that the combined effects of alpha/beta-type SASP and DPA explain the novel photochemistry of DNA in spores of Bacillus species.

The photochemistry of thymidylyl-(3'-5')-5-methyl-2'-deoxycytidine in aqueous solution.

The photochemistry of the dinucleoside monophosphate thymidylyl-(3'-5')-5-methyl-2'-deoxycytidine (Tpm5dC) has been studied in aqueous solution using both 254 nm and UV-B radiation. A variety of dinucleotide photoproducts containing 5-methylcytosine (m5C) have been isolated and characterized. These include two cyclobutane dimers (CBD) (the cis-syn [c,s]and trans-syn forms), a (6-4) adduct and its related Dewar isomer, and two isomers of a product in which the m5C moiety was converted into an acrylamidine. Small amounts of thymidylyl-(3'-5')-thymidine (TpT) were also formed, presumably as a secondary photoreaction product. In addition, a photoproduct was characterized in which the m5C moiety was lost, thus generating 3'-thymidylic acid esterified with 2'-deoxyribose at the 5-hydroxyl on the sugar moiety. The c,s CBD of Tpm5dC readily undergoes deamination to form the corresponding CBD of TpT. The kinetics of this deamination process has been studied; the corresponding enthalpy and entropy of activation for the reaction have been evaluated at pH 7.4 as being, respectively, 73.4 kJ/mol and -103.5 J/K mol. Deamination was not observed for the other characterized photoproducts of Tpm5dC.

Alpha-anomeric deoxynucleotides, anoxic products of ionizing radiation, are substrates for the endonuclease IV-type AP endonucleases.

Alpha-anomeric 2'-deoxynucleosides (alphadN) are one of the products formed by ionizing radiation (IR) in DNA under anoxic conditions. Alpha-2'-deoxyadenosine (alphadA) and alpha-thymidine (alphaT) are not recognized by DNA glycosylases, and are likely removed by the alternative nucleotide incision repair (NIR) pathway. Indeed, it has been shown that alphadA is a substrate for the Escherichia coli Nfo and human Ape1 proteins. However, the repair pathway for removal of alphadA and other alphadN in yeast is unknown. Here we report that alphadA when present in DNA is recognized by the Saccharomyces cerevisiae Apn1 protein, a homologue of Nfo. Furthermore, alphaT is a substrate for Nfo and Apn1. Kinetic constants indicate that alphadA and alphaT are equally good substrates, as a tetrahydrofuranyl (THF) residue, for Nfo and Apn1. Using E. coli and S. cerevisiae cell-free extracts, we have further substantiated the role of the nfo and apn1 gene products in the repair of alphadN. Surprisingly, we found that bacteria and yeast NIR-deficient mutants are not sensitive to IR, suggesting that DNA strand breaks with terminal 3'-blocking groups rather than alphadN might contribute to cell survival. We propose that the novel substrate specificities of Nfo and Apn1 play an important role in counteracting oxidative DNA base damage.

Independent generation of the 5-hydroxy-5,6-dihydrothymidin-6-yl radical and its reactivity in dinucleoside monophosphates.

Hydroxyl radical is a major reactive oxygen species produced by gamma-radiolysis of water or Fenton reaction. It attacks pyrimidine bases and gives the 5-hydroxy-5,6-dihydropyrimidin-6-yl radical as the major product. Here we report the synthesis of all four stereoisomers of 5-hydroxy-6-phenylthio-5,6-dihydrothymidine (T*), which, upon 254 nm UV irradiation, give rise to the 5-hydroxy-5,6-dihydrothymidin-6-yl radical (I). We also incorporated the photolabile radical precursors into dinucleoside monophosphates d(GT*) and d(TT*) and characterized major products resulting from the 254-nm irradiation of these dinucleoside monophosphates. Our results showed that, under anaerobic conditions, the most abundant product emanating from the 254-nm irradiation of d(GT*) and d(TT*) is an abasic site lesion. Products with the thymine portion being modified to thymine glycol and 5-hydroxy-5,6-dihydrothymine were also observed. In addition, we demonstrated that radical I can attack the C8 carbon atom of its 5' neighboring guanine and give rise to a novel cross-link lesion. Moreover, LC-MS/MS results showed that gamma-radiation of d(GT) under anaerobic condition yielded the same type of cross-link lesions.

A powerful antiradiation compound revealed by a new high-throughput screening method.

We present a new high-throughput screening method for the selection of powerful water-soluble antiradiation compounds. This method, which uses conventional immunoassay techniques, allowed the capacity of a given compound to protect thymidine from irradiation to be evaluated. By applying this assay to an antioxidant library, we showed for the first time that norbadione A, a well-known mushroom pigment, has pronounced atypical antiradiation properties.

Glycosidic bond cleavage of thymidine by low-energy electrons.

Thymidine was exposed to low-energy electrons (LEE) as a thin solid film under a high vacuum. Nonvolatile radiation products, remaining on the irradiated surface, were analyzed by HPLC/UV and GC/MS. Here, we show that exposure of thymidine to 3-100 eV electrons gives thymine as a major product with a yield of 3.2 x 10-2 per electron (about one-third of the total decomposition of thymidine). The formation of thymine indicates that LEE induces cleavage of the glycosidic bond separating the base and sugar moieties, suggesting a nonionizing resonant process involving dissociative attachment (<15 eV). In contrast, this reaction is not very efficient by DNA base ionization and does not occur by the reaction of solvated electrons with DNA. These studies introduce a new mechanism of DNA damage involving the interaction of LEE.

In vitro studies on DNA-photosensitization by different drug stereoisomers.

The possible stereoselectivity in DNA-photosensitization by carprofen (a NSAID drug) and ofloxacin (a fluoroquinolone agent) was investigated. The different drug stereoisomers or racemic mixtures were UVA-irradiated and the relaxation of the supercoiled circular pBR322 quantified by electrophoresis. Formation of single strand breaks was compared for each group of compounds. Moreover a mechanistic study by means of repair enzymes: T4 endonuclease V (specific of cyclobutane pyrimidine dimers), E. coli endonuclease III (revealing oxidized pyrimidines) and E. coli Formamidopyrimidine-DNA glycosylase (revealing oxidized purines) provided further insights into a possible stereoselectivity of the different reaction pathways in drug photosensitized-DNA damage. Ofloxacin and levofloxacin (its S stereoisomer) were responsible of single strand breaks formation as well as oxidation of pyrimidine and purine bases. No pyrimidine dimers were observed. Racemic, R and S stereoisomers of carprofen were less efficient than ofloxacin in DNA single strand breaks formation and did not induce enzyme-sensitive sites. The photoproducts distribution of drug-photosensitized reactions of 2'-deoxyguanosine and thymidine were established by HPLC as fingerprints for assignment of the DNA-photosensitization mechanism. Both Type I and Type II mechanisms were assigned to nucleoside-photosensitization by ofloxacin and levofloxacin. In the case of carprofen, a weak nucleoside degradation was obtained. The data suggest that levofloxacin, the (S) stereoisomer, might be slightly more efficient than racemic ofloxacin. In the case of carprofen the (S) isomer appears to be somewhat less active than its (R) enantiomer. However, due to the small differences found, the possible stereoselectivity has to be confirmed by future studies.

Inter-strand photoproducts are produced in high yield within A-DNA exposed to UVC radiation.

Far-UV irradiation of DNA leads to the dimerization of pyrimidine bases, resulting in the formation of cyclobutane type dimers and (6-4) photoproducts. In the dry state, an additional thymine dimeric photolesion, the spore photoproduct, is also generated. While most photoproducts are expected to be produced between adjacent pyrimidines, little attention has been paid to lesions involving bases located on different DNA strands. Using HPLC- mass spectrometry analysis of enzymatically digested DNA, we observed that, in the dry state, inter-strand dimeric photoproducts represented 30% of the total yield of dimeric thymine lesions. The major inter-strand damage was found to be the spore photoproduct. Formation of inter-strand lesions in significant yield could be obtained in solution upon modification of the DNA conformation as the result of the addition of large amounts of ethanol. In both cases, DNA is in the A-form, which is characterized by a high compaction, likely to favor inter-strand photoreactions. Since the latter DNA conformation is also predominant in bacterial spores, the formation and repair of dimeric photoproducts involving thymine bases located on different DNA strands may thus be relevant in terms of deleterious effects of UV radiation to the latter microorganisms.