CHARACTERIZATION OF THE (6-4) PHOTOPRODUCT OF 2'-DEOXYCYTIDYLYL-(3'->5')-THYMIDINE AND OF ITS DEWAR VALENCE ISOMER

Far-UV irradiation of dCpT in aqueous solution was found to generate the corresponding pyrimidine (6-4) pyrimidone photoadduct and the related Dewar valence isomer. The two photoproducts of dCpT were found to undergo deamination in aqueous solution, giving rise to the corresponding dUpT derivatives at a relatively slow rate. It was also shown that the Dewar isomer is much more alkali-labile than its (6-4) precursor.     

QUANTITATIVE CONVERSION OF THE (6–4) PHOTOPRODUCT OF TpdC TO ITS DEWAR VALENCE ISOMER UPON EXPOSURE TO SIMULATED SUNLIGHT

It has been found that the (6-4) photoproduct of thymidylyl-(3'----5')-deoxycytidine (TpdC) is converted quantitatively to a further photoproduct upon exposure to Pyrex-filtered medium pressure mercury arc light. Infrared UV, FAB MS, 1H NMR, 13C NMR and 31P NMR spectra were obtained for both the (6-4) product and its photolysis product. 1H NMR assignments were made on the basis of proton decoupling and homonuclear shift correlated experiments and 13C NMR assignments were made on the basis of proton-detected heteronuclear shift correlated experiments. The Dewar pyrimidinone structure was assigned to the photolysis product by analysis of the spectral data in comparison to those of the Dewar photoproduct of TpT and other Dewar pyrimidinones. The (6-4) product of TpdC is the second member of the class of (6-4) photoproducts that has been found to photoisomerize to its Dewar valence isomer upon exposure to wavelengths greater than 280 nm, the first being that of TpT (Taylor and Cohrs, 1987, J. Am. Chem. Soc. 109, 2834-2835). These results further support the proposal that all members of the (6-4) photoproduct class are converted to their Dewar valence isomers upon exposure to sunlight.     

Far-UV photochemistry and photosensitization of 2'-deoxycytidylyl-(3'-5')-thymidine: isolation and characterization of the main photoproducts.

Far-UV irradiation of 2'-deoxycytidylyl-(3'-5')-thymidine (dCpT) gave rise to the pyrimidine (6-4) pyrimidone adduct and its Dewar valence isomer as the main photoproducts. The absolute configuration of the former adduct was determined and its photoisomerization studied. A comparison of the alkali lability of both compounds showed that hydrolysis of the phosphodiester bond occurs for the Dewar valence isomer but not for its (6-4) precursor. In addition, the trans-syn and cis-syn cyclobutane dimers of dCpT were obtained by acetophenone photosensitization and characterized. Finally, the deamination rate constants for this series of compounds were shown to be dramatically influenced by the nature and the configuration of the photoproducts.     

The sugar conformation governs (6-4) photoproduct formation at the dinucleotide level

The LNA dinucleotide mimic of TpT whose two-sugar puckers are locked in the C3'-endo conformation selectively produces the corresponding cyclobutane pyrimidine dimer under 254 nm irradiation. In the natural series (TpT) the sugar puckers are in a major C2'-endo sugar conformation and the (6-4) photoproduct is also produced. Consequently, this study demonstrates that the C2'-endo conformation of the sugar pucker is necessary for (6-4) photoproduct formation.     

DNA Binding Mode of Antibody Fragments Specific for TT Photo-Dimers

A single-chain antibody specific for the pyrimidine(6-4)pyrimidone photoproduct in DNA was constructed and its binding properties to the cognate oligonucleotides were investigated. The fluorescent property of 2-aminopurine was used to study the binding mode of the antibody fragment to the pyrimidine(6-4)pyrimidone photoproduct in double-stranded DNA. The results indicated that the single-chain antibody recognizes the lesion in the single-stranded state.     

Further insight in the photochemistry of DNA: structure of a 2-imidazolone (5-4) pyrimidone adduct derived from the mutagenic pyrimidine (6-4) pyrimidone photolesion by UV irradiation

Pyrimidine (6-4) pyrimidone photoproducts represent one of the major mutagenic and carcinogenic class of DNA damage produced by UV exposure. At present, besides their conversion to their Dewar valence isomer, (6-4) photoproducts are generally believed to be photostable, and the observed biological properties of Paterno-Büchi-derived photoproducts are, thus far, exclusively attributed to these two types of compounds. Using a model system (2) relevant to DNA photochemistry, we have observed that the 5'-base moiety of the (6-4) thymine dimer 3, under far-UV radiation, is able to undergo a ring contraction leading to a 2-oxoimidazoline, 1. This unprecedented secondary photochemical reaction constitutes the first report of a major photomodification affecting (6-4) photoproducts and strongly questions the biological stability of the (6-4) adducts under UV light with 2-imidazolone (5-4) pyrimidone adducts being possibly another source of endogenous DNA damage.     

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.     

Mechanism of the alkali degradation of (6-4) photoproduct-containing DNA

The (6-4) photoproduct is one of the major damaged bases produced by ultraviolet light in DNA. This lesion is known to be alkali-labile, and strand breaks occur at its sites when UV-irradiated DNA is treated with hot alkali. We have analyzed the product obtained by the alkali treatment of a dinucleoside monophosphate containing the (6-4) photoproduct, by HPLC, NMR spectroscopy, and mass spectrometry. We previously found that the N3-C4 bond of the 5' component was hydrolyzed by a mild alkali treatment, and the present study revealed that the following reaction was the hydrolysis of the glycosidic bond at the 3' component. The sugar moiety of this component was lost, even when a 3'-flanking nucleotide was not present. Glycosidic bond hydrolysis was also observed for a dimer and a trimer containing 5-methyl-2-pyrimidinone, which was used as an analog of the 3' component of the (6-4) photoproduct, and its mechanism was elucidated. Finally, the alkali treatment of a tetramer, d(GT(6-4)TC), yielded 2'-deoxycytidine 5'-monophosphate, while 2'-deoxyguanosine 3'-monophosphate was not detected. This result demonstrated the hydrolysis of the glycosidic bond at the 3' component of the (6-4) photoproduct and the subsequent strand break by β-elimination. It was also shown that the glycosidic bond at the 3' component of the Dewar valence isomer was more alkali-labile than that of the (6-4) photoproduct.     

THE USE OF SPECIFIC RADIOIMMUNOASSAYS TO DETERMINE ACTION SPECTRA FOR THE PHOTOLYSIS OF (6-4) PHOTOPRODUCTS

Abstract. A radioimmunoassay (RIA) was developed which specifically detects a photoproduct produced by the near-UV photolysis of pyrimidine(6-4)pyrimidone photoproducts. This assay was used in conjunction with a previously characterized RIA which specifically detects (6-4) photoproducts to determine the relative efficiency of wavelengths between 265 and 435 nm for photolysing these lesions. The rate of loss of antibody-binding sites associated with (6-4) photoproducts correlates with the production of those associated with its photolysis product. Action spectra for both the loss of (6-4) photoproducts and the induction of the photolysis product parallel the absorption spectrum of the (6-4) photoproduct.     

Relative Contributions of UVB and UVA to the Photoconversion of (6‐4) Photoproducts into their Dewar Valence Isomers

Dewar valence isomers are photoisomerization products of pyrimidine (6‐4) pyrimidone photoproducts, a major class of UV‐induced DNA lesions, which exhibits a maximal absorption around 320 nm. However, Dewar isomers are not produced in significant amounts in cells exposed to biologically relevant doses of UVB. In contrast, they are readily produced when cells are exposed to a combination of UVA and UVB. The present computational work demonstrates that, on the basis of known absorption properties and formation quantum yields, the difference in Dewar formation between the two types of radiation can be explained by the role of normal bases. In the UVB range, at the low level of (6‐4) photoproducts present in cells exposed to realistic doses, normal bases are present in overwhelming amounts and absorb the vast majority of the incident photons. In contrast, the absorption of DNA bases is much weaker in the UVA range while that of (6‐4) photoproducts is still significant, making photoisomerization possible. This two‐photon process makes it difficult to define an action spectrum for the formation of Dewar isomers.     

Sugar conformational effects on the photochemistry of thymidylyl(3'-5')thymidine

The synthesis and conformational analysis of 2'-O,5-dimethyluridylyl(3'-5')-2'-O,5-dimethyluridine (1a), the analogue of thymidylyl(3'-5')thymidine (TpT; 1b) in which a methoxy group replaces each 2'-alpha-hydrogen atom, are described. In comparison with TpT, such modification increases the population of the C3'-endo conformer of the sugar ring puckering at the 5'- and 3'-ends from 30 to 75% and from 37 to 66%, respectively. Photolyses of 1a and TpT at 254 nm are qualitatively comparable (the cis-syn cyclobutane pyrimidine dimer and the (6-4) photoproduct are formed), although it is significantly faster in the case of 1a. These results are explained by the increased propensity of the modified dinucleotide to adopt a base-stacked conformation geometry reminiscent of that for TpT.     

The relative cytotoxicity of (6-4) photoproducts and cyclobutane dimers in mammalian cells

Abstract— The significance of the pyrimidine(6-4)pyrimidone photoproduct in mammalian cell killing is considered. Photochemical data indicate that the(6–4) photoproduct is induced at a substantial frequency compared to the cyclobutane dimer and that the action spectra for the induction of both lesions are equivalent. The repair of(6–4) photoproducts in various normal and UV-hypcrsensitive mammalian cell lines, including several recently derived somatic cell hybrids and transformants, is presented. The sensitivity of these cells to ultraviolet irradiation correlates better with the capacity to repair(6–4) photoproducts than cyclobutane dimers. These data are used to support that idea that the(6–4) photoproduct is one of the major cytotoxic lesions induced in DNA by ultraviolet light.     

ESTABLISHMENT and CHARACTERIZATION OF A MONOCLONAL ANTIBODY RECOGNIZING THE DEWAR ISOMERS OF(6–4)PHOTOPRODUCTS

Abstract— We established a monoclonal antibody(DEM–1) that recognizes UV-induced DNA damage other than cyclobutane pyrimidine dimers or(6–4)photoproducts. The binding ofDEM–1 antibody to 254 nm UV-irradiated DNA increased with subsequent exposure to UV wavelengths longer than 310 nm, whereas that of the 64M-2 antibody specific for the(6–4)photoproduct decreased with this treatment. Furthermore, the increase inDEM–1 binding was inhibited by the presence of the 64M-2 antibody during the exposure. We concluded that theDEM–1 antibody specifically recognized the Dewar photoproduct, which is the isomeric form of the(6–4)photoproduct. TheDEM–1 antibody, however, also bound to DNA irradiated with high fluences of 254 nm UV, suggesting that 254 nm UV could induce Dewar photoproducts without subsequent exposure to longer wavelengths of UV. Furthermore, an action spectral study demonstrated that 254 nm was the most efficient wavelength for Dewar photoproduct induction in the region from 254 to 365 nm, as well as cyclobutane dimers and(6–4)photoproducts, although the action spectrum values in the U V-B region were significantly higher compared with those for cyclobutane dimer and(6–4)photoproduct induction.     

Ab initio study of the (5R)- and (5S)-TT pyrimidine h5(6-4) pyrimidone photoproducts. Implications on the design of new biologically relevant analogues

A computational study of a series of N(1)- and/or C(6)-alkyl-5,6-dihydrothymine diastereomers at theory levels up to MP4(SDTQ)/6-31G//HF/6-31G and MP2/6-311G//HF/6-31G has demonstrated the respective importance of the substituents at positions 1, 5, and 6 on the energetically favored conformation of each isomer. Results obtained both in the gas and condensed phase indicate that unsubstitution of the N(1)-position favors a half-chair conformation with the C(5) -and C(6)-substituents in the equatorial position. On the other hand, in the case of the (6S)-1,6-dimethyl-5,6-dihydrothymine, the C(6)-substituent adopts the axial position to minimize its van der Waals interactions with the N(1)-substituent. Furthermore, if the configuration at the C(5)-dihydrothymine position has no resultant influence on the total molecular free energy, when a pyrimidone substituent is introduced at the dihydrothymine C(6)-position, additional repulsive forces between the C(5)- and C(6)-substituents make the diaxially substituted half-chair conformation the most energetically favorable one. These results indicate that the observed C(6)-axially substituted conformation of the thymine-thymine pyrimidine h(5)(6-4) pyrimidone photoproducts is not necessarily induced by the macrocyclic structure. They also nicely explain the formation mechanism of these photoproduct derivatives, and allow the prediction of the conformation of new analogues.     

Photoreactivation of cyclobutane dimers and (6-4) photoproducts in the epidermis of the marsupial, Monodelphis domestica

Radioimmunoassays were used to investigate the repair of cyclobutane pyrimidine dimers and pyrimidine (6-4)pyrimidone photoproducts ((6-4] photoproducts) in the epidermis of the South American opossum, Monodelphis domestica. In the absence of photoreactivating light, both types of photodamage were excised with similar kinetics, 50% of the damage remaining 8 h after UV irradiation in vivo. Exposure of UV-irradiated skin to photoreactivating light resulted in removal of most of the cyclobutane dimers and an enhanced rate of (6-4) photoproduct repair. Photoenhanced excision repair of non-dimer damage increases the range of biologically effective lesions removed by in vivo photoreactivation.     

Electrospray-mass spectrometry characterization and measurement of far-UV-induced thymine photoproducts

Far-UV-induced formation of dimeric pyrimidine photoproducts within DNA is a major cause of the carcinogenic effects of solar light. The chemical structure of this class of lesion has been mostly determined by studies on model compounds. The present work is aimed at providing mass spectrometry data on the thymine-thymine photoproducts, including the diastereoisomers of the cyclobutane dimer, the (6-4) adduct, the related Dewar valence isomer and the spore photoproduct. Fragmentation mass spectra of the modified bases, nucleosides, dinucleoside monophosphates and dinucleotides were recorded following electrospray ionization with either triple-quadrupolar or ion-trap detection. The results showed differences in fragmentation pattern between the different types of photoproducts. In addition, a drastic effect of the diastereoisometry was observed for the cyclobutane dimers. A sensitive detection technique has been developed for the analysis of dinucleoside monophosphate photoproducts by high-performance liquid chromatography associated with mass spectrometry in the negative mode with multiple reaction-monitoring detection.     

Fragmentation of photomodified oligodeoxynucleotides adducted with metal ions in an electrospray-ionization ion-trap mass spectrometer

We report the effect of metal-ion adduction on the fragmentation of oligodeoxynucleotides (ODNs) bearing DNA photoproducts. When protons on backbone phosphates of ODNs are completely replaced with metal ions, cleavages occur readily within the photoproduct moiety, whereas those cleavages do not occur in photomodified ODNs in which the phosphates are associated with protons. For example, thymine/adenine (TA*) photoproducts revert to their undamaged precursors upon collisional activation, the pyrimidine(6-4)pyrimidone product and its Dewar valence isomer show a characteristic neutral loss of C4H3NO3, and dimeric adenine photoproducts show a distinctive loss of NH2CN from the adenine six-membered ring. The product-ion mass spectra of photodamaged ODNs that are adducted to metal ions are complementary in terms of structure information to those spectra of ODNs in which the phosphates are associated with protons. The results also demonstrate that the energy required for strand cleavages is higher for ODNs adducted with metal ions than that for ODNs bound with protons. Furthermore, the loss of a pyrimidine is more favorable than the loss of a purine in the fragmentation of ODNs associated with metal ions.     

Crystal structure and photochemical behavior in solution of the 3'-N-sulfamate analogue of thymidylyl(3'-5')thymidine

The 3'-N-sulfamate analogue of thymidylyl(3'-5')thymidine (TnsoT, 1) exhibits a preference for a C3'-endo conformation in the solution and solid states. Its photochemical behavior in solution is compared to that of its natural counterpart, thymidylyl(3'-5')thymidine (TpT, 2), to get further insight into the significance of the C3'-endo conformation on the photoproduct formation at the single-stranded dinucleotide level. Irradiation at 254 nm of 1 led to the same type of photoproducts as observed with 2. However, 1 was significantly more photoreactive than 2, and accordingly, the initial rate of photoproduct formation was enhanced in accordance with its propensity to base stack compared to 2. The corresponding quantum yields were determined and showed that the enhancement factor (1 compared to 2) is moderate for the cyclobutane pyrimidine dimer (CPD) (1.26) and much higher for the (6-4) photoproduct (1.8). These data strongly suggest that the CPD and (6-4) photoproduct arise from distinct minor stacked conformations.     

Photochemistry of nucleic acids in cells.

A survey of the recent aspects of the main photoreactions induced by far-UV radiation in cellular DNA is reported. This mostly includes the formation of cyclobutadipyrimidines, pyrimidine(6-4)pyrimidone photoadducts and related Dewar valence isomers in various eukaryotic and prokaryotic cells, as monitored by using either specific or more general assays. Information is also provided on mechanistic aspects regarding the formation of 5,6-dihydro-5-(alpha-thyminyl) thymine, the so-called "spore photoproduct" within far-UV-irradiated bacterial spores. The second major topic of the review deals with the effects of near-UV radiation and visible light on cellular DNA which are mostly mediated by photosensitizers. The main photoreactions of furocoumarins with DNA, one major class of photosensitizers used in the phototherapy of skin diseases, involve a [2 + 2] cycloaddition to the thymine bases according to an oxygen-independent mechanism. In contrast a second type of photosensitized reaction which appears to play a major role in the genotoxic effects of both near-UV and visible light requires the presence of oxygen. The photodynamic effects which are mediated by either still unidentified endogenous photosensitizers or defined exogenous photosensitizers lead to the formation of a wide spectrum of DNA modifications including base damage, oligonucleotide strand breaks and DNA-protein cross-links.     

FLUORESCENCE QUANTUM YIELD DETERMINATION OF PYRIMIDINE (6-4) PYRIMIDONE PHOTOADDUCTS

Abstract An extensive study of the fluorescence characteristics of pyrimidine (6-4) pyrimidone photoadducts, a major class of far-UV-induced DNA lesions, was carried out on dinucleoside monophosphate (6-4) photoadducts, including thymidylyl-(3'→ 5')-thymidine (TpT), 2'-deoxycytidylyl-(3'-5')-thymidine, thymidylyl-(3'→ 5')-2'-deoxy-cytidine, 2'-deoxyuridylyl-(3'→ 5')-thymidine, 5-methyl-2'-deoxycytidylyl-(3'-5')-thymidine (6-4) photoadducts and the corresponding base (6-4) photoadducts, 6-4'-(5'-methylpyrimidin-2'-one) thymine (TT), 5-hydroxy-6-4'-(5'-methylpyrimidin-2'-one)-5,6-dihydrothymine (CT), 5-amino-6-4'-(pyrimidin-2'-one)-5,6-dihydrothymine (UC) obtained by mild acidic hydrolysis of the former derivatives. The fluorescence quantum yield (Φ_F) of these compounds was found to depend on one hand, on the nature of the two bases involved and the base substituent and, on the other hand, on the presence of the phosphate group. The hydrolysis of the phosphodiester bond was shown to enhance Φ_F, the larger effect being observed in the case of the thymine-thymine photoadducts with a seven-fold increase of the Φ_F value in the case of TT as compared to TpT (0.21 and 0.03, respectively). These results are discussed in terms of structural considerations.