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.     

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.     

QUANTUM YIELDS AND SECONDARY PHOTOREACTIONS OF THE PHOTOPRODUCTS OF dTpdT, dTpdC AND dTpdU

Abstract— The photoproducts of the dinucleoside monophosphates, dTpdT, dTpdC and dTpdU, have been purified by high performance liquid chromatography and characterized by UV absorption spectroscopy, fast atom bombardment mass spectrometry and by secondary thermal and photoreactions. Four types of photoproducts were analyzed: (1) cyclobutane dimers including cis-syn isomers and two diastereomers of the trans-syn isomers; (2) 6-4 photoadducts and the corresponding Dewar valence isomers; (3) photohydrates comprising two diastereomers and (4) a new photoproduct resembling nucleobase amine adducts, which occurs only for dTpdC. The quantum yields of formation of these photoproducts and for some secondary photoreactions were measured by kinetic analysis of the photoproduct yield as a function of photon fluence. These results indicate that cis-syn cyclobutane dimers are the photoproducts formed with highest efficiency with dT[p]dC dimers being formed with 50–75% the efficiency of dT[p]dT dimers. The 6-4 photoadducts are formed with 5–10% the efficiency of cis-syn cyclobutane dimers and the 6-4 photoadduct of dTpdC is formed two to three times more efficiently than that of dTpdT. Photohydrates are also formed efficiently due to an equilibrium between stacked and unstacked complexes of the dinucleoside monophosphates. It is shown that three of these photoproducts, namely the cyclobutane dimers of dTpdC, the 6-4 photoadducts and the possible nucleobase amine adduct, undergo photolysis in the UV-B region resulting in either photoreversion or secondary photoreaction.     

CONFORMATIONS OF DEOXYDODECANUCLEOTIDES WITH PYRIMIDINE (6-4) PYRIMIDONE PHOTOADDUCTS

Abstract— We have carried out molecular mechanical simulations of dodecanucleotide d(CGCGAAXYCGCG).d(CGCGX'Y'TTCGCG) with XY being CC, TC, TT and CT and X'Y'being their corresponding base paired dinucleotides on the complementary strand. Simulations were also carried out with the corresponding pyrimidine (6-4)-pyrimidone photoadducts incorporated in these dodecanucleotides. As in the case of the cyclobutane dimer incorporated dodecanucleotide structures (Rao et al., 1984), those regions of the DNA modified by6–4 pyrimidine adducts are found to undergo little conformational changes except in the dimer region. The conformational characteristics of the6–4 pyrimidine adduct incorporated structures seem to be influenced by the nature of the base at the 3' end of the dimer. Specifically, favorable hydrogen bonding interactions between the 5' end base and its preceding phosphate group are present in structures which have cytosine at the 3' end of the photodimer. The energetics of these structures relative to those without incorporated dimers have been discussed and the results have been analysed in the light of the currently prevalent ideas on the role of the6–4 photoadducts in mutagenesis in various organisms.     

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.     

The Dewar valence isomer of the (6-4) photoadduct of thymidylyl-(3'-5')-thymidine monophosphate: formation, alkaline lability and conformational properties.

The formation of the Dewar valence isomer of the pyrimidine(6-4)pyrimidone photoadduct of thymidylyl-(3'-5')-thymidine monophosphate (TpT) was investigated under different irradiation conditions. This photoproduct was generated on exposure of TpT to far-UV radiation. However, no detectable amount of the Dewar isomer or its precursor (pyrimidine(6-4)pyrimidone photoadduct) was observed following acetone photosensitization of TpT. The Dewar valence isomer was much more unstable than the pyrimidine(6-4)pyrimidone photoproduct when treated with hot piperidine. A detailed conformational analysis of the TpT Dewar isomer photoproduct is reported as inferred from extensive one- and two-dimensional 300 and 620 MHz proton nuclear magnetic resonance (1H NMR) measurements and molecular mechanics calculations.     

H-NMR studies of duplex DNA decamer containing a uracil cyclobutane dimer: implications regarding the high UV mutagenecity of CC photolesions

To determine the origin of the UV-specific CC to TT tandem mutation at the CC site, we made a duplex DNA decamer containing a uracil cis-syn cyclobutane dimer (CBD) as the deaminated model of a cytosine dimer. Two-dimensional 1H-NMR spectroscopy studies were performed on this sequence where two adenines (Ade) were opposite to the uracil dimer. Two imino protons of the uracil dimer were found to retain Watson-Crick hydrogen bonding with the opposite Ade, although the 5'-U(NH) of the dimer site showed unusual upfield shift like that of the 5'-T(NH) of the TT dimer, which seemed to be associated with deshielding by the flanking base rather than with reduced hydrogen bonding. (McAteer et al. 1998, J. Mol. Biol. 282:1013-1032). Hydrogen bondings at the dimer site were also supported by detecting typical strong nuclear Overhauser effects (NOE) between two imino protons and the opposite Ade H2 or NH2. But sequential NOE interactions of base protons with sugar protons were absent at the two flanking nucleotides of the 5' side of the uracil dimer and at the intradimer site, contrasting with its thymine analog where sequential NOE was absent only at the A4-T5 step. In addition, NOE cross peak for U5(NH) <--> A4(H2) was detected, although the NOE interactions of U6(NH) with A7(H2) and A17(H2) were not observed in contrast to the thymine dimer duplex. This different local structural alteration may be affected by the induced right-hand twisted puckering mode of cis-syn cyclobutane ring of the uracil dimer in the B-DNA duplex, even though the isolated uracil dimer had left-hand twisted puckering rigidly. In parallel, these observations may be correlated with observed differences in mutagenic properties between cis-syn UU dimer and cis-syn TT dimer.     

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.     

Synthesis of the TT pyrimidine (6-4) pyrimidone photoproduct-thio analogue phosphoramidite building block

The phosphoramidite building block synthesis of the thio analogue at the 5,6-dihydropyrimidine C5 position of the thymidylyl(3'-5')thymidine (6-4) photoproduct 1 is presented. This compound was readily obtained from the appropriately protected dinucleotide P-methyl-5'-O-dimethoxytritylthymidilyl(3' --> 5')-4-thiothymidine 2 after irradiation at 366 nm, then S-sulfenylmethylation of the thiol function of the resulting (6-4) adduct, and phosphitylation of the 3'-hydroxyl group.     

PHOTOREACTION OF MONOFUNCTIONAL 3-CARBETHOXYPSORALEN WITH DNA: IDENTIFICATION AND CONFORMATIONAL STUDY OF THE PREDOMINANT cis-syn FURAN SIDE MONOADDUCT TO THYMINE

The main DNA-3-CPs furan side adduct has been isolated following acidic hydrolysis of photomodified DNA and subsequent reversed-phase high performance liquid chromatography purification. This photoadduct has been identified as a thymine-3-CPs C₄-cycloadduct Thy<^{5 4'}_{6 5'}, >3-CPs based on its optical spectroscopic features and of its plasma desorption mass spectrometric characteristics. Moreover, its reversed-phase high performance liquid chromatography retention time is in agreement with a cis-syn stereochemistry as compared to the chromatographic properties of the cis-syn model adduct Thy< ^{5 4'}_{6 5'} >3-CPs previously identified. Further support for the cis-syn stereoconfiguration assignment was provided by fluorescence quenching experiments using iodide and silver ions as external and internal quenchers respectively. These data strongly indicate that the Thy< ^{5 4'}_{6 5'} >3-CPs photoadduct is located inside the DNA helix in agreement with its cis-syn stereochemistry.     

UV INDUCED (6-4) PHOTOPRODUCTS ARE DISTRIBUTED DIFFERENTLY THAN CYCLOBUTANE DIMERS IN NUCLEOSOMES

We have compared the distributions of two stable UV photoproducts in nucleosome core DNA at the single-nucleotide level using a T4 polymerase-exonuclease mapping procedure. The distribution of pyrimidine-pyrimidone (6-4) dimers was uncovered by reversing the major UV photo-product, cis-syn cyclobutane pyrimidine dimer, with E. coli DNA photolyase and photoreactivating light. Whereas the distribution of total UV photoproducts in nucleosome core DNA forms a striking 10.3 base periodic pattern, the distribution of (6-4) dimers is much more random throughout the nucleosome core domain. Therefore, histone-DNA interactions in nucleosomes strongly modulate formation of the major class of UV-induced photoproducts, while having either a constant effect or no effect on (6-4) dimer formation.     

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.     

UV IRRADIATION OF NUCLEIC ACIDS: CHARACTERIZATION OF PHOTOPRODUCTS OF THYMIDYLYL-(3'→5')-2'-DEOXY-5-FLUOROURIDINE

Abstract— The acetone-sensitized irradiation using UV-B (ultraviolet light, 280–320 nm; sunlamps) of thymidyl-yl(3'→5')deoxyfluorouridine monophosphate produces two main photoproducts. The distribution of these photo-products is dependent on the pH of the irradiation solution. At pH 6, the cis-syn cyclobutane-type photodimer is the major product, whereas at high pH (8–10) a photoadduct is the major product. These photoproducts have been identified and structurally characterized by H-1 and C-13 NMR spectroscopy. The photoadduct arises from defluorination of the 5-fluorouracil moiety. The structure of the photoadduct maintains the sugar-phosphate backbone of the starting material (d-TpF), and contains a saturated thymine moiety with an added Thy(C6-hydroxyl) and a Thy(C5)-(C5)Ura covalent bond.     

Repair of the main UV-induced thymine dimeric lesions within Arabidopsis thaliana DNA: evidence for the major involvement of photoreactivation pathways.

The UV-B induced formation of thymine cis-syn cyclobutane dimer and related (6-4) photoproduct was monitored within DNA of cultured cells and plants of Arabidopsis thaliana. This was achieved using a sensitive and accurate HPLC-tandem mass spectrometry assay. It was found that the cyclobutane pyrimidine dimer was formed in a ninefold higher yield than the (6-4) photoproduct. The removal of the lesions was then studied by incubating irradiated cells either in the darkness, under visible light or upon exposure to UV-A radiation. Dark repair of both cyclobutane dimers and (6-4) photoproducts was found to be very ineffective. In contrast, a rapid decrease in the level of photoproducts was observed when UV-B-irradiated cells were exposed to UV-A and, to a lesser extent, to visible light. The removal of (6-4) adducts was found to occur more efficiently. These results strongly suggest that repair of UV-induced photolesions in plants is mainly mediated by photolyases.     

EXCISION OF CYCLOBUTANE DIMERS IN GENOMIC AND EPISOMAL DNA IN HUMAN CELLS

Abstract Direct determination has been made of cyclobutyl pyrimidine dimer induction and excision repair in an episomal SV40 DNA population in vivo . Maintaining SV40-transformed human (GM637) cells in confluent culture results in amplification of a mutant SV40 episome to high copy number. T4 endonuclease V was used to quantify the induction and repair of cyclobutane dimers in the SV40 episome and genomic DNA of the same cells. Differences in both parameters were observed cyclobutane dimers were induced at 1.5–2-fold greater frequency in episomal DNA and excised at a reduced rate compared to genomic DNA in the host cells.     

CORRELATION OF UVC AND UVB CYTOTOXICITY WITH THE INDUCTION OF SPECIFIC PHOTOPRODUCTS IN T-LYMPHOCYTES AND FIBROBLASTS FROM NORMAL HUMAN DONORS

Abstract— By using specific monoclonal antibodies in situ and a computer-assisted image analysis system we have determined the relative induction of cyclobutane dimers, (6–4) photoproducts and Dewar isomers in human mononuclear cells and fibroblasts following irradiation with UVC, broad-spectrum UVB and narrow-spectrum UVB. The lamps produced these lesions in different proportions, with broad-spectrum UVB inducing a greater combined yield of (6–4) photoproducts and Dewar isomers per cyclobutane dimer than UVC or narrow-spectrum UVB. The relative induction ratios of (6–4) photoproducts compared to cyclobutane dimers were 0.15, 0.21 and 0.10 following irradiation with UVC, broad- or narrow-spectrum UVB, respectively. Although Dewar isomers were induced by UVC, their relative rate of formation compared to cyclobutane dimers was significantly greater after irradiation with either broad-spectrum or narrow-spectrum UVB. These values were 0.001, 0.07 and 0.07, respectively. With each lamp source, we have determined the survival of normal human T-lymphocytes and fibroblasts at fiuences, which induce equivalent yields of cyclobutane dimers, (6–4) photoproducts or (6–4) photoproducts plus Dewar isomers. Killing of fibroblasts appears to be associated with (6–4) photoproduct formation, whereas killing of T-lymphocytes seems to be mediated by combined (6–4) plus Dewar yields. These results emphasize the need to study the biological effects of UVB because cellular responses may be different from those following UVC irradiation.     

A THEORETICAL STUDY OF THE CYTOSINE EXCIMER STATE: THE ROLE OF GEOMETRY OPTIMIZATION

Abstract A study of the lowest excited dimer (excimer) singlet state of the DNA base cytosine was performed with fully optimized geometry (near the cis-syn form) using a slightly modified version of MOPAC6 (AM1 hamiltonian) and 112 excited configurations involving an active space consisting of the four highest occupied and the two lowest empty MO of the dimer. A binding energy of 3 kcal/mol for the excimer state is demonstrated, and the excimer fluorescence is predicted to be shifted 150 nm to longer wavelengths relative to that of the monomer fluorescence. The excimer geometry has the planes nonparallel (40–45° interplanar angle) and distorted, with the C₅-C₆ bonds showing quite close contact (2.4 Å). Excitation is found to be more localized in the region of the C₅-C₆ bond than expected from the monomer wavefunction. No stable excimer was found if the planes were constrained to be planar, nor if the triplet state was selected. The results suggest that the excimer geometry found in this study may be a precursor to the cis-syn cyclobutane photodimer.     

4',5'-SUBSTITUTED METHYLANGELICINS: PHOTOCYCLOADDUCTS WITH PYRIMIDINE BASES OF DNA

The isolation and characterization of photocycloadducts with pyrimidine bases from DNA samples irradiated (365 nm) in the presence of four 4',5'-substituted methylangelicins was performed. All these furocoumarins yielded mainly the cis-syn furan-side cycloadduct with thymine. For 4',5'-dimethyl-, 5,4',5'-trimethyl- and 6,4',5'-trimethylangelicin this adduct was accompanied by two pyrone-side adducts ( cis-syn and cis-anti ), whereas the 4,4',5'-trimethyl derivative gave the furan-side adduct with cytosine.
The characterization of the regio- and stereochemistry of the adducts was accomplished by ¹H NOE (nuclear Overhauser effect) and ¹H-¹³C HMBC (heteronuclear multiple-bond connectivity) spectroscopies.
The formation of different cycloadducts in DNA by the various derivatives highlights the role of the methyl groups in determining the regio- and stereochemistry of the cycloaddition.     

4'-AMINOMETHYL-4,5',8-TRIMETHYLPSORALEN PHOTOCHEMISTRY THE EFFECT OF CONCENTRATION AND UVA FLUENCE ON PHOTOADDUCT FORMATION IN POLY(dAdT) AND CALF THYMUS DNA

Abstract-The photochemistry of 4'-aminomethyl-4,5',8-trimethylpsoralen (AMT) with poly(dA-dT) and calf thymus DNA was studied. The extent of photoadduct formation and the distribution of photoadducts (3,4– and 4',5'-monoadducts and crosslinks) were determined by liquid scintillation analysis and HPLC, respectively. The adducts were characterized on the basis of their UV absorption spectra and mass spectral analysis. The high DNA binding constant for AMT (1.5 x 10⁵ M⁻¹ ) led to a high fraction of intercalated molecules, which contributed to the high level of AMT photoadduct formation, as many as 102 adducts per kilobase pair. In addition, there is a distinct difference in the adduct distribution compared to the previously studied 8-methoxypsoralen (8-MOP). Under the conditions employed for the photochemical studies, virtually all of the AMT molecules in solution are intercalated, occupying 25% of the base pair sites. Under similar conditions, 8-MOP molecules occupied 10 times fewer sites. Thus, for AMT, DNA base pair sites other than 5'TA, the well-characterized strong binding for psoralens in general, are an additional target for photomodification, which results in the formation of a higher percentage of monoadducts. The proportion of photoadducts formed was virtually independent of AMT concentration and UVA (320–400 nm radiation) fluence.     

DNA REPAIR IN THE VARIABLE PLATYFISH (Xiphophorus variatus) IRRADIATED in vivo WITH ULTRAVIOLET B LIGHT

Abstract— Dark- and light-dependent DNA repair processes were studied in vivo in the variable platyfish, Xiphophorus vuriatus . Excision (dark) repair of the (6–4) photoproduct was more efficient than that of the cyclobutane dimer with ∼ 70% of the (6–4) photoproducts reniovcd by 24 h post-UVB radiation compared to ∼30% of the cyclobutane dimers. Exposure to photoreactivating light resulted in rapid loss of most (>90%) of the cyclobutane dimers and increased excision repair of the (6–4) photoproduct. Preexposure to photoreactivating light 8 h prior to UVB radiation increased the rate of photoreactivation two-fold.