Dewar Valence Isomers Research Articles

Chemical investigation of light induced DNA bipyrimidine damage and repair

In all organisms, genetic information is stored in DNA and RNA. Both of these macromolecules are damaged by many exogenous and endogenous events, with UV irradiation being one of the major sources of damage. The major photolesions formed are the cyclobutane pyrimidine dimers (CPD), pyrimidine-pyrimidone-(6-4)-photoproducts, Dewar valence isomers and, for dehydrated spore DNA, 5-(α-thyminyl)-5,6-dihydrothymine (SP). In order to be able to investigate how nature's repair and tolerance mechanisms protect the integrity of genetic information, oligonucleotides containing sequence and site-specific UV lesions are essential. This tutorial review provides an overview of synthetic procedures by which these oligonucleotides can be generated, either through phosphoramidite chemistry or direct irradiation of DNA. Moreover, a brief summary on their usage in analysing repair and tolerance processes as well as their biological effects is provided.

DNA (6-4) Photolyases Reduce Dewar Isomers for Isomerization into (6-4) Lesions

Repair of the Dewar valence isomers by (6-4) photolyases proceeds via an enzyme catalyzed ring-opening reaction of the Dewar lesion to the (6-4) photoproduct.

Molecular Mechanisms of Ultraviolet Radiation‐Induced DNA Damage and Repair

DNA is one of the prime molecules, and its stability is of utmost importance for proper functioning and existence of all living systems. Genotoxic chemicals and radiations exert adverse effects on genome stability. Ultraviolet radiation (UVR) (mainly UV-B: 280–315 nm) is one of the powerful agents that can alter the normal state of life by inducing a variety of mutagenic and cytotoxic DNA lesions such as cyclobutane-pyrimidine dimers (CPDs), 6-4 photoproducts (6-4PPs), and their Dewar valence isomers as well as DNA strand breaks by interfering the genome integrity. To counteract these lesions, organisms have developed a number of highly conserved repair mechanisms such as photoreactivation, base excision repair (BER), nucleotide excision repair (NER), and mismatch repair (MMR). Additionally, double-strand break repair (by homologous recombination and nonhomologous end joining), SOS response, cell-cycle checkpoints, and programmed cell death (apoptosis) are also operative in various organisms with the expense of specific gene products. This review deals with UV-induced alterations in DNA and its maintenance by various repair mechanisms.

Crystal Structure of the T(6‐4)C Lesion in Complex with a (6‐4) DNA Photolyase and Repair of UV‐Induced (6‐4) and Dewar Photolesions

UV-light irradiation induces the formation of highly mutagenic lesions in DNA, such as cis-syn cyclobutane pyrimidine dimers (CPD photoproducts), pyrimidine(6-4)pyrimidone photoproducts ((6-4) photoproducts) and their Dewar valence isomers ((Dew) photoproducts). Here we describe the synthesis of defined DNA strands containing these lesions by direct irradiation. We show that all lesions are efficiently repaired except for the T(Dew)T lesion, which cannot be cleaved by the repair enzyme under our conditions. A crystal structure of a T(6-4)C lesion containing DNA duplex in complex with the (6-4) photolyase from Drosophila melanogaster provides insight into the molecular recognition event of a cytosine derived photolesion for the first time. In light of the previously postulated repair mechanism, which involves rearrangement of the (6-4) lesions into strained four-membered ring repair intermediates, it is surprising that the not rearranged T(6-4)C lesion is observed in the active site. The structure, therefore, provides additional support for the newly postulated repair mechanism that avoids this rearrangement step and argues for a direct electron injection into the lesion as the first step of the repair reaction performed by (6-4) DNA photolyases.

Translesion synthesis across the (6-4) photoproduct and its Dewar valence isomer by the Y-family and engineered DNA polymerases

We analyzed the translesion synthesis across the UV-induced lesions, the (6-4) photoproduct and its Dewar valence isomer, by using human DNA polymerases eta and iota in vitro. The primer extension experiments revealed that pol eta tended to incorporate dG opposite the 3' component of both lesions, but the incorporation efficiency for the Dewar isomer was higher than that for the (6-4) photoproduct. On the other hand, pol iota was likely to incorporate dA opposite the 3' components of the (6-4) photoproduct and its Dewar isomer with a similar efficiency. Elongation after the incorporation opposite the UV lesions was not observed for these Y-family polymerases. We further analyzed the bypass ability of an engineered polymerase developed from Thermus DNA polymerase for the amplification of ancient DNA. This polymerase could bypass the Dewar isomer more efficiently than the (6-4) photoproduct.

Cell surface expression of melanocortin-1 receptor on HaCaT keratinocytes and α-melanocortin stimulation do not affect the formation and repair of UVB-induced DNA photoproducts

Ultraviolet (UV) exposure induces an up-regulation of melanocortin-1 receptor (MC1R) expression in human skin and the alpha-melanocyte-stimulating hormone (alpha-MSH) may reduce UVB-induced DNA damage in normal human melanocytes. Using high-performance liquid chromatography coupled to tandem mass spectrometry, we investigated the formation and repair of DNA lesions in UVB-irradiated HaCaT cells stably transfected with the wild type MC1R gene (HaCaT-MC1R). Similar levels of 8 bipyrimidine photoproducts including cyclobutane pyrimidine dimers (CPDs) (T<>T, T<>C, C<>T), (6-4) photoproducts ((6-4)PPs) (TT-(6-4)PPs, TC-(6-4)PPs) and their Dewar valence isomers together with 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodGuo) were found to be generated in both non-transfected and HaCaT-MC1R cells after UVB exposure. Time-course studies of DNA photoproduct yields indicated that the DNA repair ability depended upon radiation doses. It was shown that (6-4)PPs were removed from the DNA of UVB-irradiated cells much more efficiently than CPDs. The repair efficiency of 8-oxodGuo, CPDs and (6-4)PPs was relatively similar in both cell lines and was not modified by stimulation with alpha-MSH before UVB-exposure. In conclusion, cell surface-enforced expression of MC1Rs on HaCaT keratinocytes and alpha-MSH stimulation do not affect the formation of UVB-induced DNA photoproducts and their subsequent repair.

A model for triplet mutation formation based on error-prone translesional DNA synthesis opposite UV photolesions

A triplet mutation is defined as multiple base substitutions or frameshifts within a three-nucleotide sequence which includes a dipyrimidine sequence. Triplet mutations have recently been identified as a new type of UV-specific mutation, although the mechanism of their formation is unknown. A total of 163 triplet mutations were identified through an extensive search of previously published data on UV-induced mutations, including mutations from skin, skin cancer, and cultured mammalian cells. Seven common patterns of sequence changes were found: Type I, NTC → TTT; Type IIa, NCC → PyTT or PyCT (Py, pyrimidine); Type IIb, TCC → PuTT or PuCT (Pu, purine); Type III, NCC → NAT or NTA; Type IV, NTT → AAT; Type Va, NCT → NTX; and Type Vb, PuCT → XTT (N and X, independent anonymous bases). Furthermore, it is suggested that the type of UV lesion responsible for each of these triplet mutation classes are (a) pyrimidine(6-4)pyrimidone photoproducts for Types I, IIb, III, IV and Vb, (b) cyclobutane pyrimidine dimers for Type Va, and (c) Dewar valence isomers for Types IIa and IIb. These estimations are based primarily on results from previous studies using photolyases specific for each type of UV lesion. A model is proposed to explain the formation of each type of triplet mutation, based on error-prone translesional DNA synthesis opposite UV-specific photolesions. The model is largely consistent with the ‘A-rule’, and predicts error-prone insertions not only opposite photolesions but also opposite the undamaged template base one-nucleotide downstream from the lesions.

Synthesis of oligonucleotides containing the Dewar valence isomer of the (6–4) photoproduct and their application to (6–4) photolyase studies

The (6-4) photoproduct, which is a major UV light-induced lesion formed between adjacent pyrimidine bases, is isomerized to its Dewar valence isomer by exposure to longer wavelengths. We have synthesized a phosphoramidite building block of the Dewar photoproduct formed at the thymidylyl(3'-5')thymidine site, and incorporated it into oligonucleotides on a DNA synthesizer, aiming to use them for biological studies. An alternative activator, benzimidazolium triflate, gave better results, while by-products were detected at longer retention time in the ordinary synthesis. We characterized the synthetic oligonucleotides by UV conversion, nuclease digestion and mass spectrometry. Their use in the study of the (6-4) photolyase, a DNA repair enzyme, revealed its different recognition modes between the (6-4) photoproduct and the Dewar isomer.

Chemical synthesis of oligodeoxyribonucleotides containing the Dewar valence isomer of the (6–4) photoproduct and their use in (6–4) photolyase studies

The pyrimidine(6–4)pyrimidone photoproduct, a major UV lesion formed between adjacent pyrimidine bases, is transformed to its Dewar valence isomer upon exposure to UVA/UVB light. We have synthesized a phosphoramidite building block of the Dewar photoproduct formed at the thymidylyl(3′–5′)thymidine site and incorporated it into oligodeoxyribonucleotides. The diastereoisomers of the partially protected dinucleoside monophosphate bearing the (6–4) photoproduct, which were caused by the chirality of the phosphorus atom, were separated by reversed-phase chromatography, and the (6–4) photoproduct was converted to the Dewar photoproduct by irradiation of each isomer with Pyrex-filtered light from a high-pressure mercury lamp. The Dewar photoproduct was stable under both acidic and alkaline conditions at room temperature. After characterization of the isomerized base moiety by NMR spectroscopy, a phosphoramidite building block was synthesized in three steps. Although the ordinary method could be used for the oligonucleotide synthesis, benzimidazolium triflate as an alternative activator yielded better results. The oligonucleotides were used for the analysis of the reaction and the binding of Xenopus (6–4) photolyase. Although the affinity of this enzyme for the Dewar photoproduct-containing duplex was reportedly similar to that for the (6–4) photoproduct-containing substrate, the results suggested a difference in the binding mode.

Photochemical Ring-Opening and Intramolecular Hydrogen Shift Reactions in Sulfur Analogues of α-Pyrone

A combined matrix isolation FTIR and theoretical DFT(B3LYP)/6-311++G(d,p) study of the photochemistry of sulfur analogues of alpha-pyrone [2H-thiopyran-2-one (TP) and 2H-pyran-2-thione (PT)] was carried out. The vibrational spectra of monomers of the compounds isolated in low-temperature argon matrixes were studied experimentally and assigned completely on the basis of theoretical calculations. UV irradiation (lambda > 337 nm) of the studied compounds isolated in low-temperature matrixes results mainly in the ring-opening reaction by means of the cleavage of the alpha-bond. Other photoprocesses, not involving the alpha-bond-cleavage step (such as generation of Dewar valence isomer), correspond to the minor reaction channels in both studied compounds. The ring-opening photoreaction in PT represents the first reported case of an alpha-bond cleavage in a compound with a C=S group attached to a six-membered ring, in which the internal strain practically does not exist, whereas the corresponding reaction in TP (a cleavage of a C-S bond in the alpha position with respect to a carbonyl group) is now reported for the first time. Following the ring-opening reactions, isomerization processes and intramolecular hydrogen shift reactions were observed, enabling production of TP from PT and vice versa. A detailed study of such processes was undertaken, and kinetical and mechanistical data are presented and discussed.

Repair of the three main types of bipyrimidine DNA photoproducts in human keratinocytes exposed to UVB and UVA radiations

Induction of DNA damage by solar UV radiation is a key event in the development of skin cancers. Bipyrimidine photoproducts, including cyclobutane pyrimidine dimers (CPDs), (6-4) photoproducts (64 PPs) and their Dewar valence isomers, have been identified as major UV-induced DNA lesions. In order to identify the predominant and most persistent lesions, we studied the repair of the three types of photolesions in primary cultures of human keratinocytes. Specific and quantitative data were obtained using HPLC associated with tandem mass spectrometry. As shown in other cell types, 64 PPs are removed from UVB-irradiated keratinocytes much more efficiently than CPDs. In contrast, CPDs are still present in high amounts when cells recover their proliferation capacities after cell cycle arrest and elimination of a part of the population by apoptosis. The predominance of CPDs is still maintained when keratinocytes are exposed to a combination of UVB and UVA. Under these conditions, 64 PPs are converted into their Dewar valence isomers that are as efficiently repaired as their (6-4) precursors. Exposure of cells to pure UVA radiation generates thymine cyclobutane dimers that are slightly less efficiently repaired than CPDs produced upon UVB irradiation. Altogether, our results show that CPDs are the most frequent and the less efficiently repaired bipyrimidine photoproducts irrespectively of the applied UV treatment.

Larger yield of cyclobutane dimers than 8-oxo-7,8-dihydroguanine in the DNA of UVA-irradiated human skin cells

Exposure to solar ultraviolet light is the major cause of most skin cancers. While the genotoxic properties of UVB radiation are now well understood, the DNA damaging processes triggered by less energetic but more abundant UVA photons remain to be elucidated. Evidence has been provided for the induction of oxidative lesions to cellular DNA including strand breaks and 8-oxo-7,8-dihydro-2′-deoxyguanosine (8-oxodGuo). Formation of cyclobutane pyrimidine dimers (CPDs) has also been reported, mostly in rodent cells. In order to gain insights into the relevance of the latter photoproducts in UVA-mutagenesis of human skin, we quantified the level of 8-oxodGuo and CPDs within primary cultures of normal fibroblasts and keratinocytes using specific chromatographic assays. The yield of formation of CPDs was found to be higher than that of 8-oxodGuo in both cell types. In addition, CPDs were mostly TT derivatives, and neither (6-4) photoproducts nor Dewar valence isomers were detected. These observations are reminiscent of results obtained in rodent cells and suggest that a photosensitized triplet energy transfer occurs and that this reaction is more efficient than photooxidation of DNA components. The predominant formation of CPDs with respect to oxidative damage within normal human skin cells exposed to UVA radiation should be taken into account in photoprotection strategies.

4,6-Dimethyl-α-pyrone: a matrix isolation study of the photochemical generation of conjugated ketene, Dewar valence isomer and 1,3-dimethyl-cyclobutadiene

A combined matrix isolation and molecular orbital study of the vibrational spectra and photochemistry of 4,6-dimethyl-α-pyrone (DMAP) was undertaken. Two types of photoreactions: ring opening leading to conjugated ketene and valence isomerization to the Dewar form (1,5-dimethyl-2-oxa-3-oxobicyclo[2.2.0]hex-5-ene; DOOBH), occurred upon UV ( λ>315 nm) irradiation. The latter reaction was efficient, whereas aldehyde–ketene was produced only in little amounts. In addition to the IR spectroscopic study of DMAP, the full mid-IR spectrum of the photoproduced DOOBH is reported and interpreted. Observation of 1,3-dimethyl-cyclobutadiene (DMCB), created by shorter wavelength UV irradiation ( λ>235 nm) of DOOBH, is reported for the first time. In the matrices, DMCB forms a complex with CO 2; the structure and IR absorption features of this cage confined DMCB–CO 2 complex are also investigated.

Formation of the spore photoproduct and other dimeric lesions between adjacent pyrimidines in UVC-irradiated dry DNA.

Far-UV irradiation of DNA leads to the formation of several types of dimeric lesions between adjacent pyrimidine bases including cyclobutane dimers, (6-4) photoproducts and Dewar valence isomers In the dry state, an additional specific thymine lesion, the spore photoproduct, is produced. We designed an HPLC-tandem mass spectrometry assay for the detection of the latter lesion. This technique that does not require radio-labelling of DNA allowed the simultaneous quantification of the spore photoproduct and other pyrimidine dimeric photoproducts. Using this approach, the complete distribution of bipyrimidine lesions within UVC-irradiated dry DNA was determined.

Photoisomerization reactions of 4-methoxy- and 4-hydroxy-6-methyl-α-pyrones: An experimental matrix isolation and theoretical density functional theory study

Photoisomerization reactions were studied for 4-methoxy- and 4-hydroxy-6-methyl-α-pyrones isolated in low-temperature Ar matrices. Two types of photoproducts: Dewar valence isomer and conjugated aldehyde–ketene were photogenerated upon UV irradiation of the matrix-isolated compounds. After prolonged irradiation practically all the initial material was converted to the corresponding Dewar isomers, whereas the population of aldehyde–ketene photoproducts was marginal. The structure of the main photoproducts was positively identified by comparison of their experimental IR spectra with the spectra theoretically predicted for the Dewar isomers. Good agreement between the experimental observations and the theoretical calculations allowed for reliable assignment of the infrared bands in the spectra of the initial forms of the studied compounds as well as in the spectra of the Dewar isomers. For the 4-hydroxy-6-methyl-α-pyrone the presence of its minor tautomeric form in a low-temperature matrix was detected.

UV-induced DNA damage and repair: a review

Increases in ultraviolet radiation at the Earth's surface due to the depletion of the stratospheric ozone layer have recently fuelled interest in the mechanisms of various effects it might have on organisms. DNA is certainly one of the key targets for UV-induced damage in a variety of organisms ranging from bacteria to humans. UV radiation induces two of the most abundant mutagenic and cytotoxic DNA lesions such as cyclobutane-pyrimidine dimers (CPDs) and 6-4 photoproducts (6-4PPs) and their Dewar valence Isomers. However, cells have developed a number of repair or tolerance mechanism to counteract the DNA damage caused by UV or any other stressors. Photoreactivation with the help of the enzyme photolyase is one of the most important and frequently occurring repair mechanisms in a variety of organisms. Excision repair, which can be distinguished into base excision repair (BER) and nucleotide excision repair (NER), also plays an important role in DNA repair in several organisms with the help of a number of glycosylases and polymerases, respectively. In addition, mechanisms such as mutagenic repair or dimer bypass, recombinational repair, cell-cycle checkpoints, apoptosis and certain alternative repair pathways are also operative in various organisms. This review deals with UV-induced DNA damage and the associated repair mechanisms as well as methods of detecting DNA damage and its future perspectives.

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.

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 C 4H 3NO 3, and dimeric adenine photoproducts show a distinctive loss of NH 2CN 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.

Hexafluorobenzene photochemistry: wellspring of fluorocarbon structures.

Ultraviolet irradiation of hexafluorobenzene in the vapor phase gives exclusively its Dewar valence isomer, from which a wealth of highly fluorinated compounds has been derived. Photoexcited in solution, hexafluorobenzene is capable of cycloaddition with a variety of unsaturated molecules. Like the Dewar benzene, resulting adducts have served as intermediates en route to many new fluorocarbons and fluorocarbon derivatives.

Individual determination of the yield of the main UV-induced dimeric pyrimidine photoproducts in DNA suggests a high mutagenicity of CC photolesions.

Bipyrimidine photoproducts induced in DNA by UVB radiation include cyclobutane dimers, (6-4) photoproducts, and their related Dewar valence isomers. Even though these lesions have been extensively studied, their rate of formation within DNA is still not known for each possible bipyrimidine site (TT, TC, CT, and CC). Using a method based on the coupling of liquid chromatography to mass spectrometry, we determined the distribution of the 12 possible bipyrimidine photoproducts within isolated and cellular DNA. TT and TC were found to be the most photoreactive sequences, whereas lower amounts of damage were produced at CT and CC sites. In addition to this quantitative aspect, sequence effects were observed on the relative yield of (6-4) adducts with respect to cyclobutane pyrimidine dimers. Another interesting result is the lack of formation of Dewar valence isomers in detectable amounts within the DNA of cells exposed to low doses of UVB radiation. The photoproduct distribution obtained does not fully correlate with the UV mutation spectrum. A major striking observation deals with the low yield of cytosine-cytosine photoproducts which are likely to be associated with the UV-specific CC to TT tandem mutation.