WAVELENGTH DEPENDENCE OF DNA INCISION BY A HUMAN ULTRAVIOLET ENDONUCLEASE

The wavelength dependence of an ultraviolet irradiation of the DNA substrate for a human endonuclease was determined. Sites of DNA incision for all UVB and UVC wavelengths examined were at cytosines which were neither cyclobutane pyrimidine dimers nor 6-4'-(pyrimidin-2-one)pyrimidines. The optimal wavelengths for formation of these cytosine photoproducts were between 270 and 295 nm. This human endonuclease therefore has a similar ultraviolet substrate specificity to endonuclease III.     

Triplet-Induced Lesion Formation at CpT and TpC Sites in DNA

UV irradiation induces DNA lesions particularly at dipyrimidine sites. Using time-resolved UV pump (250 nm) and mid-IR probe spectroscopy the triplet pathway of cyclobutane pyrimidine dimer (CPD) formation within TpC and CpT sequences was studied. The triplet state is initially localized at the thymine base but decays with 30 ns under formation of a biradical state extending over both bases of the dipyrimidine. Subsequently this state either decays back to the electronic ground state on the 100 ns time scale or forms a cyclobutane pyrimidine dimer lesion (CPD). Stationary IR spectroscopy and triplet sensitization via 2′-methoxyacetophenone (2-M) in the UVA range shows that the lesions are formed with an efficiency of approximately 1.5 %. Deamination converts the cytosine moiety of the CPD lesions on the time scale of 10 hours into uracil which gives CPD(UpT) and CPD(TpU) lesions in which the coding potential of the initial cytosine base is vanished.     

Repair of (6‐4) Lesions in DNA by (6‐4) Photolyase: 20 Years of Quest for the Photoreaction Mechanism

Exposure of DNA to ultraviolet (UV) light from the Sun or from other sources causes the formation of harmful and carcinogenic crosslinks between adjacent pyrimidine nucleobases, namely cyclobutane pyrimidine dimers and pyrimidine(6–4)pyrimidone photoproducts. Nature has developed unique flavoenzymes, called DNA photolyases, that utilize blue light, that is photons of lower energy than those of the damaging light, to repair these lesions. In this review, we focus on the chemically challenging repair of the (6–4) photoproducts by (6–4) photolyase and describe the major events along the quest for the reaction mechanisms, over the 20 years since the discovery of (6‐4) photolyase.     

Repair of a Dimeric Azetidine Related to the Thymine–Cytosine (6‐4) Photoproduct by Electron Transfer Photoreduction

Photolyases are intriguing enzymes that take advantage of sunlight to restore lesions like cyclobutane pyrimidine dimers or (6‐4) photoproducts. This work focused on the photoreductive process responsible for splitting of the azetidine ring proposed to occur during (6‐4) photoproduct repair at a thymine–cytosine sequence. A model compound formed by photocycloaddition between thymine and 6‐azauracil has been designed to mimic the elusive azetidine intermediate. The photoinduced electron transfer process has been investigated by means of steady‐state and time‐resolved fluorescence using photosensitizers with oxidation potentials in the singlet excited state ranging from ?3.3 to ?2.1 V vs. SCE. Azetidine ring splitting and recovery of “repaired” bases were proven by HPLC analysis.     

Oxidative cleavage of a cyclobutane pyrimidine dimer by photochemically generated nitrate radicals (no(3)*)

[reaction: see text] Photochemically generated nitrate radicals (NO(3)(*)) cleave the stereoisomeric N,N-dimethyl-substituted uracil cyclobutane dimers 1a-d into the monomeric uracil derivative 2 as the major reaction pathway. A preferred splitting of the syn dimers 1a,b was observed. The reaction is expected to proceed through initial one-electron oxidation with formation of an intermediate cyclobutane radical cation 11. In addition to cycloreversion, competing reaction steps of 11, which lead to the observed byproducts, are suggested.     

Intrinsic lifetimes of the excited state of DNA and RNA bases

The lifetimes of the excited state of free nucleobases were measured in the gas phase for the first time. They are, respectively, 1.0 and 0.8 ps for the purine bases adenine (shown above) and guanine and 3.2, 2.4, and 6.4 ps for the pyrimidine bases cytosine, uracil, and thymine at 267 nm. The longer lifetimes of the pyrimidine bases may be associated with their higher propensity toward photodegradation, especially in the case of thymine. The ultrashort lifetime of nucleobases conventionally known in solution was found to be an intrinsic molecular property due to extremely facile internal conversion, and therefore the lifetime should be largely independent of the medium at this energy, that is, whether in vacuo, in solution, or in vivo. The evolutionary selection of nucleobases as the durable carriers of genetic information is suggested to be due to their inherent immunity from photochemical reactions.     

Contribution of Cytosine‐Containing Cyclobutane Dimers to DNA Damage Produced by Photosensitized Triplet–Triplet Energy Transfer

Mutagenic cyclobutane pyrimidine dimers (CPDs) can be induced in DNA through either direct excitation or photosensitized triplet–triplet energy transfer (TTET). In the latter pathway, thymines are expected to receive the excitation energy from the photosensitizer and react with adjacent pyrimidines. By using state‐of‐the art analytical tools, we provide herein additional information on the formation of cytosine‐containing CPDs. We thus determined the yield of all possible CPDs upon TTET in a series of natural DNAs with various base compositions. We show that the distribution of CPDs cannot be explained only by excitation of individual thymines. We propose that the mechanism for TTET involves at least dinucleotides as the minimal targets. The observation of the formation of cytosine–cytosine CPDs also suggests that additional pathways are involved in this photosensitized reaction.     

Far-UV-lnduced Dimeric Photoproducts in Short Oligonucleotides: Sequence Effects

Cyclobutane pyrimidine dimers and pyrimidine(6-4)pyrimidone adducts represent the two major classes of far-UV-induced DNA photoproducts. Because of the lack of appropriate detection methods for each individual photoproduct, little is known about the effect of the sequence on their formation. In the present work, the photoproduct distribution obtained upon exposure of a series of dinucleoside monophosphates to 254 nm light was determined. In the latter model compounds, the presence of a cytosine, located at either the 5′- or the 3′- side of a thymine moiety, led to the preferential formation of (6-4) adducts, whereas the cis-syn cyclobutane dimer was the main thymine-thymine photoproduct. In contrast, the yield of dimeric photoproducts, and particularly of (6-4) photoadducts, was very low upon irradiation of the cytosine–cytosine dinucleoside monophosphate. However, substitution of cytosine by uracil led to an increase in the yield of (6-4) photoproduct. It was also shown that the presence of a phosphate group at the 5′- end of a thymine-thymine dinucleoside monophosphate does not modify the photoproduct distribution. As an extension of the studies on dinucleoside monophosphates, the trinucleotide TpdCpT was used as a more relevant DNA model. The yields of formation of the thymine-cytosine and cytosine–thymine (6-4) photoproducts were in a 5:1 ratio, very close to the value obtained upon photolysis of the related dinucleoside monophosphates. The characterization of the two TpdCpT (6-4) adducts was based on H NMR, UV and mass spectroscopy analyses. Additional evidence for the structures was inferred from the analysis of the enzymatic digestion products of the (6-4) adducts of TpdCpT with phosphodiesterases. The latter enzymes were shown to induce the quantitative release of the photoproduct as a modified dinucleoside monophosphate in a highly sequence-specific manner.     

Self-Assembling of cytosine nucleoside into triply-bound dimers in acid Media. A comprehensive evaluation of proton-bound pyrimidine nucleosides by electrospray tandem mass spectrometry,X-rays diffractometry,and theoretical calculations

Electrospray tandem mass spectrometry (ESI-MS/MS) is used to evaluate the assembling of cytosine and thymine nucleosides in the gas phase, through the formation of hydrogen bonded supermolecules. Mixtures of cytidine analogues and homologues deliver in the gas phase proton-bound heterodimers stabilized by multiple interactions, as proven by the kinetics of their dissociation into the corresponding protonated monomers. Theoretical calculations, performed on initial structures of methylcytosine homodimers available in the literature, converged to a minimized structure whereby the two pyrimidine rings interact through the formation of three hydrogen bonds of similar energy. The crystallographic data here reported show the equivalency of the two interacting pyrimidines which is attributable to the presence of an inversion center. Thymine and uracil pyrimidyl nucleosides form, by ESI, gaseous proton-bound dimers. The kinetic of their dissociation into the related protonated monomers shows that the nucleobases are weekly interacting through a single hydrogen bond. The minimized structure of the protonated heterodimer formed by thymine and N-1-methylthymine confirmed the existence of mainly one hydrogen bond which links the two nucleobases through the O4 oxygens. No crystallographic data exists on thymine proton-bound species, nor have we been able to obtain these aggregates in the solid phase. The gaseous phase, under high vacuum conditions, seems therefore a suitable environment where vanishing structures produced by ESI can be studied with a good degree of approximation.     

PHOTOPHYSICS AND PHOTOCHEMISTRY OF PHOTOREACTIVATION

Abstract— Photoreactivating enzyme (PRE) monomerizes cyclobutyl pyrimidine dimers formed in DNA by UV light ( Λ < 300 nm). The enzyme requires near UV and visible wavelengths (300 < Λ < 600 nm) for activity. Possible mechanisms of action of the PRE are suggested by non-enzymatic processes in which pyrimidine dimers are monomerized by UV and visible light. Two such non-enzymatic processes are (a) photolysis of dimers resulting from direct absorption of UV, and (b) sensitized monomerization involving charge transfer complexes. Several lines of evidence suggest that the mechanism of action of the PRE more closely resembles (b) than (a). Recent experiments on the PRE from E. coli reveal the presence of new long wavelength absorption which may indicate the presence of a ground state complex. The known ability of PRE to monomerize dimers of thymine, cytosine and uracil suggests that the carbonyl groups at 2 position of the pyrimidine ring may be important in the interaction between enzyme and dimer.     

X-ray absorption spectroscopy of the nucleotide bases at the carbon, nitrogen, and oxygen K-edges

Near-edge X-ray absorption fine structure spectra of three pyrimidine (viz., cytosine, uracil, and thymine) and two purine (viz., adenine and guanine) nucleobases, which are the key constituents of DNA and RNA, were measured at the C, N, and O K-edges using the self-absorption-free partial electron yield mode. The nucleobase samples were prepared as highly pure native polycrystalline powder films. The spectra are analyzed in terms of the electronic structure of the nucleobases. Subtle chemical effects related to the molecular structures of these heterocyclic compounds with extended pi-electron systems are considered and discussed.     

Unified model for the ultrafast decay of pyrimidine nucleobases

Ultrafast decay processes detected after absorption of UV radiation in gas-phase pyrimidine nucleobases uracil, thymine, and cytosine are ascribed to the barrierless character of the pathway along the low-lying 1(pipi*) hypersurface connecting the Franck-Condon region with an out-of-plane distorted ethene-like conical intersection with the ground state. Longer lifetime decays and low quantum yield emission are on the other hand related to the presence of a 1(pipi*) state planar minimum on the S1 surface and the barriers to access other conical intersections. A unified model for the three systems is established on the basis of accurate multiconfigurational CASPT2 calculations, whereas the effect of the different levels of theory on the results is carefully analyzed.     

ULTRAVIOLET LIGHT IRRADIATION OF DEFINED-SEQUENCE DNA UNDER CONDITIONS OF CHEMICAL PHOTOSENSITIZATION

Abstract— The formation of cyclobutane pyrimidine dimers and UV light-induced (6-4) products was examined under conditions of triplet state photosensitization. DNA fragments of defined sequence were irradiated with 313 nm light in the presence of either acetone qr silver ion. UV irradiation in the presence of both silver ion and acetone enhanced the formation of TT cyclobutane dimers, yet no (6-4) photoproducts were formed at appreciable levels. When photoproduct formation was also measured in pyrimidine dinucleotides, only cyclobutane dimers were formed when the dinucleotides were exposed to 313 nm light in the presence of photosensitizer. The relative distribution of each type of cyclobutane dimer formed was compared for DNA fragments that were irradiated with 254, 313, or 313 nm UV light in the presence of acetone. The dimer distribution for DNA irradiated with 254 and 313 nm UV light were very similar, whereas the distribution for DNA irradiated with 313 nm light in the presence of acetone favored TT dimers. Alkaline labile lesions at guanine sites were also seen when DNA was irradiated with 313 nm light in the presence of acetone.     

Mixed-metal (platinum, palladium), mixed-pyrimidine (uracil, cytosine) self-assembling metallacalix[n]arenes: dynamic combinatorial chemistry with nucleobases and metal species

Reactions between the mononuclear mixed-nucleobase complex [Pt(en)(UH-N1)(CH2-N3)]+ (1; en: ethylenediamine; UH-N1: uracil monoanion bonded through the N1 atom; CH2-N3: neutral cytosine bonded through the N3 atom) and [Pd(II)(en)] or [Pd(II)(2,2'-bpy)] (2,2'-bpy: 2,2'-bipyridine) lead to libraries of compounds of different stoichiometries and different connectivities. In these compounds, the palladium entity binds to or cross-links either the N3 sites of uracil and/or the N1 sites of cytosine, following deprotonation of these positions to give uracil dianions (U) and cytosine monoanions (CH). Cyclic species, which can be considered as metallacalix[n]arenes, have been detected in several cases, with n being 4 and 8. The complexity of the compounds formed not only results from the possibility of the two different nucleobases in building block 1 engaging in different connectivities with the Pd entities, but also from the potential for the formation of oligomers of different sizes and different conformations; in the case of cyclic tetranuclear Pt(2)Pd(2) species, this can, in principle, lead to the various arrangements (cone, partial cone, 1,2-alternate, 1,3-alternate) known from calix[4]arene chemistry. A further complication arises from the fact that, depending on the mutual orientation of the exocyclic groups of the two nucleobases (O2 and O4 of uracil, O2 and N4 of cytosine), these sites can be engaged in additional chelation of [Pd(II)(en)] and [Pd(II)(2,2'-bpy)]. Thus, penta-, hexa-, and octanuclear complexes, Pt(2)Pd(3), Pt(2)Pd(4), and Pt(2)Pd(6), derived from cyclic Pt(2)Pd(2) tetramers have been isolated and characterized.     

Effects of cytosine methylation on pyrimidine dimer formation in DNA

The relative induction of cyclobutane pyrimidine dimers (CPD) and pyrimidine (6-4)pyrimidone photoproducts ([6-4]PD) was quantified in the duplex homopolymers polydeoxyadenosine:polydeoxythymidine, polydeoxyguanosine:polydeoxycytidine and polydeoxyguanosine:polydeoxy-5-methylcytidine irradiated with UVC or UVB radiation. Cytosine methylation significantly increased the yield of cytosine (6-4)PD after irradiation with UVC light and of cytosine CPD and (6-4)PD after irradiation with UVB light. The data suggest that CPD and (6-4)PD are preferentially induced at 5-methylcytosine bases in DNA of cells exposed to sunlight and comprise a major component of the mutation spectrum leading to the initiation of sunlight-induced skin cancer.     

SIMULTANEOUS ESTABLISHMENT OF MONOCLONAL ANTIBODIES SPECIFIC FOR EITHER CYCLOBUTANE PYRIMIDINE DIMER OR (6-4)PHOTOPRODUCT FROM THE SAME MOUSE IMMUNIZED WITH ULTRAVIOLET-IRRADIATED DNA

Six new monoclonal antibodies (TDM-2, TDM-3, 64M-2, 64M-3, 64M-4 and 64M-5) specific for ultraviolet (UV) induced DNA damage have been established. In the antibody characterization experiments, two TDM antibodies were found to show a dose-dependent binding to UV-irradiated DNA (UV-DNA), decrease of binding to UV-DNA after cyclobutane pyrimidine dimer photoreactivation, binding to DNA containing cyclobutane thymine dimers, and unchanged binding to UV-DNA after photoisomerization of (6-4)photoproducts to Dewar photoproducts. These results indicated that the epitope of TDM monoclonal antibodies was the cyclobutane pyrimidine dimer in DNA. On the other hand, four 64M antibodies were found to show a dose-dependent binding to UV-DNA, unchanged binding to UV-DNA after cyclobutane pyrimidine dimer photoreactivation, undetectable binding to DNA containing thymine dimers, and decrease of binding to UV-DNA after photoisomerization of (6-4)photoproducts. These results indicated that the epitope of 64M antibodies was the (6-4)photoproduct in DNA. This is the first report of the simultaneous establishment of monoclonal antibodies against the two different types of photolesions from the same mouse. By using these monoclonal antibodies, we have succeeded in measuring both cyclobutane pyrimidine dimers and (6-4)photoproducts in the DNA from human primary cells irradiated with physiological UV doses.     

On the Formation of Cyclobutane Pyrimidine Dimers in UV‐irradiated DNA: Why are Thymines More Reactive?¶

The reaction pathways for thermal and photochemical formation of cyclobutane pyrimidine dimers in DNA are explored using density functional theory techniques. Although it is found that the thermal [2 + 2] cycloadditions of thymine + thymine (T + T --> T x T), cytosine + cytosine (C + C --> C x C) and cytosine + thymine (C + T --> C x T) all are similarly unfavorable in terms of energy barriers and reaction energies, the excited-state energy curves associated with the corresponding photochemical cycloadditions display differences that--in line with experimental findings--unanimously point to the predominance of T x T in UV-irradiated DNA. It is shown that the photocycloaddition of thymines is facilitated by the fact that the S1 state of the corresponding reactant complex lies comparatively high in energy. Moreover, at a nuclear configuration coinciding with the ground-state transition structure, the excited-state energy curve displays an absolute minimum only for the T + T system. Finally, the T + T system is also associated with the most favorable excited-state energy barriers and has the smallest S2-S0 energy gap at the ground-state transition structure.     

Theoretical insight into the intrinsic ultrafast formation of cyclobutane pyrimidine dimers in UV-irradiated DNA: thymine versus cytosine

The higher formation yields measured in the ultrafast photoinduced formation of cyclobutane thymine dimers (T<>T) with respect to those of cytosine (C<>C) are explained, on the basis of ab initio CASPT2 results, by the existence in thymine of more reactive orientations and a less efficient photoreversibility, whereas in cytosine the funnel toward the photolesion becomes competitive with that mediating the internal conversion of the excited-cytosine monomer.     

A triplet mechanism for the formation of cyclobutane pyrimidine dimers in UV-irradiated DNA

The reaction pathways for the photochemical formation of cyclobutane thymine dimers in DNA are explored using hybrid density functional theory techniques. It is concluded that the thymine-thymine [2 + 2] cycloaddition displays favorable energy barriers and reaction energies in both the triplet and the singlet excited states. The stepwise cycloaddition in the triplet excited state involves the initial formation of a diradical followed by ring closure via singlet-triplet interaction. The triplet mechanism is thus completely different from the concerted singlet state cycloaddition processes. The key geometric features and electron spin densities are also discussed. Bulk solvation has a major effect by reducing the barriers and increasing the diradical stabilities. The present results provide a rationale for the faster cycloreaction observed in the singlet excited states than in the triplet excited states.     

Pathways for fluorescence quenching in 2-aminopurine π-stacked with pyrimidine nucleobases

Fluorescent analogues of nucleobases are very useful as probes to study DNA dynamics, because natural DNA does not fluoresce significantly. In many of these analogues, such as 2-aminopurine (2AP), the fluorescence is quenched when incorporated into DNA through processes that are not well understood. This work uses theoretical studies to examine fluorescence quenching pathways in 2AP-containing dimers. The singlet excited states of π-stacked dimer systems containing 2AP and a pyrimidine base, thymine or cytosine, have been studied using ab initio computational methods. Computed relaxation pathways along the excited-state surfaces reveal novel mechanisms that can lead to fluorescence quenching in the π-stacked dimers. The placement of 2AP on the 5' or 3' terminus of the dimers has different effects on the excitation energies and the relaxation pathways on the S(1) excited state. Conical intersections between the ground and first excited states exist when 2AP is placed at the 3' side, whereas the placement of 2AP at the 5' side leads to the switching of a bright state to a dark state. Both of these processes can lead to fluorescence quenching and may contribute to the fluorescence quenching observed in 2AP when incorporated in DNA.