Incandescent lamps can produce pyrimidine dimers in DNA

DNA molecules that have been exposed to light from a 150 W incandescent spot lamp are nicked by the Micrococcus luteus endonuclease specific for cyclobutyl-type pyrimidine dimers. The production of these enzyme-sensitive sites increases with increasing spot lamp exposure. These sites have been confirmed to be pyrimidine dimers by their property of being photoreversed by an E. coli photoreactivating enzyme. The emission spectrum of the lamp shows detectable output at wavelengths less than 320 nm. These results indicate that the sensitivity of the techniques utilized in this work can be used to detect low levels of contaminating UV radiation.     

Biological consequences of cyclobutane pyrimidine dimers.

In the skin many molecules may absorb ultraviolet (UV) radiation upon exposure. In particular, cellular DNA strongly absorbs shorter wavelength solar UV radiation, resulting in various types of DNA damage. Among the DNA photoproducts produced the cyclobutane pyrimidine dimers (CPDs) are predominant. Although these lesions are efficiently repaired in the skin, this CPD formation results in various acute effects (erythema, inflammatory responses), transient effects (suppression of immune function), and chronic effects (mutation induction and skin cancer). The relationships between the presence of CPD in skin cells and the subsequent biological consequences are the subject of the present review.     

On the photoproduction of DNA/RNA cyclobutane pyrimidine dimers

The UV photoreactivity of different pyrimidine DNA/RNA nucleobases along the singlet manifold leading to the formation of cyclobutane pyrimidine dimers has been studied by using the CASPT2 level of theory. The initially irradiated singlet state promotes the formation of excimers between pairs of properly oriented nucleobases through the overlap between the ?? structures of two stacked nucleobases. The system evolves then to the formation of cyclobutane pyrimidine dimers via a shearing-type conical intersection activating a [2?+?2] photocycloaddition mechanism. The relative location of stable excimer conformations or alternative decay channels with respect to the reactive degeneracy region explains the differences in the photoproduction efficiency observed in the experiments for different nucleobases sequences. A comparative analysis of the main structural parameters and energetic profiles in the singlet manifold is carried out for thymine, uracil, cytosine, and 5-methylcytosine homodimers. Thymine and uracil dimers display the most favorable paths, in contrast to cytosine. Methylation of the nucleobases seems to increase the probability for dimerization.     

Photorepair of pyrimidine dimers in the epidermis of the marsupial Monodelphis domestica

Abstract Induction and fate of ultraviolet radiation-induced pyrimidine dimers in DNA have been measured in the epidermis of the marsupial, Monodelphis domestica, using damage-specific endonucleases from Micrococcus luteus. Approximately 90% of the dimers are lost when irradiated animals are subjected to photoreactivating light for 180 min. No loss of dimers was detected when the animals were held for a similar period of time in the dark. The capacity of these epithelial cells to photorepair pyrimidine dimers may provide a useful whole animal system in which to determine the role of pyrimidine dimers in photobiological responses of the skin.     

Use of high-performance liquid chromatography to quantitative thymine-containing pyrimidine dimers in DNA

We have developed two high-performance liquid chromatographic systems for the measurement of pyrimidine dimers in hydrolysates of DNA. Normal-phase chromatography on an NH₂ column in methanol—ethyl acetate (3:97) at an elution rate of 2.0 ml/min allowed quantitaion of thymine-containing (thymine-thymine plus thymine-uracil) pyrimidine dimers at levels as low as 0.1% of the total radioactivity as thymine in DNA. This system was unaffected by the presence of up to 1 mg of contaminating protein (bovine serum albumin) or 40 μg of DNA in hydrolysates prepared for chromatography. Reversed-phase chromatography on a μBondapak C₁₈ column allowed measurement of thymine-thymine dimers at concentrations as low as 0.02% of the total radioactivity. With 0.1% tetrahydrofuran in wateras the solvent at a flow-rate of up to 0.6 ml/min, thymine—thymine, thymine—uracil, and uracil—uracil dimers were completely resolved. We were not able to quantitate the latter two dimeric forms, however, owing to the presence of other radioactive components of undefined origin that eluted concomitantly with the uracil-containing dimers.     

Quantification of UV-induced cyclobutane pyrimidine dimers using an oligonucleotide chip assay

A lesion-specific enzyme-induced DNA strand break assay was developed for an oligonucleotide chip for the determination of UVB-induced cyclobutane pyrimidine dimers (CPDs). A 20-mer of fluorophore-labeled and biotinylated oligonucleotide was immobilized on the chip. CPDs in DNA on the chip were formed by UVB irradiation (312 nm). T4 endonuclease V (T4N5) was used to excise the CPD site as T4N5 sensitively and specifically detects CPDs. The fluorophore-labeled DNA fragments were detected by a laser-induced fluorescence (LIF) detection system. The number of CPDs induced by UVB was determined based on a mathematical equation obtained from a predetermined calibration curve. The yield of UVB-induced CPDs was 1.73 CPDs per megabase per (kJ/m²). The reliability of this value was proved by its similarity to reference values obtained from gel electrophoresis. The developed assay has strong potential to quantify most kinds of UV-induced DNA lesions.     

Dissociative electron transfer to and from pyrimidine cyclobutane dimers: an electrochemical study

Cyclic voltammetry was used to study the reduction and oxidation behaviour of several pyrimidine cyclobutane dimers mimicking UV induced lesion in DNA strands in polar solvents (N,N-dimethylformamide and acetonitrile). Both electron injection and removal to and from the dimers, respectively, lead to their cleavage and reformation of the monomeric base. The influence of stereochemistry and substitution pattern at the cyclobutane motif on the reactivity has been studied. It appears that the repair process always proceeds in a sequential fashion with initial formation of a dimer ion radical intermediate, which then undergoes ring opening by homolytic cleavage of the two C-C bonds. Standard redox potentials for the formation of both radical anion and radical cation state of the dimers were determined. Quantum calculations on simplified model compounds reveal the reason for the finding that the exergonic homolytic cleavages of the carbon-carbon bonds are endowed with sizeable activation barriers. The consequences of these mechanistic studies on the natural enzymatic repair by photolyase enzyme are discussed.     

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.     

Purine and pyrimidine compounds in murine peritoneal macrophages cultured in vitro.

Extracts of murine peritoneal macrophages were analysed by ion-pair reversed-phase high-performance liquid chromatography during incubation at 37 degrees C in vitro. Four-step gradient elution was applied to an ODS column (250 x 4.6 mm I.D.) at a flow-rate of 1.3 ml/min, allowing the separation of hypoxanthine, inosine, guanosine, adenosine, IMP, CDP, AMP, GDP, UDP, ADP, CTP, GTP, UTP and ATP within 50 min. Samples of 0.4 . 10(6)-0.5 . 10(6) cells were washed twice with RPMI 1640 medium and extracted with perchloric acid. Nucleotide concentrations of murine peritoneal macrophages did not change during incubation for 4 days in vitro.     

Absence of photoreactivation of pyrimidine dimers in the epidermis of hairless mice following exposures to ultraviolet light

Abstract—The influence of photoreactivating light on the fate of UV-induced DNA damage has been measured in the epidermis of hairless mice using damage-specific endonuclease from Micrococcus luteus. Groups of mice were exposed to varying fluences of UV at 297nm or from an FS40 fluorescent sun lamp to induce UV photoproducts. The same fluence-dependent DNA damage was observed in high molecular weight epidermal DNA regardless of whether the mice were killed immediately, or maintained in the dark or under photoreactivating light for 20 h after UV. Thus, no detectable photoreactivation of UV-induced pyrimidine dimers could be demonstrated in mouse epithelial cells in vivo.     

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.     

Inducible stable DNA replication (iSDR) and the uvr-dependent tolerance of pyrimidine dimers in UV-irradiated Escherichia coli are two uncoupled processes

Inducible stable DNA replication (iSDR) is dependent on recombination and is supposed to play a role in DNA repair of Escherichia coli. Our previous data suggested that iSDR may be involved in the tolerance of UV lesions, which remain unexcised in excision-proficient E. coli exposed to some UV pretreatments. Now, the tolerance of unexcised lesions has been followed in E. coli recB21 and in E. coli priA1 sup mutants, incapable of iSDR. The obtained data do not confirm the previous hypothesis about the involvement of iSDR in the putative uvr-dependent lesion tolerance. They rather suggest that iSDR and the uvr-dependent lesion tolerance are two uncoupled processes.     

Photoreactivation of killing in Streptomyces. 3. Action spectra for photolysis of pyrimidine dimers and adducts in S. griseus and S. griseus PHR-1

Abstract— Photolysis of tritium-labelled thymine-derived photoproducts by 254-nm ultraviolet radiation (u.v.) in conidia of Streptomyces griseus was measured by chromatography of cell hydrolysates. The relative photolysis cross-sections of uracilthymine dimer (UT○) at various wavelengths are the same as those of thymine-thymine dimer (TT○), and their ratios at 313, 365, 405 and 436 nm are 2:1:2:3. Except at 436 nm, these relative values agree very well with cross-sections previously reported for photoreactivation of u.v. killing in this organism, leading to the conclusion that photoreactivation in the wild type is due to repair of cyclobutane-type pyrimidine dimers. In a mutant showing restricted photoreactivation (S. griseus PHR-1), post-u.v. treatments at the above wavelengths did not affect UT○ and TT○ in the conidia, supporting the earlier suggestion that this organism does not contain active PR enzyme. Another u.v. photoproduct, the precursor of a pyrimidine adduct (PO-T) that appears in cell hydrolysates, was removed from both wild-type and mutant cells very efficiently at 313 nm. This is presumably a direct photochemical reaction. In addition, in wild-type cells, the precursor of PO-T appeared to be inefficiently removed photoenzymatically at all wavelengths. Removal of the precursor of PO-T appears to be biologically significant, however, only in the mutant.     

Strong coupling in dimers

Strong coupling in dimers with inclusion of a dependence of the resonance interaction on nuclear coordinates is investigated. Some spectral consequences are discussed.     

Photoreactivation of RNA in UV-irradiated insect eggs (Smittia sp., Chironomidae, Diptera) I. Photosensitized production and light-dependent disappearance of pyrimidine dimers

Abstract. Irradiation of Smittia eggs with UV during intravitelline cleavage causes the formation of pyrimidine dimers in the (largely ribosomal) RNA of the eggs. The yield of dimers is wavelength-dependent in a way that strongly suggests the involvement of photosensitizing egg components. Illumination of UV-irradiated eggs with light (380 or 400 nm) causes both photoreactivation of the eggs and mono-merization of the pyrimidine dimers in their RNA. The photoreactivable sector of the biological damage is correlated with the amount of pyrimidine dimers present in the RNA after inactivation of the eggs with UV of different wavelengths. The data are regarded as the first direct evidence that the photoreactivation of a eukaryotic organism is correlated with the light-dependent (and apparently enzymatic) monomerization of pyrimidine dimers in RNA.     

Photoreactivation of killing in Streptomyces. II. Kinetics of formation and photolysis of pyrimidine dimers and adducts in S. coelicolor and S. griseus PHR-1

Abstract— A pyrimidine adduct, 6-4‘-[pyrimidine-2’-one] thymine (PO-T)?, observed in DNA hydrolysates of 254-nm ultraviolet (u.v.) irradiated conidia of Streptomyces coelicolor, increases linearly with u.v. dose up to 2 × 10⁵ ergs/mm². Yields of thymine dimer (T○) and uracil-thymine dimer (U○) level off at much lower doses. Initial relative rates of formation of these u.v. photoproducts are: 1:1.3:4.8 for PO-T, T○ and U○, respectively. Similar results were obtained with a Streptomyces griseus mutant, PHR-1. An equation is derived to estimate the ratio of the amount of PO-T to the total amount of thymine-derived photoproducts at low (biological) u.v. doses. The observed PO-T fractions compare well with the calculated values. Rapid photolysis of the precursor of PO-T was observed by post-u. v. treatment at 313 nm of conidia of S. coelicolor and of S. griseus PHR-1. The photolysis was much slower at 365 nm and did not occur at all at 405 nm. Pyrimidine dimers were not appreciably affected by post-u. v. treatment at the above wavelengths in these Streptomyces strains. Both of these strains are phenotypically photoreactivation-deficient, and the present results indicate that they do not possess active photoreactivating enzyme. In earlier papers[3,4,5], the pyrimidine adduct found in acid hydrolysates of DNA was loosely referred to as “uracil-thymine adduct (U-T adduct)”. Such terminology is not strictly correct. The pyrimidine adduct in acid hydrolysates is PO-T (sometimes called P₂B), which could theoretically result from removal of ammonia from a C-T adduct or removal of water from a U-T adduct (see [6]).     

The efficiency of photolyase and indole complexes to repair DNA containing dimers of pyrimidine: a theoretical analysis of the electron transfer reactions

We analyze the effects of competing reactions to the efficiency of enzymatic splitting of pyrimidine dimers formed in DNA by the incidence of ultraviolet radiation. This is accomplished with the aid of a formula that expresses the efficiency of the repair in terms of parameters that regulate the reaction rates for primary and for back long-range electron transfers taking place in the process. Comparison of experimental data with estimations on account of this formula supports early conjectures in the literature that attribute the relative high performance of the enzymatic complexes of photolyase to its ability to suppress the back reaction.     

Plasmon hybridization in nanoshell dimers

We extend the plasmon hybridization method to investigate the plasmon modes of metallic nanoshell dimers. The formalism is also generalized to include the effects of dielectric backgrounds. It is shown that the presence of dielectrics shifts the plasmon resonances of the individual nanoparticles to lower energies and screens their interaction in the dimer configuration. The net result is a redshift of dimer energies compared to the system without dielectrics and a weaker dependence of the dimer plasmon energies on dimer separation. We calculate the plasmon energies and optical absorption of nanoshell dimers as a function of dimer separation. The results are in excellent agreement with the results of finite difference time domain simulations.