Thymine Dimer Formation Research Articles

Molecular and functional analysis of the (6-4) photolyase from the hexactinellid Aphrocallistes vastus

The hexactinellid sponges (phylum Porifera) represent the phylogenetically oldest metazoans that evolved 570–750 million years ago. At this period exposure to ultraviolet (UV) light exceeded that of today and it may be assumed that this old taxon has developed a specific protection system against UV-caused DNA damage. A cDNA was isolated from the hexactinellid Aphrocallistes vastus which comprises high sequence similarity to genes encoding the protostomian and deuterostomian (6-4) photolyases. Subsequently functional studies were performed. It could be shown that the sponge gene, after transfection into mutated Escherichia coli, causes resistance of the bacteria against UV light. Recombinant sponge photolyase was prepared to demonstrate that this protein binds to DNA treated with UV light (causing the formation of thymine dimers). Finally, it is shown that the photolyase gene is strongly expressed in the upper part of the animals and not in their middle part or their base. It is concluded that sponges not only have an excision DNA repair system, as has been described earlier by us, but also a photolyase-based photo-reactivating system.

UV photoprotection by combination topical antioxidants vitamin C and vitamin E

Background: Virtually all plants and animals protect themselves from the sun using vitamins C and E. Objective: The purpose of this study was to see if a combination of topical vitamins C and E is better for UV protection to skin than an equivalent concentration of topical vitamin C or E alone. Methods: We developed a stable aqueous solution of 15% L-ascorbic acid (vitamin C) and 1% α-tocopherol (vitamin E). We applied antioxidant or vehicle solutions to pig skin daily for 4 days. We irradiated (1-5× minimal erythema dose) control- and antioxidant-treated skin using a solar simulator with a 295-nm band-pass filter. On day 5, we measured antioxidant protection factor, erythema, sunburn cells, and thymine dimers. Results: The combination of 15% L-ascorbic acid and 1% α-tocopherol provided significant protection against erythema and sunburn cell formation; either L-ascorbic acid or 1% α-tocopherol alone also was protective but the combination was superior. Application during 4 days provided progressive protection that yielded an antioxidant protection factor of 4-fold. In addition, the combination of vitamins C and E provided protection against thymine dimer formation. Conclusion: Appreciable photoprotection can be obtained from the combination of topical vitamins C and E. We suggest that these natural products may protect against skin cancer and photoaging. (J Am Acad Dermatol 2003;48:866-74.)

Epidermal reconstructs: a new tool to study topical and systemic photoprotective molecules

In this study, we compared the effects of sunscreens and antioxidants on reconstructed epidermis made with or without melanocytes 24 h after UVB, UVA or UVA+B irradiation. For this purpose, we studied sunburn cells and cyclobutane pyrimidine dimer formation, protein and lipid oxidation, catalase and superoxide dismutase activities and vitamin E levels. Topical sunscreens protected against direct cell death and thymine dimer formation whereas their protective effect against protein and lipid oxidation and antioxidant depletion was less marked partly due to the difficulty of spreading the cream. Antioxidant molecules protected against direct cell death and protein oxidation but not against thymine dimer formation. Since topical sunscreens and systemic antioxidant protected the skin differently, we speculate that their association might protect more efficiently against UV-induced damage. This model is relevant to study systemic molecules but is less practical, due to the technical limitations of studying topical molecules.

Reaction mechanism of thymine dimer formation in DNA induced by UV light

The formation of thymine dimers in DNA is investigated by means of density functional theory (DFT) techniques. While it is found that a thermally induced [2+2] cycloaddition reaction proceeds via a very high energy transition state (80–88 kcal/mol above the reactant complex), the energy barrier for UV light induced formation—explored within the time-dependent DFT formalism—is only a few kilocalories per mol. As such, these results serve as an illustrative example of how UV radiation may induce DNA lesions. For the reactant complex, the calculated vertical excitation energy corresponding to the S 1←S 0 transition ( π→ π ∗) lies in the far-UV region, in accordance with experimental data.

The role of UV-B radiation in aquatic and terrestrial ecosystems—an experimental and functional analysis of the evolution of UV-absorbing compounds

The role of UV-B radiation in aquatic and terrestrial ecosystems—an experimental and functional analysis of the evolution of UV-absorbing compounds

Epidermal p53 Response and Repair of Thymine Dimers in Human Skin after a Single Dose of Ultraviolet Radiation: Effects of Photoprotection

A cellular p53 response, DNA repair enzymes and melanin pigmentation are important strategies utilized by skin keratinocytes against impairment caused by ultraviolet radiation (UVR). In this study a double-immunofluorescence technique was used to investigate UVR-induced thymine dimers and p53 protein simultaneously. Four healthy volunteers were irradiated on both sides of their buttock skin with a single dose of solar-simulating UVR. One side was pretreated with a topical sunscreen. Biopsies from different time-points were immunostained for visualization of thymine dimers, p53 and proliferation. One single physiological dose of UVR generated widespread formation of thymine dimers throughout the epidermis 4h after irradiation. The level of thymine dimers decreased over time and was followed by a p53 response in the same cells. A late proliferative response was also found. The formation of thymine dimers, the p53 response and the late proliferative response were partially blocked by topical sunscreen. Large inter-individual differences in the kinetics of thymine dimer formation and repair as well as in the p53 response were evident in both sunscreen-protected and unprotected skin.

The sensitivity of Emiliania huxleyi (Prymnesiophyceae) to ultraviolet‐b radiation

Emiliania huxleyi (Lohm.) Hay et Miller is an important component of the phytoplankton in open ocean waters. The sensitivity of this cosmopolitan alga to natural levels of UVB radiation has never been tested. Since DNA is believed to be a major target of natural UVB radiation (UVBR: 280–315 nm) in living cells, experiments with E. huxleyi were performed using growth rate reduction and DNA damage as indicators of UVBR stress. Specific growth rate, cell volume, pigment content, and CPD (cyclobutane pyrimidine dimer) formation (a measure for DNA damage) were followed during and after prolonged exposure of a series of cultures to a range of UVBR levels. E. huxleyi was found to be very sensitive to UVBR: at a daily weighted UVBR dose of only 400 J·m−2 ·d−1 (BEDDNA300nm), growth was halted. At this UVBR level, both cell volume and contents of the major photosynthetic and photoprotective pigments had increased. The UVBR vulnerability of E. huxleyi cannot be explained by a high potential for cyclobutane thymine dimer formation (the most abundant CPD type) due to a high T content of nuclear DNA: the CG content of this E. huxleyi strain is high (68%) compared with other species. The high UVBR sensitivity may be related to the stage of the cell cycle during UVBR exposure, in combination with low repair capacity. It is concluded that E. huxleyi may experience UVBR stress through the formation of cyclobutane pyrimidine dimers, with subsequent low repair capacity and thereby arrest of the cell cycle.

Molecular dynamics of formation of TD lesioned DNA complexed with repair enzyme—onset of the enzymatic repair process

To describe the first step of the enzymatic repair process (formation of complex enzyme–DNA), in which the thymine dimer (TD) part is removed from DNA, the 500picosecond (ps) molecular dynamics (MD) simulation of TD lesioned DNA and part of repair enzyme cell (inclusive of catalytic center—Arg-22, Glu-23, Arg-26 and Thr-2) was performed. TD dimer is UV originated lesion in DNA and T4 Endonuclease V is TD specific repair enzyme. Both molecules were located in the same simulation cell and their relative movement was examined. During the simulation, the research was focused on the role of electrostatic energy in formation of complex enzyme–DNA. It is found that during the first 100ps of MD, the part of enzyme approaches the DNA surface at the TD lesion, interacts extensively by electrostatic and van der Waals interactions with TD part of DNA and forms complex that lasts stable for 500ps of MD. In the beginning of MD, the positive electrostatic interaction energy between part of enzyme and TD (∼+10kcal/mol) drives enzyme towards the DNA molecule. Water-mediated hydrogen bonds between enzyme and DNA help to keep complex stable. As a reference, the MD simulation of the identical system with native DNA molecule (two native thymines (TT) instead of TD) was performed. In this system the negative electrostatic interaction energy between part of enzyme and TT (∼−11kcal/mol), contrary to the positive one in the system with TD, does not drive enzyme towards DNA and complex is not formed.

Cell migration from the ganglionic eminence to the neocortex investigated by labeling nuclei with UV irradiation via a fiber-optic cable.

Recent studies have shown that the ganglionic eminence is one of the sources of tangentially migrating cells in the developing neocortex. Since the migration of the DiI-labeled cells from the ganglionic eminence to the neocortex was not monitored by videomicroscopy in these reports, we devised a novel method to study cell migration in vitro and in vivo. The new method involves ultraviolet (UV) irradiation of the cells through a fiber-optic cable and subsequent identification of the irradiated cells on the basis of the formation of thymine dimers in the nuclei. First, we tested the new method (UV-thymine dimer-labeling method) by applying it to monitor the cell migration of neuronal precursor cells in the rostral migratory stream in the neonatal rat telencephalon. In vitro, UV irradiation for 1 s through the fiber-optic cable resulted in the formation of sufficient thymine dimers as to allow immunohistochemical detection after 6 h of incubation; a significant proportion of the irradiated cells continued to migrate in the same direction and at the same speed as those before irradiation. There was no significant difference in the cell migration distance over 6 h between cells exposed and not exposed to the UV irradiation in vitro. In vivo, this method revealed that three times as many cells in the subventricular zone of the olfactory bulb migrated rostrally as caudally. The new method also allowed us to measure the speed of cell migration, which was estimated to be about 70 microm/h at the maximum in the rostral direction. After these examinations of reliability of the method, we applied it to the rat embryo brain. One day after UV irradiation of the ganglionic eminence, labeled migrating cells were found in the striatum, in the internal capsule, and in the intermediate zone of the neocortex. The observation period of cell migration to the neocortex was extended by the use of a xeroderma pigmentosum group A gene mutant mouse, which lacked an ability to remove thymine dimer from the UV-irradiated nuclei. Two days after the UV irradiation, labeled migrating cells from the ganglionic eminence of the mutant mouse embryos were found both in the cortical plate and in the intermediate zone of the neocortex.

Activation of Interferon Regulatory Factor 3 in Response to DNA-damaging Agents

Genotoxic stress triggers signal transduction pathways that mediate either the protection or apoptosis of affected cells. The interferon regulatory factors (IRFs) are involved in a wide range of host defense mechanisms against environmental stresses. Treatment with DNA-damaging agents, including doxorubicin and UV radiation, caused phosphorylation of the IRF3 transcription factor. Phosphorylation of IRF3 induced its interaction with the transcriptional co-activator cAMP-response element binding protein-binding protein. Furthermore, genotoxic stress-induced phosphorylation of IRF3 resulted in its movement from the cytoplasm to the nucleus, where it activated transcription from its binding site. These observations suggest that IRF3 plays a role in the defensive responses induced by genotoxic stress.

Urocanic Acid Does not Photobind to DNA in Mice Irradiated with Immunosuppressive Doses of UVB*

ABSTRACTUltraviolet B (UVB, 290–320 nm) radiation initiates in vivo a dose‐ and wavelength‐dependent down regulation of cell‐mediated immunity. An action spectrum for UV‐induced immunosuppression indicated that the photoreceptor for this effect is urocanic acid (UCA), which undergoes a trans to cis isomerization in the stratum corneum on UV exposure. An accumulation of evidence has supported this conclusion. However, evidence has a/so been presented that formation of thymine dimers in DNA is responsible for initiation of UV‐induced immunosuppression. Because photobinding of UCA to DNA in vitro forming cyclobutane‐type adducts has been shown, we sought to resolve this dilemma by investigating if UCA photobinds to DNA in vivo. The [14C]cis‐UCA, [14C]trans‐UCA or [3H]8‐MOP (8‐methoxypsoralen) was applied topically to BALB/c mice that were then irradiated with a dose of UV previously shown to cause systemic suppression of contact hypersensitivity. The DNA was prepared from epidermal cells by phenol extraction immediately after in vivo irradiation and bound radioactivity determined. Although photobinding of [3H]8‐MOP was readily demonstrable under these conditions (0.9 nmol/mg DNA), no significant binding of either isomer of UCA to DNA (between 1.2 × 10−3 and 2.1 × 10−3 ng/mg DNA) could be detected. Uptake studies in keratinocytes prepared from epidermis of untreated animals indicated that [3H]8‐MOP was taken up with a rate constant of 4.2 × 10−3 pmol/s/mg protein/μmol/L. In contrast, uptake of [14C]cis‐UCA was not statistically significant from zero and uptake of [14C]trans‐UCA was negligible (0.8 × 10−3± 0.08 × 10 3 pmol/s/mg protein/μmol/L). There was no significant difference between uptake of UCA isomers but uptake of [3H]8‐MOP was significantly greater than that of either UCA isomer (P < 0.01). These studies indicate that the photobinding of UCA to DNA does not play a role in UV‐induced immunosuppression.

Urocanic acid does not photobind to DNA in mice irradiated with immunosuppressive doses of UVB.

Ultraviolet B (UVB, 290-320 nm) radiation initiates in vivo a dose- and wavelength-dependent down regulation of cell-mediated immunity. An action spectrum for UV-induced immunosuppression indicated that the photoreceptor for this effect is urocanic acid (UCA), which undergoes a trans to cis isomerization in the stratum corneum on UV exposure. An accumulation of evidence has supported this conclusion. However, evidence has also been presented that formation of thymine dimers in DNA is responsible for initiation of UV-induced immunosuppression. Because photobinding of UCA to DNA in vitro forming cyclobutane-type adducts has been shown, we sought to resolve this dilemma by investigating if UCA photobinds to DNA in vivo. The [14C]cis-UCA, [14C]trans-UCA or [3H]8-MOP (8-methoxypsoralen) was applied topically to BALB/c mice that were then irradiated with a dose of UV previously shown to cause systemic suppression of contact hypersensitivity. The DNA was prepared from epidermal cells by phenol extraction immediately after in vivo irradiation and bound radioactivity determined. Although photobinding of [3H]8-MOP was readily demonstrable under these conditions (0.9 nmol/mg DNA), no significant binding of either isomer of UCA to DNA (between 1.2 x 10(-3) and 2.1 x 10(-3) ng/mg DNA) could be detected. Uptake studies in keratinocytes prepared from epidermis of untreated animals indicated that [3H]8-MOP was taken up with a rate constant of 4.2 x 10(-3) pmol/s/mg protein/mumol/L. In contrast, uptake of [14C]cis-UCA was not statistically significant from zero and uptake of [14C]trans-UCA was negligible (0.8 x 10(-3) +/- 0.08 x 10(-3) pmol/s/mg protein/mumol/L). There was no significant difference between uptake of UCA isomers, but uptake of [3H]8-MOP was significantly greater than that of either UCA isomer (P < 0.01). These studies indicate that the photobinding of UCA to DNA does not play a role in UV-induced immunosuppression.

Nucleotide Sequence Context Effect of a Cyclobutane Pyrimidine Dimer upon RNA Polymerase II Transcription

We have studied the role of sequence context upon RNA polymerase II arrest by a cyclobutane pyrimidine dimer using an in vitro transcription system consisting of templates containing a specifically located cyclobutane pyrimidine dimer (CPD) and purified RNA polymerase II (RNAP II) and initiation factors. We selected a model sequence containing a well characterized site for RNAP II arrest in vitro, the human histone H3.3 gene arrest site. The 13-base pair core of the arrest sequence contains two runs of T in the nontranscribed strand that impose a bend in the DNA. We hypothesized that arrest of RNAP II might be affected by the presence of a CPD, based upon the observation that a CPD located at the center of a dA6.dT6 tract eliminates bending (Wang, C.-I., and Taylor, J.-S. (1991) Proc. Natl. Acad. Sci. U. S. A. 88, 9072-9076). We examined the normal H3.3 sequence and a mutant sequence containing a T --> G transversion, which reduces bending and efficiency of arrest. We show that a CPD in the transcribed strand at either of two locations in the arrest site is a potent block to transcription. However, a CPD in the nontranscribed strand only transiently pauses RNAP II. The CPD in concert with a mutation in the arrest site can reduce the extent of bending of the DNA and improve readthrough efficiency. These results demonstrate the potential importance of sequence context for the effect of CPDs within transcribed sequences.

Inhibition of UVB induced DNA photodamage in mouse epidermis by topically applied alpha-tocopherol.

Ultraviolet B (UVB, 290-320 nm) exposure results in a variety of cellular insults including induction of cyclobutane pyrimidine dimers in DNA. Accumulation of these lesions can lead to mutations in critical genes and contribute to the development of nonmelanoma skin cancer. Topically applied alpha-tocopherol (vitamin E) has previously been shown to prevent the induction of skin tumors in UVB irradiated female C3H/HeNTac mice. We hypothesized that alpha-tocopherol, which absorbs strongly in the UVB, may act as a sunscreen to prevent photodamage. To explore possible mechanisms of photoprotection, we topically applied alpha-tocopherol dispersed in a neutral cream vehicle to the dorsal epidermis of female C3H/HeNTac mice and exposed them to 2.5 J/m2/s of UVB for 60 min. Immediately after exposure, we analyzed thymine dimer levels in DNA by capillary gas chromatography with electron capture detection. Epidermal DNA from mice receiving this UVB dose contained 247 +/- 42 pmol thymine dimers/micromol thymine. Topical application of alpha-tocopherol inhibited dimer formation in a dose-dependent manner. A 1% alpha-tocopherol dispersion inhibited the formation of thymine dimers to 43% of levels in vehicle controls. Several vitamin E compounds, including alpha-tocopherol acetate, alpha-tocopherol methyl ether, gamma-tocopherol, and delta-tocopherol also inhibited thymine dimer formation, but were five- to ten-fold less potent than alpha-tocopherol. A variety of commercially available sunscreens were also less potent than alpha-tocopherol in their ability to reduce dimer formation. These results suggest that DNA photoprotection is an important mechanism by which topically applied alpha-tocopherol can inhibit UVB induced skin cancer. Alpha-Tocopherol acetate, the most common form of vitamin E in commercial skin care products, conferred less protection, perhaps due to its lower absorptivity in the UVB. Our results further underscore the importance of determining which forms of vitamin E can inhibit specific lesions involved in photocarcinogenesis.

Molecular recognition of octadecanyl ester of 1-(2-carboxyethyl)thymine to adenine at the air/water interface and photodimerization in the Langmuir-Blodgett film matrix under ultraviolet irradiation

Abstract A new single-chain nuclei acid base-containing amphiphile, octadecanyl ester of 1-(2-carboxyethyl)thymine, was synthesized, and its surface behaviors on pure water and on aqueous solutions of adenine were investigated by the method of surface pressure-area (π-A) isotherms. Molecular recognition through complementary base pairing takes place at the air/water interface between the thymine moiety in the headgroup of the amphiphile and adenine, which results in the pick-up of the base-base complex onto quartz plate by the Langmuir-Blodgett (LB) technique. Photodimerization of thymine rings was also investigated in the multilayer LB film of the amphiphile deposited from pure water surface, and the results show that the orderly and regular packing of the molecules in the LB matrix provides favorable conditions for the formation of thymine dimers.

Electron Addition to Thymine Dimers and Related Compounds: A Mimic of Natural Repair

Solar UV light can be dangerous to cellular systems. One manifestation of this is the formation of thymine dimers, containing cyclobutane rings, in suitable regions of duplex DNA. Fortunately, these dangerous defects are rapidly recognized by a number of DNA repair enzymes, including the photolyase enzymes, whose activity is also triggered by the sun's rays. This is thought to result in rapid electron transfer from the excited chromophores therein, to the pyrimidine dimer, resulting in opening of the cyclobutane ring. The electron then moves back, reconstituting the enzyme and the two pyrimidine monomer units. In an attempt to mimic this process in the absence of the enzyme, we have added radiation-generated electrons to thymine and uracil dimers at 77 K and used EPR spectroscopy to study the initial products. The results show that for trans-syn-1,3-dimethyluracil dimer, a long-lived dimer radical anion is formed, the electron being confined to one of the uracil units. On annealing, this was converted irr...

Oligonucleotide model with non-identical complementary strands for chromatographic studies of structure-dependent photosusceptibility

In a previous work, we used a quantitative chromatographic analysis of two self-complementary oligonucleotides to correlate the conformational differences between the oligonulcleotide duplexes and photochemical susceptibilities of constituent oligomers. In this work we describe a new double-stranded oligonucleotide model with non-identical complementary strands. To separately analyze photoproducts in two strands, one of them is used in a partially protected form (the hydrophobic 5′-dimethoxytrityl group uncleaved). Using a reversed-phase column, the oligomers and products of their UV photomodification are separated into two groups of peaks. This facilitates the quantitation of photoproducts in each of the complementary strands. Three 15-mer oligonucleotides, 5′TTTTTATTAAATATA-3′ (F5), 5′-AAAAATAATTTATAT-3′ (F6) and 5′-TATATTTAATAAAAA-3′ (F7) form the parallel-stranded ( ps) F5·F6 and the ordinary antiparallel-stranded ( aps) F5·F7 duplexes. For these particular sequences, the rate of cyclobutane thymine dimer formation in the ps DNA has been estimated as ca. 1.5–2 times that in the ordinary aps DNA.

Mapping of 386 kb of genomic DNA in the human dystrophin-encoding gene ( DYS) using an ordered phage λ sublibrary of a YAC clone containing the DYS region

An integrated restriction map for HindIII and EcoRI has been constructed for 386 kb of the human dystrophinencoding gene by partial digest mapping of 35 overlapping λEMBL3 cosW phage clones derived from a yeast artificial chromosome containing this region. Map construction was simplified in two ways. Firstly, the sequence arrangement of λEMBL3 cosW is such that only map data from cloned inserts are generated using partial digests of λ phage DNA asymmetrically labelled at the left cos end with a complementary 32P-labelled oligodeoxyribonucleotide. Secondly, the degree of partial digestion was standardised for each restriction enzyme by using ultraviolet light-induced formation of thymine dimers in the recognition sequence to partially block the cleavage reaction. The map provides the basis for work on the analysis of chromosomal rearrangements in this region which give rise to Duchenne muscular dystrophy, and for studies of chromosome structure and function.

In vitro evidence that UV-induced frameshift and substitution mutations at T tracts are the result of misalignment-mediated replication past a specific thymine dimer.

A previous study of UV-induced (254 nm) mutations in the lacI gene of Escherichia coli found that frameshift mutations accounted for about 35% of the observed mutations and that these mutations occurred predominantly at An.Tn sequences [Miller, J.H. (1985) J. Mol. Biol. 182, 48-65]. Because An.Tn sequences are hotspots for cis-syn thymine dimer formation [Brash, D.E., & Haseltine, W. A. (1982) Nature 298, 189-192], it would appear that UV-induced frameshift mutations are the result of an error during replicative bypass of a thymine dimer within such a sequence. To test the validity of such a proposal, replication experiments were carried out on templates containing cis-syn thymine dimers at each of the five possible sites of a T6 tract. The 59-mer templates were prepared by ligating oligonucleotides containing an EcoRI site to the 5'-end of decamers containing the cis-syn thymine dimer and oligonucleotides containing the primer site to the 3'-end. Primer-extension reactions were then carried out on these templates with a 3'----5' exonuclease-deficient (exo-) Klenow fragment of E. coli polymerase I and an exo-T7 polymerase (Sequenase Version 2.0). The replicative bypass products were cleaved with EcoRI to rigorously establish and quantify the presence of frameshift mutations. Both polymerases were able to bypass dimers at all sites, but only the exo-T7 polymerase led to detectable frameshifts, both -1 (approximately 30%) and -2 (approximately 5%), and only with the template containing a cyclobutane dimer at the second site from the 5'-end of the T6 tract. Sequencing of the T7 polymerase-catalyzed bypass products of all templates demonstrated that within the limits of discrimination only As were introduced opposite the dimer-containing T tracts. The only exception was for the template with the dimer at the second site which led to a readily detectable amount of a substitution mutation (approximately 30%) opposite the 5'-thymine of the T6 tract. A mechanism involving a competition between reversible misalignment and realignment steps and irreversible elongation steps is proposed to explain the origin of both the frameshift and the substitution mutations. The implications of this work to the mechanism of UV-induced frameshift and substitution mutations at T tracts in vivo are discussed.

The potential carcinogenic effect of Uvinul DS49 — a common UV absorber used in cosmetics

The photosensitized production of thymine dimer at 324 nm was investigated using disodium-2,2′-dihydroxy-4,4′-dimethoxybenzophenone sulphonic acid (tradename, Uvinul DS49) as sensitizer. Yields of the dimer for various irradiation times, initial thymine concentrations and initial sensitizer concentrations were determined for the free thymine base in aqueous medium. A reaction mechanism is proposed for total thymine dimer formation together with the corresponding rate constants. The mechanism and rate constants were computer simulated to support their validity.