EXCISION REPAIR OF UVR-INDUCED PYRIMIDINE DIMERS IN CORNEAL DNA

Abstract— We measured excision repair of ultraviolet radiation (UVR)-induced pyrimidine dimers in DNA of the corneal epithelium of the marsupial, Monodelphis domestica , using damage-specific nucleases from Micrococcus luteus in conjunction with agarose gel electrophoresis. We observed that 100 J ^-2 of UVR from aFS–40 sunlamp(280–400 nm) induced an average of 2.2 ± 0.2 times 10^-2 endonuclease-sensitive sites per kilobase (ESS/kb) (pyrimidine dimers) and that ∼ 50% of the dimers were repaired within 12 h after exposure. We also determined that an exposure of 400 J m^-2 was needed to induce comparable numbers of pyrimidine dimers (2.5 times 10^-2) in the DNA of skin of M. domestica in vivo . In addition, we found that 50% of the dimers were also removed from the epidermal cells of M. domestica within 12 h after exposure. A dose of 100 J m^-2 was necessary to induce similar levels of pyrimidine dimers (2.0 ± 0.2 times 10^-2) in the DNA of the cultured marsupial cell line Pt K2 ( Potorous tridactylus ).     

FORMATION OF PYRIMIDINE DIMERS IN SIMIAN VIRUS 40 CHROMOSOMES AND DNA in vitro: EFFECTS OF SALT

Abstract— Simian virus 40 chromosomes were used to determine whether packaging of DNA into chromatin affected the yield of cyclobutane pyrimidine dimers introduced by ultraviolet light (254 nm). SV40 chromatin and purified SV40 DNA (radioactively labeled with different isotopes) were mixed and irradiated in vitro . The proteins were extracted and pyrimidine dimers detected as sites sensitive to the UV-endonuclease encoded by bacteriophage T4. When irradiation was carried out in the presence of at least 0.05 M NaCl the same number of dimers were formed in chromatin as in free DNA. Irradiation in the absence of NaCl, however, reduced the relative yield of dimers in chromatin to 89% of that in free DNA. Different methods of chromatin preparation did not influence these results.     

P-AMINOBENZOIC ACID CAN SENSITIZE THE FORMATION OF PYRIMIDINE DIMERS IN DNA: DIRECT CHEMICAL EVIDENCE

Thymine-containing photoproducts with chromatographic properties similar to those of cyclobutyl pyrimidine dimers can be formed in [³H]-thymine-labeled DNA in solution by 313 nm ultraviolet radiation in the presence of para-aminobenzoic acid (PABA), a compound used in sunscreen preparations. In the absence of PABA, similar fluences of 313 nm radiation do not produce significant numbers of these photoproducts. The thymine-containing photoproducts can be reversed by 254 nm radiation so that the tritium label migrates with the mobility of thymine monomer, a behavior characteristic of thymine-containing cyclobutyl pyrimidine dimers. This result supports previous, but less direct, data from other laboratories indicating that PABA can sensitize dimer formation in the DNA of bacterial and mammalian cells.     

THE FORMATION OF PHOTOREVERSIBLE CYCLOBUTANE-TYPE PYRIMIDINE DIMERS IN ULTRAVIOLET-IRRADIATED POTATO VIRUS X*

Abstract— Photoreversible cyclobutane-type pyrimidine dimers were found in ³²P-labeled RN A isolated from intact potato virus X irradiated with 254 nm light. The number of dimers was correlated with the biological infectivity of the ultraviolet-treated virus. Assuming that incorporated ³²P is homogeneously distributed in the viral RNA chain, it can be calculated that about 5·7±1·7 dimers are present in each strand of virus per lethal biological hit.     

PYRIMIDINE DIMER FORMATION AND GERMINATION OF UV-IRRADIATED SPORES OF DICTYOSTELIUM DISCOIDEUMNC–4 ANDys–13

Abstract— Survival, UV-photoproducts and germination of UV-irradiated spores of Dictyostelium discoi-deum were studied on two strains,NC–4 andys–13. The spores ofNC–4 are about 35 times more resistant to UV thanys–13 spores at 10% survival. Pyrimidine dimers were formed in UV-irradiated spores in both strains. No photoproducts other than pyrimidine dimers were detected. The formation of pyrimidine dimers in spores was about 2% in both strains at 800 J/m₂. In the germination of spores, the conversion of spores into swollen spores was not affected by UV in both strains, but the emergence of amoebae from the swollen spores was suppressed, which was more distinctive inys–13 spores than inNC–4 spores. The emerged amoebae from the UV-irradiatedNC–4 spores were viable, while those from theys–13 spores were inviable even when they succeeded in emergence.     

PHOTOREPAIR OF NONADJACENT PYRIMIDINE DIMERS BY DNA PHOTOLYASE

Abstract— Photolyases reverse the harmful effects of UV light on cells by converting pyrimidine dimers (Pyr[]Pyr) into two pyrimidine monomers by utilizing near-UV and visible light. Previous work has shown that photolyase repairs T[c,s]T and T[t,s]T in DNA as well as U[]U in RNA, all of which are formed by joining the two adjacent pyrimidines in a light-dependent reaction. In this report, we show that Pyr[]Pyr formed in nonadjacent pyrimidines are also substrates for DNA photolyase.     

REPAIR OF PYRIMIDINE DIMERS IN ULTRAVIOLET-IRRADIATED CHLAM YDOMONAS

Abstract— A UV-specific endonuclease was used to monitor the presence of UV-induced pyrimidine dimers in the DNA of Chlamydomonas reinhardi . All of the dimers induced by 50 J/m² of 254 nm light are removed by a 2 h exposure to photoreactivating light. Nearly all of the dimers are removed by the wild-type strain of Chlamydomonas upon incubation for 24h in the dark. Two UV-sensitive mutants, UVS 1 and UVS 6, are deficient in removal of dimers in the dark. These results are interpreted to mean that Chlamydomonas has an excision-repair pathway for coping with UV-induced damage.     

THE FATE OF PYRIMIDINE DIMERS IN THE DNA OF ULTRAVIOLET-IRRADIATED CHINESE HAMSTER CELLS

Abstract —As an aid to understanding the relationship between dimer repair and cellular recovery, we have studied dimer removal and replication of dimer-containing DNA in Chinese hamster ovary (CHO) cells irradiated with ultraviolet light (254 nm). These investigations demonstrated that (1) dimers are not excised as polynucleotides of less than 500,000 mol. wt, (2) fractionation of the ultraviolet dose does not enhance dimer excision, (3) dimer-containing DNA is replicated in ultraviolet-irradiated CHO cells, and (4) the dimers are conserved in the replicated DNA. These findings support the proposed mechanism of bypass of photoproducts during DNA replication in mammalian cells.     

PHOTOREPAIR OF PYRIMIDINE DIMERS IN HUMAN SKIN IN VIVO

Abstract— The exposure of human skin in vivo to UV radiation emitted from a sunlamp induces the formation of pyrimidine dimers. The number of dimers, as detected by UV-endonuclease, decreases following exposure of the UV–irradiated skin to visible wavelengths of light. These results suggest that humans possess a mechanism by which pyrimidine dimers are photorepaired upon illumination of human skin in vivo with visible light.     

EXCISION REPAIR OF PYRIMIDINE DIMERS IN MARSUPIAL CELLS

Monodelphis domestica was further characterized as a model for photobiological studies by measuring the excision repair capabilities of this mammal's cells both in vivo and in vitro. Excision repair capability of the established marsupial cell line, Pt K2 ( Potorous tridactylus ), was also determined. In animals held in the dark, we observed that ˜50% of the dimers were removed by 12 and 15 h after irradiation with 400 J m⁻² and 600 J m⁻², respectively, from an FS-40 sunlamp (280–400 nm). Cells from primary cultures of M. domestica excised ˜50% of the dimers by 24 h after irradiating with 50 J m⁻² and 36 h after exposure to 100 J m⁻² with no loss of dimers observed 24 h following a fluence of 300 J m⁻². Pt K2 cells were observed to have removed -50% of the dimers at -12 h after 50 J m⁻² with only -10% of the dimers removed at 24 h following 300 J m⁻². The observed loss of pyrimidine dimers from epidermal DNA of UV-irradiated animals and from fibroblasts in culture, held in the dark, suggests that these marsupial cells are capable of DNA excision repair.     

THE FATE OF PYRIMIDINE DIMERS IN ULTRAVIOLET-IRRADIATED CHLAMYDOMONAS

Abstract— We have developed a chromatographic technique for the separation of ³²P-labeled pyrimidine nucleotide dimers of the form Py_pPy from ³²P-phosphate in enzymatic hydroly sates of ultraviolet-irradiated DNA. Application of this technique to ³²P-labeled Chlamydomonas reinhardii shows that ultraviolet irradiation of this organism induces pyrimidine dimers in both nuclear and chloroplast DNA. We have found no evidence that these dimers are excised from either DNA species after several hours incubation under non-photoreactivating conditions. A function has been derived to permit the pyrimidine-dimer content determined from radioactive-thymine-labeled cells to be conveniently compared to that obtained from ³²P-phosphate-labeled cells.     

PHOTOSENSITIZED SPLITTING OF PYRIMIDINE DIMERS

Abstract— The photosensitized monomerization of cis-syn and trans-syn cyclobutane-type thymine dimers, and the cis-syn thymine-uracil dimer, using anthraquinone derivatives as sensitizers, is described.     

PHOTOSENSITIZED SPLITTING OF PYRIMIDINE DIMERS IN DNA BY INDOLE DERIVATIVES AND TRYPTOPHAN-CONTAINING PEPTIDES

Abstract —Indole derivatives, such as serotonin or the oligopeptide Lys-Trp-Lys, are able to photosensitize the splitting of thymine dimers in DNA. These indole derivatives have to be bound to DNA in order to efficiently photosensitize the splitting reaction. Serotonin may also induce the photosensitized formation of thymine-containing dimers in native DNA. In this case, an equilibrium is reached when 5 per cent of the total thymines are dimerized. In both cases (splitting and dimer formation), the formation of electron donor-acceptor complexes between either dimers or two adjacent thymine monomers, and excited indole rings, could be an intermediate step in the reactions. Thymine-dimer splitting would then result from an electron transfer reaction involving the indole ring as the electron donor. These results are discussed with respect to the mechanism of action of the photoreactivating enzyme.     

QUANTIFICATION OF PYRIMIDINE DIMERS AND APURINIC SITES IN DNAs OF UNIFORM LENGTH

Abstract— A new simple in vitro assay for the determination of pyrimidine dimers and/or apurinic/apyrimidinic sites in non-radioactive DNA has been developed. In this procedure, DNA substrates of uniform length-which may be supercoiled, partially relaxed, relaxed or linear-are treated with agents which produce specific single strand nicks at the site of the lesion. The number of lesions per molecule can be expressed as a function of the amount of single-stranded molecules left intact after the specific nicking treatment. Unreacted molecules, which retain the original uniform length, are separated from the other smaller reaction products by electrophoresis on an alkaline agarose gel. In the case of circular molecules, the substrate is linearized by the use of an appropriate restriction endonuclease before loading on the gel. The amount of intact DNA molecules is obtained by integrating the corresponding peak of absorption after densitometric scan of the negative of the gel picture. This assay can detect up to eight damaged sites per duplex molecule. This method could be particularly useful when dealing with mixtures of DNA with different degrees of supercoiling or for comparisons between linear and circular DNA substrates.     

PHOTOREACTIVATION OF PYRIMIDINE DIMERS IN DNA FROM THYROID CELLS OF THE TELEOST, POECILIA FORMOSA

Abstract— We have developed and used a simple technique to estimate the quantity of pyrimidine dimers in unlabeled cellular DNA. DNA is extracted from cells, treated with an endonuclease specific for dimers, and its molecular weight estimated by its electrophoretic mobility on alkaline agarose slab gels. The technique is used to show that cells from thyroid tissue of the fish Poecilia formosa have photoreactivating activity towards dimers in the cellular DNA.