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.     

ENHANCEMENT OF PHOTOREPAIR OF ULTRAVIOLET-DAMAGE BY PREILLUMINATION WITH FLUORESCENT LIGHT IN CULTURED FISH CELLS

Fluorescent light (FL) illumination of RBCF-1 cells, derived from a goldfish, prior to 254 nm UV-irradiation enhanced their ability to photorepair. The cells were illuminated with FL for 1 h (29 W/M2) and incubated for 8 h in the dark before being irradiated with 10 J/m2 UV. The surviving fraction of FL-treated cells after UV-irradiation rose about 7-fold (from 3 to 20%) by 20 min photorepair treatment with the same FL source, whereas 4-fold (from 1.6 to 6%) in the FL non-treated cells. Flow cytometric analysis showed that FL treatment did not affect the distribution of cell cycle phase at the time of UV-irradiation (8 h after FL treatment). Pyrimidine dimers induced by UV were measured by the use of UV endonuclease of Micrococcus luteus and alkaline agarose gel electrophoresis. Initial yields of dimers after exposure to 10 J/m2 UV were almost the same (about 0.11 dimer/kb) between FL treated and non-treated cells. But after 20 min photorepair treatment, about 70% of dimers were removed in the FL treated samples, while less than 20% were removed in the non FL-treated ones.     

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 ).     

MORE EFFICIENT EXCISION REPAIR OF PYRIMIDINE DIMERS IN THE SPECIFIC DNA SEQUENCE THAN IN THE GENOME OVERALL IN GOLDFISH CELLS

Excision repair of pyrimidine dimers induced by 254 nm UV was examined in the genome overall and in a specific sequence containing a transfected gene for hygromycin B resistance, in RBCF-1 cells derived from a goldfish, by the use of UV endonuclease of Micrococcus luteus and alkaline agarose gel electrophoresis. More than 40% of dimers were removed from the specific sequence, while about 20% were removed from the genome overall, within 24 h after exposure to UV (2.5-7.5 J/m2).     

MULTIPLE EFFECTS OF FLUORESCENT LIGHT ON REPAIR OF ULTRAVIOLET-INDUCED DNA LESIONS IN CULTURED GOLDFISH CELLS

Abstract— It is known that fluorescent light illumination prior to UV irradiation (FL preillumination) of cultured fish cells increases photorepair (PR) ability. In the present study, it was found that FL preillumination also enhanced UV resistance of logarithmically growing cells in the dark. This enhancement of UV resistance differs from induction of PR because it was not suppressed by cycloheximide (CH) and it occurred immediately after FL preillumination. The effects of FL preillumination on repair of UV-induced DNA lesions in the dark were examined by an endonuclease-sensitive site assay to measure the repair of cyclobutyl pyrimidine dimers, and by enzyme-linked immunosorbent assay to quantitate the repair of (6-4) photoproducts. It was found that excision repair ability for (6-4) photoproducts in the genome overall was increased by FL preillumination. Moreover, a decrease in (6-4) photoproducts by FL illumination immediately after UV irradiation of the cells was found, the decrement being enhanced by FL preillumination with or without CH.     

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.     

UNSCHEDULED DNA SYNTHESIS: A QUANTITATIVE INDICATOR OF RESIDUAL IMMUNODETECTABLE PYRIMIDINE DIMERS IN HUMAN FIBROBLASTS AFTER ULTRAVIOLET-B IRRADIATION

We have addressed the question whether the level of UV-B induced DNA damage can be accurately assessed by the measurement of the rate of unscheduled DNA synthesis (UDS). Cultured human fibroblasts were irradiated with UV radiation at 290, 313 or 365 nm. The LD50 was 85 J/m2 at 290 nm, 4500 J/m2 at 313 nm, and 70 kJ/m2 at 365 nm. The analysis of UDS measurements indicate complete arrest of repair processes within 24 h after irradiation, irrespective of the dose (in the range 10-60 J/m2 at 290 nm, and 250-1000 J/m2 at 313 nm). Irradiation at 365 nm failed to yield detectable evidence of UDS. Incubation of irradiated cells with an antiserum directed against both 6-4 type and cyclobutane-type pyrimidine dimers shows a clear parallelism between the disappearance of the antibody-binding determinants and the variation of the rate of UDS vs time after the end of the irradiation. Thus it is concluded that in UV-B irradiated normal cultured human fibroblasts, the lack of UDS reflects the absence of immunodetectable pyrimidine dimers.     

PHOTOREACTIVATION OF ULTRAVIOLET LIGHT-INDUCED DAMAGE IN CULTURED FISH CELLS AS REVEALED BY INCREASED COLONY FORMING ABILITY AND DECREASED CONTENT OF PYRIMIDINE DIMERS

Abstract— Cultured cells derived from a goldfish were irradiated with 254nm ultraviolet light. Cell survival and splitting of pyrimidine dimers after photoreactivation treatment with white fluorescent lamps were examined by colony forming ability and by a direct dimer assay, respectively. When UV-irradiated (5 J/m²) cells were illuminated by photoreactivating light, cell survival was enhanced up to a factor of 9 (40min) followed by a decline after prolonged exposures. Exposure of UV-irradiated (15 J/m²) cells to radiation from white fluorescent lamps reduced the amounts of thymine-containing dimers in a photoreactivating fluence dependent manner, up to about 60% reduction at 120 min exposure. Keeping UV-irradiated cells in the dark for up to 120min did not affect either cell survival or the amount of pyrimidine dimers in DNA, indicating that there were not detectable levels of a dark-repair system in the cells under our conditions. Correlation between photoreactivation of colony forming ability and photoreactivation of the pyrimidine dimers was demonstrated, at least at relatively low fluences of photoreactivating light.     

QUANTITATION OF PYRIMIDINE DIMERS BY IMMUNOSLOT BLOT FOLLOWING SUBLETHAL UV-IRRADIATION OF HUMAN CELLS

An immunoslot blot assay was developed to detect pyrimidine dimers induced in DNA by sublethal doses of UV (254 nm) radiation. Using this assay, one dimer could be detected in 10 megabase DNA using 200 ng or 0.5 megabase DNA using 20 ng irradiated DNA. The level of detection, as measured by dimer specific antibody binding, was proportional to the dose of UV and amount of irradiated DNA used. The repair of pyrimidine dimers was measured in human skin fibroblastic cells in culture following exposure to 0.5 to 5 J m^-2 of 254 nm UV radiation. The half-life of repair was approximately 24, 7 and 6 h in cells exposed to 0.5, 2 and 5 J m^-2 UV radiation, respectively. This immunological approach utilizing irradiated DNA immobilized to nitrocellulose should allow the direct quantitation of dimers following very low levels of irradiation in small biological samples and isolated gene fragments.     

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.     

THE RATE OF REMOVAL OF SUNLIGHT-INDUCED PYRIMIDINE DIMERS FROM THE DNA OF CULTURED HUMAN DIPLOID FIBROBLASTS

Abstract The rate of excision of sunlight-induced pyrimidine dimers in DNA of exposed human cells was determined. Two normal excision repair-proficient human diploid fibroblast strains (WS-1 and KD) and a repair-deficient strain (XP12BE, group A) maintained in a nondividing state were exposed to summer noon-time sunlight for times (5 and 20 min) that induced numbers of dimers equivalent to far UV (254 nm) exposures of 1 and 4 J/m². Pyrimidine dimers were quantified in extracted DNA using a U V-endonuclease-alkaline sedimentation assay. The excision rates of these dimers were similar to those observed for the excision of UV-induced pyrimidine dimers. No sunlight-induced inhibition or stimulation of DNA repair was observed in either strain at these low exposures.     

Repair of (6-4) Photoproducts in Cultured Goldfish Cells at Confluence or Treated with H2O2

Previously we reported that fluorescent light (FL) illumination prior to UV irradiation (FL preillumination) had multiple effects on the repair of cyclobutane pyrim-idine dimers (CPD) and (6-4) photoproducts ([6-4] PD) in cultured goldfish cells (RBCF-1) at the exponentially growing phase. In this study, it is shown that even under the confluent condition of RBCF-1 cells, FL preillumination increased the disappearance of (6-4) PD in the dark. In addition, both at confluence and at the exponentially growing phase, the disappearance of (6-4) PD after PR treatment was increased by FL preillumination to RBCF-1 cells. Moreover, it was found that H₂O₂ pre-treatment, followed by UV irradiation, of the exponentially growing cells also enhanced the disappearance of (6-4) PD in the dark and by photorepair treatment. The degree of enhancement by H₂O₂ pretreatment was almost the same as that by FL preillumination.     

In situ PYRIMIDINE DIMER DETERMINATION BY LASER CYTOMETRY

By using antiserum against pyrimidine dimers and argon-laser imaging microspectrofluorometry, we established a new method to determine UV-induced pyrimidine dimers and their repair in individual human cells. The method was sensitive enough to determine dimers induced by UV dose as low as 2 J/m2. Normal human cells repaired 50 and 60% of total damage within 8 and 24 h after UV irradiation (20 J/m2), but Xeroderma pigmentosum cells (complementation group A) were unable to repair any within the same period. Therefore, the method proved to be a quick, easy, sensitive and accurate means to determine pyrimidine dimers in situ.     

THE EFFECTS OF GROWTH-INHIBITING TREATMENTS ON PHOTOREPAIR IN CULTURED FISH CELLS

Abstract Goldfish cells (RBCF-1) cultured at different cell densities were harvested and their photorepair (PR) abilities were examined in terms of survival. Photorepair ability gradually increased during the phase of logarithmic growth, reaching a maximum in cells at the confluent state. This enhancement of PR ability disappeared 12 h after replating of cells in fresh medium. A number of growth-inhibiting treatments (serum depletion, UVC, hydroxyurea [HU], change in incubation temperature) were tested for their ability to induce PR. The treatment of cells with HU and serum depletion induced PR while the other treatments did not. The increase in the ability to perform PR after treatment with HU or serum depletion returned to normal levels more rapidly than that after fluorescent light treatment.     

INTRODUCTION OF PYRIMIDINE DIMERS INTO DIFFERENT INTRACELLULAR FORMS OF SIMIAN VIRUS 40

We examined the production of pyrimidine dimers by UV radiation in different intracellular forms of simian virus 40 DNA. Virus and chromatin or previrions were selectively labeled with [^l4C]-thymidine and [³H]-thymidine, respectively, in the same monolayer of infected cells. Viral DNA was extracted immediately after irradiation, and pyrimidine dimers were detected as sites sensitive to the UV-endonuclease encoded by bacteriophage T4. No difference in the number of dimers introduced into chromatin, previrions. or virions was detected.     

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.     

PHOTOREACTIVATION and EXCISION REPAIR OF UV INDUCED PYRIMIDINE DIMERS IN THE UNICELLULAR CYANOBACTERIUM GLOEOCAPSA ALPICOLA (SYNECHOCYSTIS PCC 6308)

Abstract— The survival curve obtained after UV irradiation of the unicellular cyanobacterium Synecho-cystis is typical of a DNA repair competent organism. Inhibition of DNA replication, by incubating cells in the dark, increased resistance to the lethal effects of UV at higher fluences. Exposure of irradiated cells to near ultraviolet light(350–500 nm) restored viability to pre-irradiation levels. In order to measure DNA repair activity, techniques have been developed for the chromatographic analysis of pyrimidine dimers in Synechocystis. The specificity of this method was established using a haploid strain of Sacchar-omyces cerevisiae. In accordance with the physiological responses of irradiated cells to photoreactivating light, pyrimidine dimers were not detected after photoreactivation treatment. Incubation of irradiated cells under non-photoreactivating growth conditions for 15 h resulted in complete removal of pyrimidine dimers. It is concluded that Synechocystis contains photoreactivation and excision repair systems for the removal of pyrimidine dimers.     

A SENSITIVE METHOD FOR MEASURING PYRIMIDINE DIMERS IN SITU

Abstract. –An immunocytological method for detecting pyrimidine dimers in situ has been developed. The technique is based on the fixation in cell nuclei of radiolabelled antibodies directed against ultraviolet irradiated DNA. The relative amount of bound antibodies was estimated by radioautography. Pyrimidine dimers induced by a dose as low as 2 J m^-2 can easily be detected. With this technique, the dose response of DNA photoproducts and their elimination by excision and photoreactivation were followed in eukaryotic cell cultures.     

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.     

STUDY OF PYRIMIDINE DIMERS IN MAMMALIAN CELLS SURVIVING LOW DOSES OF ULTRAVIOLET RADIATION*

Abstract— Ultraviolet-induced pyrimidine dimers were not found to be excided from the DNA of Chinese hamster cells in small oligounucleotides. At doses whereby many cells survive the radiation, the dimers were still associates with the large polynucleotides even after 48 hr of postirradiation incubation.