IRRADIATION OF CIRCULAR DNA WITH 254 nm RADIATION OR SENSITIZATION IN THE PRESENCE OF Ag+ EVIDENCE FOR UNWINDING BY PHOTOPRODUCTS OTHER THAN PYRIMIDINE DIMERS

Abstract— Structural alterations of DNA irradiated with UV light were analyzed by the agarose gel technique. Relaxed, circular pAT 153 DNA molecules were sensitized by broad band radiation with a maximum at 313 nm in the presence of silver ions or irradiated with 254 nm light in buffer only. In both cases the electrophoretic mobility of DNA topoisomers was altered as a linear function of UV exposure. For DNA irradiated in the sensitized reaction the unwinding angle per site sensitive to Micrococcus luteus pyrimidine dimer endonuclease was found tobe–11.4°. This value is significantly smaller thanthe–14.3° already known for DNA topoisomers irradiated with 254 nm light. The irradiated DNAs were a very good substrate for the Escherichia coli photoreactivating enzyme (PRE). However, the photoenzymic removal of all sites sensitive to the endonuclease specific for pyrimidine dimers was not coupled to a full restoration of the original electrophoretic mobility. Thirty and 23% of the unwinding were still present in the photoreactivated topoisomers and the unwinding angles per pyrimidine dimer were then recalculatedas–10.1°and–8.7° for DNAs irradiated with 254 nm and sensitized, respectively. The limited difference between these two values could result from the different base composition of the pyrimidine dimers generated in the conditions of irradiation used. These results show that the tertiary structure of DNA is measureably altered by UV photodamages other than pyrimidine dimers.     

REMOVAL OF THYMINE-CONTAINING PYRIMIDINE DIMERS FROM UV LIGHT-IRRADIATED DNA BY S1 ENDONUCLEASE

Abstract— S₁ endonuclease was shown to remove thymine-containing pyrimidine dimers from UV-irradiated human DNA, although efficient removal could be demonstated only by using long digestion times, relatively high enzyme concentrations, and irradiation sufficient to yield dimer substitutions in DNA of 1 per 1W300 (dimers/base pair). Neutral and alkaline sucrose gradient analysis of strand break induction by S, of UV-irradiated DNA suggests that recognition of the dimer by S, is the limiting factor in its removal and dimer removal usually results from attack on the dimer containing DNA strand without the induction of a double-strand break.     

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.     

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.     

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.     

COMPARATIVE STUDIES ON THE CORRELATION BETWEEN PYRIMIDINE DIMER FORMATION and TYROSINASE ACTIVITY IN CLOUDMAN S91 MELANOMA CELLS AFTER ULTRAVIOLET-IRRADIATION

We compared the induction of pyrimidine dimer densities after UV-irradiation in mouse melanoma cells before and after treatment with cholera toxin. Treatment with cholera toxin stimulated tyrosinase activity up to 50-fold, leading to a marked, visually apparent increase in cellular melanin concentrations. Irradiation of treated and untreated cells was therefore designed to establish whether intracellular melanin protected cells from UV-induced DNA damage. In experiments described here, we determined cytosine-thymine (C-T) as well as thymine-thymine dimer levels (T-T) by high pressure liquid chromatography in cholera toxin-treated and untreated Cloudman S91 mouse melanoma cells after irradiation with UVC (less than 290 nm) and UVB light (290-320 nm). Surprisingly, induction of melanization had no effect on the formation of pyrimidine dimers by UVC or UVB irradiation. These results indicate that de novo melanin pigmentation induced via the c-AMP pathway is not involved in protection against UV-induced thymine-containing pyrimidine dimers. In separate experiments, irradiation of toxin-treated and untreated mouse melanoma cells with UVC or UVB light produced a 20-30% lower dimer density compared to irradiated human skin fibroblasts. This finding suggests that melanin has some protection properties against UV-induced pyrimidine dimers, although the exact defense mechanism seems highly complex.     

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.     

THE EFFECT OF TEMPERATURE AND WAVELENGTH ON PRODUCTION AND PHOTOLYSIS OF A UV-INDUCED PHOTOSENSITIVE DNA LESION WHICH IS NOT REPAIRED IN XERODERMA PIGMENTOSUM VARIANT CELLS

Abstract— Ultraviolet light causes a type of damage to the DNA of human cells that results in a DNA strand break upon subsequent irradiation with wavelengths around 300 nm. This DNA damage disappears from normal human fibroblasts within 5 h, but not from pyrimidine dimer excision repair deficient xeroderma pigmentosum group A cells or from excision proficient xeroderma pigmentosum variant cells. The apparent lack of repair of the ultraviolet light DNA damage described here may contribute to the cancer prone nature of xeroderma pigmentosum variant individuals. These experiments show that the same amount of damage was produced at 0^°C and 37^°C indicating a photodynamic effect and not an enzymatic reaction. The disappearance of the photosensitive lesions from the DNA is probably enzymatic since none of the damage was removed at 0^°C. Both the formation of the lesion and its photolysis by near ultraviolet light were wavelength dependent. An action spectrum for the formation of photosensitive lesions was similar to that for the formation of pyrimidine dimers and(6–4) photoproducts and included wavelengths found in sunlight. The DNA containing the lesions was sensitive to wavelengths from 304 to 340 nm with a maximum at 313 to 317 nm. This wavelength dependence of photolysis is similar to the absorption and photolysis spectra of the pyrimidine(6–4) photoproducts     

INHIBITION OF TRANSIENT GENE EXPRESSION IN CHINESE, HAMSTER OVARY CELLS BY TRIPLET-SENSITIZED UV-B IRRADIATION OF TRANSFECTED DNA

The biological effectiveness of thymine-thymine cyclobutane dimers specifically induced by photosensitized ultraviolet-B irradiation was analyzed by host-cell reactivation of triplet-sensitized, UV-B irradiated plasmid pRSV beta gal DNA transfected into normal and repair-deficient Chinese hamster ovary cells. For comparison, pRSV beta gal DNA was also UV-C irradiated and transfected into the same cell lines. Ultraviolet endonuclease-sensitive site induction was determined after UV-C irradiation or acetophenone-sensitized UV-B irradiation of plasmid pRSV beta gal DNA. These data were used to calculate the number of cyclobutane pyrimidine dimers required to inactivate expression of the lacZ reporter gene in each irradiation condition. Transfection with UV-C-irradiated plasmid DNA resulted in a significantly greater reduction of reporter gene expression than did transfection with acetophenone-sensitized UV-B-irradiated pRSV beta gal DNA at equivalent induction of enzyme-sensitive sites. Since only a fraction of the inhibition could be accounted for by noncyclobutane dimer photoproducts, these results suggest that cytosine-containing pyrimidine cyclobutane dimers may be more effective than thymine-thymine dimers in inhibiting transient gene expression as measured in such host-cell reactivation experiments in mammalian cells.     

NETROPSIN: INTERACTION WITH ULTRAVIOLET IRRADIATED DNA

Abstract—Ultraviolet irradiation of double-stranded DNA reduces the circular dichroism (i < 300 nm) induced when the basic peptide antibiotic netropsin (Nt) is added to DNA subsequent to thc irradiation compared to the CD induced by the same concentrations of Nt added to unirradiated DNA. Nt is known to bind to A T base pairs in duplex DNA but will not bind to single-stranded DNA. The reduction in the maximum induced CD observed with saturating concentrations of Nt is a linear function of the concentration of pyrimidine dimers which. along with other dinucleotide photoproducts. form short disrupted regions in duplex DNA. The decrease in the CD of Nt bound to irradiated DNA could be due to elimination of potential Nt sites in the vicinity of a dimer. reduction in the average magnitude of the CD of Nt bound near a dimer or various combinations of these effects. In addition there is a reduction in the average binding constant for Nt bound to irradiated DNA compared to unirradiated DNA suggesting that formation of dinucleotide photoproducts either tends to preferentially eliminate the tighter binding sites or that tighter sites are converted to weaker ones. A simple model suggests that no more than one-third to one-half of the pyrimidine dimcrs formed in DNA completely eliminate a Nt site.     

CHANGE IN THE BASE COMPOSITION OF MESSENGER RNA OF ESCHERICHIA COLI BY ULTRAVIOLET IRRADIATION AND ITS RECOVERY

Abstract— The base composition of messenger RNA in Escherichia coli B/r and B _8–1 irradiated with ultraviolet (u.v.) light has been examined. The experimental results are as follows: (1) the synthesis of rapidly labeled RNA does not stop in ultraviolet irradiated bacteria. (2) The rapidly labeled RNA in irradiated cells shows a change in base composition corresponding to the formation of pyrimidine dimers in DNA molecules. The mole per cent of adenine component is increased with ultraviolet dose. The ratio of purine/pyrimidine becomes larger and the GC content smaller. (3) The base composition of the rapidly labeled RNA in irradiated bacteria reversed to that in unirradiated cells, when the irradiated cells were reactivated by experimental procedures for photoreactivation or dark reactivation. The reversion in the base composition corresponds well to the decrease in the amount of thymine dimers in DNA molecules. (4) The mechanism of the change in the base composition of rapidly labeled RNA caused by ultraviolet irradiation is discussed.     

PYRIMIDINE DIMER SITES ASSOCIATED WITH THE DAUGHTER DNA STRANDS IN UV-IRRADIATED HUMAN FIBROBLASTS

Abstract. Pyrimidine dimer sites associated with the newly-synthesized DNA were detected during post-replication repair of DNA in UV-irradiated human fibroblasts. These pyrimidine dimer sites were inferred from a decrease in the molecular weight of pulse-labelled DNA after treatment with an extract of Micrococcus luteus containing UV-specific endonuclease activity. In DNA synthesized immediately after irradiation the frequency of these daughter strand dimer sites was 7–20% of that in the parental DNA. Such sites were found in fibroblasts from normal donors and from xeroderma pigmentosum patients (with defects in excision-repair or post-replication repair). They were excised from the DNA of normal cells. As the time between UV-irradiation and pulse-labelling was increased, the frequency of dimer sites associated with the labelled DNA decreased. If the pulse-label was delivered 6 h after irradiation of normal cells or excision-defective xeroderma pigmentosum cells, no dimer sites were detected in the labelled DNA. It has usually been assumed that daughter-strand dimer sites were the result of recombinational exchanges. The assay procedure used in these experiments and in similar experiments of others did not distinguish between labelled DNA containing pyrimidine dimers within the labelled section, and labelled DNA which did not contain pyrimidine dimers but was attached to unlabelled DNA which did contain dimers. The latter structures would arise during normal replication immediately following UV-irradiation of mammalian cells. Calculations are presented which suggest that a significant proportion and conceivably all of the dimer sites associated with the daughter strands may have arisen in this way, rather than from recombinational exchanges as has been generally assumed.     

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.     

Plant Responses to Changing Environmental Stress: Cyclobutyl Pyrimidine Dimer Repair in Soybean Leaves

Abstract— We have determined the capacity of soybean seedlings to repair DNA damage by UV doses that do not produce apparent injury in the plants. They remove cyclobutane pyrimidine dimers by both excision and photoreactiva-tion. The rates and relative contributions of these repair processes were determined as a function of initial level of cyclobutyl pyrimidine dimers. Photoreactivation was detected in seedlings at all initial dimer levels. Although excision was not observed at the lowest dimer frequencies, at higher initial dimer levels it was quite effective in dimer removal. The rates of repair in soybean were substantially higher than in alfalfa seedlings at the same DNA damage levels.     

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.     

Structure and dynamics of poly(T) single-strand DNA: implications toward CPD formation

The formation of cyclobutane pyrimidine dimers between adjacent thymines by UV radiation is thought to be the first event in a cascade leading to skin cancer. Recent studies showed that thymine dimers are fully formed within 1 ps of UV irradiation, suggesting that the conformation at the moment of excitation is the determining factor in whether a given base pair dimerizes. MD simulations on the 50 ns time scale are used to study the populations of reactive conformers that exist at any given time in T18 single-strand DNA. Trajectory analysis shows that only a small percentage of the conformations fulfill distance and dihedral requirements for thymine dimerization, in line with the experimentally observed quantum yield of 3%. Plots of the pairwise interactions in the structures predict hot spots of DNA damage where dimerization in the ssT18 is predicted to be most favored. The importance of hairpin formation by intra-strand base pairing for distinguishing reactive and unreactive base pairs is discussed in detail. The data presented thus explain the structural origin of the results from the ultrafast studies of thymine dimer formation.     

SKH-1 hairless mice repair UV-induced pyrimidine dimers in epidermal DNA

The SKH-1 hairless mouse strain has been used extensively as a model for human photocarcinogenesis, photoimmunology and photoaging, but little is known about DNA repair in living mouse skin. Mice were irradiated with UV-B light at doses which produce mild to severe sunburn, and the frequency of pyrimidine dimers in epidermal DNA was measured immediately and 6 h after irradiation using T4 endonuclease V treatment and alkaline agarose gel electrophoresis. The results demonstrate significant removal of pyrimidine dimers in mouse skin in vivo, with a dimer half-life of 7.4 h. These findings are similar to the repair of dimers in human skin in vivo. The SKH-1 hairless mouse is thus a useful model for pyrimidine dimer repair in human skin.     

DNA光复活作用机理的研究进展*

"环丁烷型嘧啶二聚体(Pyr< > Pyr) 是太阳光中紫外线造成DNA 损伤的主要光化学产物。DNA 光复活酶(或称光解酶) 能够利用可见光裂解二聚体的环丁烷环而修复DNA。本文对DNA 光复活过程中的光解酶对Pyr< > Pyr 的识别和光催化Pyr< > Pyr 裂解反应进行了综述, 介绍了DNA 光解酶的结构、DNA 的主要UV 光化学产物。较详尽地评述了国际上在光解酶催化二聚体裂解的途径以及模型研究方面的最新进展, 并预测了该领域的发展前景。     

PHOTOCHEMISTRY OF KETONES IN SOLUTION-49* A STUDY OF PHOTOSENSITIZED SPLITTING OF DIMETHYLTHYMINE DIMERS

Abstract— Several high energy ketone triplet sensitizers, e.g. carvone, camphor, 3-methylcyclohexanone, benzoin and 3-methylindanone, were studied as photosensitizers for the splitting of dimethylthymine dimers. The absence of splitting in all cases and the lack of quenching of benzoin and 3-methylindanone triplets by the trans-anti dimer of dimethylthymine strongly suggests that cleavage of dimethylthymine dimers cannot be achieved by a triplet mechanism on irradiation at Λ> 300 nm. The absence of optical rotation in the recovered chiral cis-anti dimethylthymine dimer after sensitized photolysis (12% splitting) in the presence of (—)-tryptophan suggests that. in highly polar solvents, such as methanol, where reaction probably takes place according to an electron transfer mechanism involving ion-pair intermediates, close approach of the sensitizer and substrate does not occur. To the extent that these results can be extrapolated to sensitized cleavage of cis-syn pyrimidine dimers in DNA brought about by action of photoreactivating enzyme or conventional photosensitizers, a mechanism involving dimer triplet states appears highly unlikely.     

RESTRICTION ENZYME CLEAVAGE OF UV-IRRADIATED DNA: A COMPARISON OF FAR-UV AND MID-UV WAVELENGTHS

UV-irradiated DNA is less susceptible to restriction by Type II endonucleases than unirradiated DNA presumably due to photolesions formed in the recognition sites. Previous reported studies have used 254 nm radiation or 313 nm plus acetophenone, both treatments which introduce pyrimidine dimers in preference to other photolesions. To assess the effect of a longer wavelength, at which the ratio of pyrimidine dimer formation to the formation of other photolesions is reduced, two different DNAs were irradiated with UV of either 254 or 313 nm and restricted with suitable restriction endonucleases. Restriction patterns were analysed for novel fragments resulting from UV-induced alteration of enzyme recognition sites. EcoRI restriction of 254 nm irradiated lambda DNA produced six novel bands, only three of which were observed following restriction of 313 nm irradiated lambda. These three represented the largest fragments resulting from single site blocks. Novel fragments involving adjacent site blocks observed at 254 nm were not found with 313 nm radiation. Comparison of 254 nm irradiated pSV₂gpt to that irradiated at 313 nm, both restricted with Dral, revealed a more complex pattern. Although all sites were singly blocked by radiation of both wavelengths, multiple site blocks produced by 313 nm radiation did not occur in the order predicted by the 254 nm radiation dose response. These data suggest that certain sites in pSV₂gpt may be more refractory to multiple site blocks than others when irradiated at 313 nm.