Effects of temperature on the photoreactivation of ultraviolet-b–induced dna damage in Palmaria palmata (Rhodophyta)

The accumulation of DNA damage (thymine dimers and 6-4 photoproducts) induced by ultraviolet-B radiation was studied in Palmaria palmata (L.) O. Kuntze under different light and temperature conditions, using specific monoclonal antibodies and subsequent chemiluminescent detection. Both types of damage were repaired much faster under ultraviolet-A radiation (UVAR) plus photosynthetically active radiation (PAR) than in darkness, which indicates photoreactivating activity. At 12° C, all thymine dimers were repaired after 2 h irradiation with UVAR plus PAR, whereas 6-4 photoproducts were almost completely repaired after 4 h. After 19 h of darkness, almost complete repair of 6-4 photoproducts was found, and 67% of the thymine dimers were repaired. In a second set of experiments, repair of DNA damage under UVAR plus PAR was compared at three different temperatures (0, 12, and 25° C). Again, thymine dimers were repaired faster than 6-4 photoproducts at all three temperatures. At 0° C, significant repair of thymine dimers was found but not of 6-4 photoproducts. Significant repair of both thymine dimers and 6-4 photoproducts occurred at 12 and 25° C. Optimal repair efficiency was found at 25° C for thymine dimers but at 12° C for 6-4 photoproducts, which suggests that the two photorepair processes have different temperature characteristics.     

Repair of Pyrimidine Dimer Damage Induced in Yeast by Ultraviolet Light

Crude extracts from ultraviolet (UV)-irradiated yeast cells compete with UV-irradiated transforming deoxyribonucleic acid (DNA) for photoreactivating enzyme. The amount of competition is taken as a measure of the level of cyclobutyl pyrimidine dimers in the yeast DNA. A calibration of the competition using UV-irradiated calf thymus DNA indicates that an incident UV dose (1,500 ergs/mm(2)) yielding 1% survivors of wild-type cells produces between 2.5 x 10(4) to 5 x 10(4) dimers per cell. Wild-type cells irradiated in the exponential phase of growth remove or alter more than 90% of the dimers within 220 min after irradiation. Pyrimidine dimers induced in stationary-phase wild-type cells appear to remain in the DNA; however, with incubation, they become less photoreactivable in vivo, although remaining photoreactivable in vitro. In contrast, exponentially growing or stationary-phase UV-sensitive cells (rad2-17) show almost no detectable alteration of dimers. We conclude that the UV-sensitive cells lack an early step in the repair of UV-induced pyrimidine dimers.     

No detectable DNA excision repair in UV-exposed hepatocytes from two catfish species

DNA repair is a critical process in protecting cellular genetic information from mutation. Nucleotide excision repair (NER) is a mechanism by which cells correct DNA damage caused by agents that form bulky covalent adducts and UV photoproducts such as thymine dimers and 6-4 photoproduct. NER, sometimes called dark repair, is generally accepted as being low in fish compared to mammals. This study was designed to quantitate NER in two related catfish species that have known differential sensitivities to liver carcinomas. The original hypothesis was that the more cancer resistant species, channel catfish (Ictalurus punctatus), would have more efficient DNA repair compared to the more sensitive brown bullhead (Ameriurus nebulosus). In order to measure NER, primary cultured hepatocytes of both species were exposed to UV light (10-40 J/m2) and collected at 0, 24, 48 and 72 h after exposure. Total DNA was extracted from the cells and incubated with T4 endonuclease V. Using alkaline gel electrophoresis, endonuclease sensitive sites (ESS) were quantified. Results from the ESS assay indicated there was a UV dose-response increase in thymine dimers from 0 to 40 J/m2. However, no repair (decrease in number of ESS) occurred in either fish species over a 72-h time period. When cells were exposed to photoreactivating fluorescent light, repair was detected. These studies highlight the difficulty of measuring NER in fish and are consistent with the low levels of NER reported by other researchers in fish.     

Loss of thymine dimers from mammalian cell DNA. The kinetics for antibody-binding sites are not the same as that for T4 endonuclease V sites

Antiserum specific for thymine-containing dimers was used to assay DNA isolated from ultraviolet-irradiated cells following different repair periods. A 50% loss in antibody-binding sites was evident 1 h post-irradiation, and within 4 h 80% of the sites were removed. This result contrasts with data obtained with dimer-specific T4 endonuclease V and does not appear to be due to masking of the dimers by repair enzymes. T4 endonuclease V treatment of ultraviolet-irradiated DNA at 0 degree C resulted in conversion of the thymine dimers to apyrimidinic sites. This did not result in loss of antigenicity in either PM2 or CHO cell DNA. Likewise, treatment of ultraviolet-irradiated CHO cell DNA with T4 endonuclease at 37 degrees C did not change its antigenicity. These results suggest that aglycosylation of the dimers is not responsible for their inability to bind dimer-specific antibody 2-4 h post-irradiation. The possibility that T4 endonuclease V and the antiserum have different specificities for different dimers is discussed.     

Increased efficiency of photoreversal of UV-induced dimers in the DNA of chick embryo fibroblasts with post-UV dark time

The kinetics of photoreversal of UV-induced dimers in the DNA of early passage chick embryo fibroblasts was studied by monitoring disappearance of UV-endonuleae-sensitive sites. Photorepair was found to increase in efficiency when cells were incubated in the dark for several hours at 37°C following the dimer-inducing short-wavelength (254 nm) UV treatment, but prior to the photoreactivating black light (365 nm). Folllowing a UV dose of 10 J/m² it took at least 4 h in the dark to saturate this effect. This UV dose inserts roughly 2.4 dimer/10⁷ daltons of DNA. Dark repair removes about 0.08 dimers /h/10⁷ daltons. After 6 h in the dark, exposure to black light removes an additional 1.4 dimers /10⁷ daltons leaving about 0.5 dimers unaffected by this treatment. After saturation of the dark effect, the amount of photoreactivation depends only on total black light fluence and not on fluence rate for the range of rates studied. This indicates that during 30 min, the maximum time of black light exposure, no appreciable reattachment of the photorepair molecule to additional unrepaired dimer sites occurs. We estimate that the number of effective photorepair molecules per chick chick cell is at least of the order of 2 × 10⁵.     

Reduced formation of bipyrimidine photoproducts in DNA UV irradiated at high intensity

DNA was irradiated using an excimer laser (248 nm) at low intensity (3.15 x 10(7) watts/m2) or high intensity (1.25 x 10(11) watts/m2). Fluences up to 30 kJ/m2 were delivered at either intensity. Following irradiation, DNA damage products were measured, yielding the following findings: 1) the rate of formation of thymine-thymine and thymine-cytosine cyclobutane dimers and the bipyrimidine photoadduct 6-4'-[pyrimidine-2'-one]thymine were reduced at high intensity by about 2-fold and 2) extensive release of free thymine and thymine decomposition fragments occurred at high intensity, but not at low intensity. The effects of high intensity UV are due to promotion of low-lying excited state(s) by absorption of a second photon, producing higher excited state(s) with consequent ionization and base loss. Possible excited state intermediates in this process are the lowest triplet state of DNA bases and prolonged singlet states associated with excimer formation. The depletion of these excited states via promotion may be the cause of the diminished yield of bimolecular pyrimidine photoproducts, suggesting that these photoproducts are formed at low UV intensity in part from long-lived excited states. Long-lived excited states present at conventional UV intensities may contribute to formation of some photoproducts that occur rarely, but are of potential biologic importance, such as dimers between nonadjacent pyrimidines on the same strand and interstrand dimers forming DNA cross-links.     

Deoxyribonucleic Acid Repair Replication after Ultraviolet Light or X-Ray Exposure of Bacteria

A comparison of repair synthesis after ultraviolet light (UV) or X-ray exposure was made in Escherichia coli strains 15T(-) (555-7) and B/r by use of a D, (15)N, (13)C density labeling system. During the initial 15 min of incubation after UV irradiation, both a "repair" synthesis and a reduced semiconservative deoxyribonucleic acid (DNA) synthesis occurred. In the so-called "physiological" dose range used, the latter was greater than the former. X-irradiation of cells, at doses producing similar levels of cell death as in the UV-exposed cultures, did not lead to a similar repair replication process. However, a density heterogeneity of the DNA synthesized in the initial 10 min after exposure was observed. This is interpreted in terms of X ray-induced DNA degradation. Normal cells showed only a semiconservative type of replication and, therefore, within the limits of resolution of the system used (the incorporation of 1,000 to 5,000 nucleotides per replicating chromosome could be measured), DNA in normal cells did not appear to undergo a repair synthesis involving thymine exchange. These results indicate that not all repair mechanisms mimic that found after UV exposure.     

The elimination of mutagenic lesions induced by alkylating agents and UV in V79 Chinese hamster cells

The mutagenicity of MNU, EMS, BMS and UV light was compared by analyzing the dose-response curve just before and after the replicative process of the HGPRT gene in synchronized V79 Chinese hamster cells. This system makes it possible to compare a 10-h period for repair of different mutagenic lesions with no time for repair. Additional time for repair in synchronized V79 cells resulted in a reduced response for MNU and UV, but not for EMS and BMS. This result suggests that an error-free repair process operates on mutagenic lesions in methylated DNA and on thymine dimers, but not on ethylated and butylated DNA. Based on these results, it is concluded that the repair capacity of V79 cells to remove mutagenic lesions is characterized as low for UV, moderate for MNU and not detectable for the mutagenic lesions induced by EMS and BMS.     

Kinetics of unscheduled DNA synthesis in UV-irradiated chicken embryo fibroblasts

Unscheduled DNA synthesis induced by 254-nm UV radiation in chicken embryo fibroblasts was examined for 24 h following irradiation, while cells were kept in the dark. The effect on this repair process of a 2-4 h exposure to photoreactivating light immediately after UV was studied. Initial [3H]thymidine incorporation in the light-treated cells was only slightly different from that in cells not exposed to light, but a distinct difference in rate and cumulative amount of unscheduled DNA synthesis was seen several hours after irradiation. By varying the UV dose and the time allowed for photoreactivation, the amount of dimers (determined as sites sensitive to a M. luteus UV-endonuclease) and non-dimers could be changed. The results of these experiments suggest that excision repair of dimers, rather than non-dimer products, is responsible for the unscheduled DNA synthesis seen after UV irradiation.     

Effect of Thymine Starvation on Deoxyribonucleic Acid Repair Systems of Escherichia coli K-12

Thymine starvation of Escherichia coli K-12 results in greatly increased sensitivity to ultraviolet light (UV). Our studies, using isogenic strains carrying rec and uvr mutations, have shown the following. (i) Common to all strains tested is a change from multihit to single-hit kinetics of survival to UV after 60 min of thymine starvation. However, the limiting slope of UV survival curves decreases in the rec(+)uvr(+) strain and changes very little in several rec mutant strains and one uvrB mutant strain. Thus, when either the rec or uvr system is functioning alone, the limiting slopes of the UV survival curves are relatively unaffected by thymine starvation. (ii) Thymine starvation does not significantly inhibit repair processes carried out by either repair system alone; i.e., host cell reactivation of irradiated phage (carried out by the uvr system), excision of thymine dimers (uvr), or X-ray repair (rec). (iii) In a rec(+)uvr(+) strain, repair appears to be a synergistic rather than additive function of the two systems. However, after thymine starvation, repair capacity is reduced to about the sum of the repair capacities of the independent systems. (iv) The kinetics of thymineless death are not changed by rec and uvr mutations. This indicates that the lesions responsible for thymineless death are not repaired by rec or uvr systems. (v) Withholding thymine from thy rec(+)uvr(+) bacteria not undergoing thymineless death has no effect on UV sensitivity. Under these conditions one sees higher than normal UV resistance in the presence or absence of thymine. This is due to increased repair carried out by the uvr system. To explain these results we postulate that thymine starvation does not inhibit either the rec or uvr repair pathway directly. Rather it appears that thymine starvation results in increased UV sensitivity in part by inhibiting a function which normally carries out efficient coordination of rec and uvr pathways.     

Effect of serotonin on the yield of UV- and x ray-induced DNA damage

Using two-dimensional thin-layer chromatography, the effect of serotonin on the yield of thymine dimers and on cleavage of the N-glycosidic bond in the DNA irradiated with ultraviolet (UV) light and X-ray was studied. Bound serotonin was shown to reduce the synthesis of UV-induced thymine dimers but had no effect on the number of X-ray-induced breaks in the N-glycoside bonds in thymidine residues. The data obtained are discussed in terms of the mechanisms of serotonin involvement in the photoprotection of yeast cells from the lethal action of UV and X-ray irradiations.     

Correlation among the rates of dimer excision, DNA repair replication, and recovery of human cells from potentially lethal damage induced by ultraviolet radiation.

The kinetics of excision repair in confluent cultures of diploid human fibroblasts after ultraviolet irradiation at varying doses was measured by three different methods: (a) removal of thymine-containing dimers, (b) DNA excision repair synthesis, and (c) biological recovery of cells from the potentially lethal effects of the irradiation. Each method gave similar results and indicated that the excision rate was dependent upon the number of thymine-containing dimers induced (substrate concentration). For example, at a dose of 40 J/m2 (0.2% dimerization), the repair rate was 1.6 J/m2 per h as determined by a modified method to measure the number of thymine-containing dimers remaining in DNA and 1.65 J/m2 as measured by excision repair synthesis. At a dose of 7.5 J/m2, the repair rate was 0.5 J/m2 per h as measured by biological recovery, and at a dose of 7 J/m2, the repair rate was 0.46 J/m2 per h as measured by excision repair synthesis.     

Purified scrapie prions resist inactivation by UV irradiation.

The development of effective purification protocols has permitted evaluation of the resistance of isolated scrapie prions to inactivation by UV irradiation at 254 nm. Prions were irradiated on ice with doses of UV light ranging up to 120,000 J/m2. UV dosimetry experiments, performed with Saccharomyces cerevisiae plasmid DNA or eucaryotic cells, indicated that under these experimental conditions an incident UV dose of 10 J/m2 formed 2 thymine dimers per 5.1 X 10(6) daltons of eucaryotic cell DNA. The D37 values for scrapie prions ranged from 17,000 to 22,000 J/m2; D37 values were also determined for virus, viroid, and enzyme controls. The number of pyrimidine dimers formed was correlated with the D37 values obtained for irradiated prions and target nucleic acids. The D37 value for bacteriophage M13, 6.5 J/m2, occurred at a dose that would form 0.56 dimers per target genome; the D37 for potato spindle tuber viroid, 4,800 J/m2, occurred at a dose that would form about 24 dimers per target viroid. The D37 value for an EcoRI restriction site, a target of 12 bases, occurred at a dose that would correspond to the formation of 0.89 thymine dimers per target site. The D37 value for prions occurred at a dose that would form 1 dimer in every 4 bases of single-stranded target nucleic acid. If the putative scrapie nucleic acid were double-stranded and readily repairable after UV damage, then the prion D37 value could reflect a nucleic acid molecule of 30 to 45 base pairs. While the D37 value for prions fell within the range of pure protein targets, our experiments cannot eliminate the possibility that a prion contains a small, highly protected nucleic acid molecule.     

Excision repair of UV-damaged plasmid DNA in Xenopus oocytes is mediated by DNA polymerase alpha (and/or delta).

We studied DNA repair by injecting plasmids containing random pyrimidine dimers into Xenopus oocytes. We demonstrated excision repair by recovering plasmids and analyzing them with T4 UV endonuclease treatment and alkaline agarose gel electrophoresis. The mechanism for excision repair of these plasmids appears to be processive, rather than distributive, since repair occurs in 'all or none' fashion. At less than 4-5 dimers/plasmid, nearly all repair occurs within 4-6 hours (approximately 10(10) dimers repaired per oocyte); the oocyte, therefore, has abundant repair activity. Specific antibodies and inhibitors were used to determine enzymes involved in repair. We conclude that DNA polymerase alpha (and/or delta) is required because repair is inhibited by antibodies to human DNA polymerase alpha, as well as by aphidicolin, an inhibitor of polymerases alpha (and/or delta). Repair was not inhibited by hydroxyurea, cytosine beta-D-arabinofuranoside, or inhibitors of topoisomerase II (novobiocin). Oocyte repair does not activate semi-conservative DNA replication, nor is protein synthesis required. Photoreactivation cannot account for repair because dimer removal is independent of exogenous light.     

Repair rate in human fibroblasts measured by thymine dimer excorporation

Summary The UV photoproduct, thymine dimer ( ), is excorporated with a remarkably low rate from the DNA of human fibroblasts grown in cell culture. An UV dose of 18 J/m² creates 0.045% (related to thymine). Within the first two days of repair logarithmically growing and quiescent fibroblasts exhibit the same repair rates; thereafter, the proportion of is lower in growing cells due to recovery of DNA replication. Only about 50% of the lesions are excised within 24 h. In quiescent cells, 13% of the thymine dimers originally present can be detected as late as a week after UV-irradiation. Two distinct first-order rate constants indicate that approximately half of the dimers are less accessible to repair. Repair measured by the nucleoid decondensation technique corresponds to the faster repair rate, whereas the slow repair rate cannot be detected by this method. Saturation of repair is found beyond 27 J/m². The remarkably slow rate of excision indicates that thymine dimers are not lethal lesions in human fibroblasts.     

The broth effect and mutation frequency decline in cells ofEscherichia coli after irradiation with UV-light

Dependence of the broth effeot and the phenomenon of mutation frequency decline on dose of the applied UV radiation was investigated in the strainEscherichia coli B/r Hcr+ thy trp. Reversions to Trp⁺ were followed. The degree of the broth effect and the mutation frequency decline is minimal within the range of UV doses corresponding to a survival of cells lower than 10-1. In connection with the two effects, excision of thymine dimers, initiation of synthesis, synthesis and degradation of DNA were also investigated. It was found that stimulation or inhibition of an inaccurate postreplication repair mechanism, rather than inhibition or stimulation of excision of thymine dimers, are responsible for the broth effect and the mutation frequency decline, respectively.     

Sensitivity to UV radiation of small nuclear RNA synthesis in mammalian cells.

It was demonstrated previously that the synthesis of small nuclear RNA (snRNA) species U1 and U2 in human cells is very sensitive to UV radiation. In the present work, the UV sensitivity of U3, U4, and U5 snRNA synthesis is shown to be also high. The synthesis of U1, U2, U3, U4, and U5 snRNAs progressively decreased during the first 2 h after UV irradiation (this was not observed in polyadenylated RNA) and had not returned to normal rates 6 h after UV exposure. In contrast, the restoration of 5.8S rRNA synthesis began immediately after UV irradiation and was essentially complete 6 h later. A small fraction of U1 and U5 (and possibly U2 and U3) snRNA synthesis remained unaffected by high UV doses, when cell radiolabeling began 10 min after UV irradiation. The present data suggest that a factor other than the level of pyrimidine dimers in DNA (possibly, steps in the post-irradiation DNA repair process) plays an important role in the mechanism of UV-induced inhibition of U1-U5 snRNA synthesis.     

Repair,replication and survival in UV-irradiatedEscherichia coli

The influence of dimer removal through excision or photoreactivation on the kinetics of DNA synthesis, sedimentation profiles of DNA molecules and survival of cells was investigated in excision-deficient and excision-proficientEscherichia coli K-12 after a flux of 20 J m⁻². In excision-deficient cells photoreactivation did not influence the kinetics of DNA synthesis for a long period and the sedimentation properties of DNA synthesized immediately after photoreactivation were influenced only slightly. However, survival was increased remarkably. In excision-proficient cells where dimers were removed through excision, the kinetics of DNA synthesis increased rapidly, normal-sized DNA molecules were synthesized 60 min after irradiation and survival was substantially higher than in the above-mentioned case. This can hardly be interpreted as a more complete repair of dimers by excision because the persistence of dimers in these cells did not significantly influence either the kinetics of DNA synthesis or normalization of DNA molecules and/or survival of cells. It is concluded that persisting dimers play an important role in excision-deficient but not in excision-proficient cells, that a non-dimer damage to DNA causes inhibition of DNA synthesis after UV and that this damage ia of primary importance for excision-proficient cells which can easily cope with persisting dimers.     

Mechanisms and implications of the age-associated decrease in DNA repair capacity.

Skin cancer incidence is clearly linked to UV irradiation and increases exponentially with age. We studied the rate of removal of thymine dimers and (6-4) photoproducts in UV-irradiated human dermal fibroblasts derived from donors of different ages. There was a significant decrease with aging in the repair rates of both thymine dimers and (6-4) photoproducts (P<0.001). In addition, there was an age-associated decrease in the protein levels of ERCC3, PCNA, RPA, XPA, and p53 that participate in nucleotide excision repair. Moreover, the mRNA levels of XPA, ERCC3, and PCNA were significantly reduced with aging, suggesting that these decreases are often regulated at the mRNA level. Furthermore, with age induction of p53 after UV irradiation was significantly reduced. Taken together, our data suggest that the age-associated decrease in the repair of UV-induced DNA damage results at least in part from decreased levels of proteins that participate in the repair process.