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.     

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.     

NATURE OF THE INTERACTION BETWEEN THE PHOTOACTIVE COMPOUND PHENYLHEPTATRIYNE AND ANIMAL VIRUSES

Abstract— The levels of DNA excision repair, as measured by unscheduled DNA synthesis (UDS) and the UV-endonuclease sensitive site assay, were compared in cells derived from human fetal brain and dermal tissues. The level of UDS induced following ultraviolet (UV) irradiation was found to be lower (approx. 60%) in the fetal brain cells than in fetal dermal cells. It was determined, using the UV-endonuclease sensitive site assay to confirm the UDS observation, that 50% of the dimers induced by UV in fetal dermal cells were repaired in 8h, while only 15% were removed in the fetal brain cells during the same period of time. Even after 24 h, only 44% of the dimers induced by UV in the fetal brain cells were repaired, while 65% were removed in the dermal cells. These data suggest that cultured human fetal brain cells exhibit lower levels of excision repair compared to cultured human fetal dermal cells.     

LETHALITY AND THE INDUCTION AND REPAIR OF DNA DAMAGE IN FAR, MID OR NEAR UV-IRRADIATED HUMAN FIBROBLASTS: COMPARISON OF EFFECTS IN NORMAL, XERODERMA PIGMENTOSUM AND BLOOM'S SYNDROME CELLS

Abstract Using normal human fibroblasts we have determined the ability of far (254 nm), mid (310 nm) or near (365 nm) UV radiation to: (i) induce pyrimidine dimers (detected as UV endonuclease sensitive sites) and DNA single-strand breaks (detected in alkali); (ii) elicit excision repair, monitored as unscheduled DNA synthesis (UDS); and (iii) reduce colony-forming ability. Unscheduled DNA synthesis studies were also performed on dimer excision-defective xeroderma pigmentosum (XP) cells, and the survival studies were extended to include XP and Bloom's syndrome (BS) strains. UV-induced cell killing in normal, BS and XP cells was found to relate to an equivalent dimer load per genome after 254 or 310 nm exposure, whereas at 365 nm the lethal effects of non-dimer damage appeared to predominate. Lethality could not be correlated with DNA strand breakage at any wavelength. The two XP strains examined showed the same relative UDS repair deficiency at the two shorter wavelengths in keeping with a predominant role for pyrimidine dimer repair in the expression of UDS. However, UDS was not detected in 365 nm UV-irradiated normal and XP cells despite dimer induction; this effect was due to the inhibition of DNA repair functions since 365 nm UV-irradiated normal cells showed reduced capacity to perform UDS subsequent to challenge with 254 nm UV radiation.
In short, the near UV component of sunlight apparently induces biologically important non-dimer damage in human cells and inhibits DNA repair processes, two actions which should be considered when assessing the deleterious actions of solar UV.     

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.     

SATURATION OF DNA REPAIR IN MAMMALIAN CELLS*

Abstract—Excision repair seems to reach a plateau in normal human cells at a 254 nm dose near 20J/m². We measured excision repair in normal human fibroblasts up to 80J/m². The four techniques used (unscheduled DNA synthesis, photolysis of BrdUrd incorporated during repair, loss of sites sensitive to a UV endonuclease from Micrococcus luteus , and loss of pyrimidine dimers from DNA) showed little difference between the two doses. Moreover, the loss of endonuclease sites in 24 h following two 20J/m² doses separated by 24 h was similar to the loss observed following one dose. Hence, we concluded that the observed plateau in excision repair is real and does not represent some inhibitory process at high doses but a true saturation of one of the, rate limiting steps in repair.     

REPAIR OF CYCLOBUTANE DIMERS AND (6–4) PHOTOPRODUCTS IN ICR 2A FROG CELLS

Abstract— The removal of cyclobutane dimers and Pyr(6–4)Pyo photoproducts from the DNA of UV-irradiated ICR 2A frog cells was determined by radioimmunoassay. In the absence of photoreactivat-ing light, 15% of the cyclobutane dimers and 60% of the (6–4) photoproducts were removed 24 h post-irradiation with 10 J m⁻², Exposure to 30 kJ m⁻² photoreactivating light resulted in removal of 80% of the cyclobutane dimers and an enhanced rate of repair of (6–4) photoproducts, resulting in a loss of 50% of these lesions in 3 h. The preferential removal of (6–4) photoproducts by excision repair resembles previously published data for mammalian cells.     

Lack of correlation between DNA strand breakage and p53 protein levels in human fibroblast strains exposed to ultraviolet lights

The contribution of DNA strand breaks accumulating in the course of nucleotide excision repair to upregulation of the p53 tumor suppressor protein was investigated in human dermal fibroblast strains after treatment with 254 nm ultraviolet (UV) light. For this purpose, fibroblast cultures were exposed to UV and incubated for 3 h in the presence or absence of l-beta-D-arabinofuranosylcytosine (araC) and/or hydroxyurea (HU), and then assayed for DNA strand breakage and p53 protein levels. As expected from previous studies, incubation of normal and ataxia telangiectasia (AT) fibroblasts with araC and HU after UV irradiation resulted in an accumulation of DNA strand breaks. Such araC/HU-accumulated strand breaks (reflecting nonligated repair-incision events) following UV irradiation were not detected in xeroderma pigmentosum (XP) fibroblast strains belonging to complementation groups A and G. Western blot analysis revealed that normal fibroblasts exhibited little upregulation of p53 (approximately 1.2-fold) when incubated without araC after 5 J/m2 irradiation, but showed significant (three-fold) upregulation of p53 when incubated with araC after irradiation. AraC is known to inhibit nucleotide excision repair at both the damage removal and repair resynthesis steps. Therefore, the potentiation of UV-induced upregulation of p53 evoked by araC in normal cells may be a consequence of either persistent bulky DNA lesions or persistent incision-associated DNA strand breaks. To distinguish between these two possibilities, we determined p53 induction in AT fibroblasts (which do not upregulate p53 in response to DNA strand breakage) and in XP fibroblasts (which do not exhibit incision-associated breaks after UV irradiation). The p53 response after treatment with 5 J/m2 UV and incubation with araC was similar in AT, XPA, XPG and normal fibroblasts. In addition, exposure of XPA and XPG fibroblasts to UV (5, 10 or 20 J/m2) followed by incubation without araC resulted in a strong upregulation of p53. We further demonstrated that HU, an inhibitor of replicative DNA synthesis (but not of nucleotide excision repair), had no significant impact on p53 protein levels in UV irradiated and unirradiated human fibroblasts. We conclude that upregulation of p53 at early times after exposure of diploid human fibroblasts to UV light is triggered by persistent bulky DNA lesions, and that incision-associated DNA strand breaks accumulating in the course of nucleotide excision repair and breaks arising as a result of inhibition of DNA replication contribute little (if anything) to upregulation of p53.     

XERODERMA PIGMENTOSUM COMPLEMENTATION GROUP F: MORE ASSIGNMENTS AND REPAIR CHARACTERISTICS

Abstract— The specific heterodikaryon complementation method enabled us to assign three patients with mild xeroderma pigmentosum (XP) symptoms (XP25KO, XP27KO, XP28KO) to complementation group F. UV-induced unscheduled DNA synthesis (UDS) remained unnormalized in the heterodikaryons between either of the above three XP strains and the reference group F XP3YO. All these particular XP strains as well as XP3YO exhibited an equally low level of10–15% UDS by a 3 h [³H]-thymidine labeling following 10 J/m² 254 nm UV, while they attained 60% UDS of normal at an extended time of 25 h. The present group F strains were 3 and 1.5 times as sensitive to the lethal effect of UV as normal and XP group E cells, respectively, based on the mean lethal dose ( Do ) comparison. Normal cells had the biphasic time-UDS kinetics of early rapid and late slow repair. Characteristically, however, all of the present group F strains were defective in only early rapid repair, but normally proficient in slow repair.     

POSTIRRADIATION PROPERTIES OF A UV-SENSITIVE VARIANT OF CHO

Abstract A UV-hypersensitive mutant of Chinese hamster ovary (CHO) cells, termed 43-3B, has been used in a comparative study with the wild type CHO in order to determine the involvement of repair in several postirradiation phenomena. 43-3B has the same growth rate and chromosome number as the wild type CHO-9. It is hypersensitive to UV irradiation (D₀ of 0.3 J/m² as compared to 3.2 J/m² for the wild type). 43-3B shows only about 17% of the UV-stimulated unscheduled DNA repair synthesis of CHO-9 as measured by autoradiography. When breaks in supercoiled chromatin are measured after UV by the nucleoid sedimentation method, the mutant appears to be capable of carrying out only limited incision. A much reduced ability to recover control rates of semiconservative DNA synthesis after UV irradiation was observed in the repair-deficient 43-3B cell line, suggesting that the removal of UV-induced replication blocks by excision repair is the most important factor in allowing recovery of UV-inhibited DNA synthesis. Recovery of colony-forming ability between fractionated UV exposures was observed in the wild type CHO-9, but little recovery was seen in 43-3B. This indicates that excision repair capability can also be important in split-fluence recovery.     

DNA EXCISION REPAIR IN THE VERY UV-SENSITIVE XERODERMA PIGMENTOSUM COMPLEMENTATION GROUP A STRAIN XP4LO

Abstract— XP4L0, a xeroderma pigmentosum complementation group A strain, exhibits very limited DNA repair activity. It has extreme sensitivity to UV (254 nm) as determined by colony forming ability. The rate of loss of UV (1 J/m²)-induced pyrimidine dimers from populations of quiescent, nondividing XP4LO cells was determined and found to be slower than that observed for other group A strains (XP25R0, XP12BE, XP8LO). The extreme UV-sensitivity is also exhibited by the nondividing cells in a survival assay that employs nondividing cell populations and does not involve cell reproduction. This result suggests that the extreme sensitivity measured previously by colony-forming ability (a cell-reproduction assay) is due to the excision repair defect alone and not to an additional post-replication repair defect. The very limited excision allows for an accurate definition of target size for inactivation of nondividing cells, about 1 pyrimidine dimer per 10⁵ base pairs, and when compared to results observed for other XP-A strains, provides further evidence that even though excision repair in group A is severely limited, it has biological significance.     

UV-induced unscheduled DNA synthesis in human skin: dose response, correlation with erythema, time course and split dose exposure in vivo

Unscheduled DNA synthesis (UDS) has been shown to be saturated above a threshold dose of UV-C in human fibroblasts in vitro. We have investigated by autoradiography whether a similar saturation occurs in human skin in vivo with UV-B and whether this phenomenon correlates with the erythemal response. In addition, we determined the time course of UDS at 24 h after exposure and the effect of dual exposures separated by 24 h. The dose-response curve was established by exposure to 1/16, 1/8, 1/4, 1/2, 1, 2, 3, 4 and 6 MEDs UV-B. For the time-course study, areas exposed to 1/2 and 2 MEDs were biopsied after 1, 3, 6, 12 and 24 h. Autoradiography was performed in vitro. The dose-response curve showed a significant increase in UDS from 1/16 to 1 minimal erythema dose (MED), whereas no significant difference was observed between 1 MED and the higher UV-B doses tested. The 24 h time sequence revealed a gradual decrease in UDS activity. The 1/2 MED curve declined more rapidly and reached the zero-level between 12 h and 24 h, whereas about 50% of the initial UDS value was still retained 24 h after 2 MEDs. The dual-dose study revealed that a second hit of fractions of the MED resulted in lower levels of UDS than induced by these fractions alone in previously untreated areas. UDS increases with the erythemal dose between 1/16 and 1 MED. It reaches a plateau after 1 MED and cannot be increased by doses up to 6 MEDs, suggesting a saturation of excision repair in vivo. Time course studies support such a saturation phenomenon. The failure to increase significantly UDS by a second irradiation 24 h after the first exposure needs further clarification. Since persistence of DNA lesions may lead to an accumulation after repeated exposures, additional mechanisms other than excision repair may protect human skin by error-free removal of possibly mutagenic sites. Photoreactivation may be important in this respect.     

PYRIMIDINE DIMER INDUCTION AND REMOVAL IN THE EPIDERMIS OF HAIRLESS MICE: INEFFICIENT REPAIR IN THE GENOME OVERALL AND RAPID REPAIR IN THE H-ras SEQUENCE

Excision repair of pyrimidine dimers was examined at the genome overall in three strains of hairless ( hr/hr ) and congenic wild-type mice, as well as in the expressed H- ras gene in hairless mice. The assay used a pyrimidine dimer-specific endonuclease from Micrococcus luteus and alkaline agarose gel electrophoresis. From 0 to 25% of endonuclease-sensitive sites were removed at the genome level in either hairy or hairless mice but about 50% were removed in the H- ras gene in hairless mice by 24 h after exposure to 5.4 J/cm² UV (290-400 nm) irradiation. No differences were observed in the repair capacity between hairy and hairless mice, thus eliminating defective DNA repair as the explanation for the greater susceptibility to UV carcinogenesis in hairless mice.     

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 ).     

EXCISION OF CYCLOBUTANE DIMERS IN GENOMIC AND EPISOMAL DNA IN HUMAN CELLS

Abstract Direct determination has been made of cyclobutyl pyrimidine dimer induction and excision repair in an episomal SV40 DNA population in vivo . Maintaining SV40-transformed human (GM637) cells in confluent culture results in amplification of a mutant SV40 episome to high copy number. T4 endonuclease V was used to quantify the induction and repair of cyclobutane dimers in the SV40 episome and genomic DNA of the same cells. Differences in both parameters were observed cyclobutane dimers were induced at 1.5–2-fold greater frequency in episomal DNA and excised at a reduced rate compared to genomic DNA in the host cells.     

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.     

EFFECTS OF DOSE FRACTIONATION ON ULTRAVIOLET SURVIVAL OF ESCHERICHIA COLI

Abstract— Exposure of E. coli B/r and B at low average dose rates of u.v. radiation (2537 Å), produced either by fractionated doses or by continuous irradiation at a very low dose rate (80 ergs/mm²/hr), results in much increased survival compared to single exposure at high dose rate. This increase is attributed to repair taking place during the irradiation period. The effect is small in the repair-deficient strains E. coli B_8-1_, and C syn- , and is absent in phage T1 and T4, which cannot undergo repair in the extracellular state. However, the prolonged time available for repair in these experiments accounts for only a very minor part of the increase in survival. The principal factor apparently is that the number of lesions present at any time remains relatively low. Presumably complete repair, not only the excision step, can occur in buffer during the irradiation period. This interpretation is supported by experiments in which cells were exposed to combinations of highly fractionated irradiation and single-dose irradiation. We therefore propose that mutual interference in repair, possibly by overlapping of repair regions in complementary DNA strands, reduces considerably the repair efficiency if many lesions are present. This hypothesis explains the 'shouldered' survival curves of B/r and possibly other E. coli strains as due to decreasing repair efficiency with increasing u.v. dose     

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.     

Nucleotide excision repair proteins rapidly accumulate but fail to persist in human XP-E (DDB2 mutant) cells

The xeroderma pigmentosum (XP-E) DNA damage binding protein (DDB2) is involved in early recognition of global genome DNA damage during DNA nucleotide excision repair (NER). We found that skin fibroblasts from four newly reported XP-E patients with numerous skin cancers and DDB2 mutations had slow repair of 6-4 photoproducts (6-4PP) and markedly reduced repair of cyclobutane pyrimidine dimers (CPD). NER proteins (XPC, XPB, XPG, XPA and XPF) colocalized to CPD and 6-4PP positive regions immediately (<0.1 h) after localized UV irradiation in cells from the XP-E patients and normal controls. While these proteins persist in normal cells, surprisingly, within 0.5 h these repair proteins were no longer detectable at the sites of DNA damage in XP-E cells. Our results indicate that DDB2 is not required for the rapid recruitment of NER proteins to sites of UV photoproducts or for partial repair of 6-4PP but is essential for normal persistence of these proteins for CPD photoproduct removal.