The use of metabolic inhibitors to compare the excision repair of pyrimidine dimers and nondimer DNA damages in human skin fibroblasts exposed to 254-nm and sunlamp-produced greater than 310-nm ultraviolet radiation

Normal human skin fibroblasts were exposed to either 0-5 J/m2 of 254-nm ultraviolet (UV) radiation or 0-50 kJ/m2 of the Mylar-filtered UV (greater than 310 nm) produced by a fluorescent sunlamp. These cells were then incubated for 0-20 min in medium containing 10 mM hydroxyurea (HU) and 0.1 mM 1-beta-D-arabinofuranosyl cytosine (ara C), and the yield of DNA strand breaks was measured by means of the alkaline elution technique. For cells irradiated with 254-nm UV, which results primarily in the formation of cyclobutane pyrimidine dimers, a rapid increase in DNA strand breaks was detected following incubation with these metabolic inhibitors. In contrast, only a low level of strand breaks formed in cells incubated with HU and ara C after irradiation with approximately equitoxic fluences of sunlamp UV greater than 310 nm, which mainly causes the induction of nondimer DNA lesions. Hence, these results are consistent with the conclusion that the pathways involved in the repair of nondimer DNA damages induced by UV wavelengths greater than 310 nm differ from the repair of pyrimidine dimers.     

Isolation of a mutant cell line derived from ICR 2A frog cells hypersensitive to the induction of nondimer DNA damage by solar ultraviolet radiation

A mutant cell line DRP 36, hypersensitive to nondimer DNA damage induced by exposure of cells to the Mylar-filtered solar ultraviolet (UV) radiation produced by a fluorescent sunlamp plus photoreactivating light (PRL) was isolated from the haploid ICR 2A frog cell line. The D₀ for mutant cells exposed to this solar UV source was 3.3 kJ/m² compared with a D₀ of 7.3 kJ/m² for the parental ICR 2A cells. In contrast, DRP 36 and ICR 2A cells exhibited similar levels of survival following 254-nm irradiation which causes the induction primarily of pyrimidine dimers. The cross-sensitivity to additional DNA damaging agents was examined, and it was determined that the DRP 36 cells are also hypersensitive to treatment with -rays, ethyl methane sulfonate (EMS), cis-dichlorodiammine platinum (II) (DDP), and 4-nitroquinoline oxide (4-NQO) while exhibiting normal sensitivity tol-phenylalanine mustard (L-PAM), 1,3-bis-(2-chloroethyl)-1-nitrosourea (BCNU) and mitomycin C (MMC).     

DNA repair by articular chondrocytes. II. Direct measurements of repair of ultraviolet and X-ray damage in monolayer cultures

The abilities of human and rabbit articular chondrocytes to repair ultraviolet (UV) and X-ray damage were measured in terms of the removal of UV—endonuclease-sensitive sites (pyrimidine dimers) and single-strand breaks, respectively. The initial 3-h rate of dimer repair in human cells, incubated in medium containing 10% human serum, was about 2.5 times as large as in rabbit cells incubated in medium containing 10% or 20% fetal bovine serum. Similar results have been previously reported for unscheduled DNA synthesis (UDS), indicating that UDS is a valid quantitative measure of repair in this cell system. The repair of single-strand breaks was rapid (approx. 50% completed in <10 min). An estimate, from the measured numbers of lesions and patch sizes, indicated that the amount of UDS following 20 krad would be 100 to 300 times less than that in 3 h following to J/m² of 254 nm and hence would not be detectable radioautographically.     

DNA damage,photorepair, and survival in fish and human cells exposed to UV radiation

The effect of various wavelengths of UVB radiation on the induction of cyclobutane pyrimidine dimers in fish cells and human fibroblasts and the repair of these lesions were studied using an UV-endonuclease to measure dimers (endonuclease sensitive sites) by sedimentation of radioactive DNA, by gel electrophoresis of unlabeled DNA, and by cell survival. The data show that fish cells have an efficient photoreactivation system at wavelength > 304 nm that reverses cytotoxicity and dimer formation after exposure to filtered sunlamp irradiation of a shorter wave-length (λ > 290 nm). Shorter wavelengths in UVB (<304 nm) are more effective in photoreversal than longer ones (<320 nm). As a consequence, 50–85% of dimers induced by these wavelengths in fish are photoreactivated while they are being formed. A major cytotoxicological lesion is the cyclobutane pyrimidine dimers. Cultured human fibroblasts do not possess such a repair system. These results indicate that sun-lamp irradiation has wavelengths that both damage and repair DNA.     

DNA-protein crosslinking in normal and solar UV-sensitive ICR 2A frog cell lines exposed to solar UV-radiation

DNA-protein crosslinks (DPC) were measured following exposure to the solar UV wavelengths produced by a fluorescent sunlamp in ICR 2A frog cells and two solar UV-sensitive mutants derived from this cell line. Approx. 5–7 DPC per 10¹⁰ dalton were induced in these cells by either 150 kJ/m² of sunlamp UV > 315 nm plus photoreactivating light (PRL) or 10 kJ/m² of sunlamp UV > 295 nm. The irradiated cells were then incubated for 0–24 h and the level of DPC measured using alkaline elution. It was found for the ICR 2A cells exposed to sunlamp UV > 315 nm that the level of DPC increased about 3-fold during a 2-h postirradiation incubation and then decreased. The mutant cell lines also showed an enhancement in the level of DPC following irradiation, although it was much less pronounced and the levels decreased much more rapidly. In a similar fashion, the level of DPC increased in ICR 2A cells exposed to sunlamp UV > 295 nm with more than a 5-fold enhancement after a 4-h incubation. Once again, the mutant cell lines showed an increase in the level of DPC that was smaller and more transient than the effect in the ICR 2A cells. These results suggests that this enhancement in DPC may be indicative of a process that plays a role in cellular survival following solar UV-irradiation.     

Effect of arsenite on the DNA repair of UV-irradiated Chinese hamster ovary cells.

Arsenite, an ubiquitous human carcinogen, has been shown to enhance the cytotoxicity, mutagenicity and clastogenicity of UV light in mammalian cells. Arsenite may exert its co-genotoxic effects by inhibiting DNA repair. Results from alkaline sucrose gradient sedimentation show that arsenite did not accumulate UV-induced DNA strand breaks in Chinese hamster ovary (CHO) K1 cells as aphidicolin plus hydroxyurea (HU) did. These data indicate that arsenite did not inhibit the activity of DNA polymerase alpha in UV repair. Treatment with arsenite before UV irradiation slightly reduced the DNA strand breaks accumulated by cytosine beta-D-arabinofuranoside (AraC) plus HU. This effect implies that arsenite only slightly inhibited the incision of UV-induced DNA adducts. The low molecular weight DNA accumulated by post-UV incubation with AraC plus HU shifted to high molecular weight upon the incubation of cells in drug-free medium, but this shifting was prohibited by the presence of arsenite. This suggests that arsenite inhibited the rejoining of DNA strand breaks. When a pulse-chase labelling procedure was applied on UV-irradiated cells, the chain elongation of nascent DNA was strongly inhibited by post-incubation with arsenite. These data show that arsenite inhibited post-replication repair in UV-irradiated cells. Therefore, the steps inhibited by arsenite in UV-induced DNA repair in CHO K1 cells are different from human fibroblasts in which the inhibition of excision of pyrimidine dimers by arsenite was reported to be the major target.     

Differential effects of luminol,nickel, and arsenite on the rejoining of ultraviolet light and alkylation-induced DNA breaks

When Chinese hamster ovary cells were treated with ultraviolet (UV) light or methyl methanesulfonate (MMS), a large number of DNA strand breaks could be detected by alkaline elution. These strand breaks gradually disappeared if the treated cells were allowed to recover in a drug-free medium. The presence of nickel or arsenite during the recovery incubation retarded the disappearance of UV-induced strand breaks, whereas the disappearance of MMS-induced strand breaks was retarded by the presence of arsenite or of luminol, a new inhibitor for poly(ADP-ribose) synthetase. Luminol, however, had no apparent effect on the repair of UV-induced DNA strand breaks, and nickel had no effect on the repair of MMS-induced DNA strand breaks. When UV- or MMS-treated cells were incubated in cytosine arabinofuranoside (AraC) plus hydroxyurea (HU), a large amount of low molecular weight DNA was detected by alkaline sucrose sedimentation. The molecular weight of these DNAs increased if the cells were further incubated in a drug-free medium. This rejoining of breaks in cells pretreated with UV plus AraC and HU was inhibited by nickel and by arsenite, but not by luminol. The rejoining of breaks in cells pretreated with MMS plus AraC and HU was inhibited by luminol and by arsenite, but not by nickel. These results suggest that different enzymes may be used in DNA resynthesis and/or ligation during the repairing of UV- and MMS-induced DNA strand breaks, and that nickel, luminol, and arsenite may have differential inhibitory effects on these enzymes. © 1994 Wiley-Liss, Inc.     

Biochemical and cytogenetical characterization of Chinese hamster ovary X-ray-sensitive mutant cells xrs 5 and xrs 6 VI. The correlation between UV-induced DNA lesions and chromosomal aberrations,and their modulations with inhibitors of DNA repair synthesis

The role of UV-induced DNA lesions and their repair in the formation of chromosomal aberrations in the xrs mutant cell lines xrs 5 and xrs 6 and their wild-type counterpart, CHO-K1 cells, were studied.The extent of induction of DNA single-strand breaks (SSBs) and DNA double-strand breaks (DSBs) due to UV irradiation in the presence or absence of 1-β-d-arabinofuranosylcytosine (ara-C) and hydroxyurea (HU) was determined using the alkaline and neutral elution methods. Results of these experiments were compared with the frequencies of induced chromosomal aberrations in UV-irradiated G₁ cells treated under similar conditions.Xrs 6 cells showed a defect in their ability to perform the incision step of nucleotide repair after UV irradiation. Accumulation of breaks 2 h after UV irradiation in xrs 6 cells in the presence of HU and ara-C remained at the level of incision breaks estimated after 20 min, which was about 35% of that found in wild-type CHO-K1 cells. In UV-irradiated CHO-K1 and xrs 5 cells, more incision breaks were present after 2 h compared with 20 min post-treatment with ara-C, a further increase was evident when HU was added to the combined treatment. The level of incision breaks induced under these conditions in xrs 5 was about 80% of that observed in CHO-K1 cells. UV irradiation itself did not induce any detectable DNA strand breaks. Accumulation of SSBs in UV-irradiated cells post-treated with ara-C and HU coincides with the increase in the frequency of chromosomal aberrations. These data suggest that accumulated SSBs when converted to DSBs in G₁ give rise to chromosome-type aberrations, whereas strand breaks persisting until S-phase result in chromatid-type aberrations.Xrs 6 appeared to be the first ionizing-radiation-sensitive mutant with a partial defect in the incision step of DNA repair of UV-induced damage.     

Preferential excision repair and non-preferential photoreactivation of pyrimidine dimers in the c-ras sequence of cultured goldfish cells

The time courses of excision repair and photoreactivation of pyrimidine dimers induced by 254-nm UV were examined in the genome overall and in the c-ras sequence of RBCF-1 cells derived from a goldfish, by the use of UV endonuclease of Micrococcus luteus and alkaline agarose gel electrophoresis. Excision repair was more efficient in the ras sequence than in the genome overall, whereas no differences in efficiency of photoreactivation were detected. These results suggest that excision repair is affected by the accessibility of chromatin, while photoreactivation is not.     

Transient appearance of photolyase-induced break-sensitive sites in the DNA ultraviolet light-irradiated Syrian hamster feta cells

Syrian hamster fetal fibroblasts (HFC) were examined for photolyase-induced break-sensitive sites after ultraviolet light (UV) exposure and growth. These sites, observed in excision-defective human xeroderma pigmentation (XP) cells, are due to cleavage of the internal phosphdiester bond of UV-induced pyrimidine dimers. Excision-inefficient HFC required photolyase-induced break-sensitive sites during incubation after UV (10 J/m²). However, these were observed transiently, with a maximum of 5% of the pyrimidine dimers at 9 h post UV; by 18 h they were undetectable. Caffeine (1 mM) delayed the peak of photolyase-induced break-sensitive sites by 2 h. In human XP cells photolyase-induced break-sensitive sites accumulate to a plateau level of about 20% of the pyrimidine dimers. The present results extend to rodent cells the observation that cleavage of the internal phosphodiester bond of pyrimidine dimers may be an early in their excision repair. Furthermore, the data suggest that photolyase-induced break-sensitive sites might be necessary for replication bypass at pyrimide dimers.     

UV endonuclease-mediated enhancement of UV survival in Micrococcus luteus: evidence revealed by deficiency in the Uvr homolog

Unlike its phage T4 counterpart (also known as endonuclease V), Micrococcus luteus UV endonuclease (pyrimidine dimer DNA glycosylase/apurinic-apyrimidinic endonuclease) has suffered from lack of genetic evidence to implicate it in the promotion of UV survival of the cell, i.e., mutants with its deficiency are no more UV-sensitive than the wild type. On the assumption that the contribution of UV endonuclease is obscured by the presence of a homolog of Escherichia coli UvrABC endonuclease, which has recently been identified in this bacterium, survival studies were carried out in its absence. With 254-nm UV irradiation, which generates not only pyrimidine dimers but also 6-4 photoproducts as lethal lesions, a double mutant defective in both UV endonuclease and the Uvr homolog was shown to be more sensitive than a single mutant defective only in the latter, with a dose reduction factor of approximately 2 at the survival level of 37%. Furthermore, molecular photosensitization, which produces only pyrimidine dimers, revealed an even greater difference in sensitivity, the dose reduction factor being about 3.4. These results indicate that the contribution to cell survival of UV endonuclease, an enzyme specific for pyrimidine dimers, is manifest if the backup by the Uvr homolog is absent.     

Paracetamol inhibits UV-induced DNA repair in resting human mononuclear blood cells in vitro

The effects of paracetamol on the repair of DNA damage in restinghuman peripheral mononuclear blood cells (MNC) in vitro wereinvestigated by means of the alkaline elution technique. Lowdoses of UV light (254 nm, 3 J/m²) caused a transient increasein the amount of DNA singlestrand breaks and alkali-labile sites(SSBs). Paracetamol (0.1–1.0 mM) present during post-irradiationincubation approximately doubled the maximum level of UV-induced(1–3 J/m²) SSBs and delayed the completion of repair.Although there were considerable variations between cells preparedfrom different donors, the level of UV-induced DNA SSBs wasalways higher with paracetamol. Hydroxyurea (0.3 mM), an inhibitorof ribonucleotide reductase, caused a similar increased accumulationand slow removal of SSBs, whereas cytosine-l-ß-D-arabinofuranoside(Ara C) (10 µM), an inhibitor of DNA polymerases, ledto a steady accumulation of DNA SSBs. The increased levels ofSSBs caused by paracetamol or hydroxyurea were both completelysuppressed by concomitant addition of deoxyribonucleosides;this supports the notion that paracetamol as well as hydroxyureainhibits ribonucleotide reductase. About the same rates of formationand removal of UV-induced SSBs were observed in T lymphocytes,B lymphocytes and monocytes. In both isolated T lymphocytesand B lymphocytes, paracetamol (0.3 mM) markedly increased thelevel of DNA SSBs induced by UV, whereas monocytes seemed tobe less sensitive to the effect of paracetamol. It is concludedthat the inhibition of DNA repair may contribute to the clastogeniceffects of paracetamol.     

Excision repair characteristics of denV-transformed xeroderma pigmentosum cells

Introduction of the denV gene if phage T4, encoding the pyrimidine dimer-specific endonuclease V, into xeroderma pigmentosum cells XP12RO(M1) was reported to result in partial restoration of colony-forming ability and excision repair synthesis. We have further characterized 3 denV-transformed XP clones in terms of rates of excision of pyrimidine dimers and size of the resulting resynthesized regions following exposure to 100 J/M² from an FS-40 sunlamp. In the denV-transformed XP cells were observed 50% dimer removal within 3–6 h after UV exposure as compared to no measurable removal in the XP12RO(M1) line and 50% dimer excision after 18 h in the GM637A human, control cells. Dimer removal was assayed with Micrococcus luteus UV-endonucleasse in conjunction with sedimentation of treated DNA in alkaline sucrose gradients. The size of the resulting repaired regions was determined by the bromouracil photolysis technique. Based on the photolytic sensitivity of DNA repaired in the presence of bromodeoxyuridine, we calculated that the excision of a dimer in the GM637A cells to be accompanied by the resynthesis of a region ∼ 95 nucleotides in length. Conversely, the resynthesized regions in the denV-transformed clones were considerably smaller and were estimated to be between 13 and 18 nucleotides in length. These results may indicate that either the endonuclease that initiated dimer repair dictated the size of the resynthesized region or that the long-patch repair observed in the normal cells resulted from the repair of non-dimer DNA lesions.     

Repair of 2 um Plasmid DNA in Saccharomyces cerevisiae

Summary We have developed a system for assaying pyrimidine dimers in the 2 m DNA plasmid of Saccharomyces cerevisiae, using Micrococcus luteus UV endonuclease to nick dimer-containing plasmid molecules and measuring percentages of nicked and covalently closed circles on agarose gels. UV-irradiation induced dimers in plasmid DNA, in vivo, at the same rate as in chromosomal DNA. After a dose of 20 Joules·m^–2, approximately 86% of plasmid molecules had. at least one dimer. After 3 h incubation under normal growth conditions only 4% still retained dimers in a wild-type strain. In a rad1 (excision-defective) mutant 81% of plasmid molecules still had dimers after 3 h, suggesting that excision repair operates to remove dimers from plasmid DNA in wild-type yeast. Dimers can be removed from 2 ,um DNA in a rad1 mutant by photoreactivation.     

Hypersensitivity to mutation and sister-chromatid-exchange induction in CHO cell mutants defective in incising DNA containing UV lesions

Five UV-sensitive mutant strains of CHO cells representing different genetic complementation groups were analyzed for their ability to perform the incision step of nucleotide excision repair after UV exposure. The assay utilized inhibitors of DNA synthesis to accumulate the short-lived strand breaks resulting from repair incisions. After 6 J/m², each of the mutants showed <10% of the incision rate of the parental AA8 cells. After 50 J/m², the rate in AA8 was similar to that at 6 J/m², but the rates in the mutants were significantly higher (20% of the rate of AA8). Thus by this incision assay the mutants were phenotypically indistinguishable. Each of the mutants were hypersensitive to mutation induction at both thehprt andaprt loci by a factor of 10, and in the one strain tested ouabain resistance was induced sevenfold more efficiently than in AA8 cells. Sister chromatid exchange was also induced with sevenfold increased efficiency in the two mutant strains examined. Thus, these CHO mutants resemble xeroderma pigmentosum cells in terms of their incision defects and their hypersensitivity to DNA damage by UV.     

The kinetics of repair of oxidative DNA damage (strand breaks and oxidised pyrimidines) in human cells

Single cell gel electrophoresis is a sensitive method for detecting DNA strand breaks. Cells embedded in agarose are converted to nucleoids by treating with detergent and high salt. DNA breaks render the nucleoid DNA susceptible to extension by electrophoresis, forming ‘comets’. We find that when DNA breakage resulting from H₂O₂ treatment is examined, freshly isolated normal human lymphocytes are relatively resistant compared with transformed human cells. When incubated after treatment with H₂O₂, HeLa cells repair most strand breaks within 1 h, and a substantial fraction of the oxidised pyrimidines (detected by converting them to DNA breaks with endonuclease III) within 4 h. However, lymphocytes are less proficient at repair; during incubation for 4 h after treatment with H₂O₂, no detectable removal of endonuclease III-sensitive sites is seen. While the addition of deoxyribonucleosides promotes completion of repair of UV damage by lymphocytes, it has no significant effect on repair of oxidative damage.     

(6-4) Photoproducts and not cyclobutane pyrimidine dimers are the main UV-induced mutagenic lesions in Chinese hamster cells

A partial revertant (RH1–26) of the UV-sensitive Chinese hamster V79 cell mutant V-H1 (complementation group 2) was isolated and characterized. It was used to analyze the mutagenic potency of the 2 major UV-induced lesions, cyclobutane pyrimidine dimers and (6-4) photoproducts. Both V-H1 and RH1–26 did not repair pyrimidine dimers measured in the genome overall as well as in the active hprt gene. Repair of (6-4) photoproducts from the genome overall was slower in V-H1 than in wild-type V79 cells, but was restored to normal in RH1–26. Although V-H1 cells have a 7-fold enhanced mutagenicity, RH1–26 cells, despite the absence of pyrimidine dimer repair, have a slightly lower level of UV-induced mutagenesis than observed in wild-type V79 cells. The molecular nature of hprt mutations and the DNA-strand specificity were similar in V79 and RH1–26 cells but different from that of V-H1 cells. Since in RH1–26 as well as in V79 cells most hprt mutations were induced by lesions in the non-transcribed DNA strand, in contrast to the transcribed DNA strand in V-H1, the observed mutation-strand bias suggests that normally (6-4) photoproducts are preferentially repaired in the transcribed DNA strand. The dramatic influence of the impaired (6-4) photoproduct repair in V-H1 on UV-induced mutability and the molecular nature of hprt mutations indicate that the (6-4) photoproduct is the main UV-induced mutagenic lesion.     

UV-sensitive rodent mutant cell lines of complementation groups 6 and 8 differ phenotypically from their human counterparts

Rodent UV-sensitive mutant cell lines of complementation groups 6 and 8 are the genetic counterparts of human Cockayne syndrome CS-B and CS-A, respectively. The original mutant in this group, UV61, was described as defective in cyclobutane pyrimidine dimer removal after high doses of UV. We have examined the responses of several cell lines from group 6 to low doses of UV irradiation, and find that these mutants have wild-type capacity for DNA repair as indicated by incision, cyclobutane pyrimidine dimer, and (6-4) photoproduct removal. ERCC6, the product of the gene defective in CS-B and group 6 mutants, is implicated in the regulation of repair of actively transcribed genes in Cockaynesyndrome; however, this protein clearly is not required for the processing of low levels of damage in CHO cells, which occurs remarkably efficiently, 40–50% of dimers being removed in both wild-type and group 6 mutants in 5 hours following 0.1 Jm^-2 of UV. The group 8 mutant cell line US31, on the other hand, is very deficient in repair of UV damage, showing a more extreme phenotype than is seen in the corresponding human syndrome CS-A. In both complementation groups, expression of mutations in a gene involved in regulation of DNA repair takes very different forms in human and rodent cells. Environ. Mol. Mutagen. 29:152–160, 1997. © 1997 Wiley-Liss, Inc.     

Effects of adenine arabinoside and coformycin on the kinetics of G2 chromatid aberrations in X-irradiated human lymphocytes.

The kinetics of chromatid aberrations have been studied in human lymphocytes exposed to X-rays in the G2 phase of the cell cycle and incubated with or without the nucleoside analogue 9-beta-D-arabinofuranosyladenine (ara A), known to inhibit the repair of DNA double-strand breaks. In the absence of ara A an exponential decrease in frequencies of chromatid breaks occurred which we interpret as repair. Few breaks were observed if samples were harvested immediately following irradiation. The frequency of chromatid breaks at 1 h after X-irradiation (442 per 100 cells/Gy) was similar to that previously observed in Chinese hamster ovary (CHO) K1 cells. However, the exponential decrease of chromatid breaks between X-irradiation and sampling occurred with a t1/2 of 0.87 h, a faster rate than we have previously observed in CHO K1 cells and was not inhibited by 200 microM ara A alone, in contrast with our previous findings in a human fibroblast line. However, in the presence of the ADA inhibitor coformycin, inhibition of break repair was already observed at an ara A concentration of 100 microM indicating that the apparent unresponsiveness to ara A of lymphocyte chromatid break rejoining results from the deamination of this nucleoside analogue. This deamination effect was confirmed by measurements of DNA synthesis which showed stable inhibition of synthesis by ara A only when coformycin was present. Frequencies of chromatid exchanges in irradiated cells remained constant except at the sampling time directly after irradiation, consistent with the view that chromosomal radiosensitivity remained constant throughout the G2 phase, except for the period immediately prior to mitosis.(ABSTRACT TRUNCATED AT 250 WORDS)