Fluorometric analysis of DNA unwinding (FADU) as a method for detecting repair-induced DNA strand breaks in UV-irradiated mammalian cells

Fluorometric analysis of DNA unwinding (FADU assay) was originally designed to detect X-ray-induced DNA damage in repair-proficient and repair-deficient mammalian cell lines. The method was modified and applied to detect DNA strand breaks in Chinese hamster ovary (CHO) cells exposed to ionizing radiation as well as to UV light. Exposed cells were allowed to repair damaged DNA by incubation for up to 1 h after exposure under standard growth conditions in the presence and in the absence of the DNA synthesis inhibitor aphidicolin. Thereafter, cell lysates were mixed with 0.15 M sodium hydroxide, and DNA unwinding took place at pH 12.1 for 30 min at 20 degrees C. The amount of DNA remaining double-stranded after alkaline reaction was detected by binding to the Hoechst 33258 dye (bisbenzimide) and measuring the fluorescence. After exposure to X-rays DNA strand breaks were observed in all cell lines immediately after exposure with subsequent restitution of high molecular weight DNA during postexposure incubation. In contrast, after UV exposure delayed production of DNA strand break was observed only in cell lines proficient for nucleotide excision repair of DNA photoproducts. Here strand break production was enhanced when the polymerization step was inhibited by adding the repair inhibitor aphidicolin during repair incubation. These results demonstrate that the FADU approach is suitable to distinguish between different DNA lesions (strand breaks versus base alterations) preferentially induced by different environmental radiations (X-rays versus UV) and to distinguish between the different biochemical processes during damage repair (incision versus polymerization and ligation).     

Only complete rejoining of DNA strand breaks after UVC allows K562 cell proliferation and DMSO induction of erythropoiesis

DNA strand breaks are early intermediates of the repair of UVC-induced DNA damage, however, since they severely impair cellular activities, their presence should be limited in time. In this study, the effects of incomplete repair of UVC-induced DNA strand breaks are investigated on K562 cell growth and the induction of erythroid differentiation by addition of DMSO to the cell culture medium. The kinetics were followed after UV irradiation by single cell gel electrophoresis, and in total cell population by alkaline or neutral agarose gel electrophoresis. Shortly after exposure, an extensive fragmentation occurred in DNA; DNA double strand breaks were negatively correlated with recovery time for DNA integrity. DNA damage induced by UVC 9J/m2 rapidly triggered necrosis in a large fraction of irradiated K562 cells, and only 40% of treated cells resumed growth at a very low rate within 24h of culture. The addition of DMSO to the culture medium of cells 15min after UVC, when DNA strand break repair was not yet complete, produced apoptosis in >70% of surviving cells, as determined by TUNEL assay. Conversely, if DMSO was added when the resealing of DNA strand breaks was complete, surviving K562 cells retained full growth capacity, and their progeny underwent erythroid differentiation with normal levels of erythroid proteins, delta-aminolevulinic acid dehydrase and hemoglobin. This study shows that the extent of DNA strand break repair influences cell proliferation and the DMSO induced erythroid program, and the same UVC dose can have opposite effects depending on cellular status.     

Energy deposition mechanisms and biochemical aspects of DNA strand breaks by ionizing radiation

A theoretical model based on physical, chemical, and biochemical mechanisms has been presented to evaluate the yields of DNA strand breaks (single and double) as a function of linear energy transfer (LET ) or ?dE/dx. Energetic heavy charged particles are considered explicitly to provide a general theory for low- as well as for high-LET radiation. There are three main features of the calculation: (a) track structure considerations for the energy deposition pattern, (b) three-dimensional structure of DNA molecules to provide information on the exact location of damage, and (c) a Monte-Carlo scheme to simulate the diffusion processes of water radicals. To avoid the complexities of a cellular medium, an aqueous solution of DNA is considered in the calculation. When the results of the calculations are compared with experimental measurements of the yields of strand breaks in mammalian DNA (exposed in a cellular complex), reasonable agreement is obtained. However, only those experimental data have been compared where there were no enzyme repair processes. The method of calculation has also been extended to study breaks in higher-order structures of DNA molecules such as chromatin. Specific limitations of the present model have been pointed out for making further improvements.     

UV-mediated DNA strand breaks in corneal epithelial cells assessed using the comet assay procedure

Ultraviolet (UV)-mediated DNA damage in various tissues has been well documented. However, research on the damaging effect of UV irradiation on the DNA of corneal epithelium is scarce, even though this is of interest because the cornea is directly exposed to damaging solar (UV) radiation. In this study, we developed a corneal epithelium Comet assay model to assess the background DNA damage (as strand breaks) in cells retrieved from different layers of the porcine corneal epithelium, and to investigate the effect of UV irradiation on DNA damage in corneal epithelial cells. Results show that the background DNA strand breaks decreased significantly (P < 0.001) toward deeper layers of the epithelium. Exposure to the same intensity (0.216 J/cm2) of UVA, UVB and UVC caused a significant (P < 0.001) increase in DNA strand breaks of deeper-layer cells: mean +/- SD %DNA scores (10 gels per treatment, with 100 irradiated cells scored per gel) were 10.2% +/- 1.4% for UVA, 27.4% +/- 4.6% for UVB, and 14.7% +/- 1.8% for UVC compared with 4.2% +/- 0.5% for controls (ambient room light). This study has shown for the first time that the Comet assay for DNA strand breaks can be used successfully with corneal epithelial cells. This report will support future studies investigating environmental influences on corneal health and the assessment of possible protective strategies, and in applying DNA lesion-specific versions of the Comet assay in this corneal epithelial cell model.     

Effect of hydration on the induction of strand breaks and base lesions in plasmid DNA films by gamma-radiation

The yields of gamma-radiation-induced single- and double-strand breaks (ssb's and dsb's) as well as base lesions, which are converted into detectable ssb by the base excision repair enzymes endonuclease III (Nth) and formamidopyrimidine-DNA glycosylase (Fpg), at 278 K have been measured as a function of the level of hydration of closed-circular plasmid DNA (pUC18) films. The yields of ssb and dsb increase slightly on increasing the level of hydration (Gamma) from vacuum-dried DNA up to DNA containing 15 mol of water per mole of nucleotide. At higher levels of hydration (15 < Gamma < 35), the yields are constant, indicating that H2O*+ or diffusible hydroxyl radicals, if produced in the hydrated layer, do not contribute significantly to the induction of strand breaks. In contrast, the yields of base lesions, recognized by Nth and Fpg, increase with increasing hydration of the DNA over the range studied. The maximum ratios of the yields of base lesions to that of ssb are 1.7:1 and 1.4:1 for Nth- and Fpg-sensitive sites, respectively. The yields of additional dsb, revealed after enzymatic treatment, increase with increasing level of hydration of DNA. The maximum yield of these enzymatically induced dsb is almost the same as that for prompt, radiation-induced dsb's, indicating that certain types of enzymatically revealed, clustered DNA damage, e.g., two or more lesions closely located, one on each DNA strand, are induced in hydrated DNA by radiation. It is proposed that direct energy deposition in the hydration layer of DNA produces H2O*+ and an electron, which react with DNA to produce mainly base lesions but not ssb. The nucleobases are oxidized by H2O*+ in competition with its conversion to hydroxyl radicals, which if formed do not produce ssb's, presumably due to their scavenging by Tris present in the samples. This pathway plays an important role in the induction of base lesions and clustered DNA damage by direct energy deposition in hydrated DNA and is important in understanding the processes that lead to radiation degradation of DNA in cells or biological samples.     

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.     

Never Cared for What They Do: High Structural Stability of Guanine-Quadruplexes in the Presence of Strand-Break Damage

DNA integrity is an important factor that assures genome stability and, more generally, the viability of cells and organisms. In the presence of DNA damage, the normal cell cycle is perturbed when cells activate their repair processes. Although efficient, the repair system is not always able to ensure complete restoration of gene integrity. In these cases, mutations not only may occur, but the accumulation of lesions can either lead to carcinogenesis or reach a threshold that induces apoptosis and programmed cell death. Among the different types of DNA lesions, strand breaks produced by ionizing radiation are the most toxic due to the inherent difficultly of repair, which may lead to genomic instability. In this article we show, by using classical molecular simulation techniques, that compared to canonical double-helical B-DNA, guanine-quadruplex (G4) arrangements show remarkable structural stability, even in the presence of two strand breaks. Since G4-DNA is recognized for its regulatory roles in cell senescence and gene expression, including oncogenes, this stability may be related to an evolutionary cellular response aimed at minimizing the effects of ionizing radiation.     

γH2Ax: biomarker of damage or functional participant in DNA repair "all that glitters is not gold!"

The phosphorylation of H2Ax on its S139 site, γH2Ax, is important for the assembly of repair complexes at DNA double strand breaks (DSBs). The formation and functional role of γH2Ax after other kinds of DNA damage, especially UV light, where DSBs are rare, is less clear. Following UV light in the UVB and UVC ranges, complex distributions of γH2Ax can be identified, quite unlike the discrete enumerable foci seen after ionizing radiation. Several distinct distributions of γH2Ax occur: a low level nuclear-wide distribution of γH2Ax occurs during nucleotide excision repair; irregular focal distributions occur at arrested replication forks; high intensity nuclear-wide γH2Ax occurs in association with S-phase apoptosis. The intensity and distributions of γH2Ax vary according to the activity of excision repair, bypass polymerase and apoptotic caspases. The frequency of DSBs at arrested replication forks is low but highly variable in different cell types, and probably caused by enzymatic action. Despite the prominence of S139 phosphorylation following UV damage, mutation of this site has no influence on the UV damage response indicating that γH2Ax is a biomarker but not a participant in the UV-DNA damage response.     

ALKALINE ELUTION STUDIES OF HEMATOPORPHYRIN-DERIVATIVE PHOTOSENSITIZED DNA DAMAGE AND REPAIR IN CHINESE HAMSTER OVARY CELLS

Abstract We have used alkaline elution to study DNA damage produced by the photosensitizer hematoporphyrin derivative (HPD) in cultured Chinese hamster cells. Dosimetry was performed by measuring fluence and calculating photon absorption by intracellular HPD. HPD photosensitization causes DNA strand breakage. These breaks are repaired by the cell, although their fractional rate of repair is smaller than that for X-ray induced strand breaks at equivalent levels of strand breakage. The combined DNA polymerase inhibitors cytosine arabinoside and hydroxyurea suppress the repair of HPD-photosensitized breaks more strongly than they suppress repair of X-ray induced breaks. Addition of novobiocin to the aforementioned inhibitors causes almost total suppression of photosensitized break repair. A nucleotide excision repair system with inhibitor susceptibility similar to that of the system which removes pyrimidine dimers thus does not act upon HPD-photosensitized damage. The repair rate and inhibitor sensitivity findings together suggest biologically important differences in the chemical nature of X-ray induced and HPD-photosensitized strand breaks. In addition to strand breaks, HPD photosensitization produces covalent DNA-protein crosslinks, some of which persist through at least 90 min incubation, but which are repaired within 180 min.     

Single,double, and multiple double strand breaks induced in DNA by 3-100 eV electrons

Nonthermal secondary electrons with initial kinetic energies below 100 eV are an abundant transient species created in irradiated cells and thermalize within picoseconds through successive multiple energy loss events. Here we show that below 15 eV such low-energy electrons induce single (SSB) and double (DSB) strand breaks in plasmid DNA exclusively via formation and decay of molecular resonances involving DNA components (base, sugar, hydration water, etc.). Furthermore, the strand break quantum yields (per incident electron) due to resonances occur with intensities similar to those that appear between 25 and 100 eV electron energy, where nonresonant mechanisms related to excitation/ionizations/dissociations are shown to dominate the yields, although with some contribution from multiple scattering electron energy loss events. We also present the first measurements of the electron energy dependence of multiple double strand breaks (MDSB) induced in DNA by electrons with energies below 100 eV. Unlike the SSB and DSB yields, which remain relatively constant above 25 eV, the MDSB yields show a strong monotonic increase above 30 eV, however with intensities at least 1 order of magnitude smaller than the combined SSB and DSB yields. The observation of MDSB above 30 eV is attributed to strand break clusters (nano-tracks) involving multiple successive interactions of one single electron at sites that are distant in primary sequence along the DNA double strand, but are in close contact; such regions exist in supercoiled DNA (as well as cellular DNA) where the double helix crosses itself or is in close proximity to another part of the same DNA molecule.     

Age-dependent DNA repair and cell cycle distribution of human skin fibroblasts in response to UVA irradiation

Ageing process in cells is associated with oxidative stress. Ultraviolet A produces reactive oxygen species responsible for accumulation of DNA and cellular damage. After the evaluation of antioxidant enzyme activities and oxidative stress markers at the basal state, we have studied the responses to UVA stress of coetaneous fibroblasts, isolated from different male donors (2-88 years, n=23) in terms of cytotoxicity, genotoxicity and DNA repair capacities. For this purpose, we have determined level of DNA damage using the comet assay (single strand breaks and alkali-labile sites) and the cell cycle distribution after a 5 J/cm2 irradiation. No differences with age were observed for antioxidant enzyme activities and oxidative stress markers. DNA strand breaks after UVA irradiation (5-20 J/cm2), was found to be age-dependent. DNA repair was slow and also significantly affected by ageing. The cell cycle distribution analysis showed that high repair correlated with high proliferative capacities at basal level. Twenty-four hours after the stress, fraction of young fibroblasts blocked in G1 phase was significantly increased whereas significant modifications concerned the G2-M phase for adult and older fibroblasts. These results indicate an age-dependent decline in the DNA repair capacities correlated with modifications of the cell cycle parameters.     

Detection of telomere damage as a result of strand breaks in telomeric and subtelomeric DNA

Telomeres are the tandem repetitive DNA sequences at both ends of a chromosome with a repeating unit of TTAGGG. The integrity of a telomere is crucial to chromosomal stability and cellular viability. Damages to telomere DNA disrupt telomere integrity and accelerate telomere shortening. We describe a method for the assessment of strand breaks in the telomere/subtelomere region in cultured cells. Cells were embedded in agarose plugs and subjected to lysis and alkaline treatment to relax the DNA double helix. The telomere fragments as the result of strand breaks in the telomere/subtelomere region were then separated from the genomic DNA by electrophoresis, blotted onto membranes, and detected by a probe specific to the telomere sequence. Because of the large content of the telomere in human cells and the fact that telomere DNA is much more prone to damage than the bulk genomic DNA, the analysis may serve as a good indication of general DNA damage as well.     

Low-energy electron-induced single strand breaks in 2'-deoxycytidine-3'-monophosphate using the local complex potential based time-dependent wave packet approach

Recent experimental and theoretical investigations on resonant electron scattering off DNA and DNA fragments using low-energy electrons (LEEs), to propose the mechanism for single strand breaks (SSBs) and double strand breaks (DSBs), have received considerable attention. It is our purpose here to understand theoretically the comprehensive route to SSB in a selected DNA fragment, namely, 2'-deoxycytidine-3'-monophosphate (3'-dCMPH), induced by LEE (0-3 eV) scattering using the local complex potential based time-dependent wave packet (LCP-TDWP) approach. To the best of our knowledge, there is no time-dependent quantum mechanical study that has been reported in the literature for this DNA fragment to date. Initial results obtained from our calculation in the gas phase provide a good agreement with experimental observation and show the plausibility of SSB at 0.75 eV, which is very close to the highest SSB yield reported from the experimental measurement (0.8 eV) on plasmid DNA in the condensed phase.     

SIRT1 promotes DNA repair activity and deacetylation of Ku70

Human SIRT1 controls various physiological responses including cell fate, stress, and aging, through deacetylation of its specific substrate protein. In processing DNA damage signaling, SIRT1 attenuates a cellular apoptotic response by deacetylation of p53 tumor suppressor. The present study shows that, upon exposure to radiation, SIRT1 could enhance DNA repair capacity and deacetylation of repair protein Ku70. Ectopically over-expressed SIRT1 resulted in the increase of repair of DNA strand breakages produced by radiation. On the other hand, repression of endogenous SIRT1 expression by SIRT1 siRNA led to the decrease of this repair activity, indicating that SIRT1 can regulate DNA repair capacity of cells with DNA strand breaks. In addition, we found that SIRT1 physically complexed with repair protein Ku70, leading to subsequent deacetylation. The dominant-negative SIRT1, a catalytically inactive form, did not induce deacetylation of Ku70 protein as well as increase of DNA repair capacity. These observations suggest that SIRT1 modulates DNA repair activity, which could be regulated by the acetylation status of repair protein Ku70 following DNA damage.     

THE REPAIR OF DNA STRAND BREAKS IN HUMAN LYMPHOCYTES EXPOSED TO NEAR UV-RADIATION (UVA) AND FAR UV-RADIATION (UVC)

Abstract— UVA irradiation of human lymphocytes induces DNA strand breaks and a portion of these breaks are closed at a slower rate than X-ray induced DNA strand breaks and the strand breaks generated during repair of UVC induced DNA lesions. In addition, the yield of DNA strand breaks in lymphocytes pretreated with UVA radiation and given a subsequent exposure with UVC radiation is higher and shows a slower decrease with increasing repair time in comparison with the expected yield based on additivity between UVA and UVC induced DNA strand breaks. This indicates that UVA delays the closure of the intermediate strand breaks formed in the repair process of UVC induced DNA lesions.     

DNA DAMAGE AND REPAIR IN HUMAN CELLS EXPOSED TO SUNLIGHT

Cultured human cells were treated with direct sunlight under conditions which minimised the hypertonic, hyperthermic and fixative effects of solar radiation. Sunlight produced similar levels of DNA strand breaks as equitoxic 254 nm UV in two fibroblast strains and a melanoma cell line, but DNA repair synthesis and inhibition of semiconservative DNA synthesis and of DNA chain elongation were significantly less for sunlight-exposed cells. DNA breaks induced by sunlight were removed more rapidly. Thus, the repair of solar damage differs considerably from 254 nm UV repair. Glass-filtered sunlight (> 320 nm) was not toxic to cells and did not induce repair synthesis but gave a low level of short-lived DNA breaks and some inhibition of DNA chain elongation; thymidine uptake was enhanced. Filtered sunlight slightly enhanced UV-induced repair synthesis and UV toxicity; photoreactivation of UV damage was not found. Attempts to transform human fibroblasts using sunlight, with or without phorbol ester, were unsuccessful.     

The effect of photofrin on DNA strand breaks and base oxidation in HaCaT keratinocytes: a comet assay study

Photodynamic therapy (PDT) kills cells via the production of singlet oxygen and other reactive oxygen species. PDT causes chromosomal damage and mutation to cultured cells. However, DNA damage does not contribute to the phototoxic effect. To study the effect of Photofrin-PDT-induced DNA damage, we used the comet assay in combination with endonuclease III and formamidopyrimidine DNA glycosylase and a human keratinocyte cell line to investigate photogenotoxicity and its prevention by tocopherol (TOC). This study shows that PDT induced DNA damage in HaCaT cells at doses allowing cells to survive 7 days after irradiation. alpha-TOC did not prevent the acute cell lysis caused by Photofrin-PDT but did prevent Photofrin-PDT-induced DNA damage. However, the concentration of TOC that conferred protection (100 microM) was higher than is detected in human serum. Base oxidation was also measured using the comet assay. Although TOC could prevent frank DNA strand breaks caused by PDT, it was unable to decrease the level of base oxidation as revealed by enzyme-sensitive sites. It is suggested that the potential genotoxic risk from laser-PDT could be low, and that topical micro-TOC at a high concentration may be useful in preventing some types of DNA damage without preventing acute photolysis after Photofrin-PDT.     

A minor groove binding copper-phenanthroline conjugate produces direct strand breaks via beta-elimination of 2-deoxyribonolactone

Copper-phenanthroline complexes and their conjugates are useful reagents for studying nucleic acid interactions. Although DNA cleavage by such complexes was discovered more than 20 years ago, significant questions remain unanswered regarding the chemical mechanism(s) by which DNA is damaged. Kinetic evidence is provided, which demonstrates that the major pathway for DNA damage by a minor groove binding molecule conjugated to copper phenanthroline (6) involves C1'-oxidation. Additional experiments using 6 and a DNA substrate containing 2-deoxyribonolactone (1) show that direct strand breaks are produced via beta-elimination from 1. These studies support the original mechanism for DNA damage by copper phenanthroline put forth by Sigman and a more recent proposal concerning the mechanism for direct strand break formation.     

Electron attachment-induced DNA single strand breaks: C3'-O3' sigma-bond breaking of pyrimidine nucleotides predominates

A detailed understanding of DNA strand breaks induced by low energy electrons (LEE) is of crucial importance for the advancement of many areas of molecular biology and medicine. To elucidate the mechanism of DNA strand breaks by LEEs, theoretical investigations of the electron attachment-induced C3'-O3' sigma-bond breaking of the pyrimidine nucleotides have been performed. Calculations of 2'-deoxycytidine-3'-monophosphate and 2'-deoxythymidine-3'-monophosphate in their protonated form (denoted as 3'-dCMPH and 3'-dTMPH) have been carried out with the reliably calibrated B3LYP/DZP++ theoretical approach. Our results demonstrate that the transfer of the negative charge from the pi*-orbital of the radical anion of pyrimidines to the DNA backbone does not pass through the N1-glycosidic bond. Instead, the migration of the excessive negative charge through the atomic orbital overlap between the C6 of pyrimidine and the C3' of ribose most likely represents a pathway that subsequently leads to the strand breaks. The proposed mechanism of the LEE-induced single strand breaks in DNA assumes that the formation of the base-centered radical anions is the first step in this process. Subsequently, these electronically stable radical anions may undergo either C-O bond breaking or N-glycosidic bond rupture. The present investigation of 3'-dCMPH and 3'-dTMPH yields an energy barrier of 6.2-7.1 kcal/mol for the C3'-O3' sigma-bond cleavage. This is much lower than the energy barriers required for the C5'-O5' sigma-bond and the N1-glycosidic bond break. Therefore, we conclude that the C3'-O3' sigma-bond rupture dominates the LEE-induced single strand breaks of DNA.