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.     

DNA strand break: structural and electrostatic properties studied by molecular dynamics simulation

Due to their lethal consequences and a relatively high probability of introduction of repair errors and mutations, single and double strand breaks are among the most important and dangerous DNA lesions. However, the mechanisms of their recognition and repair processes are only poorly known at present. This work defines and analyzes a DNA with single strand break as a template study for future complex analyses of biologically serious double strand break damage and its enzymatic repair mechanisms. Besides a non-damaged DNA serving as a reference system with no surprising results, system with open valences of the atoms at the strand break ends as well as a system with filled valences were simulated. In both cases during the first few nanoseconds the broken ends of strand breaks are significantly exposed to the outside of the molecule. However, with increasing time, the system with single strand break with open valences is partially disrupted. On the contrary, the system with filled valences shows stable conformation with newly created hydrogen bond between the two strand break endings. Moreover, these endings are steadily situated in the inner part of the molecule, thus making the recognition and docking process of a repair enzyme more complicated in the case of filled valences.     

Induction of persistent double strand breaks following multiphoton irradiation of cycling and G1-arrested mammalian cells-replication-induced double strand breaks

DNA double strand breaks (DSBs) are amongst the most deleterious lesions induced within the cell following exposure to ionizing radiation. Mammalian cells repair these breaks predominantly via the nonhomologous end joining pathway which is active throughout the cell cycle and is error prone. The alternative pathway for repair of DSBs is homologous recombination (HR) which is error free and active during S- and G2/M-phases of the cell cycle. We have utilized near-infrared laser radiation to induce DNA damage in individual mammalian cells through multiphoton excitation processes to investigate the dynamics of single cell DNA damage processing. We have used immunofluorescent imaging of gamma-H2AX (a marker for DSBs) in mammalian cells and investigated the colocalization of this protein with ATM, p53 binding protein 1 and RAD51, an integral protein of the HR DNA repair pathway. We have observed persistent DSBs at later times postlaser irradiation which are indicative of DSBs arising at replication, presumably from UV photoproducts or clustered damage containing single strand breaks. Cell cycle studies have shown that in G1 cells, a significant fraction of multiphoton laser-induced prompt DSBs persists for > 4 h in addition to those induced at replication.     

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.     

The use of silver-stained "comets" to visualize DNA damage and repair in normal and Xeroderma pigmentosum fibroblasts after exposure to simulated solar radiation

The alkaline and neutral comet assays have been widely used to assess DNA damage and repair in individual cells after in vivo or in vitro exposure to chemical or physical genotoxins. Cells processed under neutral conditions generate comets primarily from DNA double strand breaks, whereas under alkaline conditions, comets arise from DNA single and double strand breaks and alkali-labile lesions. A modified version of the alkaline comet assay, as described here, used silver stain to visualize the comets and a Gelbond base to facilitate the manipulation and processing of samples. To demonstrate how these modifications improve the assay, fibroblasts derived from both normal and Xeroderma pigmentosum (Xp) individuals were exposed to simulated solar radiation and the resulting DNA damage and repair evaluated and compared with results from the relevant literature. Comets from normal fibroblasts reached their maximum length at about an hour after irradiation. Dose-dependent increases in comet length were observed up to at least 360 mJ/cm2. In contrast, comet lengths from repair deficient Xp fibroblasts were shorter than normal cells reflecting their reduced capacity to generate single strand breaks by the excision of DNA dimers. For incubation times of more than 1 h, comet lengths from normal fibroblasts underwent a time-dependent decrease, supporting the contention that this change was related to the ligation step in the DNA repair process. These changes were compatible with the model of DNA damage and repair established by others for ultraviolet radiation.     

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.     

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).     

Effects of high-energy-pulse-electron beam radiation on biomacromolecules

To study the molecular mechanism of high mutation frequency induced by high-energy-pulse-electron (HEPE) beam radiation, the effects of HEPE radiation on yeast cells, plasma membrane, plasmid DNA, and protein activity were investigated by means of cell counting, gel electrophoresis, AO/EB double fluorescent staining, etc. The results showed that the viability of yeast cells declined statistically with increase of absorbed doses. The half lethal dose (LD50) was 134 Gy. HEPE beam radiation had little influence on the function of plasma membrane and protein, while it could induce much DNA damage of single strand breaks (SSB) and double strand breaks (DSB) that were required for gene mutation. The G-value for DSB formation of HEPE beam radiation in aqueous solution was 5.7 times higher than that caused by 60Co gamma rays. HEPE can be a new effective method for induced mutation breeding and deserves further research in the future.     

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.     

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.     

Continuing damage to rat retinal DNA during darkness following light exposure

The damaging effects of visible light on the mammalian retina can be detected as functional, morphological or biochemical changes in the photoreceptor cells. Although previous studies have implicated short-lived reactive oxygen species in these processes, the termination of light exposure does not prevent continuing damage. To investigate the degenerative processes persisting during darkness following light treatment, rats were exposed to 24 h of intense visible light and the accumulation of DNA damage to restriction fragments containing opsin, insulin 1 or interleukin-6 genes was measured as single-strand breaks (ssb) on alkaline agarose gels. With longer dark treatments all three DNA fragments showed increasing DNA damage. Treatment of rats with the synthetic antioxidant dimethylthiourea prior to light exposure reduced the initial development of alkali-sensitive strand breaks and allowed significant repair of all three DNA fragments. The time course of double-strand DNA breaks was also examined in specific genes and repetitive DNA. Nucleosomal DNA laddering was evident immediately following the 24 h light treatment and increased during the subsequent dark period. The increase in the intensity of the DNA ladder pattern suggests a continuation of enzymatically mediated apoptotic processes triggered during light exposure. The protective effects of antioxidant suggests that the light-induced DNA degradative process includes both early oxidative reactions and enzymatic processes that continue after cessation of light exposure.     

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.     

DNA DAMAGE IN RAT 9L CELLS TREATED WITH 8-METHOXYPSORALEN AND NEAR-UV LIGHT ASSAYED BY VISCOELASTOMETRY AND S1 NUCLEASE

–The techniques of viscoelastometry and S1 nuclease digestion were applied to the analysis of DNA damage in rat 9L cells treated with the combination of 8-MOP (8-methoxysporalen) and near-UV light. Treatment of cells with near-UV light alone resulted in a decrease in the viscoelastic retardation time under both denaturing and nondenaturing conditons. Exposure of cells to 8-MOP alone yielded a maximum in the plot of retardation time vs dose under nondenaturing conditions, similar to that found with ionizing radition. This observation suggests that treatment with 8-MOP alone leads to DNA strand breaks. Viscoelastic analysis of cell lysates under denaturing conditions demonstrated that treatment of cells with 8-MOP and UV radiation led to substantial increases in both the viscoelastic retardation time and recoil, consistent with formation of DNA interstrand cross-links. Viscoelastic analysis of cell lysates under nondenaturing conditions showed that exposure to long wavelength UV light in the presence of 8-MOP produced a decrease in retardation time. This decrease reflects the combined effect of strand breaks and interstrand cross-links. Results from the S1 nuclease assay confirmed these observations and permitted quantitation of DNA damage arising from single-strand breaks and DNA interstrand crosslinks. The importance of including the effect of strand breaks in the quantitation of cross-link formation is discussed.     

Gel electrophoresis method for quantitation of gamma ray induced single- and double-strand breaks in DNA irradiated in vitro

We describe a method based on gel electrophoresis for the quantitation of strand breaks in DNA and demonstrate its application to the measurement of single- and double-strand breaks formed by gamma-rays for DNA irradiated in vitro. For single-strand breaks, our data span the dose range from 0.1 to 1 Gy, while for double-strand breaks doses were from 3 to 15 Gy. In agreement with results obtained using other techniques, we find that the dose response function for single-strand breaks is linear while the dose response function for double-strand breaks is curved, indicating that it is the sum of both linear and quadratic components. We discuss factors that determine the sensitivity of the method and indicate approaches to make possible the quantitation of strand breaks in the DNA of cells irradiated with sublethal doses.     

Induction of Chromosome Aberrations and Delayed Genomic Instability by Photochemical Processes

Exponentially growing cells cultured in medium containing bromodeoxyuridine, then exposed to UVA light in the presence of the dye Hoechst 33258, show significant levels of DNA strand breaks and base damage. This dye–bromodeoxyuridine–UVA photolysis treatment is markedly cytotoxic. We now demonstrate that exposure of cells to the agents used in photolysis leads directly to the formation of chromosome aberrations. Furthermore, we demonstrate that this photochemical treatment induces delayed chromosomal instability in clonal populations derived from single progenitor cells surviving photolysis. These results suggest that photolysis-induced DNA damage leads to chromosome rearrangements that could account for the observed cytotoxicity. Furthermore, in those cells surviving photolysis, the delayed effects of this treatment can be observed several generations after exposure and are manifested as compromised genomic integrity.     

PSORALENS CLEAVE pBR322 DNA UNDER ULTRAVIOLET RADIATION

Supercoiled (SC) pBR322 was used to probe the recent claim that 5-geranoxylpsoralen (5-GOP) did not photoreact with DNA. Contrary to expectations, 5-GOP was found to damage DNA in the presence of UV-A through two competing pathways: (a) single strand breaks, identified by the conversion of supercoiled into open circular and linear DNA, and (b) cross-linking, revealed by the fluence-dependent decrease in the extent of denaturation of the double stranded supercoiled DNA to single stranded circular DNA. In addition, a fluence-dependent modification reduced the ability of the restriction enzyme EcoR I to linearize the photosensitized DNA, and alkali-labile lesions were generated. Psoralen, 5-methoxypsoralen, and 8-methoxypsoralen, which are well-known to undergo cycloaddition to DNA, had a more pronounced effect on supercoiled DNA. Single strand breaks occurred more readily than with 5-GOP, and the surviving SC form remaining had reduced electrophoretic mobility in agarose gels. In all cases, the DNA damage was more prominent when oxygen was absent.     

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.     

Correlation of free radical yields with strand break yields produced in plasmid DNA by the direct effect of ionizing radiation

The purpose of this study was to determine how free radical formation (fr) correlates with single strand break (ssb) and double strand break (dsb) formation in DNA exposed to the direct effects of ionizing radiation. Chemical yields have been determined of (i) total radicals trapped on DNA at 4 K, G(Sigmafr), (ii) radicals trapped on the DNA sugar, Gsugar(fr), (iii) prompt single strand breaks, Gprompt(ssb), (iv) total single strand breaks, Gtotal(ssb), and (v) double strand breaks, G(dsb). These measurements make it possible, for the first time, to quantitatively test the premise that free radicals are the primary precursors to strand breaks. G(fr) were measured by EPR applied to films of pEC (10,810 bp) and pUC18 (2686 bp) plasmids hydrated to Gamma = 22 mol of water/nucleotide and X-irradiated at 4 K. Using these same samples warmed to room temperature, strand breaks were measured by gel electrophoresis. The respective values for pEC and pUC18 were G(fr) = 0.71 +/- 0.02 and 0.61 +/- 0.01 micromol/J, Gtotal(ssb) = 0.09 +/- 0.01 and 0.14 +/- 0.01 micromol/J, G(dsb) = 0.010 +/- 0.001 and 0.006 +/- 0.001 micromol/J, and Gtota)(ssb)/G(dsb) approximately 9 and approximately 20. Surprisingly, Gsugar(fr) approximately 0.06 mumol/J for pUC18 films, less than half of Gtotal(ssb). This indicates that a significant fraction of strand breaks are derived from precursors other than trapped DNA radicals. To explain this disparity, various mechanisms were considered, including one that entails two one-electron oxidations of a single deoxyribose carbon.     

Oxidatively Generated Damage to Cellular DNA by UVB and UVA Radiation,

This review article focuses on a critical survey of the main available information on the UVB and UVA oxidative reactions to cellular DNA as the result of direct interactions of UV photons, photosensitized pathways and biochemical responses including inflammation and bystander effects. UVA radiation appears to be much more efficient than UVB in inducing oxidatively generated damage to the bases and 2‐deoxyribose moieties of DNA in isolated cells and skin. The UVA‐induced generation of 8‐oxo‐7,8‐dihydroguanine is mostly rationalized in terms of selective guanine oxidation by singlet oxygen generated through type II photosensitization mechanism. In addition, hydroxyl radical whose formation may be accounted for by metal‐catalyzed Haber–Weiss reactions subsequent to the initial generation of superoxide anion radical contributes in a minor way to the DNA degradation. This leads to the formation of both oxidized purine and pyrimidine bases together with DNA single‐strand breaks at the exclusion, however, of direct double‐strand breaks. No evidence has been provided so far for the implication of delayed oxidative degradation pathways of cellular DNA. In that respect putative characteristic UVA‐induced DNA damage could include single and more complex lesions arising from one‐electron oxidation of the guanine base together with aldehyde adducts to amino‐substituted nucleobases.