Direct Double-strand Breaks Research Articles

Ligand-induced gene activation is associated with oxidative genome damage whose repair is required for transcription

Among several reversible epigenetic changes occurring during transcriptional activation, only demethylation of histones and cytosine-phosphate-guanines (CpGs) in gene promoters and other regulatory regions by specific demethylase(s) generates reactive oxygen species (ROS), which oxidize DNA and other cellular components. Here, we show induction of oxidized bases and single-strand breaks (SSBs), but not direct double-strand breaks (DSBs), in the genome during gene activation by ligands of the nuclear receptor superfamily. We observed that these damages were preferentially repaired in promoters via the base excision repair (BER)/single-strand break repair (SSBR) pathway. Interestingly, BER/SSBR inhibition suppressed gene activation. Constitutive association of demethylases with BER/SSBR proteins in multiprotein complexes underscores the coordination of histone/DNA demethylation and genome repair during gene activation. However, ligand-independent transcriptional activation occurring during heat shock (HS) induction is associated with the generation of DSBs, the repair of which is likewise essential for the activation of HS-responsive genes. These observations suggest that the repair of distinct damages induced during diverse transcriptional activation is a universal prerequisite for transcription initiation. Because of limited investigation of demethylation-induced genome damage during transcription, this study suggests that the extent of oxidative genome damage resulting from various cellular processes is substantially underestimated.

Low-Energy Ion-Species-Dependent Induction of DNA Double-Strand Breaks: Ion Energy and Fluence Thresholds.

The goal of this study was to determine the critical ion-radiation conditions under which heavy ion beams can induce DNA double-strand breaks. Helium, nitrogen and argon-ion beams in the energy range of 20 eV to 2 keV were used to irradiate naked DNA plasmid pGFP to fluences of 1, 2 and 4 × 1015 ions/cm2. The topological forms of DNA were subsequently analyzed using gel electrophoresis. The DNA forms were changed from the original supercoiled to damaged relaxed and linear forms, depending on the ion mass, energy, fluence and inertia. We found ion energy and fluence thresholds above which direct double-strand breaks can occur. The threshold is discussed in terms of the areal ion-energy density and the cross-section.

Critical low-energy Ar-ion beam conditions to induce direct DNA double strand break

Abstract Ultra-low-energy ion interaction with naked DNA has been an interesting research topic as it can reveal fundamentals involved in charged particle irradiation effect on life as well as biological evolution. This study was a detailed investigation on ultra-low-energy argon ion bombardment effects on naked DNA. Argon ion beam was decelerated by a deceleration lens to ultra low energy ranging from about 1 keV down to a few tens of eV to bombard naked DNA plasmid pGFP to fluences of 1, 2 and 4 × 1015 ions/cm2. After ion bombardment, gel electrophoresis analyzed changes in the DNA topological form. The result showed that the DNA form changed, including from original supercoiled to damaged relaxed and linear forms, certainly depending on the ion energy and fluence. The DNA form changes as functions of the ion energy and fluence were quantified. It was found that the critical Ar-ion beam conditions were the energy 750 eV to be the lowest with the fluence 2 × 1015 ions/cm2 which could induce the linear DNA form, indicating direct double strand breaks, a more severe damage of DNA which might lead to mutation.

Copper Enhances Antitumor Activity of Water Soluble Garlic Extract Components on a Human Hepatoma Cell Line

Water garlic extract combined with Copper (GE-Cu) shows specific anti-proliferative and antitumoral activity in human hepatocarcinoma (HepG2) cancer cell line, while it is not active on differentiated or primary isolated cells, normal human dermal fibroblasts (HDF) and human epidermal keratinocytes (HEK). Since other transition metals do not affect cell viability, the observed antineoplastic property, was found specific for copper (Cu), and atomic absorption analyses demonstrate an accumulation of this metal inside the cell nuclei. GE-Cu treatment of HepG2a cancer cells, induces cell differentiation, growth arrest and programmed cell death. Furthermore, GE-Cu produces a continuous and prolonged flux of oxidative radicals, which down-regulates nuclear antioxidant defenses and repair systems, thus generating direct double-strand breaks to DNA. Indeed, it was observed that in GE-Cu-treated HepG2 cancer cells, histone deacetylase (HDAC) is inhibited, and histone H2AX is early phospho-activated. DNA double strand break downstream, likely responsible for the observed cell death in HepG2 cell, was evidenced by an early over expression of p53 and p21. JNK/c-Jun and p-38 phosphorylative cascade are activated as response to oxidative stress also. The resulting apoptosis appears to be caspases-independent because, under our experimental conditions, nuclear translocation of the apoptosis inducing factor (AIF) is operative. Our data suggest that GE-Cu possess an active specific antitumor capability, which could provide valuable new tools in cancer prevention and in supporting traditional chemotherapy.

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.

Influence of chromatin condensation on the number of direct DSB damages induced by ions studied using a Monte Carlo code

The purpose of this work is to evaluate the influence of the chromatin condensation on the number of direct double-strand break (DSB) damages induced by ions. Two geometries of chromosome territories containing either condensed or decondensed chromatin were implemented as biological targets in the Geant4 Monte Carlo simulation code and proton and alpha irradiation was simulated using the Geant4-DNA processes. A DBSCAN algorithm was used in order to detect energy deposition clusters that could give rise to single-strand breaks or DSBs on the DNA molecule. The results of this study show an increase in the number and complexity of DNA DSBs in condensed chromatin when compared with decondensed chromatin.

Residual γH2AX foci as an indication of lethal DNA lesions

BackgroundEvidence suggests that tumor cells exposed to some DNA damaging agents are more likely to die if they retain microscopically visible γH2AX foci that are known to mark sites of double-strand breaks. This appears to be true even after exposure to the alkylating agent MNNG that does not cause direct double-strand breaks but does produce γH2AX foci when damaged DNA undergoes replication.MethodsTo examine this predictive ability further, SiHa human cervical carcinoma cells were exposed to 8 DNA damaging drugs (camptothecin, cisplatin, doxorubicin, etoposide, hydrogen peroxide, MNNG, temozolomide, and tirapazamine) and the fraction of cells that retained γH2AX foci 24 hours after a 30 or 60 min treatment was compared with the fraction of cells that lost clonogenicity. To determine if cells with residual repair foci are the cells that die, SiHa cervical cancer cells were stably transfected with a RAD51-GFP construct and live cell analysis was used to follow the fate of irradiated cells with RAD51-GFP foci.ResultsFor all drugs regardless of their mechanism of interaction with DNA, close to a 1:1 correlation was observed between clonogenic surviving fraction and the fraction of cells that retained γH2AX foci 24 hours after treatment. Initial studies established that the fraction of cells that retained RAD51 foci after irradiation was similar to the fraction of cells that retained γH2AX foci and subsequently lost clonogenicity. Tracking individual irradiated live cells confirmed that SiHa cells with RAD51-GFP foci 24 hours after irradiation were more likely to die.ConclusionRetention of DNA damage-induced γH2AX foci appears to be indicative of lethal DNA damage so that it may be possible to predict tumor cell killing by a wide variety of DNA damaging agents simply by scoring the fraction of cells that retain γH2AX foci.

Zinc-finger directed double-strand breaks within CAG repeat tracts promote repeat instability in human cells

Expanded triplet repeats have been identified as the genetic basis for a growing number of neurological and skeletal disorders. To examine the contribution of double-strand break repair to CAG x CTG repeat instability in mammalian systems, we developed zinc finger nucleases (ZFNs) that recognize and cleave CAG repeat sequences. Engineered ZFNs use a tandem array of zinc fingers, fused to the FokI DNA cleavage domain, to direct double-strand breaks (DSBs) in a site-specific manner. We first determined that the ZFNs cleave CAG repeats in vitro. Then, using our previously described tissue culture assay for identifying modifiers of CAG repeat instability, we found that transfection of ZFN-expression vectors induced up to a 15-fold increase in changes to the CAG repeat in human and rodent cell lines, and that longer repeats were much more sensitive to cleavage than shorter ones. Analysis of individual colonies arising after treatment revealed a spectrum of events consistent with ZFN-induced DSBs and dominated by repeat contractions. We also found that expressing a dominant-negative form of RAD51 in combination with a ZFN, dramatically reduced the effect of the nuclease, suggesting that DSB-induced repeat instability is mediated, in part, through homology directed repair. These studies identify a ZFN as a useful reagent for characterizing the effects of DSBs on CAG repeats in cells.

DNA cleavage and binding selectivity of a heterodinuclear Pt–Cu(3-Clip-Phen) complex

The synthesis and nuclease activity of a new bifunctional heterodinuclear platinum–copper complex are reported. The design of this ditopic coordination compound is based on the specific mode of action of each component, namely, cisplatin and Cu(3-Clip-Phen), where 3-Clip-Phen is 1-(1,10-phenanthrolin-3-yloxy)-3-(1,10-phenanthrolin-8-yloxy)propan-2-amine. Cisplatin is not only able to direct the Cu(3-Clip-Phen) part to the GG or AG site, but also acts as a kinetically inert DNA anchor. The nuclease activity of this complex has been investigated on supercoiled DNA. The dinuclear compound is not only more active than Cu(3-Clip-Phen), but is also capable of inducing direct double-strand breaks. The sequence selectivity of the mononuclear platinum complex has been investigated by primer extension experiments, which reveal that its interaction with DNA occurs at the same sites as for cisplatin. The Taq polymerase recognizes the resulting DNA damage as different from that for unmodified cisplatin. The sequence-selective cleavage has been investigated by high-resolution gel electrophoresis on a 36-bp DNA fragment. Sequence-selective cleavages are observed in the close proximity of the platinum sites for the strand exhibiting the preferential platinum binding sites. The platinum moiety also coordinates to the other DNA strand, most likely leading only to mono guanine or adenine adducts.Electronic supplementary materialThe online version of this article (doi:10.1007/s00775-008-0346-y) contains supplementary material, which is available to authorized users.

Multiphoton near-infrared femtosecond laser pulse-induced DNA damage with and without the photosensitizer proflavine.

The excitation of pBr322 supercoiled plasmid DNA with intense near-IR 810 nm fs laser pulses by a simultaneous multiphoton absorption mechanism results in single-strand breaks after treatment of the irradiated samples with Micrococcus luteus UV endonuclease. This enzyme cleaves DNA strands at sites of cyclobutane dimers that are formed by the simultaneous absorption of three (or more) 810 nm IR photons (pulse width approximately 140 fs, 76 MHz pulse repetition, average power output focused through 10x microscope objective is approximately 1.2 MW/cm2). Direct single-strand breaks (without treatment with M. luteus) were not observed under these conditions. However, in the presence of 6 microM of the intercalator proflavine (PF), both direct single- and double-strand breaks are observed under conditions where substantial fractions of undamaged supercoiled DNA molecules are still present. The fraction of direct double-strand breaks is 30 +/- 5% of all measurable strand cleavage events, is independent of dosage (up to 6.4 GJ/cm2) and is proportional to In, where I is the average power/area of the 810 nm fs laser pulses, and n = 3 +/- 1. The nicking of two DNA strands in the immediate vicinity of the excited PF molecules gives rise to this double-strand cleavage. In contrast, excitation of the same samples under low-power, single-photon absorption conditions (approximately 400-500 nm) gives rise predominantly to single-strand breaks, but some double-strand breaks are observed at the higher dosages. Thus, single-photon excitation with 400-500 nm light and multiphoton activation of PF by near-IR fs laser pulses produces different distributions of single- and double-strand breaks. These results suggest that DNA strand cleavage originates from unrelaxed, higher excited states when PF is excited by simultaneous IR multiphoton absorption processes.

Single- and double-strand photocleavage of DNA by YO, YOYO and TOTO

Photocleavage of dsDNA by the fluorescent DNA stains oxazole yellow (YO), its dimer YOYO) and the dimer TOTO of thiazole orange (TO) has been investigated as a function of binding ratio. On visible illumination, both YO and YOYO cause single-strand cleavage, with an efficiency that varies with the dye/DNA binding ratio in a manner which can be rationalized in terms of free dye being an inefficient photocleavage reagent and externally bound dye being more efficient than intercalated dye. Moreover, the photocleavage mechanism changes with binding mode. Photocleavage by externally bound dye is, at least partly, oxygen dependent with scavenger studies implicating singlet oxygen as the activated oxygen intermediate. Photocleavage by intercalated dye is essentially oxygen-independent but can be inhibited by moderate concentrations of beta- mercaptoethanol--direct attack on the phosphoribose backbone is a possible mechanism. TOTO causes single-strand cleavage approximately five times less efficiently than YOYO. No direct double-strand breaks (dsb) are detected with YO or YOYO, but in both cases single-strand breaks (ssb) are observed to accumulate to eventually produce double-strand cleavage. With intercalated YO the accumulation occurs in a manner consistent with random generation of strand lesions, while with bisintercalated YOYO the yield of double-strand cleavage (per ssb) is 5-fold higher. A contributing factor is the slow dissociation of the bis-intercalated dimer, which allows for repeated strand-attack at the same binding site, but the observation that the dsb/ssb yield is considerably lower for externally bound than for bis-intercalated YOYO at low dye/DNA ratios indicates that the binding geometry and/or the cleavage mechanism are also important for the high dsb-efficiency. In fact, double-strand cleavage yields with bis-intercalated YOYO are higher than those predicted by simple models, implying a greater than statistical probability for a second cleavage event to occur adjacent to the first (i.e. to be induced by the same YOYO molecule). With TOTO the efficiency of the ssb-accumulation is comparable to that observed with YOYO.

C1027 chromophore, a potent new enediyne antitumor antibiotic, induces sequence-specific double-strand DNA cleavage.

C1027, a new macromolecular antitumor antibiotic produced by a Streptomyces strain, shows highly potent cytotoxicity to cultured cancer cells and marked DNA cleaving ability. The structure of its chromophore, responsible for most of the biological activities of the antibiotic, was recently determined and found to contain a nine-membered enediyne. In contrast to other enediyne antibiotics, such as neocarzinostatin, calicheamicin, esperamicin, and recently found kedarcidin, C1027 damages duplex DNA even in the absence of thiols. The DNA damage caused by C1027 includes double-strand breaks, single-strand breaks, and abasic sites. Experiments with plasmid DNA and 32P-end-labeled restriction fragments demonstrated that the chromophore, extracted from the protein-containing holoantibiotic, interacts in the DNA minor groove and cleaves double-helical DNA with a remarkable sequence-selectivity causing direct double-strand breaks. The double-strand cleavage sites, occurring predominantly at CTTTT/AAAAG, ATAAT/ATTAT, CTTTA/TAAAG, CTCTT/AAGAG, and especially GTTAT/ATAAC, consist of five nucleotide sequences with a two-nucleotide 3'-stagger of the cleaved residues (cutting sites are underlined). The chemical structures of the damaged residues at the GTTAT/ATAAC cleavage site suggest a model in which a C1027-induced double-strand break results from abstraction, by a single molecule of the diradical form of the chromophore, of a C4' hydrogen atom from the A residue of GTTAT and a C5' hydrogen atom from the A of ATAAC on the opposite strand. Single-strand breaks, which are mainly produced at adenylate and thymidylate residues, appear to be separate events presumably resulting from different binding modes of the drug to DNA.(ABSTRACT TRUNCATED AT 250 WORDS)

Sequence-specific double-strand breakage of DNA by neocarzinostatin involves different chemical mechanisms within a staggered cleavage site.

Direct double-strand breaks in DNA have been implicated in cellular lethality of the antitumor antibiotic neocarzinostatin, but the mechanism of their formation has not been elucidated. Evidence is presented that neocarzinostatin causes sequence-specific direct double-strand breaks whose formation is strongly influenced by the activating thiol. Seven-fold more double-strand breaks result when glutathione rather than 2-mercaptoethanol is used to activate the drug to its putative diradical form, while the sequence specificity of cleavage remains the same. These data explain earlier inconsistencies in the ratios of double-strand to single-strand breaks obtained from in vitro and in vivo studies. Double-strand cleavage sites, occurring predominantly at GT steps, especially AGT.ACT, consist of trinucleotide sequences with a two-nucleotide 3'-stagger of the cleaved residues. The chemical structures of the cleavage sites suggest a model in which a neocarzinostatin-induced double-strand break results from abstraction of a C5' hydrogen atom from the T of ACT and the C4' hydrogen atom of the T of AGT by a single molecule of the diradical form of the drug. Single-strand breaks at these sites occur as separate events with attack at the C5' hydrogens. These findings permit the generalization that single-strand breaks produced by neocarzinostatin show a base preference but no clear sequence specificity, while bistranded lesions are sequence-specific in nature.