Strand breaks in whole plasmid dna produced by the decay of (125)I in a triplex-forming oligonucleotide.

DNA strand breaks produced by the decay of (125)I positioned against a specific site in plasmid DNA via a triplex-forming oligonucleotide were studied both in the immediate vicinity of the site of the decay with a single nucleotide resolution and in the whole plasmid by measuring the percentages of supercoiled, open-circular and linear forms. The localized breaks are distributed within 10 bp in each direction from the decay site with maxima in both strands just opposite the (125)I-dC residue in the triplex-forming oligonucleotide. The distributions of breaks in the two DNA strands are almost symmetrical, in agreement with the geometry of the pyrimidine motif triplex. We found that about 25% of the double-strand breaks were located outside the 90-bp fragment containing the triplex-forming oligonucleotide binding sequence. The ratio of single- to double-strand breaks in the whole plasmid was 11 for bound triplex-forming oligonucleotide compared to 26 when the triplex-forming oligonucleotide was free in solution. The number of double-strand breaks per decay of (125)I was 0.46 for bound triplex-forming oligonucleotide and 0.17 for free triplex-forming oligonucleotide. Comparing the data on the localized damage and those for the whole plasmid, we concluded that, in addition to DNA breaks that are confined to a helical turn around the (125)I atom, the decay can produce breaks hundreds of base pairs away in the plasmid molecule. This linear plasmid molecule containing radiation-induced damage at a specific DNA site should be useful in studies of the molecular mechanisms of DNA repair.     

The radiation modification of the sugar fragment in DNA: the formation of breaks, a change in polymer conformation and the transfer of the damage to the base]

Comparative analysis of the author's own data and data reported in the literature on the nature of the intermediate and end molecular products of radiolysis of DNA, its precursors and substances stimulating certain DNA fragments, allows to attribute the formation of breaks and alkaline-labile sites in DNA strands and changes in the polymer conformation, to the formation and transformations of some types of primary radicals of the sugar fragment. In order to explain certain effects induced by irradiation of DNA and its precursors (a balance of basic products of radiation destruction of DNA and ESR data concerning low temperature radiolysis of bases, nucleotides and nucleosides) the author proposes a model of the transfer of a damage (free valence) from 2-deoxyribosyl to a base within one nucleotide.     

Radioprobing of DNA: distribution of DNA breaks produced by decay of 125I incorporated into a triplex-forming oligonucleotide correlates with geometry of the triplex.

The distribution of breaks produced in both strands of a DNA duplex by the decay of 125I carried by a triplex-forming DNA oligonucleotide was studied at single nucleotide resolution. The 125I atom was located in the C5 position of a single cytosine residue of an oligonucleotide designed to form a triple helix with the target sequence duplex. The majority of the breaks (90%) are located within 10 bp around the decay site. The addition of the free radical scavenger DMSO produces an insignificant effect on the yield and distribution of the breaks. These results suggest that the majority of these breaks are produced by the direct action of radiation and are not mediated by diffusible free radicals. The frequency of breaks in the purine strand was two times higher that in the pyrimidine strand. This asymmetry in the yield of breaks correlates with the geometry of this type of triplex; the C5 of the cytosine in the third strand is closer to the sugar-phosphate backbone of the purine strand. Moreover, study of molecular models shows that the yield of breaks at individual bases correlates with distance from the 125I decay site. We suggest the possible use of 125I decay as a probe for the structure of nucleic acids and nucleoprotein complexes.     

A method of calculating initial DNA strand breakage following the decay of incorporated 125I

Two sources of individual Auger electron spectra and an electron track code were used with a simple model of the DNA to successfully simulate the single-strand DNA breakage measured by Martin and Haseltine (1981). The conditions of the calculation were then extended to examine patterns of single-strand breaks in both strands of the DNA duplex to score double-strand breaks. The occurrences of five types of break were scored. The total number of double-strand breaks (dsb) per decay at the site of the decay was 0.90 and 0.65 for the different Auger electron spectra. It was shown that for mammalian cells an additional source of double-strand breaks from low LET radiation added approximately 0.17 dsb/decay to each, giving a final total of 1.07 and 0.85 dsb/decay for mammalian cells depending on the electron spectrum. Further is is shown that the energy deposition in the DNA from the iodine decay is very complex, with a broad range of energy depositions and products. Even for a particular energy deposited in the DNA different types of strand break are produced. These are identified and their probabilities calculated.     

Use of polyethylenimine-adenovirus complexes to examine triplex formation in intact cells

Triplex-forming oligonucleotides (TFOs) show potential for sequence-specific DNA binding and inhibition of gene expression. We have applied this antigene strategy using a TFO incorporating an Auger-emitting radionucleotide, 125I, to study the production of double-strand breaks (dsb) in the rat aquaporin 5 (rAQP5) cDNA. 125I-TFO bound to the pCMVrAQP5 plasmid in vitro in a dose-dependent manner and formed stable triplexes up to 65 degrees C and in the presence of 140 mM KCl. Further, 125I-TFO resulted in a predictable dsb when analyzed by Southern hybridization. To deliver TFOs to epithelial cells, we employed 125I-TFO-polyethyleneimine-adenovirus (125I-TFO-PEI-Ad) complexes. We hypothesized that these complexes would take advantage of adenoviral characteristics to transfer 125I-TFO to the cell nucleus. Adenovirus-containing complexes brought about greater uptake and nuclear localization of TFOs compared with delivery with 125I-TFO-PEI complexes alone. No significant degradation of 125I-TFO was found after delivery into cells using PEI-Ad complexes and freezing and thawing. We next used PEI-Ad complexes to deliver 125I-TFO and pCMVrAQP5 separately to epithelial cells to determine if triplexes can form de novo within cells, resulting in the specific dsb in the rAQP5 cDNA. After delivery, cell pellets were stored at -80 degrees C for more than 60 days. Thereafter, plasmid DNA was isolated from cells and analyzed for dsb by Southern hybridization. However, none were detected. We conclude that under the experimental conditions employed, effective triplexes, with 125I-TFO and pCMVrAQP5, do not form de novo inside cells.     

Lack of interference of DNA single-strand breaks with the measurement of double-strand breaks in mammalian cells using the neutral filter elution assay.

In this study, the effect of DNA single strand breaks (ssb) on the neutral (pH 9.6) filter elution of DNA from Chinese hamster ovary (CHO K1) cells containing DNA double strand breaks (dsb) was investigated. Protein associated ssb were induced by the inhibition of DNA topoisomerase I with camptothecin (cpt). Protein associated dsb were introduced by treating cells with the DNA topoisomerase II poison; etoposide (VP-16). Protein associated ssb and dsb were converted to ssb and dsb by proteinase K present in the lysis solution. In some experiments dsb were generated by the restriction endonuclease Pvu II. It was found that elution of DNA in the presence and absence of ssb was similar under neutral conditions. This finding is consistent with the view that the fast component of the bi-phasic repair kinetics observed in irradiated mammalian cells with the neutral filter elution technique is not attributable to the interference of ssb with the measurement of dsb, and thus suggests that the two components of repair observed with the neutral filter elution elution technique may represent two different types of dsb or modes of repair of dsb.     

Poly(deoxyadenylic-deoxythymidylic acid) damage by radiolytically activated neocarzinostatin

The anaerobic reaction of poly(deoxyadenylic-deoxythymidylic acid) with neocarzinostatin activated by the carboxyl radical CO2-, an electron donor generated from gamma-ray radiolysis of nitrous oxide saturated formate buffer, has been characterized. DNA damage includes base release and strand breaks. Few strand breaks are formed prior to alkaline treatment; they bear 3'-phosphoryl termini. In contrast, most (66%) of the base release occurs spontaneously. DNA damage is highly (95%) specific for thymidine sites. Neither DNA-drug covalent adduct nor nucleoside 5'-aldehyde, which are major products in the DNA-nicking reaction initiated by mercaptans and oxygen, is formed in this reaction. Data are presented to show that the CO2(-)-activated neocarzinostatin intermediate is a short-lived free radical able to abstract hydrogen atoms from the C-1' and C-5' positions of deoxyribose. Attack occurs mostly (68%) at the C-1' position, producing a lesion whose properties are consistent with those of (oxidized) apyrimidinic sites.     

SYBR Green I staining of pulsed field agarose gels is a sensitive and inexpensive way of quantitating DNA double-strand breaks in mammalian cells.

Pulsed field gel electrophoresis (PFGE) is widely used to measure DNA double strand breaks (dsb). The DNA of cultured cells can be prelabelled with radioactivity, which helps greatly in detection and quantitation of DNA dsb. However, this approach cannot be used with non-cycling cells from biopsy material. We describe a method which uses SYBR Green I to stain DNA in dried agarose gels. DNA is detected and analysed using readily available camera equipment and image analysis software. This method is as sensitive as [3H]thymidine prelabelling of cells and allows DNA dsb to be measured simply and economically in non-cycling cells.     

Biological damage from the Auger effect,possible benefits

Summary Decay of radioactive isotopes by K-capture leads to the Auger effect and results in the loss of several orbital electrons and the emission of X-rays. Whereas radiation effects are produced from the emitted electrons, the consequences of the Auger effect are strictly localized to the site of the decaying nuclide.The paper reviews the biological consequences of the decay of¹²⁵I which produces the Auger effect. Nearly all data were obtained from DNA labeled with¹²⁵I-5-iodo-2-deoxyuridine (IUdR) in bacteria and mammalian cells. Parameters of effects were cell death, DNA strand breaks, and mutation induction. In order to recognize in a cell the contribution from the Auger effect and that of absorbed radiation, experimental data are analysed in terms of the specific energy for the nuclear volume which contains the isotope.The data indicate that decay of¹²⁵I is far more toxic than is expected on the basis of absorbed dose to the labeled nucleus. Moreover, it is emphasized that the toxicity of the¹²⁵I decay is largely determined by events immediately localized to the site of decay.Because the consequences of the Auger effect are strictly localized to the molecular site of the decay,¹²⁵I and perhaps other nuclides decaying by K-capture promise to be interesting tools in cell biology and molecular biology. First data on the Auger effect as a tool are summarized.It appears that recognizable biological damage is only observed when the Auger effect takes place in vitally important molecules, an example of which is DNA.Dedicated to Prof. Dr. H. Muth on the occasion of his 60th birthday.     

Quantitative modelling of DNA damage using Monte Carlo track structure method

This paper presents data on modelling of DNA damage induced by electrons, protons and alpha-particles to provide an insight into factors which determine the biological effectiveness of radiations of high and low linear energy transfer (LET). These data include the yield of single- and double-strand breaks (ssb, dsb) and base damage in a cellular environment. We obtain a ratio of 4–15 for ssb:dsb for solid and cellular DNA and a preliminary ratio of about 2 for base damage to strand breakage. Data are also given on specific characteristics of damage at the DNA level in the form of clustered damage of varying complexity, that challenge the repair processes and if not processed adequately could lead to the observed biological effects. It is shown that nearly 30% of dsb are of complex form for low-LET radiation, solely by virtue of additional breaks, rising to about 70% for high-LET radiation. Inclusion of base damage increases the complex proportion to about 60% and 90% for low- and high-LET radiation, respectively. The data show a twofold increase in frequencies of complex dsb from low-LET radiation when base damage is taken into account. It is shown that most ssb induced by high-LET radiation have associated base damages, and also a substantial proportion is induced by low-energy electrons. Received: 20 September 1998 / Accepted in revised form: 15 December 1998     

Fragmentation of chromatin with 125I radioactive disintegrations.

The DNA in Chinese hamster cells was labeled first for 3 h with [3H]TdR and then for 3 h with [125I]UdR. Chromatin was extracted, frozen, and stored at -30 degrees C until 1.0 X 10(17) and 1.25 X 10(17) disintegrations/g of labeled DNA occurred for 125I and 3H respectively. Velocity sedimentation of chromatin (DNA with associated chromosomal proteins) in neutral sucrose gradients indicated that the localized energy from the 125I disintegrations, which gave about 1 double-strand break/disintegration plus an additional 1.3 single strand breaks, selectively fragmented the [125I] chromatin into pieces smaller than the [3H] chromatin. In other words, 125I disintegrations caused much more localized damage in the chromatin labeled with 125I than in the chromatin labeled with 3H, and fragments induced in DNA by 125I disintegrations were not held together by the associated chromosomal proteins. Use of this 125I technique for studying chromosomal proteins associated with different regions in the cellular DNA is discussed. For these studies, the number of disintegrations required for fragmenting DNA molecules of different sizes is illustrated.     

Oxidative damage of Chinese hamster fibroblasts induced by t-butyl hydroperoxide and by X-rays

The aim of this study was to investigate the mechanism(s) of X-ray-mediated cell damage in comparison to mechanism(s) of organic hydroperoxide cytotoxicity and to find the main targets for the two different kinds of cell inactivation. Damage of Chinese hamster fibroblasts induced by tert-butyl hydroperoxide (t-BHP) or X-irradiation was measured by the colony-formation assay and the average single colony volume. DNA double-strand breaks (dsb) were determined by constant-field gel electrophoresis. The contents of peroxides, of SH-groups and the size of inactivated cells were tested for oxidative modifications.Oxidative damage of fibroblasts induced by t-BHP or by X-rays inhibits cell proliferation. Simultaneously, irradiation causes an increase of DNA dsb with the dose, while incubation with t-BHP yields only a very few DNA dsb. Neither chemically induced oxidation nor irradiation significantly changed the amount of membrane lipid peroxides. Oxidation with t-BHP but not irradiation leads to a loss of the membrane SH-groups and to an increase of cell diameter.The similar decrease of cell proliferation can be caused by DNA dsb without detectable membrane damage (X-radiation) as by membrane damage with nearly no DNA dsb (chemically induced oxidative stress).     

Determination of G-values for single and double strand break induction in plasmid DNA using agarose gel electrophoresis and a curve-fitting procedure

Covalently closed circular double-stranded DNA (CC) of native plasmids was used to determine the yield of single strand breaks (ssb) and double strand breaks (dsb) as a consequence of X-irradiation. One ssb transforms DNA of the CC form to the nicked circular form (NC), whereas one dsb produced either directly or from random coincidence of single strand breaks transforms DNA of the CC as well as of the NC form to linear DNA molecules (LI form). Plasmids with more than one dsb are cleaved to linear fragments. DNA (30-800 micrograms/ml) was irradiated in air-saturated sodium phosphate buffer. The different forms of DNA were separated by gel electrophoresis and their amounts measured fluorometrically using ethidium bromide. Large linear DNA fragments with the same electrophoretic mobility as the LI form were considered by using a curve-fitting procedure. From the quantitative changes of each conformation D37 values of ssb and dsb were calculated as a function of the DNA concentration. Finally G-values were calculated by competition plots. The following yields were determined: Gssb 3.4 X 10E-8 molJ-1, and Gdsb 3.3 X 10E-10 molJ-1. Gdsb refers only to those dsb produced directly. Yields are related to strand breaks without further treatment by heat or alkali.     

Action of gamma endonuclease on clustered lesions in irradiated DNA

Summary Irradiation of DNA in situ i.e. in phage particles or in the cell leads to alterations of single DNA nucleotides as well as to clustered lesions such as double strand breaks or unpaired DNA regions the latter being sensitive to digestion by S 1 nuclease. A contribution will be made to the configuration of such S 1-nuclease-sensitive sites (S 1 sites). DNA from irradiated lambda phage containing S 1 sites was treated with gamma endonuclease fromM. luteus which is known to split the nucleotide strand at the position of oxidized pyrimidine base. It was found that the gamma endonuclease induces double-strand breaks at some of the S 1 sites indicating double base damage within this site. However, half of the S 1 sites are not converted into a double-strand break by the gamma endonuclease, indicating base damage only on one strand within the unpaired region.Dedicated to Prof. W. Jacobi on the occasion of his 60th birthday     

Plasmid DNA breakage by decay of DNA-associated Auger electron emitters: approaches to analysis of experimental data

Plasmid DNA is a popular substrate for the assay of DNA strand breakage by a variety of agents. The use of the plasmid assay relies on the assumption that individual damaging events occur at random, which allows the application of Poisson statistics. This assumption is not valid in the case of damage arising from decay of DNA-associated Auger electron emitters, since a single decay event can generate a few breaks in the same DNA strand, which is indistinguishable from a single break in the assay. The consequent analytical difficulties are overcome by considering relaxation events rather than single-strand breaks, and linearization events rather than double-strand breaks. A further consideration is that apart from damage at the site of DNA-associated decay, which is the principal interest of the analysis, some DNA damage also arises from the radiation field created by all decay events. These two components of damage are referred to as internal and external breakage, respectively, and they can be separated in the analysis since their contribution depends on the experimental conditions. The DNA-binding ligand Hoechst 33258 labeled with 125I was used in our experiments to study breakage in pBR322 plasmid DNA arising from the decay of this Auger electron emitter. The values obtained for the efficiency (per decay) of plasmid relaxation and linearization by the 125I-labeled ligand were 0.090 +/- 0.035 and 0.82 +/- 0.04, respectively. When dimethylsulfoxide was included as a radical scavenger, the efficiency values for relaxation and linearization were 0.15 +/- 0.02 and 0.65 +/- 0.05, respectively.     

Ataxia telangiectasia-mutated gene product inhibits DNA damage-induced apoptosis via ceramide synthase.

DNA double-stranded breaks (dsb) activate surveillance systems that identify DNA damage and either initiate repair or signal cell death. Failure of cells to undergo appropriate death in response to DNA damage leads to misrepair, mutations, and neoplastic transformation. Pathways linking DNA dsb to reproductive or apoptotic death are virtually unknown. Here we report that metabolic incorporation of 125I-labeled 5-iodo-2'deoxyuridine, which produces DNA dsb, signaled de novo ceramide synthesis by post-translational activation of ceramide synthase (CS) and apoptosis. CS activation was obligatory, since fumonisin B1, a fungal pathogen that acts as a specific CS inhibitor, abrogated DNA damage-induced death. X-irradiation yielded similar results. Furthermore, inhibition of apoptosis using the peptide caspase inhibitor benzyloxycarbonyl-Val-Ala-Asp fluoromethylketone did not affect CS activation, indicating this event is not a consequence of induction of apoptosis. ATM, the gene mutated in ataxia telangiectasia, is a member of the phosphatidylinositol 3-kinase family that constitutes the DNA damage surveillance/repair system. Epstein-Barr virus-immortalized B cell lines from six ataxia telangiectasia patients with different mutations exhibited radiation-induced CS activation, ceramide generation, and apoptosis, whereas three lines from normal patients failed to manifest these responses. Stable transfection of wild type ATM cDNA reversed these events, whereas antisense inactivation of ataxia telangiectasia-mutated gene product in normal B cells conferred the ataxia telangiectasia phenotype. We propose that one of the functions of ataxia telangiectasia-mutated gene product is to constrain activation of CS, thereby regulating DNA damage-induced apoptosis.     

Strand breaks produced in X-írradiated crystalline DNA: influence of base sequence

This study reports the radiation-chemical yields for DNA single-strand breaks (SSBs) in crystals of CGCACG:CGTGCG (I) and CACGCG:CGCGTG (II) duplexes induced by direct ionization using X rays. The DNA fragmentation products, consisting of 3'- and 5'-phosphate-terminated fragments, were quantified by ion-exchange chromatography using a set of reference compounds. The yields of single-strand breaks in I and II are 0.16 +/- 0.03 micro mol/J and 0.07 +/- 0.02 micro mol/J, respectively. The probability of cleavage at a given site is relatively independent of which of the four bases is at that site. For the very small sample of base sequences studied to date, there is no obvious dependence on base sequence. However, there appears to be an increased frequency of strand breaks at the non-phosphorylated termini of the oligodeoxynucleotides. These results show that direct ionization is efficient at producing single-strand breaks in DNA and that its action is relatively indiscriminate with respect to base sequence.     

Specific herpes simplex virus-induced incorporation of 5-iodo-5'-amino-2',5'-dideoxyuridine into deoxyribonucleic acid.

5-Iodo-5'-amino-2',5'-dideoxyuridine (AIdUrd) is a novel thymidine analog which inhibits herpes simplex virus, type 1 (HS-1 virus) replication in the absence of detectable host toxicity. When murine, simian, or human cells in culture are treated with [125I]AIdUrd for up to 24 hours essentially none of the nucleoside becomes cell-associated. In contrast, upon HS-1 virus infection significant radiolabel is detected in both nucleotide pools and in DNA. The major acid-soluble metabolite has been shown by enzymic and chromatographic analysis to be the 5'-triphosphate of AIdUrd. DNA from HS-1 virus-infected Vero cells labeled with [14C]thymidine, 5-[125I]iodo-2'-deoxyuridine (IdUrd), or [125I]AIdUrd was isolated by buoyant density centrifugation and subjected to digestion by pancreatic DNase I, spleen DNase II, micrococcal nuclease, spleen, and venom phosphodiesterases. Analysis of the digestion products clearly indicate that AIdUrd is incorporated internally into the DNA structure. DNA containing AIdUrd therefore contains phosphoramidate (P-N) bonds, known to be extremely acid-labile. The selective HS-1 virus-induced phosphorylation of AIdUrd and its subsequent incorporation into DNA may account for the unique biological activity of the AIdUrd nucleoside.     

Two types of double-strand breaks in electron and photon tracks and their relation to exchange-type chromosome aberrations

Yields of DNA double-strand breaks (dsb), i. e. the average number of dsb, N–, per relative molar mass, M _r , and dose, D, produced by electrons and photons in the energy range 50 eV – 1 MeV were calculated. The experimental data of dsb induction by ultrasoft x-rays and by photons agree well with the calculated yields of dsb as a function of photon energy. The dsb are classified into simple and complex ones. Energy transfers of less than about 200 eV producing at least two ionizations generate mainly simple dsb, while low-energy electrons with an initial energy between 200 and 500 eV induce preferentially complex dsb. Assuming that dsb is the main DNA lesion leading to exchange-type chromosome aberrations (etca), three different mechanisms have to be considered: 1) complex dsb on its own; 2) interaction between two dsb induced by the same primary particle; and 3) interaction between two dsb induced by different primary particles. Mechanisms 1) and 2) produce a linear term, whereas mechanism 3) leads to a quadratic term for the yield of etca. The sum of contributions 1) and 2) to the yield of dicentrics describes fairly well the non-trivial structure of the experimental data. The results suggest that interaction between complex dsb does not contribute significantly to the formation of dicentrics via mechanism 3). Received: 30 July 1995 / Accepted in revised form: 28 March 1996     

Detecting the DNA kinks in a DNA-CRP complex in solution with iodine-125 radioprobing.

Auger-electron-emitting radioisotopes such as 125I produce DNA strand breaks within nanometer range of the decay site. Here we analyze these breaks in order to study changes in DNA conformation upon binding with cyclic AMP receptor protein (CRP) in solution. The clear difference we found in break frequency in the CRP-DNA complex, as compared to the naked DNA duplex, correlates with the increased distances between the deoxyriboses and the radioiodine atom caused by the CRP-induced kink observed in the cocrystal. Thus, we demonstrate that 125I radioprobing can be used to study fine conformational changes of DNA within DNA-protein complexes.