Single-strand and double-strand deoxyribonucleic acid breaks produced by several bleomycin analogues

Production of single-strand breaks (ssb) and double-strand breaks (dsb) of PM2 phage DNA by several structurally related bleomycin (BLM) analogues was studied by gel electrophoresis. BLM A2 and BLM B2 produced a comparable extent of dsb. In various experiments, BLM A2 and BLM B2, at 22-41 ng/mL, degraded 50% of the form I DNA into 33-38% form II and 12-17% form III DNA. BLM B1' produced ssb and dsb at a ratio similar to that of BLM A2, but both at a rate less than half that of BLM A2. Phleomycin (PLM) D1 induced an equivalent amount of ssb but only one-eighth of dsb induced by BLM B2. The relatively lower extent dsb production for PLM D1 was observed either in borate buffer (pH 9.5) or in Tris-HCl buffer (pH 7.5) and in the presence or absence of exogenous Fe(II). Deamido-BLM A2 produced ssb to an extent approximately half that of BLM A2 and dsb to less than one-eighth that of BLM A2. The following conclusions were drawn. (1) BLM analogues produced ssb and dsb to different extents and ratios. (2) The ratio of dsb to ssb varied depending on the analogue, indicating a lack of a direct correlation between ssb and dsb. (3) The extent of ssb and dsb was affected by modifications on both the C- and N-terminal half-molecules of BLM: modification of either the N-terminal amide or the bithiazole greatly reduced dsb, whereas changes in structure or charge in the C-terminal amine affected ssb and dsb to a similar extent.     

The quality of DNA double-strand breaks: A Monte Carlo simulation of the end-structure of strand breaks produced by protons and alpha particles

The quality of DNA damage induced by protons and -particles of various linear energy transfer (LET) was studied. The aim was to single out specific lesions in the DNA molecule that might lead to biological endpoints such as inactivation. A DNA model coupled with a track structure code (MOCA-15) were used to simulate the lesions induced on the two helixes. Four categories of DNA breaks were considered: single-strand breaks (ssb), bluntended double-strand breaks (dsb, with no or few overlapping bases), sticky-ended double-strand breaks (with cohesive free ends of many bases), and deletions (complex lesions which involve at least two dsb within a small number of base pairs). Calculations were carried out assuming various sets of parameters characterizing the production of these different DNA breaks. No large variations in the yields of ssb and blunt- or sticky-ended dsb were found in the LET range between 10 and 200 keV/µm. On the other hand, the yield of deletions increases up to about 100 keV/µm and seems to reach a plateau at higher LET values. In the LET interval from 30 to 60 keV/µm, protons proved to be more efficient than -particles in inducing deletions. The induction of these complex lesions is thus dependent not simply on LET but also on the characteristics of the track structure. Comparison with RBE values for cell killing shows that this special class of dsb might play an important role in radiation-induced cell inactivation.     

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.     

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     

Reduction of intracellular glutathione content and radiosensitivity

The intracellular glutathione (GSH) content of HeLa, CHO and V79 cells was reduced by incubating the cells in growth medium containing buthionine sulphoximine or diethyl maleate (DEM). Clonogenicity, single-strand DNA breaks (ssb) and double-strand DNA breaks (dsb) were used as criteria for radiation-induced damage after X- or gamma-irradiation. In survival experiments, DEM gave a slightly larger sensitization although it gave a smaller reduction of the intracellular GSH. In general, sensitization was larger for dsb than for ssb, also the reduction of the o.e.r. was generally larger for dsb than for ssb. This may be due to the higher dose rate in case of dsb experiments resulting in a higher rate of radiochemical oxygen consumption. In general, no effect was found on post-irradiation repair of ssb and dsb.     

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     

Cell inactivation and DNA single- and double-strand breaks in cultured mammalian cells irradiated by a thermal neutron beam

The effects on the cellular viability and induction and repair kinetics of DNA strand breaks in HeLa cells were examined after exposure to a thermal neutron beam and compared with those after gamma-irradiation. The thermal neutron survival curve had no initial shoulder. The relative biological effectiveness (r.b.e.) value of the neutron beam was determined to be 2.2 for cell killing (ratio of D0 values), 1.8 and 0.89 for single strand breakage (ssb) by alkaline sedimentation and alkaline elution respectively, and for double strand breakage (dsb) 2.6 by neutral elution. No difference was observed between thermal neutrons and gamma-rays in the repair kinetics of ssb and dsb. It is suggested that the effect induced by the intracellular nuclear reaction, 14N(n,p)14C is mainly responsible for the high r.b.e. values observed.     

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.     

Mouse but not human embryonic stem cells are deficient in rejoining of ionizing radiation-induced DNA double-strand breaks

Mouse embryonic stem (mES) cells will give rise to all of the cells of the adult mouse, but they failed to rejoin half of the DNA double-strand breaks (dsb) produced by high doses of ionizing radiation. A deficiency in DNA-PK(cs) appears to be responsible since mES cells expressed <10% of the level of mouse embryo fibroblasts (MEFs) although Ku70/80 protein levels were higher than MEFs. However, the low level of DNA-PK(cs) found in wild-type cells appeared sufficient to allow rejoining of dsb after doses <20Gy even in G1 phase cells. Inhibition of DNA-PK(cs) with wortmannin and NU7026 still sensitized mES cells to radiation confirming the importance of the residual DNA-PK(cs) at low doses. In contrast to wild-type cells, mES cells lacking H2AX, a histone protein involved in the DNA damage response, were radiosensitive but they rejoined double-strand breaks more rapidly. Consistent with more rapid dsb rejoining, H2AX(-/-) mES cells also expressed 6 times more DNA-PK(cs) than wild-type mES cells. Similar results were obtained for ATM(-/-) mES cells. Differentiation of mES cells led to an increase in DNA-PK(cs), an increase in dsb rejoining rate, and a decrease in Ku70/80. Unlike mouse ES, human ES cells were proficient in rejoining of dsb and expressed high levels of DNA-PK(cs). These results confirm the importance of homologous recombination in the accurate repair of double-strand breaks in mES cells, they help explain the chromosome abnormalities associated with deficiencies in H2AX and ATM, and they add to the growing list of differences in the way rodent and human cells deal with DNA damage.     

2-Chlorodeoxyadenosine inhibits the repair of DNA double-strand breaks and does not inhibit the repair of DNA single-strand breaks in X-irradiated Chinese hamster V79 cells.

The exposure of log-phase Chinese hamster V79 cells to 2-chlorodeoxyadenosine (CdA) for 3 h after X irradiation enhanced the lethal effects of X-rays in a concentration-dependent manner. The enhancement of the killing efficiency of X-rays by CdA was mainly observed in the reduction of quasi-threshold doses (Dq) of the dose-response curves. When the ability of CdA to inhibit the repair of X-ray-induced double- and single-strand breaks (dsb and ssb) of DNA was investigated by neutral- and alkaline-filter elution techniques, respectively, it was observed that 90% of dsb were rejoined in the absence of CdA within 30 min after X irradiation and 15-40% of dsb rejoining was suppressed by co-incubation of the cells with 5-10 microM of CdA for 3 h after X irradiation, whereas almost 100% of ssb were rejoined within 15 min regardless of the presence or absence of CdA. From these results it was concluded that CdA interfered exclusively with the repair of DNA dsb in X-irradiated Chinese hamster V79 cells and thereby increased the lethality of X-rays.     

Enhanced strand break induction of DNA by resonant metal-innershell photoabsorption

We determined the number of single and double strand breaks (ssb and dsb) in a DNA-chloroterpyridine platinum complex induced by resonant photoabsorption in the L(III) innershell of a platinum atom. The number of ssb and dsb were measured in supercoiled plasmids (AG30) versus the chloroterpyridine platinum concentration, i.e., the ratio of intercalated molecules to the number of phosphate sites in DNA. A significant increase in the number of ssb and dsb was observed when the DNA contained intercalated molecules. This technique is an efficient way to induce ssb and dsb triggered by the atomic Auger effect.     

Heat effects on DNA repair after ionising radiation: hyperthermia commonly increases the number of non-repaired double-strand breaks and structural rearrangements

After ionising radiation double-strand breaks (dsb) are lethal if not repaired or misrepaired. Cell killing is greatly enhanced by hyperthermia and it is questioned here whether heat not only affects dsb repair capacity but also fidelity in a chromosomal context. dsb repair experiments were designed so as to mainly score non-homologous end joining, while homologous recombination was largely precluded. Human male G₀ fibroblasts were either preheated (45°C, 20 min) or not before X-irradiation. dsb induction and repair were measured by conventional gel electrophoresis and an assay combining restriction digestion using a rare cutting enzyme (NotI) and Southern hybridisation, which detects large chromosomal rearrangements (>100 kb). dsb induction rate in an X-chromosomal NotI fragment was 4.8 × 10^–3 dsb/Gy/Mb. Similar values were found for the genome overall and also when cells were preheated. After 50 Gy, fibroblasts were competent to largely restore the original restriction fragment size. Five per cent of dsb remained non-rejoined and 14% were misrejoined. Correct restitution of restriction fragments occurred preferably during the first hour but continued at a slow rate for 12–16 h. In addition, dsb appeared to misrejoin throughout the entire repair period. After hyperthermia the fractions of non-rejoined and misrejoined dsb were similarly increased to 13 and 51%, respectively. It is suggested that heat increases the probability of dsb being incorrectly rejoined but it is not likely to interfere with one dsb repair pathway in particular.     

Mathematical models of the generation of radiation-induced DNA double-strand breaks

The double-strand break (dsb) is one of the most critical lesions leading to a variety of radiobiological effects. In this paper, we reconsider the previously constructed and generally accepted mathematical models for dsb generation, and give a concrete mathematical basis for the generation of dsbs and the calculation of the number of induced dsbs, under the assumption of randomness in the break location in DNA and in the number of breaks. Using these models based on the Poisson distribution and the binomial distribution, we calculate the dose dependence of dsb generation. We deduced from our models that the dose dependence of the number of dsbs is described approximately as a quadratic form in both distribution models where dsb generation is accounted for by two ssbs. Previously reported experimental data on the dsb generation in phage DNA was found to be in good agreement with our models. Though the widely used model, the linear quadratic (LQ) model or the molecular theory of dsb formation based on the Poisson distribution, also gives the quadratic term, in spite of rough estimates or some mathematical incompleteness, a marked feature of our formulation is the absence of a parameter like the $\beta $ in the quadratic term that requires experimental data to determine. Thus in this study we provide mathematical validity to the generally accepted models of the number of dsb.     

The influence of oxygen on the induction of radiation damage in DNA in mammalian cells after sensitization by intracellular glutathione depletion

Treatment of mammalian cells with buthionine sulphoximine (BSO) or diethyl maleate (DEM) results in a decrease in the intracellular GSH (glutathione) and non-protein-bound SH (NPSH) levels. The effect of depletion of GSH and NPSH on radiosensitivity was studied in relation to the concentration of oxygen during irradiation. Single- and double-strand breaks (ssb and dsb) and cell killing were used as criteria for radiation damage. Under aerobic conditions, BSO and DEM treatment gave a small sensitization of 10-20 per cent for the three types of radiation damage. Also under severely hypoxic conditions (0.01 microM oxygen in the medium) the sensitizing effect of both compounds on the induction of ssb and dsb and on cell killing was small (0-30 per cent). At somewhat higher concentrations of oxygen (0.5-10 microM) however, the sensitization amounted to about 90 per cent for the induction of ssb and dsb and about 50 per cent for cell killing. These results strengthen the widely accepted idea that intracellular SH-compounds compete with oxygen and other electron-affinic radiosensitizers with respect to reaction with radiation-induced damage, thus preventing the fixation of DNA damages by oxygen. These results imply that the extent to which SH-compounds affect the radiosensitivity of cells in vivo depends strongly on the local concentration of oxygen.     

Effects of trypsin on X-ray-induced cell killing, chromosome abnormalities and kinetics of DNA repair in mammalian cells

When cells are trypsinized before irradiation a potentiation of X-ray damage may occur. This is known as the 'trypsin effect'. Potentiation of X-ray damage on cell killing was seen in V79 Chinese hamster cells but was marginal in Chinese hamster ovary (CHO K1) cells and not evident in murine Ehrlich ascites tumour (EAT) cells. Trypsinization did however increase the number of X-ray-induced chromosomal abnormalities in all 3 lines. To investigate the possibility that trypsin acts by digestion of proteins in chromatin, further experiments were performed to monitor DNA damage and repair. Induction of DNA breaks by X-rays was unaffected by trypsin but trypsinized EAT (suspension) cells repaired single-strand breaks (ssb) less rapidly than controls indicating an inhibitory effect of trypsin on ssb repair. However double-strand break (dsb) repair was unaffected by trypsin. It was also found that the EDTA solution in which the trypsin was dissolved also contributes to the inhibition of dsb repair. The results show that trypsinization can enhance X-ray-induced cell killing, chromosomal damage and DNA repair, the effect varying between cell lines.     

Simulation of DNA fragment distributions after irradiation with photons

The Monte Carlo track structure code PARTRAC has been further improved by implementing electron scattering cross-sections for liquid water and by explicitly modelling the interaction of water radicals with DNA. The model of the genome inside a human cell nucleus in its interphase is based on the atomic coordinates of the DNA double helix with an additional volume for the water shell. The DNA helix is wound around histone complexes, and these nucleosomes are folded into chromatin fibres and further to fibre loops, which are interconnected to build chromosomes with a territorial organisation. Simulations have been performed for the irradiation of human fibroblast cells with carbon K and aluminium K ultrasoft x-rays, 220 kVp x-rays and ⁶⁰Co γ-rays. The ratio single-strand breaks to double-strand breaks (ssb/dsb) for both types of ultrasoft x-rays is lower than for γ-rays by a factor of 2. The contributions of direct and indirect effects to strand break induction are almost independent of photon energy. Strand break patterns from indirect effects reflect differences in the susceptibility of the DNA helix to OH^• attack inside the chromatin fibre. Distributions of small DNA fragments (<3 kbp) are determined by the chromatin fibre structure irrespective of whether direct or indirect effects are causing the breaks. In the calculated fragment size distributions for larger DNA fragments (>30 kbp), a substantial deviation from random breakage is found only for carbon K irradiation, and is attributed to its inhomogeneous dose distribution inside the cell nucleus. For the other radiation qualities, the results for larger fragments can be approximated by random breakage distributions calculated for a yield of dsb which is about 10% lower than the average for the whole genome. The excess of DNA fragments detected experimentally in the 8–300 kbp region after x-ray irradiation is not seen in our simulation results. Received: 19 October 1998 / Accepted in revised form: 14 January 1999     

Enzymatic restriction of mammalian cell DNA: evidence for double-strand breaks as potentially lethal lesions

Permeabilized Chinese hamster cells were treated with the restriction endonucleases Pvu II and Bam H1 which generate blunt-ended and cohesive-ended DNA double-strand breaks (dsb), respectively. Cells were then assayed for their clonogenic ability. These experiments were performed to test the hypothesis that mammalian cell death following X-ray exposure arises from the induction of dsb in DNA, and via the formation of chromosomal aberrations. It was shown previously that Pvu II induces chromosome aberrations whereas Bam H1 was ineffective in this respect. The results reported here show that Pvu II simulates X-ray exposure, in causing a dose-dependent loss of the reproductive integrity of mammalian cells. Dsb generated by Pvu II, i.e. with blunt ends, can therefore be regarded as potentially clastogenic as well as potentially lethal. Bam H1 was found not to reduce cell survival in the same enzyme dose range. These results support the notion that X-irradiated mammalian cells undergo a mode of death in which dsb in the DNA cause chromosomal aberrations which are lethal as a result of loss of genetic material in the form of chromosome fragments, or as a result of chromosome bridge formation.     

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.     

The repair of double-strand breaks in the nuclear DNA of Saccharomyces cerevisiae and its genetic control

Summary With the use of neutral sucrose sedimentation techniques, the size of unirradiated nuclear DNA and the repair of double-strand breaks induced in it by ionizing radiation have been determined in both wild-type and homozygous rad52 diploids of the yeast Saccharomyces cerevisiae. The number average molecular weight of unirradiated DNA in these experiments is 3.0×10⁸±0.3 Daltons. Double-strand breaks are induced with a frequency of 0.58×10^-10 per Daltonkrad in the range of 25 to 100 krad. Since repair at low doses is observed in wild-type but not homozygous rad52 strains, the corresponding rad52 gene product is concluded to have a role in the repair process. Cycloheximide was also observed to inhibit repair to a limited extent indicating a requirement for protein synthesis. Based on the sensitivity of various mutants and the induction frequency of double-strand breaks, it is concluded that there are 1 to 2 double-strand breaks per lethal event in diploid cells incapable of repairing these breaks.     

DNA repair in the extremely radioresistant bacterium Deinococcus radiodurans

Deinococcus radiodurans and other members of the same genus share extraordinary resistance to the lethal and mutagenic effects of ionizing and u.v. radiation and to many other agents that damage DNA. While it is known that this resistance is due to exceedingly efficient DNA repair, the molecular mechanisms responsible remain poorly understood. Following very high exposures to u.v. irradiation (e.g. 500 Jm⁻², which is non-lethal to D. radiodurans), this organism carries out extremely efficient excision repair accomplished by two separate nucleotide excision repair pathways acting simultaneously. One pathway requires the uvrA gene and appears similar to the UvrABC excinuclease pathway defined in Escherichia coli. The other excision repair pathway is specific for u.v. dimeric photoproducts, but is not mediated by a pyrimidine dimer DNA glycosylase. Instead, it is initiated by a second bona fide endonuclease that may recognize both pyrimidine dimers and pyrimidine-(6–4)pyrimidones. After high doses of ionizing-radiation (e.g. 1.5Mrad), D. radiodurans can mend >100 double-strand breaks (dsb) per chromosome without lethality or mutagenesis. Both dsb mending and survival are recA-dependent, indicating that efficient dsb mending proceeds via homologous recombination. D. radiodurans contains multiple chromosomes per cell, and it is proposed that dsb mending requires extensive recombination amongst these chromosomes, a novel phenomenon in bacteria. Thus, D. radiodurans may serve as an easily accessible model system for the double-strand-break-initiated interchromosomal recombination that occurs in eukaryotic cells during mitosis and meiosis.