Extent of equilibrium perturbation of the DNA helix upon enzymatic methylation of adenine residues

The extent of equilibrium perturbation of the DNA helix associated with enzymatic methylation of dA residues has been determined by the agarose gel electrophoresis band-shift method. Utilization of EcoRI methylase under conditions of reduced specificity together with Escherichia coli dam methylase permitted modification of up to 300 dA residues/plasmid pBR322 dimer. A conformational change associated with methylation was observed, with the magnitude of the transition being linear with extent of modification of relaxed DNA circles. The conformational change corresponds to an unwinding of the DNA helix by 0.5 degrees/methyl group transferred to relaxed molecules. The magnitude of the effect was independent of temperature from 5-37 degrees C indicating that it is not the consequence of a thermal transition within this range.     

Measurement of radiation-induced DNA double-strand breaks by pulsed-field gel electrophoresis

We have examined the use of pulsed-field gel electrophoresis (PFGE) to measure DNA double-strand breaks induced in CHO cells by ionizing radiation. The PFGE assay provides a simple method for the measurement of DNA double-strand breaks for doses as low as 3-4 Gy ionizing radiation, and appears applicable for the measurement of damage produced by any agent producing double-strand breaks. The conditions of transverse alternating field electrophoresis determined both the sensitivity of the assay and the ability to resolve DNA fragments with different sizes. For example, with 0.8% agarose and a 1-min pulse time at 250 V for 18 h of electrophoresis, 0.39% of the DNA per gray migrated into the gel, and only molecules less than 1500 kb could be resolved. With 0.56% agarose and a 60-min pulse time at 40 V for 6 days of electrophoresis, 0.55-0.90% of the DNA per gray migrated into the gel, and molecules between 1500 and 7000 kb could be resolved.     

Alpha-helix to random-coil transition of two-chain, coiled coils: experiments on the thermal denaturation of doubly cross-linked beta beta tropomyosin

Equilibrium thermal denaturation curves (by circular dichroism) are reported for doubly cross-linked beta beta tropomyosin two-chain coiled coils. Cross-linking was performed by reaction of sulfhydryls with either ferricyanide or 5,5'-dithiobis(2-nitrobenzoate) (NbS2). The extent of reaction was determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and either by titration of residual sulfhydryls with NbS2 (ferricyanide cross-linking) or by determination of mixed disulfide (protein-S-SbN) through reaction with dithiothreitol (NbS2 cross-linking). The results indicate approximately 90% conversion to molecules with interchain cross-links at both C-36 and C-190. Thermal unfolding curves are compared with those obtained previously for non-cross-linked species. The curves are indistinguishable up to approximately 40 degrees C. Above approximately 40 degrees C, the doubly cross-linked species is more stable, but the transition is less steep. This relationship is also compared with that found between alpha alpha tropomyosin (a similar coiled coil made of a genetic variant chain having a sulfhydryl only at C-190) and its singly cross-linked derivative. Thermal curves for alpha alpha and beta beta non-cross-linked species are very similar, alpha alpha being somewhat more stable. For cross-linked alpha alpha, however, the curve sags at temperatures somewhat below the region of principal cooperative loss of helix, the latter occurring at higher temperature but with the same steepness as in the non-cross-linked case. The sag has been ascribed to a "pretransition" in the region of C-190. Thus, doubly and singly cross-linked species differ in that the former show no pretransition and decreased steepness in the principal transition.(ABSTRACT TRUNCATED AT 250 WORDS)     

A non-radioactive, PFGE-based assay for low levels of DNA double-strand breaks in mammalian cells

A PFGE method was adapted to measure DNA double-strand breaks (DSBs) in mammalian cells after low (0-25 Gy) doses of ionising radiation. Instead of radionuclide incorporation, DNA staining in the gel by SYBR-Gold was used, which lowered the background of DNA damage and could be applied to non-cycling cells. DSB level was defined as a product of a fraction of DNA released to the gel (FR) and a number of DNA fragments in the gel (DNA(fragm)) and expressed as a percentage above control value. The slope of the dose-response curve was two-fold higher compared to that with FR alone as DSB level indicator (31.4 versus 15.6% per Gy). Two alternative ways were proposed to determine the total amount of DNA, used for FR calculation: measurement of DNA content in a plug not subjected to electrophoresis, with the use of Pico-Green, or estimation of DNA released to the gel from a plug irradiated with 600 Gy of gamma-rays. The limit of DSB detection was 0.25 Gy for human G1-lymphocytes and 0.5-1 Gy for asynchronous cultures of human glioma M059 K and J or mouse lymphoma L5178Y-R and -S cells. Specificity of our PFGE assay to DSB was confirmed by the fact that no damage was detected after treatment of the cells with H(2)O(2), an inducer of single-strand DNA breaks (SSBs). On the contrary, the H(2)O(2) inflicted damage was detected by neutral comet assay, attaining 160% above control (equivalent to 2.5 Gy of X-radiation). DSB rejoining, measured in cells after X-irradiation with a dose of 10 Gy, generally proceeded faster than that measured previously after higher (30-50 Gy) doses of ionising radiation. Clearly seen were defects in DSB rejoining in radiosensitive M059 J and L5178Y-S cells compared to their radioresistant counterparts, M059 K and L5178Y-R. In some cell lines, a secondary post-irradiation increase in DSB levels was observed. The possibility is considered that these additional DSBs may accumulate during processing of non-DSB clustered DNA damage or/and represent early apoptotic events.     

Assessment of radiation-induced DNA damage in human blood lymphocytes using the single-cell gel electrophoresis technique.

The ability of the alkaline single-cell gel (SCG) electrophoresis technique to detect single-strand breaks and alkali-labile DNA damage in human cells induced by low doses of radiation was evaluated. Peripheral blood lymphocytes were irradiated with gamma-rays from a 137Cs source at doses from 0.01 to 1 Gy and exposed to alkali (pH greater than 13) for 20, 40 or 60 min and then electrophoresed at 25 V and 300 mA for either 20 or 40 min. The extent of DNA damage that was expressed and detected as DNA migration depended directly on the dose of radiation, the duration of exposure to alkali and the length of electrophoresis. At all experimental conditions tested, it was possible to detect a significant increase in DNA damage induced by a radiation dose as low as 0.05 Gy. Based on an analysis of the ratio of the range to the standard deviation for each radiation dose and experimental condition, the distribution of damage among cells for all doses was neither excessively homogeneous nor heterogeneous. Furthermore, the distribution was independent of radiation treatment. The SCG technique is rapid and sensitive, and useful for investigations concerned with effects of low doses of radiation.     

Induction and rejoining of large DNA fragments after ion irradiation.

Radiation-induced DNA double-strand breaks (DSBs) were analyzed by separating large DNA fragments by pulsed-field gel electrophoresis. Human U-343MG glioma and K562 erythroleukemia cells were irradiated with 60Co gamma rays or nitrogen ions with high linear energy transfer (125 keV/microm). By comparing the fraction of DNA released into the gel below different size thresholds, corresponding to megabase-pair-sized DNA fragments, the relative effectiveness of the nitrogen ions was found to be dependent on both dose and the threshold size used in the evaluation. This dose dependence was most evident for the smallest threshold (6 Mbp) and was due to a linear dose response for release of the fragments for the ions compared to the curvilinear response for the gamma rays. The two curves intersected, and the relative yield of fragments (nitrogen ions/gamma rays) decreased from more than 3 below 1.5 Gy to 0.8 at 30 Gy. For the larger sizes (6-10.5 Mbp), the relative yield was constant at around 0.7. Thus the ion-induced fragments were shifted to smaller sizes compared to the 60Co gamma rays, and the data for nitrogen ions could not be fitted to random fragment distributions at doses < or =20 Gy. From these results, we conclude that a substantial fraction of the DSBs induced by heavy ions were nonrandomly distributed, correlated with DSBs within a region of < or =2 Mbp. After a dose of 20 Gy, the rejoining curves for ion-induced DSBs were different for each fragment size, resulting in different levels of unrejoined breaks after 6 h.     

Renal damage in the mouse: the effect of d(4)-Be neutrons

A further study on the response of the mouse kidney to d(4)-Be neutrons (EN = 2.3 MeV) is described. The results confirm and augment the work published previously by Stewart et al. [Br. J. Radiol. 57, 1009-1021 (1984)]; the present paper includes the data from a "top-up" design of experiment which extends the measurements of neutron RBE (relative to 240 kVp X rays) down to X-ray doses of 0.75 Gy per fraction. The mean RBE for these neutrons increases from 5.8 to 7.3 as X-ray dose per fraction decreases from 3.0 to 1.5 Gy in the kidney. This agrees with the predictions from the linear quadratic (LQ) model, based on the renal response to X-ray doses above 4 Gy per fraction. The mean RBE estimate from a single dose group at 0.75 Gy per fraction of X rays is, however, 3.9. This is below the LQ prediction and may indicate increasing X-ray sensitivity at low doses. Data from this study and from those published previously have been used to determine more accurately the shape of the underlying response to d(4)-Be neutrons; an alpha/beta ratio of 20.5 +/- 3.7 Gy was found. The best value of alpha/beta for X rays determined from these experiments was 3.04 +/- 0.35 Gy, in agreement with previous values.     

No detectable misrejoining in double-minute chromosomes.

Pulsed-field gel electrophoresis combined with Southern hybridization and rare-cutting restriction endonuclease digestion has been used recently to quantify misrejoining of DNA double-strand breaks (DSBs) resulting from exposure to ionizing radiation. Measurements are made 24 h after a high dose of radiation. These studies have suggested that a large fraction of DSBs are misrejoined to result in gross rearrangements. In the experiments described here, we show that elimination of broken DNA also eliminates "misrejoined" DNA. Mouse cells resistant to high levels of methotrexate by virtue of 100-fold amplification of the dyhydrofolate reductase (Dhfr) gene were treated with 50 and 100 Gy of ionizing radiation. The cells were allowed to repair the damage for 24 h. After the repair period, the cells were immobilized in agarose. Aliquots of each sample were pre-electrophoresed to remove linear DNA molecules smaller than 6 Mbp resulting from apoptosis or necrosis. The samples repairing damage from 50 or 100 Gy that did not receive the pre-electrophoresis showed high levels of label in a region of the lane that could be due to misrejoining DNA molecules. However, when the DNA from cells undergoing apoptosis or necrosis was removed from these samples, the levels of "misrejoined" DNA were reduced to levels far below those of unirradiated controls. These results suggest that other radiation-induced effects present 24 h after irradiation with 50 or 100 Gy are more significant than misrejoining for altering hybridization to regions of the lane outside the specific bands. Measurements of misrejoining using PFGE, rare-cutting restriction endonucleases, and Southern hybridization are likely to be compromised by nonspecific hybridization to broken and difficult-to-digest DNA resulting from apoptosis or necrosis.     

The genotoxicity of alpha particles in human embryonic skin fibroblasts

Cell inactivation and induced mutation frequencies at the hypoxanthine-guanine phosphoribosyl transferase (HGPRT) locus have been measured in cultured human fibroblasts (GM10) exposed to alpha particles from 238Pu (LET at the cell surface was 100 keV/microns) and 250 kVp X rays. The survival curves resulting from exposure to alpha particles are exponential. The mean lethal dose, D0, is approximately 1.3 Gy for X rays and 0.25 Gy for alpha particles. As a function of radiation dose, mutation induction at the HGPRT locus was linear for alpha particles whereas the X-ray-induced mutation data were better fitted by a quadratic function. When mutation frequencies were plotted against the log of survival, mutation frequency at a given survival level was greater in cells exposed to alpha particles than to X rays.     

Asymmetric field inversion gel electrophoresis: a new method for detecting DNA double-strand breaks in mammalian cells

A new method is described for detecting DNA double-strand breaks (DSBs) that utilizes asymmetric field inversion gel electrophoresis (AFIGE). DNA purified from cells in agarose plugs is subjected to AFIGE and DNA breakage quantitated by the fraction of DNA released from the plug. To test the specificity of the method for DNA DSBs, purified DNA in agarose plugs was treated for increasing times with restriction endonuclease, XhoI. After an initial time period, the fraction of DNA released increased in direct proportion to time. This correlates with the expected response for a randomly broken DNA molecule. In contrast, treatment with the single-strand breaking agent, hydrogen peroxide, over a 1000-fold range produced no release of DNA from the plug. Thus the assay appears to be specific for DNA DSBs and was used to measure DNA breaks induced by gamma radiation. Purified DNA, irradiated in agarose plugs, exhibited a log-linear dose response up to doses that release greater than 90% DNA from the plug. When live cells were irradiated in agarose, a similar linear dose response was observed up to 40 Gy and a significant signal as low as 2.5 Gy. Also in live cells, a threefold lower percentage of DNA was released from the plug over the same dose range. However, less DNA per gray is released at doses above 40 Gy and may reflect a crosslinking effect produced by the irradiation of DNA in live cells. DNA which was "pulse-labeled" was used to test the effect of DNA replication on the ability of AFIGE to detect DNA DSBs. Replicating DNA irradiated in the cell or after purification exhibited a reduced rate of release from the plug per dose of irradiation. Overall, the above results indicate that AFIGE is a sensitive method for detecting DSBs in DNA.     

A model for indication of DNA functional and structural changes at low-dose radiation

The work offers an "in vivo-in vitro" model which allows to identify DNA's both structural and functional damages caused by gamma-irradiation in doses from 0.02 to 0.25 Gy. As a donor the authors used an irradiated pTTQ 19 plasmide which had two marker genes: the ampicilline-resistant gene (amp(r)) and beta-galactosidase structural gene (lacZ alpha). E. coli GM 109 bacterial stamm transformed by the irradiated plasmide was used as a recipient. The structural damages of the irradiated plasmide were registered by DNA electrophoretic analysis in agarose gel. The plasmide DNA dysfunctions were assessed by its ability to pass on ampicilline resistance to E. coli bacterial cells as well as by beta-galactosidase level. The irradiated plasmide was found to have a tendency to decrease beta-galactosidase activity and number of E. coli ampicilline-resistant transformants depending on the received radiation dose: by 24.5% (0.05 Gy), 30.9% (0.19 Gy), and by 40.2% (0.25 Gy).     

The dose-dependent fragmentation of chromatin in human fibroblasts by 3.5-MeV alpha particles from 238Pu: experimental and theoretical considerations pertaining to single-track effects

The technique of premature chromosome condensation (PCC) was used to examine the dose-response relationship for the production of interphase (G0) chromosome fragments in noncycling normal human fibroblasts following exposure to 238Pu alpha particles, with special emphasis on the low-dose region. The dose response was convincingly linear from 0.2 to 3.0 Gy. Analysis of further data collected over a dose range of 1.1 to 22.4 cGy provided no evidence of deviation from linearity in this low-dose region. The fact that this lower dose range extends into the region where single-particle effects are dominant suggests that a linear extrapolation of this response from higher to lower doses is valid. Ratios of coefficients for the induction of fragments produced by 238Pu alpha particles versus 60Co gamma rays gave an RBE of 2.34 +/- 0.09. Distributions of fragments among 60Co gamma-irradiated cells were consistent with a Poisson expectation of random damage. In contrast, overdispersion appeared to be a general feature of 238Pu alpha-particle-induced fragmentation, a phenomenon explainable under the assumption that single-particle traversals are capable of producing multiple PCC fragments. Data obtained were used to estimate practical and theoretical lower-dose limits of detection of initial chromatin breaks provided by current PCC methodology.     

Early melting of supercoiled DNA.

Denaturing gradient gel electrophoresis (formamide with urea) has been used to study the melting of supercoiled DNA. A linear gradient of denaturant concentration proportional to a 25 degrees C linear increase of temperature (Teff) from the left to the right edge of the gel was created perpendicular to DNA migration. The mobility of supercoiled DNA molecules was shown to drop to the level of relaxed molecules a long way (5-30 degrees C) before linear DNA began to melt. The further increase of Teff, including the melting range for linear molecules, caused no appreciable changes in the mobility of relaxed molecules. The transition curves are S-shaped for all the topoisomers, and an increase of superhelicity shifts the transition towards lower Teff values. The analysis of the results indicates that the observed relaxation of superhelical molecules is due to denatured region forming in them, their size increasing with the topoisomer number.     

Acceleration of DNA-double strand rejoining during the adaptive response of Chlamydomonas reinhardtii

Newly developed constant-field low voltage electrophoresis (adapted for algae cells by us) was applied to quantify the induction and repair of nuclear DNA double-strand breaks, by measuring the movement of DNA out of the starting wells into the electrophoresis gel using a UV-gel scan and computer analysis of DNA-ethidium bromide fluorescense (Syngene; Gene tools). A cell-wall-less mutant strain of Chlamydomonas reinhardtii (CW15) was used; the DNA and proteins are easily accessible because of the lack of an outer cell wall. Our results showed that giving a small priming dose (50 Gy) led to a small acceleration of dsb rejoining. When the magnitude of the priming dose was progressively increased, there was a corresponding decrease in the fraction of damage remaining at 4 hours after radiation exposure (to a test dose of 500 Gy). This indicates an upregulated rejoining of dsb following exposure of cells to the priming dose, which may be related to the strong adaptive response in this organism. Protein synthesis inhibitors were found to reduce the rate of rejoining of dsb, and from earlier results are known to inhibit the adaptive response. Thus, the adaptive response is likely to be dependent on increased dsb rejoining and depends on de novo protein synthesis. The nature of these proteins has not yet been established. C. reinhardtii CW15 is an attractive model system in which to study the underlying mechanisms of the adaptive response to ionizing radiation, and its underlying link with dsb rejoining. The results are interesting both from a basic biological point of view, and as a means to further understand the response of tumour cells to radiation therapy since the adaptive response has been postulated to determine the shape of the "shoulder" region of the survival curve of cells at low doses of radiation.     

SFM studies of carbon ion induced damages in plasmid DNA

In this study we present for the first time detailed scanning force microscopy (SFM) investigations of carbon ion induced damages in plasmid DNA in order to obtain information about the biological effectiveness of particle radiation. For this purpose, we have combined SFM and gel electrophoresis measurements in a dose range between D = 0 Gy and 5000 Gy. After irradiation with C ions, the percentage of double-strand breaks (DSBs) increases drastically, i.e. from initially 0% for D = 0 Gy to 38% for D = 5000 Gy. Increasing the dose over the total range is accompanied by a shortening of the average fragment length from L = 1100 nm to L = 575 nm. In addition to our experiments, the average numbers of induced DSBs per irradiated plasmid and per broken plasmid have been calculated from the SFM measurements. The most important among the numerous results is that a significant amount of plasmids has suffered more than two DSBs for all applied doses, indicating multiple DSBs. The number of DSBs per broken plasmid increases from approximately 1.7 after irradiation with a dose of D = 250 Gy to 3.2 after exposure to the highest dose of D = 5000 Gy. The results provide experimental data for the spatially correlated production of DSBs after carbon irradiation, that are relevant to the understanding of its biological effectiveness.     

用脉冲电场凝胶电泳检测电离辐射所致哺乳动物细胞DNA双链断裂

 本文将反向交变电场和六角形电极电场这两种脉冲电场凝胶电泳技术应用于X线照射小鼠乳癌细胞SR-1所致DNA双链断裂的检测,在本实验条件下,用这种电泳都能检测到低至1.5Gy照射所产生的DNA双链断裂,并且用六角形电极电场电泳获得了DNA双链断裂程度与照射剂量之间的良好线性关系,此外,还用此方法观察了不同浓度自由基清除剂DMSO对X线照射SR-1细胞所致DNA双链断裂的保护作用,结果进一步证实本方法的可靠性。     

Structural changes in plasma membranes prepared from irradiated Chinese hamster V79 cells as revealed by Raman spectroscopy

The effect of gamma irradiation on the integrity of plasma membranes isolated from Chinese hamster V79 cells was investigated by Raman spectroscopy. Plasma membranes of control V79 cells show transitions between (-) 10 and 5 degrees C (low-temperature transition), 10 and 22 degrees C (middle-temperature transition), and 32 and 40 degrees C (high-temperature transition). Irradiation (5 Gy) alters these transitions markedly. First, the low-temperature transition shifts to higher temperature (onset and completion temperatures 4 and 14 degrees C). Second, the middle-temperature transition shifts up to the range of about 20-32 degrees C, but the width remains unchanged. Third, the higher temperature transition broadens markedly and shifts to the range of about 15-40 degrees C. Protein secondary structure as determined by least-squares analysis of the amide I bands shows 36% total helix, 55% total beta-strand, and 9% turn plus undefined for control plasma membrane proteins. Plasma membrane proteins of irradiated V79 cells show an increase in total helix (40 and 45% at 5 and 10 Gy, respectively) and a decrease in the total beta-strand (48 and 44% at 5 and 10 Gy, respectively) structures. The qualitative analysis of the Raman features of plasma membranes and model compounds in the 1600 cm-1 region, assigned to tyrosine groups, revealed that irradiation alters the microenvironment of these groups. We conclude that the radiation dose used in the survival range of Chinese hamster V79 cells can cause damage to plasma membrane proteins without detectable lipid peroxidation, and that the altered proteins react differently with lipids, yielding a shift in the thermal transition properties.     

Chromatographic studies on the radiolysis of DNA in aqueous solution

Radiation-induced degradation of double-stranded DNA from calf thymus in aqueous solution with sodium phosphate was studied by conventional gel chromatography and by high-performance liquid-gel permeation chromatography. Comparison of the data after radiolysis of aqueous solutions of DNA under anaerobic and aerobic conditions indicates that double-strand breakage is not enhanced by oxygen. An increase of ionic strength impedes the break-down of the DNA molecules, so that loss of DNA can only be observed at doses above 100 Gy. Only reactions of OH-radicals contribute to the fragmentation of DNA, while the presence of hydrated electrons, H.-or formate radicals does not lead to a loss of highly polymerized DNA up to doses of 1500 Gy. High-performance liquid-chromatography proved to be an excellent method of studying the degradation of macromolecules as a function of dose.     

80MeV/u20Ne10+离子对SMMC-7721细胞DNA损伤及细胞周期的影响

从辐照剂量和修复时间两个角度研究了重离子辐照对肿瘤细胞DNA损伤及细胞周期的影响,为重离子治癌的临床应用积累基础数据。不同剂量的80MeV／u^20Ne^10 辐照SMMC—7721细胞样品,利用单细胞凝胶电泳技术(Single Cell Gel Electrophoresis,SCGE)对细胞DNA损伤进行了检测,利用流式细胞技术(Flow Cytometry Methods,FCM)对细胞周期变化进行了分析。80MeV／u^20Ne^10 辐照后4小时内,SMMC—7721细胞的DNA损伤与辐照剂量呈线性关系,在0小时组其线性相关因子r为0．9621,4小时组为0．914;随着修复时间的增加,DNA损伤与辐照剂量不再线性相关,但0．5Gy,1Gy和2Gy三个剂量点的DNA损伤程度极为相近。另外,重离子辐照后SMMC—7721细胞发生S期和G2／M期阻滞现象,其随剂量变化及时间变化的规律不同于X、γ等低LET(Linear Energy Transfer)射线辐照。