Size Distribution Of DNA Fragments Research Articles

Radiation breakage of DNA: a model based on random-walk chromatin structure.

Monte Carlo computer software, called DNAbreak, has recently been developed to analyze observed non-random clustering of DNA double strand breaks in chromatin after exposure to densely ionizing radiation. The software models coarse-grained configurations of chromatin and radiation tracks, small-scale details being suppressed in order to obtain statistical results for larger scales, up to the size of a whole chromosome. We here give an analytic counterpart of the numerical model, useful for benchmarks, for elucidating the numerical results, for analyzing the assumptions of a more general but less mechanistic "randomly-located-clusters" formalism, and, potentially, for speeding up the calculations. The equations characterize multi-track DNA fragment-size distributions in terms of one-track action; an important step in extrapolating high-dose laboratory results to the much lower doses of main interest in environmental or occupational risk estimation. The approach can utilize the experimental information on DNA fragment-size distributions to draw inferences about large-scale chromatin geometry during cell-cycle interphase.

Modeling and optimization of DNA recombination

This paper discusses predictive models for quantifying the outcome of DNA recombination employed in directed evolution experiments for the generation of novel enzymes. Specifically, predictive models are outlined for (i) tracking the DNA fragment size distribution after random fragmentation and subsequent assembly into genes of full length and (ii) estimating the fraction of the assembled full length sequences matching a given nucleotide target. Based on these quantitative models, optimization formulations are constructed which are aimed at identifying the optimal recombinatory length and parent sequences for maximizing the assembly of a sought after sequence target. Computational results show that the recombination outcome is a ‘complex’ function of the recombinatory length and recombined sequences and illustrate the magnitude of improvements that can be realized.

High-throughput flow cytometric DNA fragment sizing.

The rate of detection and sizing of individual fluorescently labeled DNA fragments in conventional single-molecule flow cytometry (SMFC) is limited by optical saturation, photon-counting statistics, and fragment overlap to approximately 100 fragments/s. We have increased the detection rate for DNA fragment sizing in SMFC to approximately 2000 fragments/s by parallel imaging of the fluorescence from individual DNA molecules, stained with a fluorescent intercalating dye, as they passed through a planar sheet of excitation laser light, resulting in order of magnitude improvements in the measurement speed and the sample throughput compared to conventional SMFC. Fluorescence bursts were measured from a fM solution of DNA fragments ranging in size from 7 to 154 kilobase pairs. A data acquisition time of only a few seconds was sufficient to determine the DNA fragment size distribution. A linear relationship between the number of detected photons per burst and the DNA fragment size was confirmed. Application of this parallel fluorescence imaging method will lead to improvements in the speed, throughput, and sensitivity of other types of flow-based analyses involving the study of single molecules, chromosomes, cells, etc.

Polymer chromosome models and Monte Carlo simulations of radiation breaking DNA.

Chromatin breakage by ionizing radiation is relevant to studies of carcinogenesis, tumor radiotherapy, biodosimetry and molecular biology. This article focuses on computer analysis of chromosome irradiation in mammlian cells. Polymer physics and Monte Carlo numerical methods are used to develop a coarse-grained computational approach. Chromatin is modeled as a random walk on a cubic lattice, and the radiation tracks hitting the chromatin are modeled as straight lines hitting lattice sites. Each track can make a cluster of DSBs on a chromosome. The results obtained replace conjectured DNA fragment-size distribution functions in the recently developed RLC formalism by more mechanistically motivated distributions. The discrete lattice algorithm reproduces features of current radiation experiments relevant to chromatin on large scales. It approximates the continuous formalism and experimental data with adequate precision. It was also found that assuming either fixed chromatin with correlations among different clusters of DSBs or moving chromatin with no such correlations gives virtually identical numerical predictions.

Locations of radiation-produced DNA double strand breaks along chromosomes: a stochastic cluster process formalism

Ionizing radiation produces DNA double strand breaks (DSBs) in chromosomes. For densely ionizing radiation, the DSBs are not spaced randomly along a chromosome: recent data for size distributions of DNA fragments indicate break clustering on kbp–Mbp scales. Different DSB clusters on a chromosome are typically made by different, statistically independent, stochastically structured radiation tracks, and the average number of tracks involved can be small. We therefore model DSB positions along a chromosome as a stationary Poisson cluster process, i.e. a stochastic process consisting of secondary point processes whose locations are determined by a primary point process that is Poisson. Each secondary process represents a break cluster, typically consisting of 1–10 DSBs in a comparatively localized stochastic pattern determined by chromatin geometry and radiation track structure. Using this Poisson cluster process model, which we call the randomly located clusters (RLC) formalism, theorems are derived for how the DNA fragment-size distribution depends on radiation dose. The RLC dose-response relations become non-linear when the dose becomes so high that DSB clusters from different tracks overlap or adjoin closely. The RLC formalism generalizes previous models, fits current data adequately and facilitates mechanistically based extrapolations from high-dose experiments to the much lower doses of interest for most applications.

High resolution confocal laser scanning microscopy analysis of DNA excision repair capability in small volume marrow samples exposed to DNA directed treatment moieties.

Assessment of resistance to drug moieties in tissue culture is complicated by limited sample, clonal selection and alteration of cycling fraction and cycle duration in clonally mixed lesions. DNA damage assessment by single cell gel electrophoresis (SCGE) of excised DNA is limited by non-linear analysis in fluorescent light microscopy. Confocal Laser Scanning Microscopy (CLSM) with high N.A. magnification allows for quantitation of total excised DNA fragment size distribution but is still limited by the large volume required for labour intensive SCGE, precluding multi-exposure clinical testing. To optimise sample requirement for SCGE and CLS. Standard slide mounted bed gels were punched with multiple coded 6 mm wells and filled with suspensions of cells subjected to drug/concentration variations. After SCGE, 30 microns frozen sections were prepared of each well and mounted in ethidium bromide solution on multi-well hydrophilic slides to allow for short working distance of high resolution CLSM in a Zeiss Axiovert L410 SM. Testing for feasibility, reproducibility and consistency used both cultured standard leukaemic cell lines, normal human control marrow and clinical samples. Multiple well SCGE followed by frozen section, high resolution CLSM allows for rapid analysis of high numbers of multiple drug exposure permutations clinically required.

Investigation of Neutron-Induced Damage in DNA by Atomic Force Microscopy: Experimental Evidence of Clustered DNA Lesions

Using atomic force microscopy (AFM), we have investigated neutron-induced DNA double-strand breaks in plasmids in aqueous solution. AFM permits direct measurement of individual DNA molecules with an accuracy of a few nanometers. Furthermore, the analysis of the DNA fragment size distribution is non-parametric, whereas other methods are dependent on the model. Neutron irradiation of DNA results in the generation of many short fragments, an observation not made for damage induced by low-LET radiation. These data provide clear experimental evidence for the existence of clustered DNA double-strand breaks and demonstrate that short DNA fragments may be produced by such radiations in the absence of a nucleosomal DNA structure.

A formalism for analysing large-scale clustering of radiation-induced breaks along chromosomes

Purpose: To model intrachromosomal clustering of DSB (DNA double strand breaks) induced by ionizing radiation. That DSB are located non-randomly along chromosomes after high LET irradiation, with clustering even at extremely large scales, has been confirmed by recent pulsed field gel electrophoresis data for size distributions of DNA fragments. We therefore extend the standard random-breakage model for DNA fragment-size distributions to a more general 'clustered-breakage' formalism, which can take correlations of DSB locations along a chromosome into account. Methods: The new formalism is based mainly on a one-track probability distribution, describing the DNA fragment-size pattern due to a single primary high-energy particle, a pattern determined by track structure and chromatin geometry. Multitrack fragment-size distributions are derived mathematically from the one-track distribution, so that dose-response relations are obtained. Results: The clustered-breakage formalism is applicable to any chromosomal geometry and any radiation track structure. It facilitates extrapolations of high-dose data to the much lower doses of interest for most applications. When applied to recently published data for irradiation of mammalian cells with ions of LET 100 keV mu m -1 it indicates a pattern of Mbp-scale DSB clusters, each containing a number of DSB and corresponding to a very large-scale, multiply-damaged chromatin site. Although DSB are bunched, DSB clusters are scattered almost at random throughout the genome. Estimates of DSB yield are markedly increased by resolving such clusters into individual DSB. The dose-response relation for fragments of a given size becomes non-linear when clusters from different tracks interlace or adjoin, as can occur for high doses and large sizes. Conclusions: DSB clustering along chromosomes, which influences important radiobiological endpoints, is described quantitatively by the clustered-breakage formalism.

Migration patterns in pulsed-field electrophoresis of DNA restriction fragments from log-phase mammalian cells after irradiation and incubation for repair.

An assay system was developed to detect changes of restriction fragment profiles obtained after pulsed-field gel electrophoresis (PFGE) for DNA from mammalian cells that were irradiated and incubated for repair. DNA was prepared from irradiated log-phase human melanoma cells (MRI 221) after incubation for repair (6 h) and was digested with the rare-cutting restriction enzyme NotI (RE) prior to PFGE separation. DNA-fragment size distributions were compared to the respective PFGE profiles from unirradiated controls. After doses of 5 and 10 Gy (plus a 6-h incubation for repair), the relative amount of DNA retained in the plug during PFGE was increased. For higher doses (30 and 60 Gy), this phenomenon was superimposed by the residual fragmentation (25-30% of the initial breakage of 0.42 dsb/100 Mbp/Gy). Since irradiated cells accumulated in S phase during incubation for repair, a correction for the reduced electrophoretic migration of DNA from S-phase cells was necessary, and a 0.5% increase in DNA retention per 1% S-phase increment was found. However, the % retention for the 5 Gy plus repair sample was significantly higher than for the S-phase adjusted control (p < 0.01). Radiation induced DNA-protein crosslinks cannot account for the observed phenomenon because of the extensive proteolysis in DNA preparation, and also a loss of restriction enzyme recognition sites appear to be an unlikely explanation from simple quantitative considerations. Based on the recent observation of misrejoining during dsb repair, it is proposed that the incorrect joining of DNA ends also causes a more random distribution of replicating DNA in restriction fragments derived from cells in S phase after incubation for repair. This process would necessarily increase the proportion of DNA unable to migrate in PFGE.

Erratum: Methods for the Quantification of DNA Double-Strand Breaks Determined from the Distribution of DNA Fragment Sizes Measured by Pulsed-Field Gel Electrophoresis

Erratum: Methods for the Quantification of DNA Double-Strand Breaks Determined from the Distribution of DNA Fragment Sizes Measured by Pulsed-Field Gel Electrophoresis

Methods for the Quantification of DNA Double-Strand Breaks Determined from the Distribution of DNA Fragment Sizes Measured by Pulsed-Field Gel Electrophoresis

Different methods were used for evaluating data for DNA double-strand breaks (DSBs), as obtained by pulsed-field gel electrophoresis (PFGE) after X irradiation of Chinese hamster ovary cells. A total of 60 data points in the dose range of 0 to 116 Gy, along with repair data for 30 and 60 Gy, were analyzed by four methods: (1) percentage of DNA released from the plug, (2) specific size markers (percentage of DNA less than specific sizes, (3) fragment size distributions and (4) shape of the molecular weight (M) distributions. With the last method, both the slope and the intercept of the logarithm of the amount of radioactive DNA/delta M/M plotted as a function of M were used for calculating DSBs/100 Mbp. The slope and the intercept analyses differ in that the former is relatively independent of DNA trapped in the agarose plugs, i.e. cannot be released by doses of 100-150 Gy, whereas the intercept is dependent on the percentage of DNA trapped. Also, calculations of DSBs/100 Mbp for methods 1, 2 and 3 depend on the amount of DNA trapped in the plug. However, the slope method is unreliable for doses below about 20 Gy, and the scatter of data points is much greater than that obtained by the intercept method and by methods 1, 2 and 3. Therefore, the fragment size distribution and the specific size marker methods give the most consistent results, with 0.49 +/- 0.03 (95% CI) (DSBs/100 Mbp)/Gy. With the specific size marker method, however, care must be taken in selection of size markers in relation to the levels of DSBs of interest. Assuming randomly distributed DSBs, all four methods gave essentially the same results; i.e., the dose response was linear with a calculated level of 0.5-0.6 (DSBs/100 Mbp)/Gy, which is the same as 0.47-0.62 determined previously by calibrating with 125IdU.

Linear Induction of DNA Double-Strand Breakage with X-Ray Dose, as Determined from DNA Fragment Size Distribution

Pulsed-field gel electrophoresis has been applied to separate DNA from mouse L1210 cells exposed to X-ray doses of 1 to 50 Gy. Simultaneous separation of marker chromosomes in the range 0.1 to 12.6 Mbp allowed calculation of the size distribution of the radiation-induced fragments. The distribution was consistent with a random induction of double-strand breaks (DSBs). A theoretical relationship between the size distribution of such fragments and the average number of induced breaks was used to calculate the yield and dose response. The DNA distribution was determined by both radiolabeling and fluorescence staining. Two independent methods were used to evaluate the radiation-induced yield of DSBs, both assuming that all DNA is broken at random. In the first method we compared the theoretical and experimental fraction of DNA that is below a given size limit. By this method we estimated the yield to be 0.006-0.007 DSB/Gy per million base pairs using the radiolabel and 0.004-0.008 DSB/Gy per million base pairs by fluorescence staining. The dose response was linear in both cases. In the second method we looked only at the size distribution in the resolving part of the gel and compared it to the theoretical distribution. By this method a value of approximately 0.012 DSB/Gy/Mbp was found, using fluorescence as a measure of DNA distribution. In a normal diploid mammalian genome of size 6000 Mbp, this is equivalent to a yield of 25-50 DSBs/Gy or 70 DSBs/Gy, respectively. The second approach, which looks only at the smaller fragments, may overestimate the yield, while the first approach suffers from uncertainties about the fraction of DNA irreversibly trapped in the well. The assay has the capacity to detect a dose of less than 1 Gy.

Differential Dependence on Chromatin Structure for Copper and Iron Ion Induction of DNA Double-Strand Breaks

The induction of DNA DSB (double-strand breaks) in isolated nuclear chromatin by Cu(II) or Fe(II)-EDTA in the presence of H2O2 and ascorbate has been compared to DSB induction by gamma-radiation. V79 nuclei embedded in agarose plugs were treated with each agent on ice, and the resultant DNA fragments were analyzed by pulsed-field gel electrophoresis. In the absence of low molecular weight radical scavengers, both irradiation and treatment with iron ion induced random DSB, as judged by the size distribution of DNA fragments, and the yield of DSB in each case was enhanced by either the expansion of chromatin (approximately 5-fold) or the removal of histones (21-25-fold) before treatment. In contrast, treatment with Cu(II) produced small DNA fragments of uniform size (approximately 100-200 kbp), independent of the yield of DSB. In addition, neither the DNA fragment size nor the yield of DSB produced by Cu(II) was affected by the prior removal of histones from chromatin. Deproteinized DNA was degraded randomly by Cu(II) but at a slower rate than observed for chromatin. In the presence of ascorbate, H2O2 was found to be essential for DSB induction by Fe(II)-EDTA but not by Cu(II), possibly because H2O2 can be produced from ascorbate and Cu(II) in the presence of oxygen. Despite the above differences between the production of DSB by the two metal ions, DSB induction in native chromatin by either metal ion was blocked by 0.1 M EDTA or 0.25 M thiourea but was resistant to the hydroxyl radical scavengers 0.25 M DMSO and 0.25 M mannitol.(ABSTRACT TRUNCATED AT 250 WORDS)

An electrophoretic approach to the assessment of the spatial distribution of DNA double-strand breaks in mammalian cells.

An approach is presented making it possible to investigate whether breaks in fragmented mammalian chromosomal DNA were induced randomly and independently from each other. Genomic DNA isolated from mammalian cells irradiated with gamma-rays or restriction enzyme-treated human DNA was resolved according to size using pulsed field gel electrophoresis, and the resulting DNA mass distributions were measured in ethidium bromide-stained gels. The DNA profiles thus obtained were compared to the predictions on DNA fragment size distribution which follow from a so-called random breakage model to test whether the experimental outcome is compatible with the assumption of a random localization of breaks. Comparisons of fragment distributions may be performed utilizing two equivalent representations that are linked by an adequate transformation. Considering either directly measurable DNA mass profiles in units of migration distances along a gel lane or transformed distributions in units of molecular length, we show for gamma-irradiated samples that the predictions derived from the employed models agree well with the observed data, thus allowing an immediate quantification of double-strand breaks (DSB). Using restriction enzyme-treated DNA as a paradigm, the disagreement of predicted and observed data shows the applicability of our approach to the detection of a non-random distribution of DSB. Therefore, we suppose that our approach may also be useful to reveal a clustering of DSB, which is postulated to occur after damage induction by densely ionizing radiation. Furthermore, investigations on the spatial distribution of chemically or endogenously produced DSB, as well as residual DSB after repair, may be attempted.

Initial radiation-induced DNA damage in human tumour cell lines: a correlation with intrinsic cellular radiosensitivity.

The role of the initial DNA double-strand breaks (dsb) as a determinant of cellular radiosensitivity was studied in human breast and bladder cancer cell lines. Cell survival was measured by monolayer colony-forming assay as appropriate and differences in radiosensitivity were seen (alpha-values ranged from 0.12 to 0.54). After pulsed-field gel electrophoresis (PFGE) the initial slopes of dose-response curves were biphasic with a flattening of the curves above 30 Gy. When the frequency of DNA dsb induction was assessed using a mathematical model based on the DNA fragment size distribution into the gel lane, we found a statistically significant relationship between the number of DNA dsb induced and the corresponding alpha-values and fraction surviving after 2Gy (P = 0.0049 and P = 0.0031 respectively). These results support the view that initial damage is a major determinant of cell radiosensitivity.

Cytotoxic manifestations of the interaction between hyperthermia and TNF: DNA fragmentation.

The relationship of DNA fragmentation to the greatly enhanced cytotoxicity seen in vitro against tumour cells when recombinant human tumour necrosis factor-alpha (TNF-alpha) is combined with hyperthermia was investigated. The TNF-alpha-sensitive L929 and -resistant EMT6 cells were treated with 8.8 and 16 ng of TNF-alpha per ml, respectively, and then heated at 40.5 degrees C for 24 h (L929) or at 43 degrees C for 1 h (L929) or 1.5 h (EMT-6) beginning 1 h later. For both cell lines at both temperatures, the addition of heating to the TNF-alpha treatment significantly decreased viability and increased DNA fragmentation at earlier time points than seen with either TNF-alpha or heat alone. DNA fragmentation was further studied using agarose gel electrophoresis to examine the size distribution of the DNA fragments and the ability of intracellular calcium buffering agents BAPTA and quin-2 to inhibit fragmentation. At 4.5 h after L929 cells were treated with TNF-alpha at 43 degrees C, the size distribution of DNA fragments more closely resembled the oligonucleosome sized apoptotic DNA fragmentation, as seen in irradiated rat thymocytes, than the spectrum of DNA fragments seen in necrotic fragmentation. However, while BAPTA and quin-2 inhibited the calcium-dependent apoptotic fragmentation seen in thymocytes they did not inhibit the DNA fragmentation in L929 cells. In addition, the loss of membrane integrity in both L929 and EMT-6 cells preceded or approximated the appearance of DNA fragmentation, whereas loss of membrane integrity usually follows DNA fragmentation in apoptosis. However, morphological studies showed that apoptotic bodies were present in L929 cell cultures treated with TNF-alpha and heat, and were distinguishable from necrosing cells. We conclude that both types of DNA fragmentation are operant in some cell lines exhibiting a cytotoxic response to TNF-alpha and heat treatments, and that increased fragmentation reflects the greatly enhanced cytotoxic interactions seen with combination treatments in those cells.

The use of biphasic linear ramped pulsed field gel electrophoresis to quantify dna damage based on fragment size distribution

Purpose : The development of biphasic linear pulse ramping gel electrophoresis has permitted resolution of DNA fragments from 200 Kbp to 6 Mbp in a single gel. We used this technique to measure radiation-induced DNA damage based on fragment size. Methods and Materials : Human colon cancer cells (HT29 and LS174T) and Chinese hamster ovary cells were embedded in agarose, deproteinized, irradiated with 5–80 Gy, and assessed for DNA double strand breakage using pulsed field gel electrophoresis. The frequency of DNA double strand breakage determined using a previously published method was compared to the breakage frequency calculated using the fragment size distribution. Results : Both methods produced similar estimates for breakage frequency of approximately 5 X 10 -9 breaks Gy −1bp −1. Conclusions : These findings suggest that biphasic linear pulse ramping gel electrophoresis can yield a quantitative estimate of DNA fragment distribution resulting from irradiation. The ability to quantify the distribution of DNA fragment sizes produced by irradiation should yield important information concerning the mechanisms of both DNA double strand break induction and repair.

Dose Response in Neutral Filter Elution

In neutral filter elution a nonlinear relationship between fraction of eluted DNA and dose is usually observed, which is often interpreted as a nonlinear induction of DNA double-strand breaks (DSBs) with dose. The conclusiveness of this hypothesis is questioned here on the basis of theoretical considerations regarding the size distribution of DNA fragments. A simple hydrodynamic model is proposed which generates the typical features of the dose response of neutral filter elution: (1) the shoulder at low doses, (2) a quasilinear correlation in an intermediate dose range, (3) a saturation at high doses, and (4) a linearization of the curve in the intermediate and higher dose range in a semilogarithmic plot. These features were derived even with the assumption of a linear induction of DSBs with dose. Thus it is demonstrated that the fraction of eluted DNA could conceivably be a nonlinear function of dose even if the induction of DSBs is directly proportional to the radiation dose.

In CHEF Electrophoresis a Linear Induction of Dsb Corresponds to a Nonlinear Fraction of Extracted DNA with Dose

A theory for DNA double-strand break (dsb) analysis by pulsed field electrophoresis is presented, based on the size distribution of DNA fragments, which converts an induction of dsb linear with dose into a nonlinear fraction of extractable DNA, in agreement with experimental observations.

Quantitation of single- and double-strand DNA breaks in vitro and in vivo

This communication describes a rapid and convenient procedure for quantitation of strand breaks in bacterial DNA, both in vitro and in vivo, using agarose gel electrophoresis. The electrophoretic determination of single strand breaks is carried out in alkaline medium, followed by renaturation of the gel and intercalation of the fluorescent dye, ethidium bromide. Double-strand breaks are determined by electrophoresis in neutral medium containing the dye. The distribution of DNA fragment sizes, the determination of the number-average molecular weight, the quantitation of the average number of DNA breaks per molecule, and the ratio between the single- and double-strand breaks are evaluated from microdensitometric scanning of the gels. The application of this analysis to damage caused by a combination of ascorbate and copper is demonstrated.