Pulsed-field gel electrophoresis in the measurement of DNA double-strand break repair in xrs-6 and CHO cell lines: DNA degradation under some conditions interferes with the assessment of double-strand break rejoining.

Pulsed-field gel electrophoresis (PFGE) is a sensitive assay for DNA double-strand breaks (DSBs). The application of PFGE to the measurement of DSB rejoining requires attention to conditions after irradiation. When cells are irradiated and incubated while encapsulated in agarose, DNA DSB rejoining appears incomplete or absent due to DNA degradation artifacts. Using PFGE we have measured the DSB rejoining defect in xrs-6 cells and shown how DNA degradation can affect the results.     

Pulsed-field gel electrophoresis of chromosomal DNA of Saccharomyces pastorianus after exposure to x-rays (30–50 keV) and neutrons (14 MeV)

Chromosomes of budding yeast Saccharomyces pastorianus were used to determine the extent of DNA double-strand breaks (DSBs) induced by x-rays (30-50 keV) and 14 MeV neutrons. The yeast chromosomes were separated by pulsed-field gel electrophoresis (PFGE) and the proportion of unbroken molecules corresponding to the largest chromosome no. IV (1500 kbp) was used to calculate the DSB frequency assuming a random distribution of hits. To determine the protective contribution of the cell environment, chromosomes embedded in agarose plugs as well as intact yeast cells, were irradiated under conditions completely inhibiting DNA repair. Following irradiation, the intact cells were also embedded in agarose plugs and the chromosomes isolated to perform PFGE. All radiation experiments resulted in a linear dose-effect curve for DSBs. For both radiation qualities, the yield of DSBs for exposed isolated chromosomes exceeded that for intact yeast cells by a factor of 13. The relative biological effectiveness (RBE) of 14 MeV neutrons in the induction of DNA DSBs was about 2.5. This figure was found to be identical for the in vivo and in vitro exposure of yeast chromosomes (neutrons 36.7 and 2.8, x-rays 14.5 and 1.1 x 10(-8) DSB x Bp-1 Gy-1 for isolated DNA and intact cells, respectively).     

AT细胞DNA双链断裂重接修复效率及其忠实性

用脉冲电场凝胶电泳和双标记基因质粒ＤＮＡ转染技术研究辐射敏感的毛细血管扩张性共济失调症患者皮肤成纤维细胞（ＡＴ５ＢＩＶＡ）和正常辐射抗性的人宫颈癌细胞（ＨｅＬａＳ３）ＤＮＡ双链断裂重接修复率及其忠实性。结果表明γ射线照射诱发ＤＮＡ双链断裂的产额和重接修复率,在两株细胞间无差别．而ＡＴ细胞对导入的限制性内切酶ＥｃｏＲＶ产生双链断裂质粒ＤＮＡ的重接修复忠实性显著低于ＨｅｌａＳ３ｔｅ胞,表明ＡＴ细胞易发生ＤＮＡ错误修复,这很可能就是ＡＴ细胞高度辐射敏感性的主要原因。     

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.     

Use of pulsed-field-gradient gel electrophoresis to construct a physical map of the Caulobacter crescentus genome

The restriction enzyme DraI cleaves the Caulobacter crescentus genome into at least 35 fragments which have been resolved in agarose gels using pulsed-field-gradient gel electrophoresis (PFGE). When digests were performed using DNA from strains containing Tn5 insertion mutations, altered band migrations were observed. Using PFGE with the appropriate pulse times, size differences as small as 2% could be resolved in large fragments. Using this approach, we have constructed a partial physical map of the genome which correlates well with the C. crescentus genetic map and have shown the size of the genome to be approx. 3800 kb. Using hybridization with cloned genes, we have determined the map locations of five previously unmapped genes. In addition, we have shown that PFGE can be used to rapidly determine the map locations of new insertion mutations or the sizes of deletion mutations.     

Detection of ionizing radiation-induced DNA double-strand breaks by filter elution is affected by nuclear chromatin structure

Chinese hamster ovary cells were irradiated with 250 kVp X rays and analyzed for the presence of DNA double-strand breaks using either polycarbonate filter elution or pulsed-field agarose gel electrophoresis at neutral pH. Reduction in DNA length detected by filter elution was produced as a nonlinear function of increasing radiation dose, with a quasi-threshold at low total dose, and as a first-order function of increasing radiation dose as detected by gel electrophoresis. The quasi-threshold observed with filter elution was eliminated when nuclei were isolated from irradiated cells and their chromatin relaxed in a buffer containing low-molarity monovalent cation prior to analysis by filter elution. The results suggest either that the chemical structure of the DNA double-strand breaks produced by low-LET radiation necessitates a DNA relaxation step before they can be detected accurately by filter elution, or that at low total radiation dose a DNA complex forms on the polycarbonate filter.     

Rejoining kinetics of DNA single- and double-strand breaks in normal and DNA ligase-deficient cells after exposure to ultraviolet C and gamma radiation: an evaluation of ligating activities involved in different DNA repair processes

The repair kinetics for rejoining of DNA single- and double-strand breaks after exposure to UVC or gamma radiation was measured in cells with deficiencies in DNA ligase activities and in their normal counterparts. Human 46BR cells were deficient in DNA ligase I. Hamster EM9 and EM-C11 cells were deficient in DNA ligase III activity as a consequence of mutations in the XRCC1 gene. Hamster XR-1 cells had mutation in the XRCC4 gene, whose product stimulates DNA ligase IV activity. DNA single- and double-strand breaks were assessed by the comet assay in alkaline conditions and by the technique of graded-field gel electrophoresis in neutral conditions, respectively. 46BR cells, which are known to re-ligate at a reduced rate the DNA single-strand breaks incurred during processing of damage induced by UVC but not gamma radiation, were shown to have a normal repair of radiation-induced DNA double-strand breaks. EM9 cells exhibited a reduced rate of rejoining of DNA single-strand breaks after exposure to ionizing radiation, as reported previously, as well as UVC radiation. EM-C11 cells were deficient in the repair of radiation-induced-DNA single-strand breaks but, in contrast to EM9 cells, demonstrated the same kinetics as the parental cell line in the resealing of DNA breaks resulting from exposure to UVC radiation. Both EM9 and EM-C11 cells displayed a significant defect in rejoining of radiation-induced-DNA double-strand breaks. XR-1 cells were confirmed to be highly deficient in the repair of radiation-induced DNA double-strand breaks but appeared to rejoin DNA single-strand breaks after UVC and gamma irradiation at rates close to normal. Taken together these results indicate that: (1) DNA ligase I is involved only in nucleotide excision repair; (2) DNA ligase IV plays an important role only in repair of DNA double-strand breaks; and (3) DNA ligase III is implicated in base excision repair and in repair of DNA double-strand breaks, but probably not in nucleotide excision repair.     

Chromosome-specific identification and quantification of S1 nuclease-sensitive sites in yeast chromatin by pulsed-field gel electrophoresis

Sites that are sensitive to the single-strand-specific endonuclease S1 ('S1-sensitive sites', SSS) occur in native chromatin and, like DNA double-stranded breaks (DSB), they are induced by DNA-damaging agents, such as ionizing radiation. We have developed a method to quantify SSS and DSB in yeast chromatin by using pulsed-field gel electrophoresis (PFGE) to separate the intact chromosomal-length DNA molecules from the lower molecular-weight broken ones. Direct evaluation of the photonegatives of the ethidium bromide-stained gels by laser densitometry enabled us to calculate the numbers of DSB and SSS per DNA molecule. These numbers were determined from the bulk of the non-separated genomic DNA of yeast, corresponding to a single band in the PFGE (pulse time 10 seconds), and in each of the eight largest yeast chromosomes, corresponding to distinct bands in the PFGE gels (pulse time 50 seconds), which were not superimposed by the smear of the broken, low molecular-weight DNA. Furthermore, the induction of DSB and SSS in a specific chromosome (circular chromosome III) was determined by Southern hybridization of the PFGE gels with a suitable centromere probe, followed by densitometry of the autoradiographs. Our method allows the chromosome-specific monitoring of DSB and all those DNA structures that are processed either in vivo or in vitro into DSB and which may not be distributed randomly within the genome.     

Use of pulsed-field gel electrophoresis to measure X-ray-induced double-strand breaks in DNA substituted with BrdU

The substitution of BrdU for TdR in the DNA of Chinese hamster ovary cells caused radiosensitization for both cell killing and an increase in the rate of neutral elution of the DNA. However, no radiosensitization was observed for the amount of DNA that migrated from the plug of agarose gels subjected to pulsed-field gel electrophoresis. An unexpected observation, however, was that the migration rate of BrdU-substituted DNA was relatively independent of radiation dose and was much less than that of unsubstituted DNA which migrated at a faster rate as the radiation dose increased. This difference in migration between TdR- and BrdU-labeled DNA was observed only when electrophoresis conditions were optimized for separating DNA molecules from 1 to 7 Mb. Possibly, the increase in negative charge on BrdU-labeled DNA increases the reorientation time during each pulse, with a resulting decrease in rate of migration, or radiation effects on BrdU-labeled DNA may be responsible for the decrease in migration rate.     

Differential protection by nitroxides and hydroxylamines to radiation-induced and metal ion-catalyzed oxidative damage

Modulation of radiation- and metal ion-catalyzed oxidative-induced damage using plasmid DNA, genomic DNA, and cell survival, by three nitroxides and their corresponding hydroxylamines, were examined. The antioxidant property of each compound was independently determined by reacting supercoiled DNA with copper II/1,10-phenanthroline complex fueled by the products of hypoxanthine/xanthine oxidase (HX/XO) and noting the protective effect as assessed by agarose gel electrophoresis. The nitroxides and their corresponding hydroxylamines protected approximately to the same degree (33-47% relaxed form) when compared to 76.7% relaxed form in the absence of protectors. Likewise, protection by both the nitroxide and corresponding hydroxylamine were observed for Chinese hamster V79 cells exposed to hydrogen peroxide. In contrast, when plasmid DNA damage was induced by ionizing radiation (100 Gy), only nitroxides (10 mM) provide protection (32.4-38.5% relaxed form) when compared to radiation alone or in the presence of hydroxylamines (10 mM) (79.8% relaxed form). Nitroxide protection was concentration dependent. Radiation cell survival studies and DNA double-strand break (DBS) assessment (pulse field electrophoresis) showed that only the nitroxide protected or prevented damage, respectively. Collectively, the results show that nitroxides and hydroxylamines protect equally against the damage mediated by oxidants generated by the metal ion-catalyzed Haber-Weiss reaction, but only nitroxides protect against radiation damage, suggesting that nitroxides may more readily react with intermediate radical species produced by radiation than hydroxylamines.     

Non-homologous end-joining defect in fanconi anemia hematopoietic cells exposed to ionizing radiation

Fanconi anemia is a genetically heterogeneous recessive disease characterized mainly by bone marrow failure and cancer predisposition. Although it is accepted that Fanconi cells are highly sensitive to DNA crosslinking agents, their response to ionizing radiation is still unclear. Using pulsed-field gel electrophoresis, we have observed that radiation generates a similar number of DNA double-strand breaks in normal and Fanconi cells from three (FA-A, FA-C and FA-F) of the 11 complementation groups identified. Nonsynchronized as well as nonproliferating Fanconi anemia cells showed an evident defect in rejoining the double-strand breaks generated by ionizing radiation, indicating defective non-homologous end-joining repair. At the cellular level, no difference in the radiosensitivity of normal and FA-A lymphoblast cells was noted, and a modest increase in the radiosensitivity of Fanca-/- hematopoietic progenitor cells was observed compared to Fanca+/+ cells. Finally, when animals were exposed to a fractionated total-body irradiation of 5 Gy, a similar hematopoietic syndrome was observed in wild-type and Fanca-/- mice. Taken together, our observations suggest that Fanconi cells, in particular those having nonfunctional Fanconi proteins upstream of FANCD2, have a defect in the non-homologous end-joining repair of double-strand breaks produced by ionizing radiation, and that compensatory mechanisms of DNA repair and/or stem cell regeneration should limit the impact of this defect in irradiated organisms.     

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 small (58-nm) attached sphere perturbs the sieving of 40-80-kilobase DNA in 0.2-2.5% agarose gels: analysis of bacteriophage T7 capsid-DNA complexes by use of pulsed field electrophoresis.

Although the icosahedral bacteriophage T7 capsid has a diameter (58 nm) that is 234-fold smaller than the length of the linear, double-stranded T7 DNA, binding of a T7 capsid to T7 DNA is found here to have dramatic effects on the migration of the DNA during both pulsed field agarose gel electrophoresis (PFGE; the field inversion mode is used) and constant field agarose gel electrophoresis (CFGE). For these studies, capsid-DNA complexes were obtained by expelling DNA from mature bacteriophage T7; this procedure yields DNA with capsids bound at a variable position on the DNA. When subjected to CFGE at 2-6 V/cm in 0.20-2.5% agarose gels, capsid-DNA complexes arrest at the electrophoretic origin. Progressively lowering the electrical potential gradient to 0.5 V/cm results in migration; most complexes form a single band. The elevated electrical potential gradient (3 V/cm) induced arrest of capsid-DNA complexes is reversed when PFGE is used instead of CFGE. For some conditions of PFGE, the mobility of capsid-DNA complexes is a function of the position of the capsid on the DNA. During either CFGE (0.5 V/cm) or PFGE, capsid-DNA complexes increasingly separate from capsid-free DNA as the percentage of agarose increases. During these studies, capsid-DNA complexes are identified by electron microscopy of enzymatically-digested pieces of agarose gel; this is apparently the first successful electron microscopy of DNA from an agarose gel.(ABSTRACT TRUNCATED AT 250 WORDS)     

Microwave effects on plasmid DNA

The exposure of purified plasmid DNA to microwave radiation at nonthermal levels in the frequency range from 2.00 to 8.75 GHz produces single- and double-strand breaks that are detected by agarose gel electrophoresis. Microwave-induced damage to DNA depends on the presence of small amounts of copper. This effect is dependent upon both the microwave power and the duration of the exposure. Cuprous, but not cupric, ions were able to mimic the effects produced by microwaves on DNA.     

Evidence that DNA fragmentation in apoptosis is initiated and propagated by single-strand breaks

Apoptosis is characterised by the degradation of DNA into a specific pattern of high and low molecular weight fragments seen on agarose gels as a distribution of sizes between 50-300 kb and sometimes, but not always, a ladder of smaller oligonucleosomal fragments. Using a 2D pulsed field-conventional agarose gel electrophoresis technique, where the second dimension is run under either normal or denaturing conditions, we show that single-strand breaks are introduced into DNA at the initial stages of fragmentation. Using single-strand specific nuclease probes we further show that the complete fragmentation pattern, including release of small oligonucleosomal fragments can also be generated by a single-strand endonuclease. Three classes of sites where single-strand breaks accumulate were identified. The initial breaks produce a distribution of fragment sizes (50 kb to >1 Mb) similar to those generated by Topoisomerase II inhibitors suggesting that cleavage may commence at sites of attachment of DNA to the nuclear matrix. A second class of rare sites is also cut further reducing the size distribution of the fragments to 50-300 kb. Thirdly, single-strand breaks accumulate at the linker region between nucleosomes eventually causing double-strand scissions which release oligonucleosomes. These observations further define the properties of the endonuclease responsible for DNA fragmentation in apoptosis.     

High-resolution separation and accurate size determination in pulsed-field gel electrophoresis of DNA. 4. Influence of DNA topology

Pulsed-field gel electrophoresis is a powerful technique for the fractionation of linear DNA molecules with sizes above 50 kilobase pairs (kb). Here it is demonstrated that this technique is also effective for separating smaller DNAs including linear, circular, and supercoiled species. The mobilities of linear DNAs larger than 8 kb can be modulated by pulse times between 0.1 and 100 s. The mobility of supercoiled DNA molecules up to 16 kb is generally unaffected by these pulse times except that 10-s pulse times cause a small but distinct increase in the mobility. The general insensitivity of small supercoiled DNAs to pulse time presumably occurs because these species reorient so rapidly that they spend most of their time undergoing conventional electrophoresis. However, the mobilities of larger supercoiled DNAs are affected by pulse times of less than 1 s, and at 0.1 s the molecules are better resolved by pulsed electrophoresis than by ordinary electrophoresis. The mobility of 3-19 kb nicked and relaxed circular DNA molecules is also affected by pulse time but in a complex way.     

Unidirectional pulsed-field electrophoresis of single- and double-stranded DNA in agarose gels: analytical expressions relating mobility and molecular length and their application in the measurement of strand breaks

Unidirectional pulsed-field electrophoresis improves the separation of single-stranded DNA molecules longer than 20 kilobases (kb) in alkaline agarose gels compared to static-field electrophoresis. The greatest improvement in separation is for molecules longer than 100 kb. The improved resolution of long molecules with unidirectional pulsed-field electrophoresis makes possible the measurement of lower frequencies of single-strand breaks. The analytical function that relates the length and mobility of single-stranded DNA electrophoresed with a static field also applies to unidirectional pulsed field separations. Thus, the computer programs used to measure single-strand breaks are applicable to both undirectional pulsed- and static-field separations. Unidirectional pulsed-field electrophoresis also improves the separation of double-stranded DNA in neutral agarose gels. The function relating molecular length and mobility for double-stranded DNA separated by unidirectional pulsed-field electrophoresis is a superset of the function for single-stranded DNA. The coefficients of this function can be determined by iterative procedures.     

The fraction of DNA released on pulsed-field gel electrophoresis gels may differ significantly between genomes at low levels of double-strand breaks

A common way to use pulsed-field gel electrophoresis (PFGE) for measuring the induction and repair of DNA double-strand breaks (DSBs) in mammalian cells is by using the fraction of total DNA released, FR, from the plug. We have analyzed the general relationship between initial chromosome sizes and FR. It is shown that, because of the difference in initial chromosomal size, the discrepancy in FR values between human and rodent cells may become significant at doses of radiation producing approximately 5 DSBs/100 Mbp or less.     

Evidence for complexity at the nanometer scale of radiation-induced DNA DSBs as a determinant of rejoining kinetics

The rejoining kinetics of double-stranded DNA fragments, along with measurements of residual damage after postirradiation incubation, are often used as indicators of the biological relevance of the damage induced by ionizing radiation of different qualities. Although it is widely accepted that high-LET radiation-induced double-strand breaks (DSBs) tend to rejoin with kinetics slower than low-LET radiation-induced DSBs, possibly due to the complexity of the DSB itself, the nature of a slowly rejoining DSB-containing DNA lesion remains unknown. Using an approach that combines pulsed-field gel electrophoresis (PFGE) of fragmented DNA from human skin fibroblasts and a recently developed Monte Carlo simulation of radiation-induced DNA breakage and rejoining kinetics, we have tested the role of DSB-containing DNA lesions in the 8-kbp-5.7-Mbp fragment size range in determining the DSB rejoining kinetics. It is found that with low-LET X rays or high-LET alpha particles, DSB rejoining kinetics data obtained with PFGE can be computer-simulated assuming that DSB rejoining kinetics does not depend on spacing of breaks along the chromosomes. After analysis of DNA fragmentation profiles, the rejoining kinetics of X-ray-induced DSBs could be fitted by two components: a fast component with a half-life of 0.9+/-0.5 h and a slow component with a half-life of 16+/-9 h. For alpha particles, a fast component with a half-life of 0.7+/-0.4 h and a slow component with a half-life of 12+/-5 h along with a residual fraction of unrepaired breaks accounting for 8% of the initial damage were observed. In summary, it is shown that genomic proximity of breaks along a chromosome does not determine the rejoining kinetics, so the slowly rejoining breaks induced with higher frequencies after exposure to high-LET radiation (0.37+/-0.12) relative to low-LET radiation (0.22+/-0.07) can be explained on the basis of lesion complexity at the nanometer scale, known as locally multiply damaged sites.     

Pulsed field gel electrophoresis: Theory,instruments and application

Pulsed field gel electrophoresis (PFGE) is a technique for the fractionation of high-molecular-weight DNA ranging from 10 kb to 10 Mb by electrophoresis in agarose gel with an electric field that alternates (pulsates) in two directions. This technology plays a key role in modern genomics, as it allows manipulations with DNA of whole chromosomes or their large fragments. In this review, we discuss (1) the theory behind PFGE; (2) different instruments based on the principle of pulsed field, as well as their advantages and limitations; (3) factors affecting the DNA mobility in PFGE gel; and (4) practical applications of the technique.