Double-strand Breaks In Plasmid DNA Research Articles

Scanning force microscopy studies of X-ray-induced double-strand breaks in plasmid DNA.

We present an analysis of X-ray-induced damage in PhiX174 plasmid DNA, applying doses between D = 250 and 1,500 Gy. To analyse this damage in detail, the distribution of plasmid fragments after irradiation have been determined by scanning force microscopy. The results show that even for the lowest dose of D = 250 Gy, a significant amount of double-strand breaks are observed. For increasing dose, the percentage of small fragments increases and is accompanied by a shortening of the average fragment length from < L > = 1,400 nm for a dose of D = 250 Gy to < L > = 1,080 nm after irradiation with D = 1,500 Gy. The most crucial parameter, the average number of double-strand breaks per broken plasmid (<DSB(b)> ) has been determined for the first time for the applied doses. The results show that the average number of DSBs per broken plasmid <DSB(b)> increases almost linearly from a value of <DSB(b)> = 1.3 after irradiation with D = 250 Gy to <DSB(b)> = 1.7 after exposure to D = 1,500 Gy. The presented results show that the amount of DSBs induced by X-ray radiation in plasmid DNA can be calculated with high accuracy by means of scanning force microscopy, providing relevant information regarding the interaction of X-rays with DNA molecules.

Mechanisms Underlying Production of Double-Strand Breaks in Plasmid DNA after Decay of125I-Hoechst

Previously, the kinetics of strand break production by (125)I-labeled m-iodo-p-ethoxyHoechst 33342 ((125)IEH) in supercoiled (SC) plasmid DNA had demonstrated that approximately 1 DSB is produced per (125)I decay both in the presence and absence of the hydroxyl radical scavenger DMSO. In these experiments, an (125)IEH:DNA molar ratio of 42:1 was used. We now hypothesize that this DSB yield (but not the SSB yield) may be an overestimate due to subsequent decays occurring in any of the 41 (125)IEH molecules still bound to nicked (N) DNA. To test our hypothesis, (125)IEH was incubated with SC pUC19 plasmids ((125)IEH:DNA ratio of approximately 3:1) and the SSB and DSB yields were quantified after the decay of (125)I. As predicted, the number of DSBs produced per (125)I decay is one-half that reported previously ( approximately 0.5 compared to approximately 1, +/- DMSO) whereas the number of SSBs ( approximately 3/(125)I decay) is similar to that obtained previously ( approximately 90% are generated by OH radicals). Direct visualization by atomic force microscopy confirms formation of L and N DNA after (125)IEH decays in SC DNA and supports the strand break yields reported. These findings indicate that although SSB production is independent of the number of (125)IEH bound to DNA, the DSB yield can be augmented erroneously by (125)I decays occurring in N DNA. Further analysis indicates that 17% of SSBs and 100% of DSBs take place within the plasmid molecule in which an (125)IEH molecule decays, whereas 83% of SSBs are formed in neighboring plasmid DNA molecules.

Spatial Distribution of Radiation-Induced Double-Strand Breaks in Plasmid DNA as Resolved by Atomic Force Microscopy

Atomic force microscopy (AFM) has been used to directly visualize, size and compare the DNA fragments resulting from exposure to low- and high-LET radiation. Double-stranded pUC-19 plasmid ("naked") DNA samples were irradiated by electron-beam or reactor neutron fluxes with doses ranging from 0.9 to 10 kGy. AFM scanning in the tapping mode was used to image and measure the DNA fragment lengths (ranging from a few bp up to 2864 bp long). Double-strand break (DSB) distributions resulting from high-LET neutron and lower-LET electron irradiation revealed a distinct difference between the effects of these two types of radiation: Low-LET radiation-induced DSBs are distributed more uniformly along the DNA, whereas a much larger proportion of neutron-induced DSBs are distributed locally and densely. Furthermore, comparisons with predictions of a random DSB model of radiation damage show that neutron-induced DSBs deviate more from the model than do electron-induced DSBs. In summary, our high-resolution AFM measurements of radiation-induced DNA fragment-length distributions reveal an increased number of very short fragments and hence clustering of DSBs induced by the high-LET neutron radiation compared with low-LET electron radiation and a random DSB model prediction.

Induction of single- and double-strand breaks in plasmid DNA by 100–1500 eV electrons

Purpose : To investigate the induction of DNA strand breaks by electrons with energies ranging from 0.1 to 1.5 keV. Materials and methods : Dry supercoiled plasmid DNA was irradiated with electrons of energies ranging from 0.1 to 1.5keV and the results were compared with those obtained by gamma-irradiation of the same plasmid in solution. For electron irradiation, the plasmid was deposited on a gold substrate under a controlled atmosphere to minimize contamination of the DNA film. Electron bombardments were performed under ultra-high vacuum conditions (UHV 10 9 torr). DNA damage was detected by gel electrophoresis followed by quantitation of the DNA bands by fluorescence or by hybridization with a radioactive probe. Results : Electrons with energies from 0.1 to 1.5keV induced single, double and multiple double-strand breaks in supercoiled plasmid DNA. For equal doses, we observed a marked increase in the efficiency of induction of double- and multiple-strand breaks in supercoiled DNA as a function of electron energy. In contrast to γ-irradiation, the formation of small DNA fragments by electrons did not seem to be related to the production of the linear form of the plasmid. Conclusions : Electrons within the energy range of the secondary electrons generated by high-energy ionizing radiation induce single, double and multiple double-strand breaks in DNA. Problems associated with low-energy electron irradiation experiments and dose calculations in thin films are also discussed.

A role for p53 in DNA end rejoining by human cell extracts.

The tumor suppressor p53 is a major regulator in the response of human cells to DNA damage. In this study we assessed the role of p53 in the repair of DNA double-strand breaks in plasmid DNA using cell extracts from three human lymphoblastoid cell lines derived from the same donor. TK6, WI-L2-NS and TK6-E6-5e cells express wild-type, mutated and essentially no p53 protein, respectively. Total cellular extracts from TK6, WI-L2-NS and TK6-E6-5e cells were incubated with EcoRI linearized pUC19 DNA. Southern blot analysis of end-rejoined DNA indicated that the major products formed were linear multimers. There was approximately 2-fold greater end rejoining in WI-L2-NS and TK6-E6-5e extracts compared with TK6 extracts. Total DNA from end-rejoining reactions was purified and used to transform bacteria. Using the lacZ reporter gene as a measure of repair fidelity we found that misrepair, as indicated by white colonies, occurred at 4.1% to 6.5% of transformants, with no significant difference between the three cell lines. Gel analysis revealed that misrepair involved only deletions. Sequence analysis of 11 misrepaired products from each cell line showed 12 different deletions from 4 to 48 bp in length, but each cell line yielded similar product types. These results indicate that total cellular extracts from human lymphoblastoid cells lacking p53 or expressing mutated p53 have increased end-rejoining activity as compared with extracts from cells expressing wild-type p53. However, the p53 status does not influence the ratio of misrepair:correct repair, or the type of misrepair events.

Reduction of 3-amino-1,2,4-benzotriazine-1,4-di-N-oxide (tirapazamine, WIN 59075, SR 4233) to a DNA-damaging species: a direct role for NADPH:cytochrome P450 oxidoreductase.

3-amino-1,2,4-benzotriazine-1,4-di-N-oxide (tirapazamine, WIN 59075, SR 4233, NSC 130181) has entered phase 1 clinical trials as a bioreductive hypoxic cell cytotoxin because of its novel structure and impressive selective cytotoxicity towards hypoxic cells. Understanding the enzymology and underlying mechanism of oxidative and reductive DNA damage may allow more optimal development and use of this agent and contribute to the rational design of new bioreductive drugs. Here we provide unambiguous evidence that WIN 59075 undergoes one-electron reduction by purified rat liver NADPH:cytochrome P450 oxidoreductase to generate single- and double-strand breaks in plasmid DNA. The DNA damage caused may be important for the therapeutic toxicity of the drug. Enzyme kinetic parameters for this oxidoreductase reaction are in the range 1.01-1.61 mM for Km and 4416-5099 nmol/min/mg for Vmax. The relative levels of expression and cellular localization of target tumour NADPH:cytochrome P450 oxidoreductase may contribute to the therapeutic selectivity of WIN 59075.

Double-strand breaks in plasmid DNA and the induction of deletions

Double-strand breaks (dsbs) have been produced in plasmid DNA by various restriction endonucleases and the survival and the deletion mutation incidence have been measured in E. coli. The deletion formation is known to depend upon the occurrence of short direct repeats within the DNA molecule. In order to study the role of these repeats we constructed plasmid molecules with repeats of various lengths or with a 10-base pair repeat at different distances from each other. Furthermore the influence of the location and the structure of the dsb was studied. Repair and deletion frequencies of the linearized plasmids were measured after transformation of E. coli. The yield of the specific deletion mutation (the one which occurs between the introduced repeats) increases nearly linearly with the square of the length of the repeat, while the yield of the correctly repaired DNA and the yield of all other deletion mutants remained constant. The slope of the linear increase of the yield of the specific deletion depends on the location and the structure of the dsb. The yield of the specific deletion mutation decreases with increasing distance between the repeats. A proposal for the rate-determining step of the deletion formation is made.

Repair of double-strand breaks in plasmid DNA in the yeast Saccharomyces cerevisiae.

We studied the repair of double-strand breaks (DSB) in plasmid DNA introduced into haploid cells of the yeast Saccharomyces cerevisiae. The efficiency of repair was estimated from the frequency of transformation of the cells by an autonomously replicated linearized plasmid. The frequency of "lithium" transformation of Rad+ cells was increased greatly (by 1 order of magnitude and more) compared with that for circular DNA if the plasmid was initially linearized at the XhoI site within the LYS2 gene. This effect is due to recombinational repair of the plasmid DNA. Mutations rad52, rad53, rad54 and rad57 suppress the repair of DSB in plasmid DNA. The kinetics of DSB repair in plasmid DNA are biphasic: the first phase is completed within 1 h and the second within 14-18 h of incubating cells on selective medium.

Molecular mechanisms for the hypoxiadependent activation of 3-amino-1,2,4-benzotriazine-1, 4-dioxide (SR 4233)

The reduction of the hypoxic cell toxin 3-amino-1,2,4-benzotriazine-1,4-dioxide (SR 4233) was investigated using pulse radiolysis, radiation chemical reduction, and xanthine oxidase. Evidence was found that the one-electron reduction product of the parent compound is an oxidizing radical that caused single- and double-strand breaks in plasmid DNA and that produced a malondialdehyde-like thiobarbituric acid adduct from 2-deoxy- d-ribose. Possible forms of the reactive radical, either carbonor nitrogen-centered, are suggested. The “natural” lifetime of the radical was sufficiently long that it could diffuse over significant distances within hypoxic cells and thus inflict oxidative damage on cellular targets. The radical reacted with O 2; at a rate comparable to those of the nitroimidazoles misonidazole and metronidazole. Thus, the selectivity for hypoxic cells is probably due to the elimination of “futile” reduction when the cellular oxygen concentration is sufficiently low.

Ascorbate potentiates DNA damage by 1-methyl-1-nitrosourea in vivo and generates DNA strand breaks in vitro.

Ascorbic acid (vitamin C) is an important intracellular reducing agent. It also has been suggested to be (i) a protective agent against development of cancer, (ii) a therapeutic agent for malignancies and (iii) a mutagen. We have found that high concentrations of ascorbate leads to DNA damage in several in vivo and in vitro situations. Guinea-pigs receiving oral 1-methyl-1-nitrosourea (MNU) were used as a whole animal model. Administration of sodium ascorbate prior to MNU increased strand breakage in pancreatic DNA. Concentrations of ascorbate greater than 0.5 mM increased the frequency of DNA strand breaks caused by MNU in both L1210 murine leukemia cells and guinea-pig pancreatic cells in tissue culture; ascorbate alone led to DNA strand breaks in the latter cells. Investigations of the mechanism of DNA damage were carried out with purified DNA. Ascorbate produced single- and double-strand breaks in plasmid DNA. Cleavage was catalyzed by copper(II), inhibited by catalase and blocked by the presence of thiols. We conclude that superoxide and hydrogen peroxide produced during the oxidation of ascorbate leads to generation of hydroxyl free radicals that can mediate DNA strand scissions and potentiate the effects of alkylating carcinogens.

Production of single- and double-strand breaks in plasmid dna by ozone

Agarose gel electrophoresis and electron microscopy were used to determine the type of lesions produced in DNA by ozone. This strong oxidizing agent was found to relax, linearize, then degrade native plasmid (pAT153) DNA molecules in solution. Ozone, like ionizing radiation, thus produced DNA breakage. To ascertain this point, wild-type and radiosensitive strains of Escherichia coli were transfected with control or ozonated plasmid DNA, and the host cells were selected for antibiotic resistance. A significat reduction in the transforming ability of pAT153 was observed following ozonation. Mutants deficient in the repair of DNA single-strand breaks yielded less ampicillin- or tetracycline-resistant clones than repair-proficient strains. In E. coli, the same gene products are probably involved in the repair of both radiation- and ozone-induced DNA breaks.