Open Circular Form Research Articles

Mechanism of DNA damage by cadmium and interplay of antioxidant enzymes and agents

Cadmium is an environmental toxicant, which causes cancer in different organs. It was found that it damages DNA in the various tissues and cultured cell lines. To investigate the mechanism of DNA damage, we have studied the effect of cadmium-induced DNA damage in plasmid pBR322 DNA, and the possible ameliorative effects of antioxidative agents under in vitro conditions. It was observed that cadmium alone did not cause DNA damage. However, it caused DNA damage in the presence of hydrogen peroxide, in a dose dependent manner, because of production of hydroxyl radicals. Findings from this study show the conversion of covalently closed circular double-stranded pBR 322 DNA to the open circular and linear forms of DNA when treated with 10 muM cadmium and various concentrations of H(2)O(2). The conversion was due to nicking in DNA strands. The observed rate of DNA strand breakage was dependent on H(2)O(2) concentration, temperature, and time. Metallothionein I failed to prevent cadmium-induced DNA nicking in the presence of H(2)O(2). Of the two antioxidant enzymes (catalase and superoxide dismutase) studied, only catalase conferred significant (50-60%) protection. EDTA and DMSO exhibited protection similar to catalase, while mannitol showed only about 20% protection against DNA damage. Ethyl alcohol failed to ameliorate cadmium-induced DNA strands break. From this study, it is plausible to infer that cadmium in the presence of hydrogen peroxide causes DNA damage probably by the formation of hydroxyl ions. These results may indicate that cadmium in vivo could play a major role in the DNA damage induced by oxidative stress.

Photoinduced cleavage and DNA-binding of the Ruthenium(II) polypyridyl complex [Ru(phen)2(ipbd)](ClO4)2

A novel ruthenium(II) polypyridyl complex [Ru(phen)2{ipbd)](ClO4)2 (ipbd = 3-(1H-Imidazo[4,5-f][1,10]phenanthrolin-2-yl)-1-benzodioxane, phen = 1,10-phenanthroline} has been synthesized and characterized by elemental analysis, ES-MS and 1H-NMR spectra. The interaction of the complex with calf thymus DNA (CT-DNA) was investigated by absorption titration, fluorescence spectra, thermal denaturation, viscosity measurements, circular dichroism and photoinduced cleavage. The results suggest that the complex intercalates into the CT-DNA base pairs. Upon irradiation, this novel Ru(II) complex can cleave the plasmid pBR 322 DNA from the supercoiled form I to the open circular form II.

Efficient Photoinduced DNA Damage by Coralyne

The photoinduced DNA damage by the berberine derivative coralyne is presented. The irradiation of coralyne in the presence of plasmid DNA namely, pBR322, leads to remarkably fast DNA damage by single-strand cleavage, as determined by agarose-gel electrophoresis. Even upon exposure to sunlight, almost all of the supercoiled plasmid is converted to the open circular form in less than a minute [c(pBR322) = 3.5 × 10-9M; c(coralyne) = 4.3 × 10-5M], The efficiency of the DNA strand cleavage is not decreased in the presence of radical-trapping reagents such as tert-butanol or DMSO. Moreover, the extent of the DNA damage is the same under aerobic conditions and at reduced oxygen concentration. Thus, the formation of reactive intermediates such as hydroxyl radicals or singlet oxygen is excluded. These results show that the exposure of coralyne and derivatives thereof to light, even with moderate light intensity, needs to be avoided during experiments in which their biological activity is assessed by plasmid unwinding assays.

Analysis of plasmid DNA damage induced by melanin with capillary electrophoresis

Dilute linear poly( N-isopropylacrylamide) (PNIPAM) in Tris–Mes–EDTA (TME) buffer was used as sieving matrix for capillary electrophoresis (CE) of plasmid DNA and plasmid topological isomers induced by melanin in uncoated capillary. At the optimized condition of 0.1% (w/v) PNIPAM in TME buffer, base line separation of the plasmid DNA ladder (2–12 kbp) was achieved within 15 min. Three positive clones with inserts of 468, 1147 and 1566 bp can be distinguished from the plasmid pUC 18 vector within 13 min. The migration order of the plasmid topological isomers in the dynamic coating matrix was confirmed by the enzymatically prepared and UV-induced plasmids. The covalently closed circular form appeared firstly, followed by the linear plasmid form and then the open circular form. The effect of bacterial melanin obtained from Pseudomonas maltophilia AT18 on plasmid pUC 18 was investigated by CE in uncoated capillary in vitro. Plasmid pUC 18 incubated with either melanin or copper ions alone sustained little DNA damage. The combination of melanin with Cu(II) can cause the plasmid pUC 18 conformational changes from covalently closed circular form to open form. Understanding the damage effect of melanin with copper ions on DNA would be important for the melanin-related application, such as photoprotective antioxidant in protecting the skin from cancer, pathophysiology research in clinic. The investigation of melanin induced plasmid conformational changes by CE in uncoated capillary also revealed that the application of the dynamic coating matrix could be extended to the study of plasmid conformational changes in other plasmid-based biological technologies.

Influence of plasmid DNA topology on the transfection properties of DOTAP/DOPE lipoplexes

Plasmid DNA (pDNA) can occur in the compact supercoiled (SC) form, the relaxed open circular (OC) form and the linearized form. In this paper we investigated the transfection efficiency of SC, OC and linearized pDNA complexed to DOTAP/DOPE liposomes in Vero cells. Only DOTAP/DOPE liposomes containing SC pDNA showed protein expression while DOTAP/DOPE liposomes loaded with OC or linearized pDNA failed. First we questioned if the better transfection properties of the SC pDNA-containing lipoplexes could be due to a better transcription of SC pDNA in the nuclei of the cells, compared to OC and linearized pDNA. However, microinjecting (naked) SC, OC or linearized pDNA in the nuclei of the Vero cells revealed that the transcription efficiency was independent on the pDNA topology but did depend on the intranuclear concentration of the pDNA. As the amount of pDNA that reaches the nucleus is determined by the amount of pDNA that arrives in the cytosol it could be hypothesized that SC pDNA is more efficiently released from the DOTAP/DOPE liposomes when compared to OC and linearized pDNA. However, microinjecting comparable concentrations of the pDNA topologies in the cytoplasm still resulted in a significantly higher transfection in the case of SC pDNA, especially in cells that underwent cell division in the period after injection. It seems that, compared to OC and linearized pDNA, SC pDNA is better suited to reach the perinuclear region, a prerequisite to become entrapped in the nuclei of the cells during cell division.

The Mechanism of Plasmid Curing in Bacteria

Bacterial plasmids have a major impact on metabolic function. Lactose fermentation of E. coli or hemolysin B transporter expressed by the plasmids that carry these respective genes could be readily obviated by heterocyclic compounds that readily bind to plasmid DNA. These compounds could also reverse the resistance to antibiotics of E. coli, Enterobacter, Proteus, Staphylococcus and Yersinia strains by eliminating plasmids. However, the frequency and extent of this effect was significantly less than might have been expected based on a complex interaction with plasmid DNA. The effects of heterocyclic compounds on the plasmids responsible for the virulence of Yersinia and A. tumefaciens, or on nodulation, nitrogen fixation of Rhizobia accounted for the elimination of 0.1 to 1.0 % of plasmids present in the populations studied. Bacterial plasmids can be eliminated from bacterial species grown as pure or mixed bacterial cultures in the presence of sub-inhibitory concentrations of non-mutagenic heterocyclic compounds. The antiplasmid action of the compounds depends on the chemical structure of amphiphillic compounds having a planar ring system with substitution in the L-molecular region. A symmetrical pi-electron conjugation at the highest occupied molecular orbitals favours the antiplasmid effect. The antiplasmid effect of heterocyclic compounds is expressed differentially in accordance with the structural form of the DNA to which they bind. In this manner "extrachromosomal" plasmid DNA that exists in a superhelical state binds more compound than its linear or open-circular form; and least to the chromosomal DNA of the bacterium, that carries the plasmid. It can also be noted that these compounds are not mutagenic and their antiplasmid effects correlate with the energy of HOMO-orbitals. Plasmid elimination is considered also to take place in ecosystems containing numerous bacterial species. This opens up a new perspective in rational drug design against bacterial plasmids. The inhibition of conjugational transfer of antibiotic resistance plasmid can be exploited to reduce the spread of antibiotic resistance plasmid in the ecosystem. Inhibition of plasmid replication at various stages, as shown in the "rolling circle" model (replication, partition, conjugal transfer) may also be the theoretical basis for the elimination of bacterial virulence in the case of plasmid mediated pathogenicity and antibiotic resistance. The large number of compounds tested for antiplasmid effects provides opportunities for QSAR studies in order to find a correlation between the antiplasmid effect and the supramolecular chemistry of these plasmid curing compounds. Plasmid elimination in vitro provides a method of isolating plasmid free bacteria for biotechnology without any risk of inducing mutations.

The Two Plasmodium falciparum Nucleosome Assembly Proteins Play Distinct Roles in Histone Transport and Chromatin Assembly

The malarial parasite Plasmodium falciparum has two nucleosome assembly proteins, PfNapS and PfNapL (Chandra, B. R., Olivieri, A., Silvestrini, F., Alano, P., and Sharma, A. (2005) Mol. Biochem. Parasitol. 142, 237-247). We show that both PfNapS and PfNapL interact with histone oligomers but only PfNapS is able to deposit histones onto DNA. This property of PfNapS is divalent cation-dependent and ATP-independent. Deletion of the terminal subdomains of PfNapS abolishes its nucleosome assembly capabilities, but the truncated protein retains its ability to bind histones. Both PfNapS and PfNapL show binding to the linker histone H1 suggesting their probable role in extraction of H1 from chromatin fibers. Our data suggests distinct sites of interaction for H1 versus H3/H4 on PfNapS. We show that PfNapS and PfNapL are phosphorylated both in vivo and in vitro by casein kinase-II, and this modification is specifically inhibited by heparin. Circular dichroism, fluorescence spectroscopy, and chymotrypsin fingerprinting data together suggest that PfNapL may undergo very small and subtle structural changes upon phosphorylation. Specifically, phosphorylation of PfNapL increases its affinity 3-fold for core histones H3, H4, and for the linker histone H1. Finally, we demonstrate that PfNapS is able to extract histones from both phosphorylated and unphosphorylated PfNapL, potentially for histone deposition onto DNA. Based on these results, we suggest that the P. falciparum NapL is involved in the nucleocytoplasmic relay of histones, whereas PfNapS is likely to be an integral part of the chromatin assembly motors in the parasite nucleus.

A new dinuclear biphenylene bridged copper(II) complex: DNA cleavage under hydrolytic conditions

The dinucleating ligand, tpbpd (tetrapyridyl biphenylenediamine) forms a dicopper complex with practically no electronic coupling between the two copper (II) centres. The EPR spectrum of the complex is consistent with coordination of each copper ion to two nitrogens of the binuclear ligand. Cyclic voltammogram of the complex also reveals that the two copper (II) centres have identical ligating environment. This dimeric copper (II) complex is found to be a very efficient catalyst for the cleavage of plasmid DNA in the absence of any added cofactor. The amount of conversion of supercoiled form (Form I) of plasmid to the open circular form (Form II) depends on the concentration of the complex as well as the duration of incubation of the complex with DNA. The maximum rate of conversion of the supercoiled form to the nicked circular form at pH 7.5 in the presence of 150 μM of the complex is found to be 1.8 × 10 −3 s −1.

Ru(TAP)3]2+‐Photosensitized DNA Cleavage Studied by Atomic Force Microscopy and Gel Electrophoresis: A Comparative Study

Topological modifications of plasmid DNA adsorbed on a variety of surfaces were investigated by using atomic force microscopy (AFM). On mica modified with 3-aminopropyltriethoxysilane (APS) or poly-L-lysine, the interaction between the plasmid DNA and the surface "freezes" the plasmid DNA conformation deposited from solution, and the AFM images resemble the projection of the three-dimensional conformation of the plasmid DNA in solution. Modified mica with low concentrations of Mg(2+) leads to a decrease in the interaction strength between plasmid DNA and the substrate, and the AFM images reflect the relaxed or equilibrium conformation of the adsorbed plasmid DNA. Under these optimized deposition conditions, topological modifications of plasmid DNA were produced under irradiation in the presence of [Ru(TAP)(3)](2+) (TAP = 1,4,5,8-tetraazaphenanthrene), which is a non-intercalating complex, and were followed as a function of illumination time. The observed structural changes correlate well with the conversion of the supercoiled covalently closed circular form (ccc form) into the open circular form (oc form), induced by a single-strand photocleavage. The AFM results obtained after fine-tuning of the plasmid DNA-substrate interaction compare well with those observed from gel electrophoresis, indicating that under the appropriate deposition conditions, AFM is a reliable technique to investigate irradiation-induced topological changes in plasmid DNA.

Efficient Photoinduced DNA Damage by Coralyne

The photoinduced DNA damage by the berberine derivative coralyne is presented. The irradiation of coralyne in the presence of plasmid DNA namely, pBR322, leads to remarkably fast DNA damage by single-strand cleavage, as determined by agarose-gel electrophoresis. Even upon exposure to sunlight, almost all of the supercoiled plasmid is converted to the open circular form in less than a minute [c(pBR322) = 3.5 x 10(-9) M; c(coralyne) = 4.3 x 10(-5) M]. The efficiency of the DNA strand cleavage is not decreased in the presence of radical-trapping reagents such as tert-butanol or DMSO. Moreover, the extent of the DNA damage is the same under aerobic conditions and at reduced oxygen concentration. Thus, the formation of reactive intermediates such as hydroxyl radicals or singlet oxygen is excluded. These results show that the exposure of coralyne and derivatives thereof to light, even with moderate light intensity, needs to be avoided during experiments in which their biological activity is assessed by plasmid unwinding assays.

Extent of Supercoiling in Plasmid DNA Vaccines

Plasmid DNA is currently being considered for immunization as an alternative approach to protein-based vaccines. The prevailing opinion is that the linear and open-circular forms of plasmid DNA are less effective than the supercoiled isoform in inducing immune responses. However, it can be argued that the plasmid is likely to be nicked and relaxed during its transport into the nucleus, regardless of the level of supercoiling present. This article investigates the validity of the recommendations regarding supercoiling requirements for optimal efficiency of DNA vaccines.Some studies appear to be consistent with the notion that cell transfection with plasmids is better with the supercoiled than the relaxed open-circular isoform. Other studies have shown that there is a minimum limit of supercoiled plasmid that needs to be present for potent in vivo responses to DNA vaccines, with the limit being reported to be >70% in one study. However, other data indicate that gene transfer is not significantly affected by the level of supercoiling of the gene, and cationic lipids and microparticles can be used to deliver both isoforms without significant loss of efficiency in vivo.To further address this issue, we evaluated the in vitro and in vivo potency of DNA plasmids encoding the HIV proteins, gp140 env and p55 gag. The in vitro results showed no relationship between the degree of supercoiling and cellular transfection efficiency. Similarly, the antibody titers measured in mice immunized with fully relaxed open-circle plasmids were not significantly different from responses elicited with supercoiled plasmid. However, linearized DNA was ineffective.Based on the findings of ourselves and others that the relaxed open-circular form of a plasmid DNA is no less efficient in transfecting cells or in eliciting antibody responses than its supercoiled form, we propose that it is possible to lower the requirement for a high percentage of supercoiled plasmid in a DNA vaccine formulation.

Radiation protection by Terminalia chebula: Some mechanistic aspects

Radioprotective ability of the aqueous extract of the fruit of Terminalia chebula (TCE) was evaluated for its antioxidant and radioprotective abilities. TCE (50 microg) was able to neutralise 1,1-diphenyl-2-picrylhydrazyl, a stable free radical by 92.9%. The free radical neutralizing ability of TCE was comparable to that of ascorbate (100 microM) 93.5% and gallic acid (100 microM) 91.5% and was higher than that of the diethyldithiocarbamate (200 microM) 55.4%, suggesting the free radical activity of TCE. TCE protected the plasmid DNA pBR322 from undergoing the radiation-induced strand breaks. Radiation damage converts the supercoiled form (ccc) of plasmid to open circular form (oc); the presence of TCE during radiation exposure protected the plasmid from undergoing these damages. The administration of TCE (80 mg/kg body weight, i.p.) prior to whole body irradiation of mice (4 Gy) resulted in a reduction of peroxidation of membrane lipids in the mice liver as well as a decrease in radiation-induced damage to DNA, as assayed by single-cell gel electrophoresis (comet assay). TCE also protected the human lymphocytes from undergoing the gamma radiation-induced damage to DNA exposed in vitro to 2 Gy gamma-radiation. These results suggest the radioprotective ability of TCE.

Bis(2,2′-bipyridine)cobalt(III) complexes containing asymmetric ligands: Synthesis, DNA-binding and photocleavage studies

Three new CoIII complexes: [Co(bpy)2(pdtb)]3+ (1), [Co(bpy)2(pdta)]3+ (2) and [Co(bpy)2(pdtp)]3+ (3) have been synthesized and characterized. The DNA binding behavior of the CoIII complexes has been investigated by spectroscopic methods and viscosity measurements. The results indicate that the size and shape of the intercalated ligand have a marked effect on the binding affinity of complexes involving CT-DNA. Complexes (2) and (3) have been found to promote cleavage of plasmid pBR 322 DNA from the supercoiled form I to the open circular form II upon irradiation. Photocleavage mechanisms are proposed.

Atomic Force Microscopy Measurement of DNA Fragment Induced byHeavy Ions

Choosing 7Li and 12C heavy ions respectively with differentlinear energy transfer (LET) values, purified plasmid DNA samples inaqueous solution are irradiated with various doses. The atomic forcemicroscopy (AFM) is used for analysis of DNA fragments induced by boththe kinds of heavy ions. There is a change of three forms of DNA, i.e.supercoiled, open circular and linear form, as the dose is observed.The distribution function of DNA fragment length is obtained for thefirst time and fitted with the Tsallis entropy statistical theory. Theresult indicates that AFM is a useful tool for analysis of the shortfragment of DNA, high-LET heavy ion radiation induces DNA double strandbreaks (DSBs) more effectively, and the distributions of the DSBs aremore local and dense in comparison with low-LET radiation.

Synergistic effect of green tea polyphenols with trolox on free radical-induced oxidative DNA damage

The antioxidant effect of the principal polyphenolic components extracted from green tea leaves, namely (−)-epicatechin (EC), (−)-epigallocatechin (EGC), (−)-epicatechin gallate (ECG) and (−)-epigallocatechin gallate (EGCG), and their synergistic antioxidant effects with trolox against oxidative DNA damage were studied. The oxidative DNA damage was initiated by a water-soluble azo initiator, 2,2′-azobis (2-amidinopropane hydrochloride) (AAPH) and the ability of green tea polyphenols and/or trolox (a water-soluble analogue of α-tocopherol) to inhibit the oxidative damage of DNA was assessed, in vitro, by measuring the conversion of supercoiled pBR322 plasmid DNA to the open circular and linear forms. It was found that these green tea polyphenols could significantly inhibit the oxidative damage of DNA synergistically with trolox, with an activity sequence of EC = ECG > EGCG > EGC.

DNA-binding and photocleavage studies of [Ru(phen) 2(NMIP)] 2+

A novel ligand 2′-(2″-nitro-3″,4″-methylenedioxyphenyl)imidazo[4′,5′-f][1,10]-phenanthroline (NMIP) and its complex [Ru(phen) 2(NMIP)] 2+ have been synthesized and characterized by mass spectroscopy, 1H NMR and cyclic voltammetry. Binding of the complex with calf thymus DNA (CT DNA) has been investigated by spectroscopic methods, viscosity and electrophoresis measurements. The experimental results indicate that [Ru(phen) 2(NMIP)] 2+ binds to DNA via partial intercalative mode and the individual enantiomers of it bind to DNA in different rates. [Ru(phen) 2(NMIP)] 2+ has also been found to promote cleavage of plasmid pBR 322 DNA from the supercoiled Form I to the open circular Form II upon irradiation.

Ruthenium(II) complexes containing asymmetric 2-(pyrazin-2-yl)naphthoimidazole: syntheses, characterization, DNA-binding and photocleavage studies

A novel asymmetric bidentate ligand, 2-(pyrazin-2-yl)naphthoimidazole (PZNI), and its Ru(II) complexes [Ru(bpy) 2(PZNI)] 2+ ( 1) and [Ru(phen) 2(PZNI)] 2+ ( 2) have been synthesized and characterized by elemental analysis, mass spectra, 1H NMR, and electronic spectroscopy. The electrochemical behaviors of the novel complexes were studied by cyclic voltammetry. The DNA-binding properties of the complexes were investigated by spectroscopic methods and viscosity measurements. The experimental results indicate that the complexes 1 and 2 interact with calf thymus DNA by intercalative mode via the terminal naphthyl ring into the base pairs of DNA. The two Ru(II) complexes have also been found to promote the cleavage of plasmid pBR 322 DNA from the supercoiled form I to the open circular form II upon irradiation.

Synthesis, Characterization, and DNA Interaction Studies of the Ruthenium(II) Complexes [Ru(bpy)2(ipbp)]2+ and [Ru(ipbp)(phen)2]2+ (ipbp=3‐(1H‐Imidazo[4,5‐f][1,10]phenanthrolin‐2‐yl)‐4H‐1‐benzopyran‐2‐one; bpy=2,2′‐Bipyridine; phen=1,10‐Phenanthroline)

AbstractA novel ligand 3‐(1H‐imidazo[4,5‐f][1,10]phenanthrolin‐2‐yl)‐4H‐1‐benzopyran‐4‐one (ipbp) and its ruthenium(II) complexes [Ru(bpy)2(ipbp)]2+ (1) and [Ru(ipbp)(phen)2]2+ (2) (bpy=2,2′‐bipyridine, phen=1,10‐phenanthroline) were synthesized and characterized by elemental analysis and mass, 1H‐NMR, and electronic‐absorption spectroscopy. The electrochemical behavior of the complexes was studied by cyclic voltammetry. The DNA‐binding behavior of the complexes was investigated by spectroscopic methods and viscosity measurements. The results indicate that complexes 1 and 2 bind with calf‐thymus DNA in an intercalative mode. In addition, 1 and 2 promote cleavage of plasmid pBR 322 DNA from the supercoil form I to the open circular form II upon irradiation.

Interaction of polypyridyl ruthenium (II) complex containing asymmetric ligand with DNA

A novel asymmetric bidentate ruthenium (II) complex, [Ru(bpy) 2(PYNI)] 2+ (bpy = 2,2′-bipyridine, PYNI = 2-(2′-pyridyl)naphthoimidazole), has been synthesized and characterized by elemental analysis, ES-MS (electrospray mass spectra) and 1H NMR. The electrochemical behaviors of this complex were studied by cyclic voltammetry. DNA interaction studies suggest that [Ru(bpy) 2(PYNI)] 2+ binds to calf thymus DNA (CT-DNA) in an intercalative mode. Interestingly, this new Ru(II) complex has also been found to promote cleavage of plasmid pBR 322 DNA from the supercoiled form I to the open circular form II upon irradiation.

Impact of intrinsic DNA structure on processing of plasmids for gene therapy and DNA vaccines

Several non-Watson Crick DNA structures have been discovered to date, which may be incorporated into future plasmid constructs for gene therapy and DNA vaccine products. In this study, intrinsic DNA structures were included at a defined point in a 2.9 kb plasmid, and their effects on cell growth rate, total plasmid yield, and topology (i.e. the relative proportions of supercoiled plasmid, open circular and linear forms), were determined. The stability of the inserted sequences were assessed using gel electrophoresis. Z-DNA was shown to be unstable in a batch Escherichia coli DH1 production system grown in complex medium. Encouragingly other sequences studied (triplex, bend and quadruplex) did not cause spontaneous deletions, and no detrimental effect was found on growth rate or on total plasmid yield; indicating that such sequences could be included in future DNA products without any detrimental effect on plasmid yields; although the intra molecular triplex studied significantly decreased the proportion of supercoiled species.