Loss of transforming activity of plasmid DNA (pBR322) in E. coli caused by singlet molecular oxygen

Plasmid DNA pBR322 in aqueous solution was exposed to singlet molecular oxygen (1O2) generated by microwave discharge. DNA damage was detected as loss of transforming activity of pBR322 in E. coli (CMK) dependent on the time of exposure. DNA damage was effectively decreased by singlet-oxygen quenchers such as sodium azide and methionine. Replacement of water in the incubation buffer by D2O led to an increase in DNA damage. 9,10-Bis(2-ethylene)anthracene disulfate was used as a chemical trap for 1O2 quantitation by HPLC analysis of the endoperoxide formed.     

Mechanism of NADH-sensitized formation of DNA breaks during irradiation with near UV light]

NADH-photosensitized in vitro formation of single-stranded breaks in plasmid DNA pBR322 depends on both the concentration of the sensitizer and the influence of near-UV radiation (320-400 nm). Scavengers and inhibitors of different activated oxygen species (sodium azide, sodium benzoate, catalase and superoxide dismutase) prevent the formation of breaks in full or partly. The data obtained show that hydroxyl radical (.OH) and singlet oxygen (1O2) are directly involved in the induction of breaks. In this process hydrogen peroxide (H2O2) plays the role of an intermediate in the reaction of .OH formation from superoxide anion-radical (O2-.) which is the first NAD.H-photogenerated product.     

Inhibition of radiation-induced DNA damage in plasmid pBR322 by chlorophyllin and possible mechanism(s) of action

Naturally occurring compounds capable of protecting DNA against ionizing radiation and chemical mutagens have considerable potential for prevention of mutation-based health impairment including cancer and other degenerative diseases. Chlorophyllin (CHL), a water-soluble derivative of chlorophyll, has been examined for its ability to protect DNA against radiation induced strand breaks using an in vitro plasmid DNA system. Gamma-radiation, up to a dose of 6 Gy (dose rate 1.25 Gy/min), induced a dose-dependent increase in single-strand breaks (ssbs) in plasmid pBR322 DNA. CHL per se did not induce, but inhibited radiation-induced ssbs in a concentration-dependent manner; 500 microM giving about 90% protection. The protection afforded by CHL was comparatively less than that of trolox, a water-soluble analogue of alpha-tocopherol. To elucidate the underlying mechanism(s), reaction of CHL with the radiation-derived hydroxyl radical (.OH) and deoxyribose peroxyl radical (ROO.) was studied by pulse radiolysis. CHL exhibited a rate constant of 6.1+/-0.4x109 M-1 s-1 with.OH and 5.0+/-1.3x107 M-1 s-1 with ROO. To our knowledge, this is the first report providing direct evidence of free radical-scavenging properties of CHL. The results showed that CHL, effectively protects plasmid DNA against ionizing radiation, in an in vitro system independent of DNA repair or other cellular defense mechanisms. The ability of CHL to scavenge. OH and ROO., may contribute to its protective effects against radiation induced DNA damage in the pBR322 system.     

DNA interactions of a functionalized ruthenium(II) mixed-polypyridyl complex [Ru(bpy)2ppd]2+

A novel polypyridyl ligand pteridino[7,6-f][1,10]phenanthroline-1,13(10H,12H)-dione (ppd) and its ruthenium(II) complex [Ru(bpy)2ppd]2+ have been synthesized and characterized by elemental analysis, electrospray mass spectra and 1H NMR. The interaction of the complex with calf thymus DNA was investigated by spectroscopic and viscosity measurements. The results suggest that the complex binds to DNA via an intercalative mode and serves as a molecular "light switch" for DNA. Moreover, the complex has been found to promote the photocleavage of plasmid DNA pBR322 under irradiation at 365 nm. The mechanism studies reveal that singlet oxygen (1O2) and superoxide anion radical (O2*(-))play a significant role in the photocleavage.     

Escherichia coli Fpg protein and UvrABC endonuclease repair DNA damage induced by methylene blue plus visible light in vivo and in vitro.

pBR322 plasmid DNA was treated with methylene blue plus visible light (MB-light) and tested for transformation efficiency in Escherichia coli mutants defective in either formamidopyrimidine-DNA glycosylase (Fpg protein) and/or UvrABC endonuclease. The survival of pBR322 DNA treated with MB-light was not significantly reduced when transformed into either fpg-1 or uvrA single mutants compared with that in the wild-type strain. In contrast, the survival of MB-light-treated pBR322 DNA was greatly reduced in the fpg-1 uvrA double mutant. The synergistic effect of these two mutations was not observed in transformation experiments using pBR322 DNA treated with methyl methanesulfonate, UV light at 254 nm, or ionizing radiation. In vitro experiments showed that MB-light-treated pBR322 DNA is a substrate for the Fpg protein and UvrABC endonuclease. The number of sites sensitive to cleavage by either Fpg protein or UvrABC endonuclease was 10-fold greater than the number of apurinic-apyrimidinic sites indicated as Nfo protein (endonuclease IR)-sensitive sites. Seven Fpg protein-sensitive sites per PBR322 molecule were required to produce a lethal hit when transformed into the uvrA fpg-1 mutant. These results suggest that MB-light induces DNA base modifications which are lethal and that these modifications are repaired by Fpg protein and UvrABC endonuclease in vivo and in vitro. Therefore, one of the physiological functions of Fpg protein might be to repair DNA base damage induced by photosensitizers and light.     

Investigation of DNA binding mechanism, photoinduced cleavage activity, electrochemical properties and biological functions of mixed ligand copper(II) complexes with benzimidazole derivatives: synthesis and spectral characterization

The benzimidazole derivative Schiff bases and their copper(II) (Cu(II)) mixed-polypyridyl complexes (1-4) have been synthesized and characterized by the spectral and analytical techniques. DNA binding/cleavage studies indicate a stronger binding capability for the complex 4 which is confirmed by the absorbance, viscometric and gel-electrophoresis studies. The photocleavage of plasmid pBR322 DNA reveals that hydroxyl radical (OH(?)) and singlet oxygen ((1)O(2)) are likely to be the reactive species. Analysis of the growth activity shows that the antimicrobial effect of these Schiff bases on Gram-negative bacteria is higher than that on Gram-positive. Furthermore, the complexes having nitro group show an increased antimicrobial effect.     

Relaxation of supercoiled plasmid DNA by oxidative stresses in Escherichia coli.

The relaxation of plasmid DNA was observed after the visible light irradiation of Escherichia coli AB1157 harboring plasmid pBR322 or some other plasmids in the presence of a photosensitizing dye, such as toluidine blue or acridine orange, and molecular oxygen. Treatment of the cells with hydroperoxides, such as tert-butyl hydroperoxide, cumene hydroperoxide, and hydrogen peroxide, also caused the plasmid DNA relaxation in vivo. Relaxation was not observed in these treatments of purified pBR322 DNA in vitro. Plasmid DNA relaxation was also detected after near-UV irradiation. Far-UV irradiation did not induce such relaxation.     

Plasmid DNA acquires immunogenicity on exposure to singlet oxygen

In the present study, the effect of singlet oxygen (1O2) (generated by ultraviolet (UV) irradiation of methylene blue) on plasmid DNA has been analyzed by UV spectroscopy, fluorescence spectroscopy, and S1 nuclease digestibility. Both native and 1O2-modified plasmid DNA were treated with a number of restriction enzymes to map out the sites damaged by 1O2. It was also observed that, on exposure to 1O2, native plasmid DNA that is non-immunogenic acquired the ability to elicit an immune response in experimental animals. However, the induced antibodies exhibited appreciable cross reactivity with various polynucleotides and nucleic acids. The data indicate that the antibodies, though cross-reactive, preferentially bind 1O2-modified epitopes on plasmid DNA. Gel retardation assay further substantiated the enhanced recognition of 1O2-modified plasmid DNA over the native form. The antibodies developed were then subjected to competition ELISA with sera from various diseases such as systemic lupus erythematosus, rheumatoid arthritis, and cancer. These results suggest that upon exposure of DNA to 1O2, neo-epitopes are generated, which may be one of the factors for the induction of circulating autoantibodies in the three diseases.     

DNA protective properties of vanillin against gamma-radiation under different conditions: possible mechanisms

Ionizing radiation is an important genotoxic agent. Protecting against this form of toxicant, especially by a dietary component, has several potential applications. In the present study, we have examined the ability of vanillin (4-hydroxy-3-methoxybenzaldehyde), a naturally occurring food flavouring agent, to inhibit radiation-induced DNA damage measured as strand breaks under in vitro, ex vivo and in vivo conditions besides the possible mechanisms behind the observed protection. Our study showed that there was a concentration-dependent inhibition of the disappearance of super-coiled (ccc) form of plasmid pBR322 (in vitro) upon exposure to 50 Gy of gamma-radiation. Presence of 0.5 mM vanillin has a dose-modifying factor (DMF) of 6.75 for 50% inactivation of ccc form. Exposure of human peripheral blood leucocytes (ex vivo) to gamma-radiation causes strand breaks in the cellular DNA, as assessed by comet assay. When leucocytes were exposed to 2 Gy of gamma-radiation there was an increase in parameters of comet assay such as %DNA in tail, tail length, 'tail moment' and 'Olive tail moment'. The presence of 0.5 mM vanillin during irradiation significantly reduced these parameters. Damage to DNA in mouse peripheral blood leucocytes after whole-body exposure of mice (in vivo) to gamma-radiation was studied at 1 and 2 h post-irradiation. There was recovery of DNA damage in terms of the above-mentioned parameters at 2 h post-irradiation. This was more than that observed at 1 h. The recovery was more in vanillin treated mice. Hence our studies showed that vanillin offers protection to DNA against radiation-induced damage possibly imparting a role other than modulation of DNA repair. To examine the possible mechanisms of radioprotection, in terms of radiation-derived radicals, we carried out the reaction of vanillin with ABTS*(+) radical spectrophotometrically besides with DNA peroxyl and carbonyl radicals by using pulse radiolysis. Our present investigations show that vanillin has ability to protect against DNA damage in plasmid pBR322, human and mouse peripheral blood leucocytes and splenic lymphocytes besides enhancing survival in splenic lymphocytes against gamma-radiation, and that the possible mechanism may involve scavenging of radicals generated during radiation, apart from modulation of DNA repair observed earlier.     

Methylene blue photosensitized oxidation of cysteine sulfinic acid and other sulfinates: the involvement of singlet oxygen and the azide paradox

The methylene blue photosensitized oxidation of cysteine sulfinic acid is investigated. Enhancement of the oxygen consumption rate in deuterium oxide suggests the involvement of singlet oxygen ((1)O(2)) in oxidation. Addition of the (1)O(2) quencher azide produced an unusual enhancement of the oxidation rate of all the sulfinates assayed. It is assumed that azide works as a one-electron carrier between (1)O(2) and the sulfur compounds. Analyses of the products indicate that the photochemical oxidation of cysteine sulfinic acid proceeds through two simultaneous mechanisms. The Type II (singlet oxygen) mechanism is responsible for oxidation of the sulfinic group to the sulfonic group with production of cysteic acid, stable to the photooxidation system, whereas the Type I (electron transfer) mechanism is involved in the degradation of cysteine sulfinic acid to acetaldehyde. Other products detected were ammonia, sulfate, and hydrogen peroxide which account for the degradation of cysteine sulfinic acid and for the excess of oxygen consumption detected during the oxidative reaction.     

Vanillin as an antioxidant in rat liver mitochondria: Inhibition of protein oxidation and lipid peroxidation induced by photosensitization

Using rat liver mitochondria, as model systems, we have examined the ability of the natural compound and the food-flavoring agent, vanillin to protect membranes against oxidative damage induced by photosensitization at concentrations normally used in food preparations. Vanillin, at a concentration of 2.5 mmol/L, has afforded significant protection against protein oxidation and lipid peroxidation in hepatic mitochondria induced by photosensitization with methylene blue plus light. The effect observed was both time- and concentration-dependent. The inhibitory effect is similar to ascorbic acid and the singlet oxygen quencher, diazabicyclo[2.2.2]octane (DABCO) but less effective than sodium azide and glutathione. Examination of possible mechanisms responsible for the observed protection, showed that vanillin has a significant ability to quench singlet oxygen (¹O₂), a reactive species responsible for damage induced during photosensitization by Type II mechanism. Hence, this flavoring compound, due to its antioxidant ability, may have potential to prevent oxidative damage to membranes in mammalian tissues and thereby the ensuing diseased states.     

Effects of Tris and Hepes buffers on the interaction of palladium-diaminopropane complexes with DNA

The Pd(II) complexes, [PdCl(2)(1,2-pn)] and [PdCl(2)(1,3-pn)] (pn is diaminopropane), were synthesized and characterized by analytical and spectroscopic (FT-IR, (1)H NMR and (13)C NMR) techniques. UV difference spectral study performed on Pd-pn/DNA systems, indicate a pronounced interaction of palladium complexes with DNA in cell-free media; comparison of lambda(max), Abs(max) and %H values observed for the two compounds might be attributed to structural differences of the chelated ligand rings. Results obtained from electrophoretic analysis of Pd complexes in presence of pBR322 plasmid DNA show a clear decreasing of the supercoiled (SC) DNA form mobility, that could be attributed to unwinding of the double helix; a parallel increasing of the open-circular (OC) DNA form mobility is also noted, this fact implying that the binding of complexes either shortens or condenses the DNA helix. Interaction studies of Pd complexes with plasmid DNA in different buffer systems indicate that DNA binding efficiency capable of modifying the tertiary structure of pBR322 decreased from NaClO(4) to Hepes 2, Hepes 1 [Hepes=4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid], and Tris [(hydroxymethyl)aminomethane] buffers, in this order. Moreover, the level of DNA modifications produced by palladium complexes in 10 mM NaClO(4) remains unchanged after transferring the samples into the medium required for subsequent biophysical or biochemical analyses.     

Directed modification of the Tcr gene region of the plasmid pBR322 using complementary single-stranded DNA fragments carrying alkylating groups

A method is suggested for chemical modification of preselected regions of plasmid DNA by complementary single-stranded restriction fragments of DNA (srf DNA), carrying alkylating reagents. The gene coding for tetracycline resistance of plasmid pBR322 was used as a target. Srf DNA was prepared by a partial digestion of a double-stranded EcoRI-BamHI restriction fragment (377 base pairs) from Tcr by E. coli exonuclease III. The residues of an alkylating reagent N,N,N'-tri(beta-chlorethyl)-N'-(p-formylphenyl) propylenediamine 1,3 (TFP) were attached covalently to 4-5% of sfr DNA bases. The alkylating derivative of the sfr DNA was hybridized with supercoiled pBR322 plasmid DNA. The hybridization conditions (37 degrees C, 40% formamide, 0,2 M NaCl, 0,1 M Tris-HCl pH 7,5, 0,001 M EDTA) under which the bases carrying TFP residues are not eliminated from the sfr DNA, and transforming activity of pBR322 DNA does not decrease were established. It was shown that about 20% of plasmid pBR322 molecules form D-loops with alkylating sfr DNA under these conditions. It was shown that sfr DNA, carrying TFP can alkylate the complementary region of plasmid DNA, forming cross-linked D-loops. A method for the site-directed mutagenesis of switching off the preselected genes or non-transcribed DNA functional regions (promotors, introns etc) integrated into plasmids of other vectors is suggested.     

Strand scission in DNA by Gossypol and Cu(II): Role of Cu(I) and oxygen-free radicals

Gossypol, a polyphenolic binaphthyl dialdehyde found in cotton seeds, is a dietary mutagen and a potential male contraceptive. In the presence of Cu(II), gossypol caused breakage of supercoiled plasmid pBR322 DNA. The products were relaxed circles or a mixture of these and linear molecules. Other metal ions tested [Ni(II), Co(II), Mn(II), and Fe(II)] were ineffective or less effective in the DNA breakage reaction. In the case of gossypol-Cu(II) mediated cleavage, (Cu(I) was shown to be an essential intermediate by using the Cud) sequestering reagent bathocuproine. By using job plots, it was established that in the absence of DNA, eight Cu(II) ions can be reduced by one gossypol molecule. The involvement of active oxygen species, such as singlet oxygen and H₂O₂, was established by the inhibition of DNA breakage by catalase and by sodium azide. It was further shown that gossypol is capable of directly producing H₂O₂.     

"In vitro" protection of DNA from Fenton reaction by plant polyphenol verbascoside

The protection effect of verbascoside (Ver) against Fenton reaction on plasmid pBR322 DNA was studied using agarose gel electrophoresis and UV-visible spectroscopy. The pBR322 plasmid DNA is damaged by hydroxyl radical (OH*) generated from the Fenton reaction with H2O2 and Fe(II) or Fe(III). This DNA damage is characterized by the diminution of supercoiled DNA forms or by the increase of relaxed or linear DNA forms after oxidative attack. The UV spectrum study showed that verbascoside can form complexes with Fe(II) or Fe(III), and the complexation can be reversed by the addition of EDTA. The formation constants of verbascoside-Fe complexes were estimated as 10(21.03) and 10(31.94) M(-2) for Fe(II) and Fe(III) respectively. The inhibition of Fenton reaction by verbascoside could be partially explained by the sequestration of Fe ions.     

Methylene blue photosensitized oxidation of hypotaurine in the presence of azide generates reactive nitrogen species: formation of nitrotyrosine

In our previous study on the hypotaurine (HTAU) oxidation by methylene blue (MB) photochemically generated singlet oxygen (1O2) we found that azide, usually used as 1O2 quencher, produced, instead, an evident enhancing effect on the oxidation rate [L. Pecci, M. Costa, G. Montefoschi, A. Antonucci, D. Cavallini, Biochem. Biophys. Res. Commun. 254 (1999) 661-665]. We show here that this effect is strongly dependent on pH, with a maximum at approximately pH 5.7. When the MB photochemical system containing HTAU and azide was performed in the presence of tyrosine, 3-nitrotyrosine was produced with maximum yield at pH 5.7, suggesting that azide, by the combined action of HTAU and singlet oxygen, generates nitrogen species which contribute to tyrosine nitration. In addition to HTAU, cysteine sulfinic acid, and sulfite were found to induce the formation of 3-nitrotyrosine. No detectable tyrosine nitration was observed using taurine, the oxidation product of HTAU, or thiol compounds such as cysteine and glutathione. It is shown that during the MB photooxidation of HTAU in the presence of azide, nitrite, and nitrate are produced. Evidences are presented, indicating that nitrite represents the nitrogen species involved in the production of 3-nitrotyrosine. A possible mechanism accounting for the enhancing effect of azide on the photochemical oxidation of HTAU and the production of nitrogen species is proposed.     

DNA Interaction and Photocleavage Properties of Ru (II) Complexes [Ru(bpy)2(pibi)]2+ and [Ru(phen)2(pibi)]2+

A new asymmetry ligand pibi (pibi = 2-(pyridine-2-yl)-1-H-imidazo[4,5-f]benzo[d]imidazolone) and its ruthenium complexes with [Ru(L)₂(pibi)]²⁺ (L = bpy (2, 2′-bipyridine), phen (1, 10-phenanthroline)), have been synthesized and characterized. The binding of two complexes with calf thymus DNA has been investigated by spectroscopic and viscosity measurement. The results indicate that both complexes can bind to CT-DNA through intercalative mode. Under irradiation at 365 nm, both complexes can partly promote the photocleavage of plasmid pBR322DNA. The low singlet oxygen generation abilities of the two complexes may be the factor for the low DNA photocleavage abilities.     

Supercondensed structure of plasmid pBR322 DNA in an Escherichia coli DNA topoisomerase II mutant

An unusual structural component, supercondensed pBR322 DNA, has been found in plasmid pBR322 DNA samples isolated from a DNA topoisomerase II mutant of Escherichia coli, SD108 (topA+, gyrB225). The supercondensed pBR322 DNA moved faster than supercoiled pBR322 DNA as a homogeneous band in agrose gels when the DNA samples were analysed by electrophoresis. The mobility of the supercondensed DNA was not substantially affected by chloroquine intercalation. The supercondensed pBR322 DNA migrated as a high density "third DNA band" when the samples were subjected to caesium chloride/ethidium bromide gradient equilibrium centrifugation. The unusual pBR322 DNA visualized by electron microscopy was a globoid-shaped particle. These observations suggest that the pBR322 plasmid can assume a tertiary structure other than a supercoiled or relaxed structure. DNA topoisomerases may be involved in the supercondensation of plasmid DNA and chromosomal DNA.     

Rifampicin-induced replication of the plasmid pBR322 in Escherichia coli strains carrying dnaA mutations

The replication pattern of the plasmid pBR322 was examined in the dnaA mutants of Escherichia coli. The rate of pBR322 DNA synthesis is markedly decreased after dnaA cells are shifted to the restrictive temperature of 42 degrees C. However, addition of rifampicin (RIF) to cultures of dnaA strains incubated at 42 degrees C after a lag of 90 min results in a burst of pBR322 synthesis. This RIF-induced pBR322 replication remains dependent on DNA polymerase I activity. Efficient plasmid pBR322 replication is observed at 42 degrees C in the double mutant dnaA46cos bearing an intragenic suppressor of dnaA46. Though replication of pBR322 in dnaA46cos growing at 42 degrees C is initially sensitive to RIF plasmid synthesis is restored after 90 min incubation in the presence of the drug. RIF-induced replication of the plasmid pBR327, lacking the rriB site implicated in RIF-resistant synthesis of the L strand of ColE1-like plasmids (Nomura and Ray 1981; Zipursky and Marians 1981), was observed also in dnaA46 at 42 degrees C.