Specific interactions between DNA left-handed supercoils and actinomycin D

The interactions between the natural cyclopentapeptide antibiotic actinomycin D (ACT) and circular pBR322 DNA have been studied by freezing the topological state of the DNA in the complex by topoisomerase I reaction. Both supercoiled and relaxed DNAs, in the complexes at low antibiotic/DNA base-pair ratios, showed a dramatic decrease in linking number that cannot be explained by taking into account only the generally accepted unwinding of 28 degrees for each ACT molecule bound. Recent results derived from the crystallographic analysis of the complex between GpC and ACT suggest that ACT could mediate non-covalent cross-links between distant sections of DNA. Bridges between ACT and different sections of the pBR322 double helix could also explain our results. Two-dimensional gel electrophoresis of ACT-relaxed pBR322 DNA complexes reveals that all supercoils induced by ACT are negative. Two models of the complexes which correspond to the stabilization of DNA crossing by one or two molecules of ACT are proposed. In both cases the ability of ACT to stabilize only DNA left-handed supercoils is derived from the chirality of ACT, when it interacts with DNA.     

A specific DNA unwinding activity associated with SV40 large T antigen

The incubation of highly purified large T antigen with relaxed, circular SV40 DNA in the presence of topoisomerase I (nicking closing enzyme) resulted in the introduction of negative superhelical turns in the DNA. ATP was not required for this reaction. A similar introduction of superhelical turns could also be obtained when a recombinant plasmid DNA (Y182), which contains sequences from both SV40 DNA and pBR322, was used. However, no effect was observed when relaxed pBR322 DNA, which does not contain SV40 DNA sequences, was incubated with T antigen in the presence of topoisomerase. These results are consistent with the hypothesis that large T antigen can recognize and unwind specific sequences on SV40 DNA.     

Effect of 2-chloroethylnitrosoureas on plasmid DNA including formation of strand breaks and interstrand cross-links

Plasmid [3H]pBR 322 was incubated with various alkylating agents including chlorozotocin, N,N'-bis(2-chloroethyl)-N'-nitrosourea (BCNU), N-ethyl-N-nitrosourea (Enu) and dimethylsulfate (DMS). Formation of DNA strand breaks was followed by separation of the various forms of DNA on agarose gels and liquid scintillation counting of the bands. All alkylating agents examined were capable of rapidly producing strand breaks in time and concentration dependent fashion. Bands migrating as relaxed circular and supercoiled forms of the plasmid disappeared, and extensive alkylation resulted in formation of a band that migrated faster than the linear form of DNA. Electron microscopy of this band showed that it consisted of relaxed circles. Prolonged storage of alkylated plasmid resulted in fragmentation of the DNA, possibly due to strand scission at apurinic sites. A new neutral denaturation technique was developed, which allowed for the detection of DNA interstrand cross-links with minimal effects on other potentially labile sites of the alkylated DNA. The level of alkylation was quantitated by incubating [3H]pBR 322 with [2-chloroethyl-U-14C]chlorozotocin and was shown to be independent of DNA concentration but have a linear relationship with drug concentration. Linear and relaxed circular forms of the plasmid were alkylated to a somewhat higher extent than supercoiled DNA. Alkylation of pBR 322 with defined superhelical densities showed no preferential loss in DNA with a specific superhelical density, indicating that alkylation-induced unwinding is independent of superhelicity under the experimental conditions used.     

Construction and characterization of new cloning vehicles. III. Derivatives of plasmid pBR322 carrying unique Eco RI sites for selection of Eco RI generated recombinant DNA molecules.

In vitro recombinant DNA techniques were used to construct two new cloning vehicles, pBR324 and pBR235. These vectors, derived from plasmid pBR322, are relaxed replicating elements. Plasmid pBR324 carries the genes from pBR322 coding for resistance to the antibiotics ampicillin (Apr) and tetracycline (Tcr) and the colicin E1 structural and immunity genes derived from plasmid pMBI. Plasmid pBR325 carries the Apr and Tcr genes from pBR322 and the cloramphenicol resistance gene (Cmr) from phage P1Cm. In these plasmids the unique EcoRI restriction site present in the DNA molecule is located either in the colicin E1 structural gene (pBR324) or in the Cmr gene (pBR325). These vectors were constructed in order to have a single EcoRI site located in the middle of a structural gene which when inactivated would allow, for the easy selection of plasmid recombinant DNA molecules. These plasmids permit the molecular cloning and easy selection of EcoRI, BamHI, HindIII, PstI, HincII, SalI, (XamI), Smal, (XmaI), BglII and DpnII restriction generated DNA molecules.     

Interaction of (+/-)-7r,8t-dihydroxy-9t,10t-epoxy-7,8,9,10-tetrahydrobenzo(a)pyrene with relaxed circular pBR322 DNA

The interaction of (+/-)-BPDE (1) with DNA at neutral pH was investigated by the application of relaxed circular pBR322 DNA. (+/-)-BPDE causes a rapid positive supercoiling of this DNA followed by a slower spontaneous relaxation. The results indicate that there are two clearly discernible types of chemical interactions between 1 and DNA, a rapid intercalative covalent binding and a slower strand breakage. The implications of these findings are discussed.     

The recF-dependent endonuclease from Escherichia coli K12. Formation and resolution of pBR322 DNA multimers

Summary The instability of supercoiled pBR322 DNA obtained from different cells has been investigated. Partially purified plasmid DNA species from rec ⁺, recA and recBC sbcB cells are converted in vitro first to relaxed and then to linear molecules. The recA and recBC sbcB cells produce the best conditions for the monomerization of the pBR322 DNA and the stable maintenance of plasmids. The supercoiled pBR322 DNA from the recBC sbcB recF144 cells has been isolated preferentially in multimeric from (circular oligomers). These DNA forms are not converted to plasmid monomers and are converted to linear molecules three-fold slower than the monomer linearization in the case of the recBC sbcB cells.On the other hand, incubation of the pure pBR322 DNA with the recF-dependent protein Z (Krivonogov and Novitskaja 1982) results in the ATP-independent conversion of supercoiled plasmid DNA to relaxed and linear molecules. These results demonstrate an endonuclease activity of the recF-controlled protein Z, which may be involved in general recA-dependent recombination and formation of the pBR322 monomers in the cell.The results also show that the recF144 mutation in recBC sbcB recF and recF cells leads to the absence of detectable amounts of a 49,000 molecular weight protein.     

超螺旋DNA的碱变构研究

对超螺旋ＤＮＡ（ＤＮＡⅠ）的碱处理产物进行了琼脂糖凝胶电泳,氯化铯－溴化乙锭密度梯度超离心分析,紫外吸收光谱分析和电镜观察。实验结果表明超螺旋ＤＮＡ在碱性环境中的结构改变发生在很窄的ｐＨ范围内（ｐＨ１２．８８─１３．００）．超过ｐＨ临界点的超螺旋ＤＮＡ碱变构产物紫外吸收高于同浓度天然ＤＮＡ紫外吸收的２９％。变构产物在ＣｓＣｌ－ＥＢ密度梯度超离心中的高密度区形成稳定的区带．用透射电镜的观察表明碱变构的超螺旋ｐＢＲ３２２ＤＮＡ具有高电子密度并呈中空颗粒状,以上事实表明,ＤＮＡ在高ｐＨ下可产生一种结构有序的相对稳定的产物．这些结果意味着在碱处理过程中,超螺旋ＤＮＡ在构象上发生了改变,使其分子由扭曲线形变成球形颗粒状。根据实验事实本文对超螺旋ＤＮＡ的碱变构产物（ＤＮＡⅣ）提出一个新的结构模型──压缩模型。这个模型能更合理地解释一些实验现象。     

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.     

Study of mechanisms of electric field-induced DNA transfection. IV. Effects of DNA topology on cell uptake and transfection efficiency.

Electric parameters and solvent conditions are known to influence the efficiency of DNA transfection of cells by a pulsed electric field (PEF). A previous study (Neumann, E., M. Schaefer-Ridder, Y. Wang, and P. H. Hofschneider. 1982. EMBO (Eur. Mol. Biol. Organ.) J. 1:841-845) has indicated that DNA topology is also an important determinant. We report an investigation of the PEF induced uptake, stability, and expression of three different topological isomers, circular supercoiled (scDNA), circular relaxed (crDNA), and linearized (lnDNA) forms of the plasmid pBR322, by Escherichia coli strain JM105. Monomeric pBR322 prepared by the electroelution from an agarose gel was in the supercoiled form. Treatment of the scDNA with wheat germ topoisomerase I removed the superhelicity and the DNA assumed the relaxed circular form. Treatment of scDNA by a restriction endonuclease, EcoRI or Hind III, linearized the DNA. The MgCl2-dependent bindings of all three forms of DNA to the cell surface were indistinguishable. So was the PEF induced cell uptake. In contrast, the transfection efficiency (TE) for the scDNA and the crDNA were high (approximately 2 x 10(8) micrograms-1 DNA at neutral pH), whereas that for the lnDNA was approximately five orders of magnitude lower (less than 1 x 10(3) micrograms-1 DNA). Analysis by agarose gel electrophoresis indicated that the PEF loaded lnDNA was degraded by the host cell within 3 h. However, the loaded scDNA and the crDNA were stable and expressed in the cytoplasm. We conclude that first, the PEF induced DNA entry into E. coli did not depend on the topology of the DNA.(ABSTRACT TRUNCATED AT 250 WORDS)     

Atomic force microscopy study of the structural effects induced by echinomycin binding to DNA

Atomic force microscopy (AFM) has been used to examine the conformational effects of echinomycin, a DNA bis-intercalating antibiotic, on linear and circular DNA. Four different 398 bp DNA fragments were synthesized, comprising a combination of normal and/or modified bases including 2,6-diaminopurine and inosine (which are the corresponding analogues of adenine and guanosine in which the 2-amino group that is crucial for echinomycin binding has been added or removed, respectively). Analysis of AFM images provided contour lengths, which were used as a direct measure of bis-intercalation. About 66 echinomycin molecules are able to bind to each fragment, corresponding to a site size of six base-pairs. The presence of base-modified nucleotides affects DNA conformation, as determined by the helical rise per base-pair. At the same time, the values obtained for the dissociation constant correlate with the types of preferred binding site available among the different DNA fragments; echinomycin binds to TpD sites much more tightly than to CpG sites. The structural perturbations induced when echinomycin binds to closed circular duplex pBR322 DNA were also investigated and a method for quantification of the structural changes is presented. In the presence of increasing echinomycin concentration, the plasmid can be seen to proceed through a series of transitions in which its supercoiling decreases, relaxes, and then increases.     

Early stages in RecA protein-catalyzed pairing. Analysis of coaggregate formation and non-homologous DNA contacts.

RecA protein will catalyze the in vitro pairing of homologous DNA molecules. To further explore the events involved in the search for homology, we have applied a nitrocellulose filter binding assay to follow pairing, and a sedimentation assay to follow the generation of aggregates (termed coaggregates) formed between RecA-complexed single-stranded (ss) DNA and double stranded (ds) DNA. Electron microscopy (EM) was used to visualize the structures involved. RecA protein promoted the pairing of circular M13 ssDNA and linear M13mp7 dsDNA efficiently in the absence of coaggregates. Indeed, pairing of homologous ss- and dsDNAs involved coaggregate formation only if the dsDNA was circular. For DNAs containing only a few hundred base-pairs of homology, for example pUC7 dsDNA and M13mp7 ssDNA, pairing and joint formation was observed if the dsDNA was superhelical but not if it was topologically relaxed or linear with the homology internal to an end of the dsDNA. The effect of non-covalently attached heterologous dsDNA on the RecA-promoted joining of M13 ssDNA and linear M13mp7 dsDNA (with non-M13 sequences at both ends) was found to depend on the topology and concentration of the heterologous DNA. A tenfold excess of superhelical pBR322 DNA strongly inhibited pairing. However, addition of relaxed or linear pBR322 DNA to the pairing reaction had little effect. As seen by EM, superhelical pBR322 DNA inhibited joint formation by excluding the homologous dsDNA form the coaggregates. EM also revealed heterologous DNA interactions presumably involved in the search for homology. Here the use of EM has provided a direct visualization of the form and architecture of coaggregates revealing a dense interweaving of presynaptic filaments and dsDNA.     

Studies of interaction of dichloro[eta2-dimethyl-(2-methylidene-cyclohexylmethyl)-amino]platinum(II) with DNA: effects on secondary and tertiary structures of DNA - cytotoxic assays on human cancer cell lines Capan 1 and A431

The interaction with DNA and the cytotoxic activity of a new organometallic platinum(II) compound were studied. Different techniques were used to evaluate changes in secondary and tertiary DNA structures, and to obtain images of DNA morphological changes. The ability of platinum complex to modify secondary DNA structure was explored by circular dichroism (CD). Electrophoretic mobility showed changes in tertiary DNA structure. Finally, Atomic Force Microscopy (AFM) revealed morphological changes of plasmid DNA (pBR322). This compound breaks the traditional structure-activity rules for cis-platinum compounds, but it could be of interest because of its different kinetics. An organometallic bond normally shows a higher trans-effect than an amine ligand, and that fact, a priori, could contribute to a higher DNA binding rate. Human A431 and Capan-1 cells (vulvae carcinoma and pancreatic carcinoma, respectively) were exposed to increasing concentrations of cisplatin and complex 6 for 24 h, after which time the cell number/viability was determined by the colorimetric MTT assay. A low cytotoxicity of organometallic compound 6 against A431 and Capan-1 cancer cell lines was observed and this result is consistent with the low interaction with DNA observed in previous studies.     

Nicking activity on pBR322 DNA of ribosome inactivating proteins from Phytolacca dioica L. leaves

Ribosome-inactivating proteins isolated from Phytolacca dioica L. leaves are rRNA-N-glycosidases, as well as adenine polynucleotide glycosylases. Here we report that some of them cleave supercoiled pBR322 dsDNA, generating relaxed and linear molecules. PD-L1, the glycosylated major form isolated from the winter leaves of adult P . dioica plants, produces both free 3'-OH and 5'-P termini randomly distributed along the DNA molecule, as suggested by labelling experiments with [alpha- 32P]dCTP and [gamma- 32 P]dATP. Moreover, when the reaction is carried out under low-salt conditions, cleavage is observed mainly at a specific site, located downstream of the ampicillin resistance gene (close to position 3200), ending with the deletion of a fragment of approximately 70 nucleotides. This cleavage pattern is similar to that obtained under the same conditions with mung bean nuclease, a single-strand endonuclease. Furthermore, pBR322 DNA treated with PD-L1 shows reduced transforming activity with E . coli HB101 competent cells in comparison to untreated control plasmid DNA.     

Fluorescence studies of Hoechst 33342 with supercoiled and relaxed plasmid pBR322 DNA

The fluorescence properties of Hoechst 33342 (HO 33342) were examined with plasmid pBR322 in the supercoiled (Form I) or relaxed covalently closed circular (Form Io) conformation in order to determine whether qualitative or quantitative differences in fluorescence properties might provide an assay for topological states of DNA. It was found that HO 33342 exhibited a 30% greater fluorescence intensity with Form I pBR322, independent of the dye or DNA concentration. As the dye to DNA ratio was increased, a red shift of approximately 8 nm was observed for HO 33342 complexed with Form I or Form Io. The red shift in fluorescence emission occurred at higher HO 33342 concentrations with Form I vs. Form Io DNA; however, when Form I and Form Io were mixed in various proportions, neither the fluorescent intensity differences nor the HO 33342 concentration at which the wavelength shift occurred could be used to quantitate the relative proportions of topological states present. These results suggest that although the fluorescence properties of HO 33342 complexed with Form I DNA are different than those of HO 33342 complexed with Form Io DNA, the fluorescence assay is not sufficiently sensitive to quantitatively discriminate among a mixture of DNA in various topological states.     

Understanding apoptotic signaling pathways in cytosine deaminase-uracil phosphoribosyl transferase-mediated suicide gene therapy in vitro

The present work was carried out to evaluate the antioxidant activity of hesperidin and to study its protective effect on H₂O₂ induced oxidative damage on pBR322 DNA and RBC cellular membrane. The in vitro assays were performed with different concentrations (2, 4, 6, 8, and 10 μg/ml, which were equivalent to 3.27, 6.55, 9.83, 13.10, and 16.38 μM) of hesperidin and the results clearly indicate that hesperidin at 10 μg/ml exhibited radical scavenging activity greater than that of standards like ascorbic acid and trolox. The protective effect of hesperidin on pBR322 DNA and RBC cellular membrane on treatment with different concentrations of H₂O₂ shows that hesperidin at 2.5 mM converts the open circular form (oc) of pBR322 DNA that is an indication of damage to super coiled (ccc) form and at 10 μg/ml it prevents membrane damage. Thus, our result proves hesperidin to be a valuable antioxidant that protects pBR322 DNA and RBC cellular membrane from free radical induced oxidative damage.     

DNA Topoisomerase in Nuclei Purified from Root Meristems of Pisum sativum

DNA topoisomerase is present in nuclei purified from the rootmeristems of Pisum sativum seedlings. The DNA topoisomeraseis solubilized from nuclei by 500 mol m^–3 NaCl and relaxessupercoiled pBR322 DNA forming a series of DNA topoisomers whichmigrate electrophoretically between the supercoiled and opencircular forms. The presence of ATP in the incubation mixtureincreases the number of DNA topoisomers migrating electrophoreticallyin the region with slightly greater mobility than the open circularform. The formation of topoisomers with different linking numbersmight be the result of the activation of a different DNA topoisomerasewhich has a peculiar relaxing activity or introduces supercoilsinto the open circular form of pBR322 DNA. A low unknottingactivity with knotted P4 DNA is also present in the same nuclearpreparation. The hypothesis is made that both DNA topoisomerase I and IImight be present contemporaneously in these nuclei. The DNArelaxing activity seems to be stable and is activated by KC1.Partial purification by ion-exchange chromatography is not sufficientto separate these two DNA topoisomerases. Key words: Pisum sativum, pea, DNA topoisomerase, nuclei, cell proliferation     

Antioxidant potential of aminothiazole derivative and its protective effect on H2O2-induced oxidative damage on pBR322 DNA and RBC cellular membrane

The present work was carried out to evaluate the antioxidant and free radical scavenging activity of aminothiazole derivative by performing various in vitro assays; to study its protective effect on H(2)O(2)-induced oxidative damage on pBR322 DNA and on RBC cellular membrane. The in vitro assays were performed with different concentrations of aminothiazole derivative (6.15, 12.29, 18.44, 24.59, and 30.73 microM) and the results were compared with standards like ascorbic acid and trolox. Our results clearly indicated that aminothiazole derivative at a dose of 18.44 microM exhibited radical scavenging activity greater than that of ascorbic acid and trolox. The DNA protective effect on pBR322 DNA showed that there was a concentration-dependent inhibition of the disappearance of supercoiled (ccc) form of DNA on incubation with 30 mM H(2)O(2) in the presence of different concentrations of aminothiazole derivative. Thus our compound at 1.5 mM prevents the conversion from supercoiled (ccc) form to open circular form (oc) form of pBR322 DNA. Pretreatment with aminothiazole derivative at a dose of 18.44 microM prevents membrane damage and exhibits an IC(50) value, which is the concentration of the sample required to inhibit 50% of the radical formed greater than that of the standards (ascorbic acid and trolox). Thus our compound of interest aminothiazole derivative exhibits antioxidant and free radical scavenging properties greater than that of standards like ascorbic acid and trolox and thereby protects pBR322 DNA and RBC cellular membrane from free radical induced oxidative damage.