DNA binding proteins of rat thigh muscle: Purification and characterization of an endonuclease

Two major DNA binding proteins of molecular weights 34,000 and 38,000 have been identified in the 30,000 g supernatant (S-30) fraction of rat thigh muscle extracts. The presence of 38 KD DNA binding protein in the muscle S-30 could be demonstrated only if Triton X-100 treated extracts were used for Afinity chromatography suggesting that this protein may be a membrane associated DNA binding protein. The 38 KD DNA binding protein differed from the 34 KD DNA binding protein also in its chromatographic behaviour in DE-52 columns in which the 38 KD protein was retained, while the 34 KD protein came out in the flow-through in an electrophoretically pure form. The 34 KD DNA binding protein can also be purified by precipitation with MgCl₂. Incubation of 0 15 M NaCl eluates (containing the 38 KD and/or 34 KD DNA binding protein) in the presence of 100 mM Mg²⁺ resulted in the specific precipitation of the 34 KD protein. Prolonged incubation (30 days) of the 0.15 M NaCl eluates containing the two DNA binding proteins at 4°C led to the preferential degradation of the 34 KD DNA binding protein. Nitrocellulose filter binding assays indicated selective binding of purified 34 KD protein to ss DNA. Purified 34 KD DNA binding protein cleaved pBR 322 supercoiled DNA, and electrophoresis of the cleavage products in agarose gels revealed a major DNA band corresponding to the circular form of DNA.     

竹红菌甲素敏化质粒pBR 322 DNA光氧化——使封闭环DNA转变为开环DNA

甲素可敏化质粒pBR 322 DNA光氧化断链的使其封闭环DNA转变为开环DNA。甲素敏化pBR 322 DNA光氧化反应可被单线态氧淬灭剂-NaN_3抑制,证明此光敏氧化机制属Ⅱ型过程。     

pBR322 contains glucocorticoid regulatory element DNA consensus sequences

A computer search of the pBR322 DNA sequence identified five sites matching reported glucocorticoid regulatory element (GRE) DNA consensus sequences and three related sites. A pBR322 DNA fragment containing one GRE site was shown to bind immobilized HeLa S3 cell glucocorticoid receptor and to compete for receptor binding in a competitive binding assay. Conversely, a pBR322 DNA fragment devoid of GRE sites showed barely detectable interaction with glucocorticoid receptor in either of these assays. These results demonstrate the importance of GRE consensus sequences in glucocorticoid receptor interactions with DNA, and further identify a cause for high background binding observed when pBR322 DNA is used as a negative control in studies of glucocorticoid receptor-DNA interactions.     

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。     

A DNA-binding protein from Ustilago maydis prefers duplex DNA without chain interruptions.

Using a nitrocellulose filter binding assay, we have partially purified a protein from mitotic cells of Ustilago maydis that binds preferentially to covalently closed circular duplex DNA. DNA containing single- or double-strand breaks is bound poorly by the protein. Once formed, the DNA-protein complex is stable, resisting dissociation in high salt. However, when a DNA strand is broken, the complex appears to dissociate. The protein binds equally well to form I DNA of phi X174 or the plasmid pBR322, but has a higher affinity for a hybrid plasmid containing a cloned region of Drosophila melanogaster satellite DNA.     

DNA-binding proteins of human placenta: purification and characterization of an endonuclease

DNA binding proteins present in the cytoplasm and nuclei of term placenta were isolated by DNA-cellulose chromatography and analysed by electrophoresis in high resolution polyacrylamide gradient gels. A denatured DNA specific protein of approximate molecular weight 34 000 daltons was the predominant DNA binding protein of the cytoplasm; this protein consisted of over 65% of the total DNA binding proteins of the 0.15 M NaCl eluate of the cytoplasm. The cytoplasmic extracts contained two additional DNA binding proteins of molecular weight 24 000 and 18 000 daltons and these proteins bound preferentially to ds DNA. All the three DNA binding proteins were also present in the nuclei and electrophoresis of histones in adjacent lanes indicated that they are not histones. The 34 000-dalton DNA binding protein has been purified by ammonium sulphate fractionation followed by phosphocellulose (PC) chromatography. The DBP eluted from the PC column between 0.125–0.15M potassium phosphate. PC fractions containing electrophoretically pure 34KD DBP showed an endonuclease activity capable of converting plasmid pBR 322 DNA to the linear form. Maximum endonucleolytic activity was observed in the presence of 3–5 mM Mg²⁺ and the enzyme activity was completely inhibited by 3 mM ethylenediamine tetraacetate.     

Atomic force microscopic study on topological structures of pBR322 DNA

Plasmid pBR322 DNA (0.5mg/mL) isolated from Escherichia coli HB101 was suspended in Tris-HCl-EDTA (1 mol/L - 0.1 mol/L, pH8.5); then a drop of the above solution was deposited on freshly cleaved mica substrate. After adsorption for about 1 min, the sample was stained with phosphotungstic acid. The residua] solution was removed with a piece of filter paper. Afterwards the sample was imaged with a home-made atomic force microscope (AFM) in air. The AFM images of pBR322 DNA with a molecular resolution have been obtained. These images show that pBR322 DNA exists in several different topological structures: (i) relaxed circular DNA with a different diameter; (ii) supercondensed DNA with different particle sizes; (iii) dimeric catenane connected by one relaxed circular molecule and another dose-compacted molecule which might be either supercoiled or intramolecular knotted form; (iv) oligomeric catenane with multiple irregular molecules in which DNA is interlocked into a complex oligomer; (v) possibly-existing     

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.     

Ozonolysis of supercoiled pBR322 DNA resulting in strand scission to open circular DNA.

Treatment of supercoiled pBR322 DNA with ozone resulted in the conversion of closed circular DNA to open circular DNA. Restriction analysis of the resulting open circular DNA showed that ozonolysis in the absence of salt caused single strand cleavage at specific sites.     

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.     

The cleavage of DNA at phosphorothioate internucleotidic linkages by DNA gyrase.

We have constructed a plasmid which contains 22 copies of a 147 bp DNA fragment which contains the major DNA gyrase cleavage site from plasmid pBR322 (located at base-pair 990). We have found that this fragment is efficiently bound and cleaved by gyrase. The selectivity for the sequence corresponding to position 990 in pBR322 is maintained even when this site is located only 15 bp from one end of the 147 bp fragment. A strategy for the specific incorporation of a single thiophosphoryl linkage into the 147 bp fragment has been developed, and gyrase has been shown to catalyse efficient cleavage of fragments bearing phosphorothioate linkages at the gyrase cleavage site in one or both strands.     

Naphthalimide intercalators with chiral amino side chains: effects of chirality on DNA binding, photodamage and antitumor cytotoxicity

Several novel heterocyclic-fused naphthalimides intercalators with chiral amino side chains were investigated. Their side chains' chiral configuration determines DNA binding activities in the order: S-enantiomers > R-enantiomers. And their DNA photodamaging activities were in good agreement with their DNA binding constants, the S-enantiomers could photocleave circular supercoiled pBR322 DNA more efficiently than their R-enantiomers. S-enantiomer B(3) could photodamage DNA at 0.2 microM and cleave supercoiled plasmid DNA from form I to form II completely at 50 microM. Almost all of these intercalators showed effective cytoxicities against human lung cancer cells and murine leukemia cells. S-enantiomers showed different antitumor cytotoxicity by comparison with R-enantiomers. This work may provide additional information for the role of amino side chains on intercalators as antitumor agents.     

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.     

DNA-binding proteins of the malaria vector Anopheles stephensi: purification and characterization of an endonuclease

DNA-binding proteins present in fourth instar larvae of Anopheles stephensi were isolated by affinity chromatography on native and denatured DNA cellulose columns and analyzed by electrophoresis on polyacrylamide gels. A denatured DNA-specific protein with an approximate molecular weight of 30 kDa was the predominant DNA binding protein of larvae. This protein was purified to electrophoretic homogeneity by ammonium sulfate fractionation followed by phosphocellulose chromatography. The purified 30 kDa binding protein showed an endonucleolytic activity capable of converting pBR 322 supercoiled DNA to the circular form. Maximum endonucleolytic activity was observed in the presence of 5 mM Mg(2+) at pH 7.4. Enzyme activity was completely inhibited by EDTA.     

DNA gyrase complex with DNA: determinants for site-specific DNA breakage.

DNA gyrase catalyses DNA supercoiling by making a transient double-stranded DNA break within its 120-150 bp binding site on DNA. Addition of the inhibitor oxolinic acid to the reaction followed by detergent traps a covalent enzyme-DNA intermediate inducing sequence-specific DNA cleavage and revealing potential sites of gyrase action on DNA. We have used site-directed mutagenesis to examine the interaction of Escherichia coli gyrase with its major cleavage site in plasmid pBR322. Point mutations have been identified within a short region encompassing the site of DNA scission that reduce or abolish gyrase cleavage in vitro. Mapping of gyrase cleavage sites in vivo reveals that the pBR322 site has the same structure as seen in vitro and is similarly sensitive to specific point changes. The mutagenesis results demonstrate conclusively that a major determinant for gyrase cleavage resides at the break site itself and agree broadly with consensus sequence studies. The gyrase cleavage sequence alone is not a good substrate, however, and requires one or other arm of flanking DNA for efficient DNA breakage. These results are discussed in relation to the mechanism and structure of the gyrase complex.     

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.