Uptake of circular deoxyribonucleic acid and mechanism of deoxyribonucleic acid transport in genetic transformation of Streptococcus pneumoniae.

Deoxyribonucleic acid (DNA) from the covalently closed circular DNA molecules of Pseudomonas phage PM2 was found to enter normally transformable cells of Streptococcus pneumoniae as readily as linear bacterial DNA. In a mutant of S. pneumoniae that lacks a membrane nuclease and is defective in DNA entry, as many molecules of PM2 DNA as of linear DNA were bound on the outside of cells at equivalent DNA concentrations. Bound DNA suffered single-strand breaks, but circular DNA with preexisting breaks was bound no better than closed circles. In the presence of divalent cations, DNA bound to cells of a leaky nuclease mutant showed double-strand breaks. At least the majority of PM2 DNA that entered normal cells was single stranded. These results are consistent with a mechanism for DNA entry in which DNA is first nicked on binding, then a double-strand break is formed by cleavage of the complementary strand, and continued processive action of the membrane nuclease facilitates entry of the originally nicked strand. Although the bulk of circular donor DNA appeared to enter in this way, the results do not exclude entry of a small amount of donor DNA in an intact form.     

Strand breaks of mammalian mitochondrial DNA induced by carcinogens.

Closed circular mitochondrial DNA in mammalian cells was degradated to the open circular form by exposure of the cells to the carcinogens N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) and 4-nitroquinoline 1-oxide (4NQO). MNNG caused more strand scission of mitochondrial DNA than 4NQO at the same concentration. The action of the carcinogens on mitochondrial DNA did not parallel that with nuclear DNA which was damaged by 4NQO more markedly than by MNNG. Mitochondrial DNA damaged by carcinogens was not repaired during 4-20 h of post-treatment incubation of the cells. Incorporation of labeled thymidine into the closed circular mitochondrial DNA, decreased by the treatment of cells with carcinogens, recovered during post-treatment incubation.     

Platinum complexes of p- and m-aminobenzamidine. Synthesis, characterization, and cytotoxic activity.

Four platinum(II) aminobenzamidine complexes have been prepared and characterized by IR and 1H and 13C NMR spectroscopy, and tested for their ability to interact with the nicked and closed circular forms of the pUC8 plasmid DNA. The results show that the complexes of formula [Pt(LH)2Cl2]2X have a cis- geometry with an amino-Pt bonding, where L is either p- or m-aminobenzamidine and where 2X is 2Cl- or PtCl4(2-). It was observed that these complexes significantly alter the electrophoretic mobility of nicked and closed circular forms of DNA and that the alteration in electrophoretic mobility due to Pt(II)-p-aminobenzamidine binding is higher than that due to Pt(II)-m-aminobenzamidine. No difference in mobility was observed whether the DNA interacted with complexes having as counteranion Cl- or PtCl4(2-). The synthesized compounds were, in addition, assayed for antitumor activity in vitro against colon (CX-1), lung (LX-1), and mammary (MX-1) human tumor cells. The results show that these complexes inhibited the multiplication of the tumor cells and that they show higher specificity for lung cells.     

Separation of closed circular DNA from linear DNA by electrophoresis in two dimensions in agarose gels

A method that provides an easy, rapid, and reproducible way for separating closed circular DNA species from linear DNA and nicked circles is described. The method is based on the difference in mobility of form I (supercoiled) DNA and form II (nicked circles), and the differential mobility of relaxed circular DNA in the presence and absence of ethidium bromide (EtdBr). It can be used for detection or for purification of plasmid, episomal, or viral DNA from the bulk of cellular DNA, or from other DNA mixtures.     

Covalently closed circular duplex DNA of Epstein-Barr virus in a human lymphoid cell line.

A non-integrated form of Epstein-Barr virus DNA was purified from the Burkitt lymphoma-derived human lymphoid cell line Raji by CsCl density gradient centrifugation and neutral glycerol gradient centrifugation. This intracellular form of the virus DNA sediments at a rate typical of a covalently closed circular DNA molecule of the size of the virus genome in both neutral and alkaline solution. Treatment with low doses of X-rays leads to a discontinuous conversion of the molecules to a form with the sedimentation properties of open circular DNA (a circular duplex molecule containing one or more single-strand breaks). The direct observation of large circular DNA molecules by electron microscopy further confirms the covalently closed circular duplex structure of part of the intracellular viral DNA. Such circular molecules were not detected in corresponding DNA fractions from Epstein-Barr virus-negative human lymphoid cell lines. In ethidium bromide/CsCl density gradient centrifugation experiments, the purified non-integrated virus DNA behaves as twisted, covalently closed DNA circles with the same initial superhelix density as polyoma virus DNA. The latter additional purification technique permits the isolation of intracellular Epstein-Barr virus DNA in > 90% pure form from non-producer cells. The molecular weight of the circular virus DNA from Raji cells, determined by contour length measurements, is the same within experimental error as that of the linear DNA from virus particles.     

RNase and alkali sensitivity of closed circular mitochondrial DNA of rat ascites hepatoma cells

A preparation of the closed circular DNA duplex was obtained from whole rat ascites hepatoma cells, AH66, by lysis of cells with SDS and purification by CsCl-dye buoyant-density centrifugation. RNase A converted the closed circular mitochondrial DNA to open circular molecules. The closed circular DNA was also sensitive to alkali. The conversion to the open form was shown from the results of centrifugal analyses on neutral and alkaline sucrose density gradients and CsCl-ethidium bromide. These results indicate the presence of at least one RNA region in closed circular double stranded mitochondrial DNA.     

Involement of supertwisted DNA in integrative recombination of bacteriophage lambda.

Negatively supertwisted closed circular DNA is the primary substrate for integrative recombination of phage λ DNA in vitro. Closed circular λ DNA without supertwists must be converted to the supertwisted form by the action of Escherichia coli DNA gyrase before efficient recombination can occur. When negatively supertwisted substrate is provided, E. coli DNA gyrase and its cofactors are dispensable components of recombination reaction mixtures. In the absence of DNA gyrase activity, circular DNA considerably less negatively twisted than naturally occurring supercoils is an effective substrate, but positively supertwisted DNA appears to be an ineffective substrate.The predominant products of integrative recombination in vitro are covalently closed circles. The closure of the recombined sites appears to occur without appreciable DNA synthesis and without the action of E. coli DNA ligase. No detectable difference can be observed between the degree of supertwisting of product DNA and that of unrecombined DNA. These facts suggest that the resealing of broken DNA strands is an integral part of the recombination reaction mechanism and is closely coupled with the breakage and realignment steps of recombination.     

Adriamycin-mediated introduction of a limited number of single-strand breaks into supercoiled DNA

It was reported previously that Adriamycin converts form I covalently closed circular, supercoiled bacteriophage PM2 DNA to the relaxed circular form II DNA; no form III linear DNA was produced as a result of the extracellular action of Adriamycin in the presence of NADH-dehydrogenase. When form II DNA, produced by the action of Adriamycin, was treated with the BAL 31 nuclease, a single sharp DNA band after agarose gel electrophoresis indicated the presence of only full-length linear form III DNA. As one of its activities, the BAL 31 nuclease introduces a single-strand break in the complementary strand opposite a preexisting single-strand break. When form II DNA, produced by the action of gamma irradiation, was reacted with the BAL enzyme, the resulting linear DNA molecules exhibited a broad range of molecular weights, indicating the presence of many single-strand breaks in the substrate form II DNA. When the Adriamycin-produced form II DNA was treated with restriction endonucleases that cleave PM2 DNA at a single site, either with or without pretreatment with the BAL enzyme, the formation of only full-length linear DNA was observed. Thus, the drug is capable of introducing one or only a very limited number of single-strand breaks into supercoiled DNA; furthermore, these breaks are introduced at random sites along the DNA molecules.     

Rapid screening for plasmid DNA

Summary A procedure is described for demonstrating plasmid DNA and its molecular weight, based on rate zonal centrifugation of unlabelled DNA in neutral sucrose gradients containing a low concentration of ethidium bromide. Each DNA species is then visualized as a discrete fluorescent band when the centrifuge tube is illuminated with ultra-violet light. Plasmids exist as closed circular and as relaxed circular molecules, which sediment separately, but during preparation of lysates, closed circular molecules are nicked so that each plasmid forms only a single band of relaxed circles within the gradient.     

Amplification of closed circular DNA in vitro.

The polymerase chain reaction is a powerful technique used to amplify nucleic acids in vitro . The reaction produces linear products, and as of yet, closed circular products have not been possible. Since the replicatively competent form of many DNA molecules is the closed circular form, it would be adventitious to amplify closed circular DNA as closed circular molecules. Until now, these molecules could only be amplified in vivo in appropriate host cells. Here, we describe an in vitro procedure, ligation-during-amplification (LDA), for selective amplification of closed circular DNA using sequence-specific primers. LDA is useful for site-directed mutagenesis, mutation detection, DNA modification, DNA library screening and circular DNA production.     

F deoxyribonucleic acid superinfected into phenocopies of donor strains.

When F+ donor cells of Escherichia coli are conjugated with F-, F+, or Hfr recipients under the conditions of phenocopy mating, the male recipients are found capable of accepting the F episome as effectively as the F- recipients. The F deoxyribonucleic acid (DNA) superinfected into the male recipients is converted to the covalently closed, circular duplex form, as in the F- recipients. It is also found that the synthesis of the strand complementary to the transferred single strand and its subsequent conversion to the covalently closed, circular duplex occur effectively in male recipients as well as in female recipients. Under these mating conditions, F-ilv+ episome superinfected to F+ and Hfr cells is diluted out during growth, whereas F-ilv+ transferred into F-cells is replicated and established in almost all progeny cells. These results suggest that the incompatibility of the F episome is not due to the reduction in the rate of the conversion of transferred single-straned F DNA to covalently closed, circular duplex, but, rather, to an inhibition of further replication of the covalently closed, circular F DNA.     

Melting experiment concerning the topological structure of closed circular double-stranded DNA

A melting experiment was performed on the whole set of populations of the replicative form of ?X174 DNA, which can be obtained treating this DNA with rat liver nicking-closing enzyme in the presence of ethidium bromide. Gel electrophoresis performed by loading the DNA samples at neutral and alkaline pH allows separation of these populations in discrete sets of bands, which can then be compared. The outcome of the experiments indicates that in the range of electrophoretic mobilities which can be explored, no band is formed exclusively by circular complementary strands which can be separated by alkaline denaturation. These results are compared with what would be expected if double-stranded closed circular DNA had structures other than the canonical double helix. Under nonrestrictive hypotheses, the experiments reported allow one to obtain a minimum estimate of the absolute value of the linking number of a closed circular double-stranded DNA: for native ?X174 RF DNA, the linking number appears to be greater than 12 (in absolute value). Some data on the electrophoretic mobility of denatured closed circular duplexes are reported, which still wait for a physicochemical interpretation.     

Infecting bacteriophage mu DNA forms a circular DNA-protein complex

Upon superinfection of immune (lysogenic) cells with bacteriophage Mu, a form of Mu DNA accumulates that sediments about twice as fast as the linear phage DNA marker in neutral sucrose gradients. This form is also detected upon infection of sensitive cells with Mu. We have purified it and examined its physical nature. Under the electron microscope it appears circular and supertwisted. Upon treatment with Pronase, phenol or sodium dodecyl sulfate, however, it is converted to a linear Mu-length form, indicating that the circle is not covalently closed. The linear DNA still has heterogeneous host sequences at its termini. The circular DNA is resistant to the action of Escherichia coli exonuclease III and T7 exonuclease, but becomes sensitive to these nucleases after treatment with Pronase showing the presence of a protein that binds non-covalently to the ends of the DNA to circularize it as well as protect it from digestion with exonucleases. The complex is resistant to high salt (up to 6 M-NaCl) but can undergo transitions between forms that are partially open, open circular, linear and circular dimers and trimers. Examination of DNA from mature phage particles reveals that a circular DNA species is present in at least 0.1 to 1% of the population. The purified complex is extremely efficient in transfection of E. coli spheroplasts. We estimate the molecular weight of the protein in this DNA-protein complex to be approximately 64,000, and suggest that this complex might represent the integrative precursor of infecting Mu DNA.     

Restriction endonuclease cleavage of linear and closed circular murine leukemia viral DNAs: discovery of a smaller circular form.

Both linear (form III) and closed circular (form I) viral DNAs obtained from mouse cells infected with Moloney murine leukemia virus were cleaved by Sal I, Sma I, Bam HI and Pst I restriction endonucleases. DNA fragments generated by these cleavages were ordered with respect to the 5' and 3' ends of the RNA genome by several techniques, including comparisons of the DNA fragments from cleavages of the linear and closed circular forms, double digestions using different combinations of enzymes and the use of an RNA probe specific for the 3' end. DNA from Hirt extractions of infected cells yielded a discrete species of linear viral DNA whose size was determined by agarose gel electrophoresis to be 5.7 x 10(6) daltons. In the course of characterizing the closed circular DNA, we observed two form I DNA molecules. The larger molecule was the same size as the linear DNA. The second molecule migrated faster on agarose gels and was the predominant species of the two closed circular DNAs. Using the restriction endonuclease maps which we derived, we demonstrate that this novel form I DNA is a smaller homogeneous species of viral DNA, missing about 600 nucleotides found in the linear and larger closed circular DNA molecules. We have localized the site of this missing DNA piece to be at either one or both ends of the linear viral DNA.     

三阴性乳腺癌中环状RNA的表达特点及功能意义

环状RNA是由前体RNA通过反向剪接形成的一类共价闭合环状分子.在过去,环状RNA被认为是DNA转录的"噪音",不参与生物代谢过程.然而,最近研究表明,环状RNA的异常表达可影响包括三阴性乳腺癌在内的多种恶性肿瘤的发生发展.该文综述了环状RNA在肿瘤中的分子机制及其在三阴性乳腺癌细胞增殖、凋亡、迁移、侵袭和药物抗性中的...     

Discontinuous DNA synthesis by purified mammalian proteins

Five proteins purified from mouse cells acting together efficiently convert a single-stranded circular DNA template to covalently closed duplex circle by a discontinuous mechanism. DNA polymerase alpha/primase with the assistance of alpha accessory factor covers the single-stranded circle with RNA-primed DNA fragments. Primers are removed by a combination of RNase H-1 and a 5'-exonuclease that was identified by its ability to complete this in vitro system. The 5'-exonuclease is required to remove residual one or two ribonucleotides at the primer/DNA junction that are resistant to RNase H-1. Gap filling is by the DNA polymerase alpha/primase, and DNA ligase I converts the DNA fragments to continuous strand. The concerted action of the five proteins emulates synthesis of the staging strand at the replication fork.     

S1核酸酶作用与微环DNA分子的克隆策略

米曲霉来源的S1 核酸酶具有降解单链DNA或RNA的作用。在适当的条件下 ,该酶能将不同的环形DNA分子从超螺旋转变成开环和线形结构 ,对质粒pUC19的实验证明 ,S1 核酸酶的这种转变作用与加入的酶量呈正相关。在 2 5 μL总反应体积中 ,按 10 0ngDNA加入 5u至 17u的S1 核酸酶 ,能获得较高比例的线形DNA。由于微环DNA分子太小 ,单酶切位点的出现率较低 ,很难用常规方式进行克隆 ,以S1 核酸酶进行线形化是微环DNA克隆的途径。pC3是已知最小的真核生物线粒体DNA类质粒 (5 37bp) ,经S1 核酸酶线形化后 ,成功地克隆到pMD18 T载体上。     

Preparation and melting of single strand circular DNA loops.

A method for preparation of single strand DNA circles of almost arbitrary sequence is described. By ligating two sticky ended hairpins together a linear duplex is formed, closed at both ends by single stranded loops. The melting characteristics of such loops are investigated using optical absorbance and NMR. It is shown by comparison with the corresponding linear sequence (closed circle minus the end loops) that the effects of end fraying and the strand concentration dependence of the melting temperature are eliminated in the circular form. Over the concentration range examined (0.5 to 2.0 micromolar strands), the circular DNA has a monophasic melting curve, while the linear duplex is biphasic, probably due to hairpin formation. Since effects of duplex to single strands dissociation do not contribute to melting of the circular molecules (dumbells), these DNAs present a realistic experimental model for examining local thermal stability in DNA.