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At least 307 records · Page 17Linked to original sources

PubMed1991

Circular DNA of human immunodeficiency virus: analysis of circle junction nucleotide sequences.

During infection of cells by retroviruses, some of the nonintegrated viral DNA can be found as a circular form containing two tandem, directly repeated long terminal repeats. The nucleotide sequence at the point where the long terminal repeats join (the circle junction) can be used to deduce the terminal nucleotides of the linear form of the viral DNA. Comparison of the termini of linear viral DNA with sequences at the junctions between the integrated provirus and the host chromosome has revealed that for most retroviruses 2 bp are removed from each end of the linear viral DNA during integration. For human immunodeficiency virus type 1 (HIV-1), however, sequence considerations involving primer-binding sites had suggested that only 1 bp is removed during integration. We obtained the nucleotide sequences at the ends of HIV-1 DNA by using the polymerase chain reaction to amplify fragments corresponding to the HIV-1 circle junction. Of 17 clones containing amplified sequences, 10 had identical circle junctions that contained an additional 4 bp (GTAC) relative to the integrated provirus. This indicates that, as for other retroviruses, 2 bp are removed from each end of the linear HIV-1 viral DNA during integration. The remaining seven isolates contained insertions or deletions at the circle junction.

Base Sequence↗

PubMed1979

Extracellular nucleases of Alteromonas espejiana BAL 31.IV. The single strand-specific deoxyriboendonuclease activity as a probe for regions of altered secondary structure in negatively and positively supercoiled closed circular DNA.

The dependence of the initial rate of introduction of the first single-chain scission (initial nicking rate) into covalently closed circular phage PM2 DNA by the single strand-specific nuclease from Alteromonas espejiana BAL 31 upon the superhelix density (sigma) of the DNA has been examined. The initial nicking rate decreases with decreasing numbers of negative superhelical turns (decreasing values of -sigma), which behavior is characteristic of other single strand-specific nucleases as reported earlier. In contrast to earlier work, the initial nicking rates of closed circular DNAs by the action of the Alteromonas nuclease have been shown to be readily measurable at values of -sigma as low as 0.02. However, even at the elevated concentrations of enzyme and extended digestion periods required to cause nicking at an appreciable rate at near-zero values of sigma, closed circular DNA containing very few superhelical turns (form IO DNA) is not cleaved at a detectable rate. When this DNA is rendered positively supercoiled by ethidium bromide (EtdBr), it is not affected by the nuclease until very high positive values of sigma are attained, at which low rates of cleavage can be detected at elevated enzyme concentrations. The effects of EtdBr on the enzyme activity have been tested and are entirely insufficient to allow the interpretation of zero nicking rates as the result of inhibition of the nuclease activity by the dye. Positively supercoiled DNA is concluded not to contain regions having significant single-stranded character until values of sigma are reached which are very much higher than the values of -sigma for which negatively supercoiled DNAs behave as if they contain unpaired or weakly paired bases.

DNA, Circular↗

PubMed2001

Initial amplification of duck hepatitis B virus covalently closed circular DNA after in vitro infection of embryonic duck hepatocytes is increased by cell cycle progression.

The relationship between the cell cycle and early amplification of duck hepatitis B virus covalently closed circular (CCC) DNA was studied after in vitro infection of fetal hepatocytes. We first showed that embryonic hepatocytes proliferated for at least 6 days after plating and that complete viral replication including CCC DNA amplification occurred in these proliferating cells. Addition of sodium butyrate or aphidicolin reversibly blocked cells in the G1 phase and diminished CCC DNA synthesis, which was restored after drug withdrawal, concomitantly with the entry of cells into S phase. Cell cycle progression of fetal hepatocytes can be triggered by stimulation with epidermal growth factor (EGF), hepatocyte growth factor (HGF), and tumor growth factor alpha (TGF-alpha). CCC DNA synthesis increased with progression to the S phase induced by EGF, HGF, and TGF-alpha alone or in combination. By contrast, tumor growth factor beta (TGF-beta) alone or in combination with EGF inhibited cell proliferation and viral DNA synthesis. By double labeling, viral nucleocapsids were found predominantly in bromodeoxyuridine-positive hepatocytes, indicating that high viral replication occurs preferentially in proliferating hepatocytes. CCC DNA was also detected mainly in cells in the S and G2/M phases separated from cells in the G1 phase by cell sorting. Taken together, these results show that hepatocyte proliferation may positively regulate the initial amplification of CCC DNA of avian hepadnaviruses, and may explain why mitosis is not necessarily associated with loss of CCC DNA.

Animals↗

PubMed1982

Mechanism of linear DNA circularization: formation of 'lasso'-like structures of pre-mRNA DNA copies.

An electron microscopic investigation of a DNA product synthesized on the pre-mRNA template using AMV reverse transcriptase in the absence of actinomycin D revealed linear single-stranded and double-stranded molecules, and, also, double-stranded branched as well as 'lasso'-like and circular structures. Models for the formation of such DNA structures are proposed.

Animals↗

PubMed1998

The post-incision steps of the DNA base excision repair pathway in Escherichia coli: studies with a closed circular DNA substrate containing a single U:G base pair.

The DNA base excision repair pathway is responsible for removal of oxidative and endogenous DNA base damage in both prokaryotes and eukaryotes. This pathway involves formation of an apurinic/apyrimidinic (AP) site in the DNA, which is further processed to restore the integrity of the DNA. In Escherichia coli it has been suggested that the major mode of repair involves replacement of a single nucleotide at the AP site, based on repair synthesis studies using oligonucleotide substrates containing a unique uracil base. The mechanism of the post-incision steps of the bacterial base excision repair pathway was examined using a DNA plasmid substrate containing a single U:G base pair. Repair synthesis carried out by repair-proficient ung, recJ and xon E.coli cell extracts was analyzed by restriction endonuclease cleavage of the DNA containing the uracil lesion. It was found that replacement of the uracil base was always accompanied by replacement of several nucleotides ( approximately 15) 3' of the uracil and this process was absolutely dependent on initial removal of the uracil base by the action of uracil-DNA glycosylase. In contrast to findings with oligonucleotide substrates, replacement of just a single nucleotide at the lesion site was not detected. These results suggest that repair patch length may be substrate dependent and a re-evaluation of the post-incision steps of base excision repair is suggested.

Base Composition↗

PubMed1983

Bacteriophage Mu DNA circularizes following infection of Escherichia coli.

Mu DNA, isolated from infected cells or minicells, has been shown to be held by proteins in twisted and open circular forms. Circularization does not require protein synthesis in the infected cells. A 64,000-dalton polypeptide is injected into the infected cell with Mu DNA and co-sediments with Mu DNA through sucrose gradients. Circularization of the infecting Mu DNA does not require removal of the Escherichia coli DNA sequences which are attached to both ends of the Mu genome in the viral particle.

Bacteriophage mu↗

PubMed1976

Circular DNA and cardiolipin in hydrogenosomes, microbody-like organelles of trichomonads.

Circular molecules of DNA approximately 3 mum in length were revealed by electron microscopy in deproteinized extracts prepared from purified hydrogenosomal fraction of a protozoan Tritrichomonae foetus. This fraction contained also cardiolipin amounting to approximately 14.4% of its total phospholipids, as detected by thin-layer chromatography and quantitative phosphorus measurement. These characteristics extend a number of biochemical properties of hydrogenosome shared also by mitochondria and by prokaryotic cells.

Cardiolipins↗

PubMed1997

A novel transcriptional element in circular DNA monomers of the duck hepatitis B virus.

We report the presence of two elements, pet and net, that are required for proper transcription of the duck hepatitis B virus (DHBV). These regions were previously identified by using plasmid clones of the virus in transient expression assays (M. Huang and J. Summers, J. Virol. 68:1564-1572, 1994). In this study, we further analyzed these regions by using in vitro-synthesized circular DHBV DNA monomers to mimic the authentic transcriptional template. We observed that pet was required for pregenome transcription from circular viral monomers, and in the absence of pet-dependent transcription, expression of the viral envelope genes was increased. We found that deletion of net in circularized DNA monomers led to the production of abnormally long transcripts due to a failure to form 3' ends during transcription. In addition, we report the presence of a net-like region in the mammalian hepadnavirus woodchuck hepatitis virus. These results are consistent with a model that net is a region involved in transcription termination and that in DHBV, pet is required for transcription complexes to read through this region during the first pass through net.

Animals↗

PubMed1990

Lupus-inducing drugs alter the structure of supercoiled circular DNA domains.

We analyzed the effects of procainamide (PROC), hydralazine (HYD), N-acetylprocainamide (NAPA), and L-canavanine (CAN) on circular supercoiled plasmids as models for chromosomal loop domains. The supercoil-dependent B-Z equilibrium in recombinant plasmids was used as an indicator of structural changes induced in circular DNA. Two-dimensional gel electrophoresis showed that PROC and HYD strongly inhibited supercoil-induced Z-DNA formation, whereas NAPA caused less pronounced changes in the B-Z equilibrium, and CAN had no effect. Gel retardation assays showed that the binding of a Z-DNA-specific autoimmune antibody to a Z-DNA-containing plasmid was strongly perturbed by HYD, but not influenced by CAN. Both PROC and NAPA showed moderate inhibition of antibody binding. Our results demonstrate the different potentials of these 4 drugs to interact with DNA and to alter the tertiary topology of DNA domains. It is conceivable that the in vivo capacity of PROC and HYD to induce antinuclear antibodies may be related to their ability to influence structural features in chromosomal DNA domains or nucleosomes, thus liberating antigenic structural epitopes in DNA and/or DNA-associated proteins.

Acecainide↗

PubMed2004

cis-Acting sequences that contribute to the synthesis of relaxed-circular DNA of human hepatitis B virus.

Synthesis of the relaxed-circular (RC) genome of hepadnaviruses is a multistep process that requires template switching during reverse transcription. Studies of duck hepatitis B virus indicated the presence of cis-acting sequences, distinct from the donor and acceptor sequences for the template switches, which contribute to the synthesis of RC DNA. However, knowledge about cis-acting requirements distinct from the donor and acceptor sites for human hepatitis B virus (HBV) was lacking. In this study, we searched for cis-acting sequences for synthesis of HBV RC DNA by analyzing a set of deletion variants that collectively represent most of the HBV genome. Sequences of epsilon, DR1, DR2, 5'r, and 3'r were not analyzed in the study. Results from Southern blotting showed that multiple cis-acting sequences were involved in the synthesis of HBV RC DNA. Analysis of several HBV/woodchuck hepatitis virus chimeras corroborated the findings from the analysis of deletion variants. This study represents a comprehensive and quantitative analysis of cis-acting sequences that contribute to the synthesis of HBV RC DNA.

DNA Replication↗

PubMed1985

The KpnI family of long interspersed nucleotide sequences is present on discrete sizes of circular DNA in monkey (BSC-1) cells.

Discretely sized molecules of small circular DNAs in African green monkey kidney (BSC-1) cells contain nucleotide sequences homologous to the KpnI family of long interspersed repetitive nucleotide sequences. The size distribution of these KpnI family-containing circular DNAs differs markedly from those of BSC-1 cell circular DNAs containing either the Alu family of short interspersed nucleotide sequences or the alpha-satellite family of tandemly repeated sequences. The structures of several cloned, apparently whole, KpnI family-related circular DNAs of varying sizes were analyzed and compared with a compilation of chromosomal KpnI sequences. In general, it was found that the cloned DNAs all contained only KpnI sequences, and that the recombination events given rise to them did not involve any noticeable gain of nucleotides.

Animals↗

PubMed1971

Nature of the complementary strands synthesized in vitro upon the single-stranded circular DNA of bacteriophage phiX174 after ultraviolet irradiation.

This paper describes experiments intended to decide whether UV lesions in DNA act as absolute blocks to chain elongation by the Escherichia coli DNA polymerase or only slow down the polymerization process. Ultraviolet (UV)-irradiated, single-stranded (SS) circular DNA of bacteriophage øX174 was used as template for the polymerase in a reaction mixture in vitro, under conditions allowing synthesis of not more than one complementary strand per template molecule. The mean length of the newly synthesized complementary strands (as determined by velocity sedimentation in alkaline CsCl gradients), as well as the over-all template activity (as measured by deoxyadenosine monophosphate [dAMP] incorporation) was found to decrease with the number of biologically lethal hits sustained by the irradiated templates. With the increase of time or temperature of reaction, the net synthesis of complementary strands increased (as a consequence of increased initiation), but their mean length remained constant. The mean length of synthesized strands was greater than would be expected if all biologically lethal hits were to block the polymerization process. The lethal hits which serve as blocking lesions are inferred to be pyrimidine dimers because it is possible to obtain synthesis of full-length complementary strands if, when heat-denatured, UV-irradiated, double-stranded replicative form (RF II) DNA of bacteriophage øX174 is used as a template, it is pretreated with yeast photoreactivating enzyme (YPRE) in presence of visible light.

Adenine Nucleotides↗

PubMed2001

Hepatitis B virus (HBV) virion and covalently closed circular DNA formation in primary tupaia hepatocytes and human hepatoma cell lines upon HBV genome transduction with replication-defective adenovirus vectors.

Hepatitis B virus (HBV), the causative agent of B-type hepatitis in humans, is a hepatotropic DNA-containing virus that replicates via reverse transcription. Because of its narrow host range, there is as yet no practical small-animal system for HBV infection. The hosts of the few related animal viruses, including woodchuck hepatitis B virus and duck hepatitis B virus, are either difficult to keep or only distantly related to humans. Some evidence suggests that tree shrews (tupaias) may be susceptible to infection with human HBV, albeit with low efficiency. Infection efficiency depends on interactions of the virus with factors on the surface and inside the host cell. To bypass restrictions during the initial entry phase, we used recombinant replication-defective adenovirus vectors, either with or without a green fluorescent protein marker gene, to deliver complete HBV genomes into primary tupaia hepatocytes. Here we show that these cells, like the human hepatoma cell lines HepG2 and Huh7, are efficiently transduced by the vectors and produce all HBV gene products required to generate the secretory antigens HBsAg and HBeAg, replication-competent nucleocapsids, and enveloped virions. We further demonstrate that covalently closed circular HBV DNA is formed. Therefore, primary tupaia hepatocytes support all steps of HBV replication following deposition of the genome in the nucleus, including the intracellular amplification cycle. These data provide a rational basis for in vivo experiments aimed at developing tupaias into a useful experimental animal system for HBV infection.

Adenoviridae↗

PubMed2001

Controlling electrophoretic trapping of circular DNA by addition of starch preparations to agarose gels.

Starch preparations were added to agarose gels to enhance the electrophoretic trapping of circular plasmid DNA. The critical voltages required to trap the open circular (OC) and the supercoiled (SC) forms of a 13.1-kbp plasmid were measured in gels composed of agarose and added starch preparations. Modified starch preparations reduced the critical voltage required to trap the OC form of the plasmid to approximately one-third of the control value (in 1% agarose gels). Amylose (a fraction of starch with a low amount of branching) also reduced the critical voltage to trap the OC form in a similar manner. The critical voltage to trap the SC form of the plasmid was not significantly reduced by the starch preparations. The capacity to trap OC DNA was increased by the addition of higher amounts of the starch preparations added to the gels. Field inversion gel etectrophoresis was used to characterize the length of the traps in the gels. The starch preparations and amylose increased the trap lengths approximately twofold. The increased trap length correlated with the decreased critical voltage required to trap the OC form of the 13.1-kbp plasmid.

Amylose↗

PubMed1975

Action of nicking-closing enzyme on supercoiled and nonsupercoiled closed circular DNA: formation of a Boltzmann distribution of topological isomers.

Highly purified nicking-closing enzyme from mouse cells in 20-fold enzyme/substrate excess converts closed circular native PM2, ColE1, and Minicol DNA into limit product sets of DNAs. Each set has a mean degree of supercoiling of approximately zero. The individual species in the sets differ by deltatau = +/-1, +/-2, etc., and the relative masses fit a Boltzmann distribution. It was also demonstrated that "nonsupercoiled" closed circular duplex molecules serve as substrates for the nicking-closing enzyme, and that a distribution of topological isomers is generated. Polynucleotide ligase, acting on nicked circular DNA, forms under the same conditions, the same set of closed DNAs. The latter enzyme freezes the population into sets of molecules otherwise in configurational equilibrium in solution.

Animals↗

PubMed2002

Construction and characterization of mismatch-containing circular DNA molecules competent for assessment of nick-directed human mismatch repair in vitro.

The ability of cell-free extracts to correct DNA mismatches has been demonstrated in both prokaryotes and eukaryotes. Such an assay requires a template containing both a mismatch and a strand discrimination signal, and the multi-step construction process can be technically difficult. We have developed a three-step procedure for preparing DNA heteroduplexes containing a site-specific nick. The mismatch composition, sequence context, distance to the strand signal, and the means for assessing repair in each strand are adjustable features built into a synthetic oligonucleotide. Controlled ligation events involving three of the four DNA strands incorporate the oligonucleotide into a circular template and generate the repair-directing nick. Mismatch correction in either strand of a prototype G.T mismatch was achieved by placing a nick 10-40 bp away from the targeted base. This proximity of nick and mismatch represents a setting where repair has not been well characterized, but the presence of a nick was shown to be essential, as was the MSH2/MSH6 heterodimer, although low levels of repair occurred in extract defective in each protein. All repair events were inhibited by a peptide that interacts with proliferating cell nuclear antigen and inhibits both mismatch repair and long-patch replication.

Base Pair Mismatch↗

PubMed1992

Circular DNA molecules imaged in air by scanning force microscopy.

Routine and reproducible imaging of DNA molecules in air with the scanning force microscope (SFM) has been accomplished. Circular molecules of plasmid DNA were deposited onto red mica and imaged under various relative humidities. In related experiments, the first images of the Escherichia coli RNA polymerase-DNA complex have also been obtained. This has been possible by (1) the use of specially modified SFM tips with a consistent radius of curvature of 10 nm or less, to minimize the amount of image distortion introduced by the finite dimensions of commercially available tips, (2) the optimization of a method to deposit and bind DNA molecules to the mica surface in a stable fashion, and (3) careful control of the sample humidity, to prevent solvation of the molecules and detachment from the surface by the scanning tip or stylus. Contact forces in the range of a few nanonewtons are routinely possible in air and in the presence of residual humidity. The spatial resolution of the images appears determined by the radius of curvature of the modified styli, which can be estimated directly from the apparent widths of the DNA molecules in the images.

Air↗

PubMed2005

Viral covalently closed circular DNA in a non-transgenic mouse model for chronic hepatitis B virus replication.

BACKGROUND/AIMS: The lack of small animal models supporting chronic hepatitis B virus (HBV) infection impedes the assessment of anti-viral drugs in the whole animal. Although transgenic mice have been used for this purpose, these models are clearly different from natural infection, because HBV is produced from the integrated HBV sequence harbored in all hepatocytes. METHODS: Balb/cA nude mice were hydrodynamically injected with a plasmid having 1.5-fold over-length of HBV DNA and analyzed for HBV replication. RESULTS: Hydrodynamically injected mice showed substantial levels of antigenemia and viremia for more than 1 year. Covalently closed circular DNA (cccDNA), the template of viral replication in natural infection, was produced in the livers and was critically involved in the long-term HBV production, because disruption of the pol gene of the inoculated DNA resulted in transient expression of HBV genes for less than 2 months. Administration of the IFNalpha gene transiently suppressed HBV DNA replication, but was not capable of eliminating HBV in this model. CONCLUSIONS: In vivo gene transfer of a plasmid encoding HBV DNA can establish chronic viral replication in mice, which involves, at least in part, new synthesis of the HBV cccDNA episome, thus recapitulating a part of human HBV infection.

Animals↗