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PubMed1997

Lack of effect of antiviral therapy in nondividing hepatocyte cultures on the closed circular DNA of woodchuck hepatitis virus.

The template for synthesis of hepadnaviral RNAs is a covalently closed circular (ccc) DNA located in the nucleus of the infected hepatocyte. Hepatocytes are normally long-lived and nondividing, and antiviral therapies in chronically infected individuals face the problem of eliminating not only the replicative forms of viral DNA found in the cytoplasm but also the cccDNA from the nucleus. Because cccDNA does not replicate semiconservatively, it is not an obvious target for antiviral therapy. However, elimination of cccDNA might be facilitated if its half-life were short in comparison to the generation time of hepatocytes and if new cccDNA formation were effectively blocked. We have therefore measured cccDNA levels in woodchuck hepatocyte cultures following in vitro infection with woodchuck hepatitis virus and treatment with inhibitors of viral DNA synthesis. The viral reverse transcriptase inhibitors lamivudine (3TC) [(-)-beta-L-2',3'-dideoxy-3'-thiacytidine), FTC (5-fluoro-2',3'-dideoxy-3'-thiacytidine) and ddC (2',3'-dideoxycytidine) were added to the cultures beginning at 4 days postinfection. Treatment for up to 36 days with 3TC reduced the amount of cccDNA in the cultures not more than twofold compared to that of an untreated control. Treatment with ddC for 36 days and with FTC for 12 days resulted in effects similar to that of treatment with 3TC. Moreover, the declines in cccDNA appeared to reflect the loss of hepatocytes from the cultures rather than of cccDNA from hepatocytes. These results emphasize the important role of the longevity of the infected hepatocytes in the persistence of an infection.

Animals↗
PubMed1989

Incorporation of exogenous circular DNA into large catenated networks in isolated nuclei. Evidence for involvement of the nuclear scaffold.

Circular plasmid DNA was efficiently converted into huge catenated intranuclear networks by incubation with isolated nuclei in the presence of ATP. The network production is abolished by omission of ATP, strongly inhibited by etoposide (VP-16), but only slightly inhibited by antibody to topoisomerase I, indicating that the major enzyme responsible for catenation is DNA topoisomerase II. Under optimal conditions, a single nucleus incorporates about 4.2 x 10(4) DNA rings into its networks. Under the light microscope, networks retrieved from nuclei appear like spheres of various sizes. Sedimentation analysis showed that most of the networks are composed of thousands of catenated rings, which was confirmed by electron microscopy. Data from experiments that caused partial disruption of the networks were submitted to analysis based on probable models of catenane structure. The results suggest that the predominant pattern is a linear alignment of catenated rings. Similar networks are formed when the nuclear scaffold is incubated with circular DNA in the presence of nuclear extract containing topoisomerase II. Titration experiments showed that the scaffold binds a stoichiometric amount of the substrate and that a critical level of DNA is required for network formation. The results are consistent with the idea that DNA-binding sites are fixed on the scaffold and mediate catenation of bound DNA circles by holding them in close proximity to each other. We propose that catenation by the nuclear scaffold also occurs in intact nuclei, suggesting additional roles for the scaffold in vivo.

Adenosine Triphosphate↗
PubMed1994

Computer simulation of protein-induced structural changes in closed circular DNA.

The effect of protein-induced wrapping on overall DNA folding is studied using Monte Carlo computer simulation techniques. A new modeling scheme is devised to represent configurations of closed circular DNA containing fragments of the double helix partially wrapped around a core of proteins. The DNA consists of two regions, a fragment wrapped in a left-handed superhelical path around a 'phantom' protein core and a free connecting loop. The loop has at least one single-stranded scission so that it can assume a torsionally relaxed state. The configuration of the loop is varied during the course of the computer simulations and the three-dimensional spatial arrangements of lowest total energy are identified. The axis of the DNA loop is represented by a finite three-dimensional Fourier series perturbation of an initial Bézier curve, making it possible to fix the position and orientation of the chain ends as well as the contour length of the free loop. The energy is approximated by elastic terms for the bending and twisting of the DNA and an excluded volume contribution that prevents the self-intersection of sequentially distant chain segments. The proportions of the protein-DNA complex, the number of superhelical turns, the chain length and the imposed linking number difference of the closed DNA are varied in the calculations. The resulting minimum energy structures are consistent with physical models and suggest new ways to think about how proteins add and remove supercoils from DNA. Of special note in this regard is the sudden collapse of three-dimensional structure that accompanies small incremental wrapping of the DNA around the idealized protein core. These observations offer new structural insight into the mechanisms of action of proteins which add or remove supercoils from DNA and provide a first step in thinking about the activity of such systems at the chemical level whereby small fluctuations in local molecular structure are translated into large-scale macromolecular changes. The configurations identified in the simulations can also be examined in the context of the well known "linking number paradox" associated with nucleosome formation on closed circular plasmids. The findings bear relevance to DNA with natural curvature as well as to protein-induced bending and deformations of the double helix.

Computer Simulation↗
PubMed1993

The free energy, enthalpy and entropy of native and of partially denatured closed circular DNA.

We have used gel electrophoresis to measure the progress of local denaturation in closed circular pBR322 DNA as a function of temperature and linking deficiency, delta Lk. Local denaturation is closely coupled to supercoiling in closed DNA, requiring statistical mechanical methods for analysis. We have applied these methods to the experimental data to evaluate the free energies for three associated molecular processes. These processes are changes in the residual linking deficiency, delta Lkr, initiation of local denaturation, and twisting of denatured strands about one another. Our results confirm the quadratic dependence of the supercoiling free energy upon delta Lk, with a free energy coefficient of 740/N kcal/mol at 37 degrees C, where N is the number of base-pairs. The free energy of initiation of denaturation is 10.2(+/- 0.7) kcal/mol. The free energy of interstrand twisting of denatured regions varies with the square of the twist density, with proportionality coefficient C tau = 1.62 (+/- 0.11) kcal/rad2 at 37 degrees C. We have also calculated the entropy and enthalpy of these three processes, using the temperature dependence of the respective free energies. We find that both the entropy and the enthalpy of supercoiling are positive and vary quadratically with delta Lk. The free energy of initiation of denaturation is independent of temperature, hence arises primarily from a change in enthalpy. The entropy and enthalpy of interstrand twisting of denatured regions are both positive, and the twisting force constant decreases with temperature. These results differ considerably from expectations based solely upon considerations of chain configuration in vacuo, indicating the importance of solvent-dependent factors in determining the structure of closed circular DNA.

DNA, Circular↗
PubMed1972

Circular DNA molecules in the genus Drosophila.

The satellite DNA's from the embryos of five species of Drosophila (D. melanogaster, D. simulans, D. nasuta, D. virilis and D. hydei) have been analyzed for the presence of closed circular duplex DNA molecules, as determined by CsCl-EBr gradients. Circular DNA molecules were found in every species but D. melanogaster. Analyses of cell fractions from adult Drosophila and organ fractions from Drosophila larvae show that fractions containing mitochondria are highly enriched in these molecules.

Animals↗
PubMed1999

Equilibrium distributions of topological states in circular DNA: interplay of supercoiling and knotting.

Two variables define the topological state of closed double-stranded DNA: the knot type, K, and DeltaLk, the linking number difference from relaxed DNA. The equilibrium distribution of probabilities of these states, P(DeltaLk, K), is related to two conditional distributions: P(DeltaLk|K), the distribution of DeltaLk for a particular K, and P(K|DeltaLk) and also to two simple distributions: P(DeltaLk), the distribution of DeltaLk irrespective of K, and P(K). We explored the relationships between these distributions. P(DeltaLk, K), P(DeltaLk), and P(K|DeltaLk) were calculated from the simulated distributions of P(DeltaLk|K) and of P(K). The calculated distributions agreed with previous experimental and theoretical results and greatly advanced on them. Our major focus was on P(K|DeltaLk), the distribution of knot types for a particular value of DeltaLk, which had not been evaluated previously. We found that unknotted circular DNA is not the most probable state beyond small values of DeltaLk. Highly chiral knotted DNA has a lower free energy because it has less torsional deformation. Surprisingly, even at |DeltaLk| > 12, only one or two knot types dominate the P(K|DeltaLk) distribution despite the huge number of knots of comparable complexity. A large fraction of the knots found belong to the small family of torus knots. The relationship between supercoiling and knotting in vivo is discussed.

DNA, Circular↗
PubMed1975

Epstein-Barr virus genomes with properties of circular DNA molecules in carrier cells.

A high-density fraction of high-molecular-weight DNA was isolated from the human lymphoid cell line Raji. This cell line contains 50 to 60 virus genome equivalents of Epstein-Barr virus DNA per cell, and the high-density DNA fraction was 10-fold enriched in such viral sequences. Sedimentation analysis on neutral glycerol gradients, followed by hybridization experiments with viral complementary RNA, showed that most of the intracellular viral DNA sequences in this material did not cosediment with the cellular DNA, but were recovered as two distinct species with sedimentation coefficients of 100 S and 65 S. These two forms sediment 1.70-1.75 and 1.10-1.12 times as fast as the linear Epstein-Barr virus DNA from virus particles, and thus have the hydrodynamic properties of a covalently closed circular form and a nicked (containing single-strand breaks) circular form of the virus genome. The 100S form also behaved as a covalently closed circular EBV DNA molecule on gradient centrifugation in CsC1/propidium diiodide, It would appear that latent Epstein-Barr virus DNA has the properties of a mammalian episome, and that both nonintegrated and integrated viral DNA sequences can be isolated from carrier cells.

Base Sequence↗
PubMed1969

Abnormal circular DNA molecules induced by ethidium bromide in the kinetoplast of Trypanosoma cruzi.

The trypanocidal drug ethidium bromide induced abnormal circular DNA molecules with high frequency in the kinetoplast of Trypanosoma cruzi. These molecules are circular dimers, trimers, or higher circular forms with a contour length which is a multiple of the monomer length usually found in the kinetoplastic DNA of normal trypanosomes. These abnormal molecules represent more than 30 per cent of the kinetoplastic DNA, while in normal kinetoplastic DNA we only observed three circular dimers out of several thousand molecules measured.

Bromides↗
PubMed1991

DNA topoisomerase III from extremely thermophilic archaebacteria. ATP-independent type I topoisomerase from Desulfurococcus amylolyticus drives extensive unwinding of closed circular DNA at high temperature.

A second type I topoisomerase was purified from the extremely thermophilic archaebacterium Desulfurococcus amylolyticus. In contrast to the previously described reverse gyrase from this organism, the novel enzyme designated as Dam topoisomerase III is an ATP-independent relaxing topoisomerase. It is a monomer with Mr 108,000, as determined by electrophoresis under denaturing conditions and by size exclusion chromatography. Dam topoisomerase III, like other bacterial type I topoisomerases, absolutely requires Mg2+ for activity and is specific for single-stranded DNA. At 60-80 degrees C, it relaxes negatively but not positively supercoiled DNA and is inhibited by single-stranded M13 DNA. At 95 degrees C, the enzyme unwinds both positively and negatively supercoiled substrates and produces extensively unwound form I* and I** DNA. The peculiarities of DNA topoisomerization at high temperatures are discussed.

Adenosine Triphosphate↗
PubMed1985

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.

Base Sequence↗
PubMed1989

Escherichia coli helicase II (uvrD) protein can completely unwind fully duplex linear and nicked circular DNA.

We have examined the duplex DNA unwinding (helicase) properties of the Escherichia coli helicase II protein (uvrD gene product) over a wide range of protein concentrations and solution conditions using a variety of duplex DNA substrates including fully duplex blunt ended and nicked circular molecules. We find that helicase II protein is able to initiate on and completely unwind fully duplex DNA molecules without the requirement for a covalently attached 3' single-stranded DNA tail. This DNA unwinding activity is dependent upon Mg2+ and ATP and requires that the amount of protein be in excess of that needed to saturate the resulting single-stranded DNA. Unwinding experiments on fully duplex blunt ended DNA with lengths of 341, 849, 1625, and 2671 base pairs indicate that unwinding occurs at the same high ratios of helicase II protein/nucleotide, independent of DNA length (50% unwinding requires approximately 0.6 helicase II monomers/nucleotide in 2.5 mM MgCl2, 10% glycerol, pH 7.5, 37 degrees C). Helicase II protein is also able to unwind completely a nicked circular DNA molecule containing 2671 base pairs. At lower but still high molar ratios of helicase II protein to DNA, duplex DNA molecules containing a single-stranded (ss) region attached to a 3' end of the duplex are preferentially unwound in agreement with the results obtained by S. W. Matson [1986) J. Biol. Chem. 261, 10169-10175). This preferential unwinding of duplex DNA with an attached 3' ssDNA most likely reflects the availability of a high affinity site (ssDNA) with the proper orientation for initiation; however, this may not reflect the type of DNA molecule upon which helicase II protein initiates DNA unwinding in vivo. The effects of changes in NaCl, NaCH3COO, and MgCl2 concentration on the ability of helicase II protein to unwind fully duplex DNA and duplex DNA with a 3' ssDNA tail have also been examined. Although the unwinding of fully duplex and nicked circular DNA molecules reported here occurs at higher helicase II protein to DNA ratios than have been previously used in most studies of this protein in vitro, this activity is likely to be relevant to the function of this protein in vivo since very high levels of helicase II protein accumulate in E. coli during the SOS response to DNA damage (approximately 2-5 x 10(4) copies/cell).(ABSTRACT TRUNCATED AT 400 WORDS)

Adenosine Triphosphatases↗
PubMed2005

Intrahepatic hepatitis B virus covalently closed circular DNA can be a predictor of sustained response to therapy.

BACKGROUND & AIMS: This study aimed to determine whether intrahepatic hepatitis B virus (HBV) covalently closed circular (ccc) DNA and total HBV DNA levels at the end of therapy would predict sustained response to therapy. METHODS: Hepatitis B e antigen (HBeAg)-positive chronic hepatitis B patients receiving either lamivudine monotherapy or combination of peginterferon and lamivudine had liver biopsy at the end of 1 year therapy and were followed for 52 more weeks after cessation of therapy. Serum HBV DNA, intrahepatic HBV ccc DNA, and total HBV DNA levels were determined. RESULTS: Forty-seven patients, including 34 males and 13 females, were studied. Twenty-seven patients received combination therapy, and 20 patients received lamivudine monotherapy. Twenty-nine patients had end-of-treatment virologic response, and 15 patients had sustained response 52 weeks after therapy. At the end of treatment, log serum HBV DNA levels correlated well with log intrahepatic HBV cccDNA and log intrahepatic total HBV DNA levels. Log intrahepatic cccDNA and log intrahepatic total DNA levels were significantly lower among patients with sustained virologic response. The adjusted odds ratio for log cccDNA was 5.3 (95% CI: 1.5-18.2, P = .009) and, for log intrahepatic HBV DNA, was 4.4 (95% CI: 1.3-14.7, P = .015) to predict sustained virologic response. Using log cccDNA at -0.80 copies/genome equivalent as cutoff, the sensitivity, specificity, and positive and negative predictive values and accuracy of predicting sustained virologic response were 73%, 78%, 56%, 86%, and 77% respectively. CONCLUSIONS: Intrahepatic HBV cccDNA and intrahepatic total HBV DNA levels at the end of therapy are superior to serum HBV DNA as surrogates of sustained virologic response.

Adult↗
PubMed1977

[Effect of liver chromatin DNAse on closed circular DNA of PM-2 phage and simian virus 40].

Comparative effect of the DNAse from rat liver chromatin and Neurospora crassa endonuclease S1 on closed circular superhelical DNA of PM-2 phage and Simian Virus 40 is studied. It is shown that both of them--the DNAse from chromatin proteins and endonuclease S1--are specific to single-stranded regions in DNA molecular. It is suggested that chromatin protein DNAse participates in reparation processes.

Animals↗
PubMed2005

Covalently closed circular DNA is the predominant form of duck hepatitis B virus DNA that persists following transient infection.

Residual hepatitis B virus (HBV) DNA can be detected in serum and liver after apparent recovery from transient infection. However, it is not known if this residual HBV DNA represents ongoing viral replication and antigen expression. In the current study, ducks inoculated with duck hepatitis B virus (DHBV) were monitored for residual DHBV DNA following recovery from transient infection until 9 months postinoculation (p.i.). Resolution of DHBV infection occurred in 13 out of 15 ducks by 1-month p.i., defined as clearance of DHBV surface antigen-positive hepatocytes from the liver and development of anti-DHBV surface antibodies. At 9 months p.i., residual DHBV DNA was detected using nested PCR in 10/11 liver, 7/11 spleen, 2/11 kidney, 1/11 heart, and 1/11 adrenal samples. Residual DHBV DNA was not detected in serum or peripheral blood mononuclear cells. Within the liver, levels of residual DHBV DNA were 0.0024 to 0.016 copies per cell, 40 to 80% of which were identified as covalently closed circular viral DNA by quantitative PCR assay. This result, which was confirmed by Southern blot hybridization, is consistent with suppressed viral replication or inactive infection. Samples of liver and spleen cells from recovered animals did not transmit DHBV infection when inoculated into 1- to 2-day-old ducklings, and immunosuppressive treatment of ducks with cyclosporine and dexamethasone for 4 weeks did not alter levels of residual DHBV DNA in the liver. These findings further characterize a second form of hepadnavirus persistence in a suppressed or inactive state, quite distinct from the classical chronic carrier state.

Adrenal Glands↗
PubMed1989

Pulsed-field gel electrophoresis of circular DNA.

Mobility of supercoiled (form I) and nicked circular (form II) plasmid DNAs was determined on two major forms of pulsed-field electrophoresis, CHEF and OFAGE. Plasmids with molecular lengths ranging from 2.30 to 17.8 kilobase pairs (kb) were used with Saccharomyces cerevisiae chromosomes as standards. Agarose gel concentrations were varied from 0.3 to 2.0 percent, with higher percentage gels resolving forms I and II of smaller plasmids. The pulsing range of 3.7 to 240 seconds resulted in quite variable Saccharomyces chromosomal mobilities on both 0.5 and 1.0 percent gels, while both form I and II of all plasmid DNAs showed relatively constant mobilities with some increase at the shortest pulse times. Using a 30 second pulse time and gel concentrations of at least 1.0 percent, the usual order of migration of plasmid forms for a 17.8 kb plasmid could be changed. We interpret this result as an increase in the relative mobility of form II in our pulsed-field gel conditions.

Chromosomes↗
PubMed1995

Analysis of recombination junctions in extrachromosomal circular DNA obtained by in-gel competitive reassociation.

Essentially all eukaryotic cells contain circular extrachromosomal DNA as a result of excision from the chromosomes. To obtain insight into the nature of recombination associated with the occurrence of such DNA species and its biological significance, we analyzed a library enriched in recombination junctions which was constructed by a novel DNA subtraction technique; in-gel competitive reassociation (IGCR). Furthermore, we also introduced inverse PCR to characterize chromosomal DNA fragments containing the recombination junctions. At least 45% of the clones in the library constructed by the IGCR procedure comprised DNA with recombination junctions. Nucleotide sequence analysis of the recombination junctions indicated that three of four extrachromosomal DNAs thus analyzed were produced through recombination between sequences with a 3-5 bp homology in the chromosomes. One extrachromosomal DNA was apparently generated through non-homologous recombination, possibly by end-to-end joining. These results have demonstrated the usefulness of IGCR in concentrating recombination junctions, which provide the most direct evidence for the mechanism of the recombinational events involved, from highly complex genomes.

Base Sequence↗
PubMed1986

Molecular cloning and characterization of small polydisperse circular DNA from mouse 3T6 cells.

We have isolated, cloned and analyzed small polydisperse circular (spc) DNA from mouse 3T6 cells. The representation of highly repeated mouse genome sequence families in spcDNA has been examined, and the B1 repeat appears overrepresented in spcDNA by two criteria. The majority of spcDNA clones, however, is made out by as yet uncharacterized middle repetitive sequences. We have investigated the increase in the spcDNA population upon cycloheximide treatment of individual sequences, which are found to amplify differentially.

Animals↗