Isolation of in Vitro Synthesized Covalently Closed Circular Double-Stranded DNA by Selective Denaturation and Filtration

Based upon their resistance to irreversible denaturation, covalently closed circular (CCC) DNA (non-nicked, double-stranded circular molecules) can be purified by alkaline denaturation, neutralization, and filtration through a nitrocellulose membrane. This procedure offers a simple means of isolating in vitro synthesized CCC DNA molecules. The preparations of molecules obtained by this method consisted of 91-97% CCC DNA and contained no detectable inhibitors of biological activity or enzymatic digestion.     

Characterization of the Host Factors Required for Hepadnavirus Covalently Closed Circular (ccc) DNA Formation

Synthesis of the covalently closed circular (ccc) DNA is a critical, but not well-understood step in the life cycle of hepadnaviruses. Our previous studies favor a model that removal of genome-linked viral DNA polymerase occurs in the cytoplasm and the resulting deproteinized relaxed circular DNA (DP-rcDNA) is subsequently transported into the nucleus and converted into cccDNA. In support of this model, our current study showed that deproteinization of viral double-stranded linear (dsl) DNA also took place in the cytoplasm. Furthermore, we demonstrated that Ku80, a component of non-homologous end joining DNA repair pathway, was essential for synthesis of cccDNA from dslDNA, but not rcDNA. In an attempt to identify additional host factors regulating cccDNA biosynthesis, we found that the DP-rcDNA was produced in all tested cell lines that supported DHBV DNA replication, but cccDNA was only synthesized in the cell lines that accumulated high levels of DP-rcDNA, except for NCI-H322M and MDBK cells, which failed to synthesize cccDNA despite of the existence of nuclear DP-rcDNA. The results thus imply that while removal of the genome-linked viral DNA polymerase is most likely catalyzed by viral or ubiquitous host function(s), nuclear factors required for the conversion of DP-rcDNA into cccDNA and/or its maintenance are deficient in the above two cell lines, which could be useful tools for identification of the elusive host factors essential for cccDNA biosynthesis or maintenance.     

Isolation of Covalently Closed Circular Deoxyribonucleic Acid from Bacteria Which Produce Exocellular Nuclease

Reproducible yields of covalently closed circular (plasmid) deoxyribonucleic acid were obtained from mutants defective for extracellular nuclease but not from the corresponding wild-type strain of Serratia marcescens     

Generation of Covalently Closed Circular DNA of Hepatitis B Viruses via Intracellular Recycling Is Regulated in a Virus Specific Manner

Persistence of hepatitis B virus (HBV) infection requires covalently closed circular (ccc)DNA formation and amplification, which can occur via intracellular recycling of the viral polymerase-linked relaxed circular (rc) DNA genomes present in virions. Here we reveal a fundamental difference between HBV and the related duck hepatitis B virus (DHBV) in the recycling mechanism. Direct comparison of HBV and DHBV cccDNA amplification in cross-species transfection experiments showed that, in the same human cell background, DHBV but not HBV rcDNA converts efficiently into cccDNA. By characterizing the distinct forms of HBV and DHBV rcDNA accumulating in the cells we find that nuclear import, complete versus partial release from the capsid and complete versus partial removal of the covalently bound polymerase contribute to limiting HBV cccDNA formation; particularly, we identify genome region-selectively opened nuclear capsids as a putative novel HBV uncoating intermediate. However, the presence in the nucleus of around 40% of completely uncoated rcDNA that lacks most if not all of the covalently bound protein strongly suggests a major block further downstream that operates in the HBV but not DHBV recycling pathway. In summary, our results uncover an unexpected contribution of the virus to cccDNA formation that might help to better understand the persistence of HBV infection. Moreover, efficient DHBV cccDNA formation in human hepatoma cells should greatly facilitate experimental identification, and possibly inhibition, of the human cell factors involved in the process.     

Effects of Entecavir on Hepatitis B Virus Covalently Closed Circular DNA in Hepatitis B e Antigen-Positive Patients with Hepatitis B

The aim of this study was to assess the effect of 48-week entecavir therapy on serum and intrahepatic hepatitis B virus, covalently closed circular DNA (HBV cccDNA) levels in hepatitis B e antigen (HBeAg)-positive patients. A total of 120 patients with HBeAg-positive chronic hepatitis were treated with entecavir for 48 weeks. Serum HBV markers, total HBV DNA, and HBV cccDNA levels were measured at baseline and week 48. Biopsies from 20 patients were available for both intrahepatic total HBV DNA and cccDNA testing at these timepoints. HBV cccDNA levels were decreased from a median level of 5.1×10⁶ copies/mL at baseline to a median level of 2.4×10³ copies/mL at week 48. Reduction magnitudes of HBV cccDNA in patients with normalized alanine aminotransferase levels and those undergoing HBeAg seroconversion were significantly greater than those in alanine aminotransferase-abnormal and HBeAg positive patients. Intrahepatic HBV cccDNA was decreased significantly after 48 weeks of treatment, but could not be eradicated. In conclusion, treatment of HBeAg-positive hepatitis B patients with entecavir for 48 weeks decreased serum and intrahepatic HBV cccDNA significantly, and the magnitude of HBV cccDNA reduction was related to total HBV DNA decrease, alanine aminotransferase normalization, and HBeAg seroconversion.     

New Method for Large-Scale Preparation of Covalently Closed λ DNA Molecules

A combination of mutations in bacteriophage lambda and its host Escherichia coli K-12 provides a convenient system for the isolation of large quantities of covalently closed circular DNA molecules. We describe two procedures for the large scale preparation of lambda DNA in the duplex circular form.     

Transcription Factor Binding to Replicated DNA

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On the Toroidal Condensed State of Closed Circular DNA

Abstract

The influence of double helix torsional elasticity on the compaction and structure of circular DNA compact form is studied theoretically in the case when the compact (globular) form has torus shape. For closed circular DNA the topological invariant, the linking number, yields a strict connection between conformation of the double helix considered as unifilar homopolymer and elastic energy of torsional twisting. The contribution of torsional elasticity to the free energy of the toruslike globule is calculated. This contribution is shown to be proportional to the square of superhelical density. Allowance of the torsional elasticity decreases the equilibrium radius of the toruslike globule formed by circular DNA. Closure of linear DNA into a ring widens the stability range of the relatively short DNA compact form and tightens it for long DNA.     

Replicating SV40 Molecules containing Closed Circular Template DNA Strands

SV40 DNA replicates as a closed circle and it is suggested that at least some SV40 DNA molecules replicate bidirectionally.     

The Twist,Writhe, and Linking Number Distributions in Closed Circular DNA

Abstract

We discuss the predictions which follow from the assumption of statistically independent twist and writhe distributions of given variances in circular DNA with single-strand nicks. The nature of the topoisomer distribution produced upon covalent closure of the nicks is described, as well as the nature of the twist and writhe distributions in the fully-closed molecules. In particular, we show how the distributions depend on the magnitudes of the given variances, and how the relative magnitudes of the variances can be deduced from experiment. One additional consequence of the theory is the prediction of a necessary difference between the temperature coefficient of the twist in nicked versus fully-closed circular DNA. The ratio of the two twist coefficients turns out to depend only on the ratio of the twist and writhe variances in nicked DNA.