Evidence for discontinuous replication of circular mitochondrial DNA molecules from Novikoff rat ascites hepatoma cells

Double-forked circular molecules of mitochondrial DNA (mtDNA) from rat tissues, indicated by their form and size to be replicative intermediates, are of two structurally distinct classes. Molecules of the first class are totally double stranded. Molecules of the second class are defined by one daughter segment being totally or partially single stranded. Length histograms of daughter segments measuring between 2% and 44% of the total 5-µm molecular contour were constructed from samples of both classes of replicating molecules derived from mtDNA or Novikoff rat ascites hepatoma cells. For single strand-containing molecules, the lengths fell into eight distinct, reproducible groups with mean values separated by 4.1–7.6% of the circular contour length. For totally double stranded molecules, the lengths fell into seven groups, corresponding to seven of the groups found for single strand-containing molecules. These results suggest that along at least 44% of the contour of mtDNA molecules there exist discrete points at which DNA synthesis tends to be arrested. This may indicate that there are pauses in normal mtDNA synthesis. However, as the DNA used in these experiments was isolated from mitochondrial fractions, the findings may indicate that continuation of synthesis beyond specific points on the nucleotide strands requires a factor which is not available after cell disruption.     

The visualization of amplicons after polymerase chain reaction

Linear DNA molecules amplified by the polymerase chain reaction were visualized by atomic force microscopy. The measured contour length of the PCR product of 1414 bp sequence was 435 +/- 15 nm. Considering that the calculated value of the distance between the nucleotides along the duplex axis is 0.31 nm, it was assumed that linear DNA molecules on the surface of mica, which serve as a support in the atomic force microscopy method, are in the A form. The influence of surface properties of the mica and the sample drying procedure on the conformation of adsorbed DNA molecules is discussed. Possible reasons for the Gaussian distribution of the contour length of the synthesized amplicon are considered.     

Interactive measurement and characterization of DNA molecules by analysis of AFM images.

BACKGROUND: In the past few years, computer-based analysis of atomic-force microscopic images has acquired increasing importance for studying biomolecules such as DNA. On the one hand, fully automated methods do not allow analysis of complex shapes; on the other hand, manual methods are usually time consuming and inaccurate. The semiautomated approach presented in this report overcomes the drawbacks of both methods. METHODS: Two kinds of images were analyzed: computer-generated filaments that modeled circular DNA molecules on a surface and real atomic-force microscopic images of DNA molecules adsorbed on an appropriate substrate surface. RESULTS: The algorithm was tested on a group of 140 simulated and 189 real plasmids with a nominal length of 913 nm. The accuracy of the length measurement was statistically evaluated on the ensemble of molecules, with particular attention to the influence of the noise. Mean contour lengths of 912 +/- 5 nm and 910 +/- 47 nm were found for simulated and real plasmids, respectively. The measured end-to-end distance of lambda-DNA molecules as a function of their contour length is reported, from which it is possible to estimate the stiffness of the DNA molecules adsorbed onto a surface; the value obtained for the DNA persistence length (42 +/- 5 nm) is consistent with values measured by other imaging techniques. CONCLUSIONS: An interactive algorithm for DNA molecule measurements based on the detection of the filament ridge line in a digitized image is presented. The simulation of artificial filaments combined with the experimental data demonstrates that the proposed method can be a valuable tool for the DNA contour length evaluation, especially in the case of complex shapes where the use of automatic methods is not possible.     

Figure-8 configuration of dimers of S13 and φX174 replicative form DNA

Evidence from electron microscopy of the replicative form of S13 and φX174 DNA shows the presence of a “figure-8” configuration. This species consists of two monomer length and one dimer length circular strands in covalently closed circular form and containing a fused junction that divides the molecule into two equal circular segments. Its existence is supported by the demonstration that it is converted by digestion with the restriction endonuclease of Hemophilus influenzae strain Rd to α- and X-shaped forms that retain the fused junction, and by examination by electron microscopy in the presence of ethidium bromide, which eliminates tangling and accidental overlays of parts of the DNA molecules. Kgure-8s are present to the extent of about 5% of the dimers present in replicative form DNA. They are proposed to be intermediates in genetic recombination in S13 and φX174.     

Role of genes O and P in the replication of bacteriophage lambda DNA.

DNA replication in coliphage λ occurs in two stages. The first round of replication generates mainly circular progeny DNA by a double-branched θ-type replicative form (Ogawa et al., 1968; Schnös &; Inman, 1970). In the late stage of λ DNA replication, however, σ-type rolling-circle replicative form DNA molecules, which produce multigenomic linear concatemers, are primarily found (Takahashi, 1974).At both early and later times, a temperature shift of λ Ots or Pts infected cells from 32 °C (permissive) to 43 °C (non-permissive temperature) caused a rapid reduction of the rate of radioactive precursor incorporation into λ DNA, showing that the gene O and P products are essential for the continuation of λ DNA synthesis. Observations on the molecular fine structure of the replicating fork after a temperature shift revealed characteristic long “single-strand connections” and single-strand “whiskers” at the branch point. These observations suggest that λ gene O and P products are directly involved in the propagation of daughter strands.     

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.     

Compaction of single molecules of supercoiled DNA immobilized on amino mica: from duplex to minitoroidal and spheroidal conformations

Supercoiled DNA pGEMEX with a length of 3993 nucleotides was immobilized on various substrates (freshly cleaved mica, standard amino mica, and modified amino mica) and visualized by atomic force microscopy. Plectonemically supercoiled DNA molecules and molecules with an extremely high level of compaction were visualized on modified amino mica, which was characterized by increased surface charge density. It was found that the length of the superhelix axis decreases two and four times to form superhelix axes of the second and third orders as the DNA compaction level increases because of the twice folding of DNA molecules. In this case, the length of the superhelix axis decreases from L approximately 470 nm to L approximately 140 nm (which corresponds to 10% contour length of a relaxed molecule on assumption of B-DNA) to form minitoroids and spheroids of approximately 50 nm diameter. Note that the previously reported experimentally measured length of the superhelix axis was equal to 35% contour length of the relaxed DNA molecule at the maximal density of the superhelix. Our data show that the significant decrease in the length of superhelix axis and the compaction of single supercoiled DNA molecules to the level of spheroids and minitoroids are caused by the screening of negatively charged DNA phosphate groups by positively charged amino groups of the modified amino mica because of its high surface charge density and increased hydrophobicity compared with standard amino mica.     

Motion and enzymatic degradation of DNA in the atomic force microscope.

The dynamics and enzymatic degradation of single DNA molecules can now be observed with the atomic force microscope. A combination of two advances has made this possible. Tapping in fluid has reduced lateral forces, which permits the imaging of loosely adsorbed molecules; and the presence of nickel ions appears to form a relatively stable bridge between the negatively charged mica and the negatively charged DNA phosphate backbone. Continuous imaging shows DNA motion and the process of DNA degradation by the nuclease DNase I. It is possible to see DNase degradation of both loosely adsorbed and tightly adsorbed DNA molecules. This method gives images in aqueous buffer of bare, uncoated DNA molecules with lengths of only a few hundred base pairs, or approximately 100 nm in length.     

Chromatin-Like Structures in Polyoma Virus and Simian Virus 40 Lytic Cycle

Nucleoprotein complexes containing viral DNA and cellular histones were extracted from nuclei of permissive cells infected with polyoma virus or simian virus 40 (SV40) and examined by electron microscopy. Polyoma and SV40 nucleoprotein complexes are almost identical. They appear as relaxed circular molecules consisting of 20 to 21 globular particles interconnected by thin filaments. Their contour length in 0.02 M salt is 2.7 times shorter than that of viral DNA form I obtained after dissociation of the proteins in 1 M NaCl. The nucleosomes have an average diameter of 12.5 nm. Each nucleosome contains 175 to 205 DNA base pairs condensed fivefold in length. The nucleosomes are regularly spaced on the circular molecule. The internucleosomal filaments are made of naked DNA, and each filament contains about 55 base pairs. The partial sensitivity of the nucleoprotein complex to cleavage by EcoR1 endonuclease suggests that the nucleosomes are not formed at specific sites on the viral genome. Faster sedimenting nucleoprotein complexes containing replicative intermediates were studied. Isopycnic centrifugation in metrizamide gradients in the absence of aldehyde fixation showed that these molecules conserved the same DNA-to-protein ratio as the form I DNA-containing complexes.     

Synthesis of mitochondrial DNA in spermatocytes of Rhynchosciara hollaenderi

During the mid to late 4th instar period of larval development, the mitochondria of Rhynchosciara spermatocytes undergo highly characteristic morphological changes. In late meiosis the enlarged mitochondria fuse to form a single mitochondrial element which will ultimately extend the length of the spermatid tail. Our studies have shown that synthesis of a circular DNA occurs during this period of mitochondrial “differentiation.” This DNA has a density of 1.681 g/cm³; and its synthesis cannot be detected in somatic tissues such as salivary gland, fat body, or gastric cecum. From analysis of DNA extracted from mitochondrial pellets, we have shown that the circular DNA is associated with the mitochondria. The contour length of the mitochondrial DNA is 9 μm, equivalent to a molecular weight of 18 × 10⁶. Although most metazoan mitochondrial DNAs exhibit contour lengths of approximately 5 μm (10 × 10⁵ daltons), there is no extractable 5 μm circular DNA in these spermatocytes. Therefore, we conclude that either Rhynchosciara spermatocytes possess a distinct 9 μm mitochondrial DNA or that the spermatocyte mitochondrial DNA represents dimers of 5 μm monomers.     

Small circular DNA complexes in eucaryotic cells

A small number of eucaryotic cells (100 to 1000 cells) were pressed by mica sheet; then the extruded contents were adsorbed on mica and processed for electron microscopy. In the absence of divalent cation, small polydisperse circular DNA molecules bound to proteins or membrane material were preferentially adsorbed. The small circular DNA complexes have been found in every eucaryotic cell, primary lymphoid tissue cells of bursa and thymus, primary cell lines of retina and liver, and established cultured cell lines of embryonal teratocarcinoma, F9 and PCC3, HeLa and 3T6. Size distribution of these DNA complexes varies, depending on the cell source. The circles less than 1 μm in contour length predominate in cultured cell lines and the larger ones in primary cell lines and cells in situ. Polydisperse covalently closed circular DNAs were recovered from thymus lymphocytes by the conventional dye-CsCl buoyant density method. Their size distribution was similar to that of the small circular DNA complexes detected by the mica-press-adsorption method. They are present in several tens to hundreds of copies per cell representing, at a maximum, 0.02% of the total cellular DNA. The possibility that small circular DNA complexes may result from gene rearrangement as well as from replicon “misfiring” (A. Varshavsky, 1981, Proc. Nat. Acad. Sci. USA 78, 3673–3677) are discussed.     

Replication process of the parvovirus H-1. VII. Electron microscopy of replicative-form DNA synthesis.

The geometry of replicative form (RF) DNA synthesis of the H-1 parvovirus was studied with the electron microscope using formamide or aqueous variations of the Kleinschmidt spreading procedure. H-1 DNA was isolated from human or hamster cells infected with a temperature-sensitive mutant, ts1, which is deficient in progeny single-stranded DNA synthesis at the restrictive temperature (S.L. Rhode, 1976), thus minimizing possible confusion between RF and progeny DNA replicative intermediates (RIs). The purity of the isolated H-1 DNA, as determined by gel electrophoresis, ethidium bromide staining, autoadiography, and digestion with endo R-EcoRI, was high. H-1 RF DNA'S WERE LINEAR DOUBLE-STRANDED MOLECULES, 1.53 MUM IN LENGTH. H-1 RIs of RF DNA replication were double-stranded, Y-shaped molecules, with the same length as RF DNAs. The replication origin was localized no more than 0.15 genome lengths from one end of the RF DNA, with replication proceeding toward the other end at a uniform rate. Similar RF and RI molecules of dimer size were also observed. The length of H-1 single-stranded DNA extracted from purified virions was measured relative to that of phiX174 and it had a very similar contour length, so that the molecular weight of H-1 single-stranded DNA would be at least 1.48 X 10(6) to 1.59 X 10(6) (Berkowitz and Day, 1974).     

Specific fragmentation of mitochondrial DNA from Neurospora crassa by restriction endonuclease Eco R I.

Linear, size-heterogenous mitochondrial DNA from $\text{Neurospora crassa}$ was cleaved by the restriction endonuclease Eco R I into eleven specific fragments. According to their contour lengths the fragments have molecular weights between 1.1 and 14 × 10⁶. The sum of the fragments lengths is identical with the contour length (19.8 μm, 41 × 10⁶ daltons) of the few circular molecules detectable in purified DNA preparations.The results suggest sequence homogeneity of mitochondrial DNA and further demonstrate that restriction enzymes can be used to establish a physical map of an unspecifically-fragmented DNA molecule.     

UV light-damaged DNA and its interaction with human replication protein A: an atomic force microscopy study

We have imaged a non-damaged and UV-damaged DNA fragment and its complexes with human replication protein A (RPA) using tapping mode atomic force microscopy (AFM). For imaging, molecules were immobilized under nearly physiological conditions on mica surfaces. Quantitative sizing of the 538 bp DNA before and after UV light treatment shows a reduction in the contour and persistence lengths and mean square end-to-end distance as a consequence of UV irradiation. Complexes of the UV-damaged DNA with RPA, an essential component of the initial steps of nucleotide excision repair, can be detected at high resolution with AFM and reveal conformational changes of the DNA related to complex formation. By phase image analysis we are able to discriminate between protein and DNA in the complexes. The DNA molecules are found to ‘wrap’ around the RPA, which in turn results in a considerable reduction in its apparent contour length.     

Electron Microscopic Evidence for Linear Insertion of Bacteriophage MU-1 in Lysogenic Bacteria

Temperate bacteriophage Mu-1 was used to generate a lysogenic derivative of the F'lac episome of Escherichia coli. Intact, covalently circular molecules of F'lac and lysogenic F'lac Mu(+) deoxyribonucleic acid (DNA) were isolated and examined by electron microscopy. The mean contour lengths of F'lac and F'lac Mu(+) molecules were 37.6 +/- 0.4 mum and 53.2 +/- 0.4 mum, respectively. The mean difference, 15.6 mum, is similar to the mean contour length of 12.9 +/- 0.1 mum obtained for linear DNA molecules released by osmotic shock from mature phage Mu-1 virions. These results provide direct physical evidence that phage Mu-1 integrates by linear insertion of its genome into the DNA of lysogenic host bacteria. Chemical and physical analyses of phage Mu-1 DNA indicate that it is similar to E. coli DNA in respect of gross base composition, buoyant density, and melting temperature.     

Imaging of individual biopolymers and supramolecular assemblies using noncontact atomic force microscopy

A variety of biopolymers is imaged using noncontact atomic force microscopy. Samples are prepared by aerosol spray deposition of aqueous solutions on freshly cleaved mica followed by air drying. The distributions of contour lengths and chain or fibril thicknesses normal to the mica substrate can be measured for individual polymer molecules or molecular assemblies. In many cases it is possible to conclude that the structures imaged and quantitatively analyzed are representative of those present in solution and not artifacts of the deposition/dessication process. Imaging of linear and cyclic triple helices of the polysaccharide scleroglucan is demonstrated. Measurements of the triple helix thickness normal to the mica surface are analyzed, and successful measurements of the molecular weight distribution and mean molar mass are described. It is demonstrated that the extent of chain association in the polysaccharide xanthan can be modulated by the addition of low molecular weight salts. The contour length and chain thickness distributions in a xanthan fraction are presented. Increases in the extent of chain association with increasing polymer concentration are documented for the gelling polysaccharide gellan, and the formation of stiff fibrillar gellan aggregates in the presence of added low molecular salt is demonstrated. Images are presented of the polysaccharide κ-carrageenan in its disordered, and presumably single-stranded, state. Biopolymers other than polysaccharides can be imaged by the same technique; this is demonstrated with the fibrous protein collagen. In general it is shown that aerosol spray deposition of biopolymer samples can be used in conjunction with noncontact atomic force microscopy to provide a fast, reliable, and reproducible method for assessing the size and shape distributions of individual biological macromolecules and macromolecular assemblies in solution with a minimum of time and effort devoted to sample preparation. © 1997 John Wiley & Sons, Inc. Biopoly 42: 133–146, 1997     

Cyclization of three satellite components of calf thymus DNA.

Cyclization of denatured and reannealed satellite components of calf thymus DNA was studied by electron microscopy. All three satellite DNA components studied (1.707g/cm-3, 1.714g/cm-3 and 1.721g/cm-3) form circular structures indicating that the sequences of the calf thymus satellite DNAs are arranged in a tandemly repetitious manner. Under appropriate annealing conditions the amount of circular structures is reproducible and practically no aggregates are formed. By comparison of cyclization experiments under defined conditions it is demonstrated that individual satellite components differ in the amount of circular structures formed during reassociation and in the distribution of linear and circular molecules. From the distribution of the contour lengths of circular molecules we conclude that the length of the repetitive sequence decreases with increasing buoyant density of the satellite components. The average lengths of the repetitive sequences calculated from electron microscopy measurements are in good agreement with those from renaturation kinetics.     

AFM studies of DNA structures on mica in the presence of alkaline earth metal ions

As counterions of DNA on mica, Mg(2+), Ca(2+), Sr(2+) and Ba(2+) were used for clarifying whether DNA molecules equilibrate or are trapped on mica surface. End to end distance and contour lengths were determined from statistical analysis of AFM data. It was revealed that DNA molecules can equilibrate on mica when Mg(2+), Ca(2+) and Sr(2+) are counterions. When Ba(2+) is present, significantly crossovered DNA molecules indicate that it is most difficult for DNA to equilibrate on mica and the trapping degree is different under different preparation conditions. In the presence of ethanol, using AFM we have also observed the dependence of B-A conformational transition on counterion identities. The four alkaline earth metal ions cause the B-A transition in different degrees, in which Sr(2+) induces the greatest structural transition.