Complementary strands of CELO virus DNA.

When alkali-denatured DNA from CELO virus (an avian adenovirus) was annealed for 15 min at 37 C in 0.1 M NaCl, 70% of the molecules formed single-stranded circles. This is probably due to base pairing of complementary sequences not more than 110 nucleotides long at the ends of the single strands and implies an inverted terminal repetition in the duplex DNA similar to that reported for the DNA from human adenoviruses. The circular molecules had a uniform length that was approximately the same as that of linear single-stranded molecules. The complementary strands of CELO virus DNA were separated on a preparative scale, and at least 40% of the heavy strands and 56% of the light strands were found to be intact as judged by the formation of single-stranded circles.     

Unwinding of Parental Strands During Simian Virus 40 DNA Replication

Pools of young (less than 60% replicated) and mature (60-90% replicated) replicating molecules of simian virus 40 (SV40) DNA have been treated at pH 12.2 in order to dissociate growing chains from the parental strands. The molecules are neutralized so that the parental strands can reassociate and they have then been isolated. They are covalently closed structures which sediment rapidly in alkaline sucrose gradients; however, the sedimentation rates are less than the sedimentation rate of SV40 DNA I. Isopycnic banding in CsCl-ethidium bromide and sedimentation velocity studies in the presence of various amounts of ethidium bromide indicate that these structures contain negative superhelical turns and several-fold-higher superhelix densities than SV40 DNA I (the covalently closed DNA molecule). These structures are those that would be predicted if nicking, unwinding, and sealing of the parental strands occurred as replication proceeded. These experiments provide a direct demonstration that there is a progressive decrease in the topological winding number which accompanies SV40 DNA replication.     

Self-annealing of 4 S strands from replicating simian virus 40 DNA

The nascent short strands (4 S) isolated from replicating Simian, virus 40 DNA hybridize specifically with denatured SV40 DNA and self-anneal extensively (70 to 92%) when incubated at 68 °C in 1 m-NaCl. Since complementary genetic sequences are present in the 4 S strands, both growing chains of SV40 DNA appear to be synthesized discontinuously at each replication fork.     

Automated forward and reverse ratcheting of DNA in a nanopore at 5-Å precision

An emerging DNA sequencing technique uses protein or solid-state pores to analyze individual strands as they are driven in single-file order past a nanoscale sensor. However, uncontrolled electrophoresis of DNA through these nanopores is too fast for accurate base reads. Here, we describe forward and reverse ratcheting of DNA templates through the α-hemolysin nanopore controlled by phi29 DNA polymerase without the need for active voltage control. DNA strands were ratcheted through the pore at median rates of 2.5-40 nucleotides per second and were examined at one nucleotide spatial precision in real time. Up to 500 molecules were processed at ～130 molecules per hour through one pore. The probability of a registry error (an insertion or deletion) at individual positions during one pass along the template strand ranged from 10% to 24.5% without optimization. This strategy facilitates multiple reads of individual strands and is transferable to other nanopore devices for implementation of DNA sequence analysis.     

Molecular Recombination in T4 Bacteriophage Deoxyribonucleic Acid: III. Formation of Long Single Strands During Recombination

Evidence was presented to support the hypothesis that long single strands appearing at late times (15 min after infection) are produced as a result of recombination and not as a continuous elongation during the replication process. The production of long strands does not depend on the multiplicity of infection, and the first long strands appear at the time when 20 to 50 phage equivalent units of deoxyribonucleic (DNA) are synthesized, and not earlier. The addition of chloramphenicol at 5 min, which prevents molecular recombination but allows replication of DNA, prevents the formation of long, single strands. Chloramphenicol added between 8 and 10 min after infection, a time at which molecular recombination is fully expressed and covalent repair of recombinant molecules is allowed, does not prevent formation of long single strands. Cutting of single-strand DNA with a limited amount of endonuclease I allows confirmation that the fast-sedimenting characteristic of intracellular denatured DNA is caused primarily by the length of the strands, and not by the formation of aggregates. The computer simulation of two recombination models indicates the feasibility of random breakage and rejoining of molecules in generating long concatenates.     

Repair Replication and Degradation of Bromouracil-Substituted DNA in Mammalian Cells after Irradiation with Ultraviolet Light

Ultraviolet (UV) light irradiation of HeLa cells in which bromouracil (BU) is substituted for thymine in one strand of the DNA, elicits a number of responses that occur predominantly in the BU strand. A small amount of degradation of both strands occurs, but the BU strand is degraded to a greater extent than the normal strand. Large UV doses (1000 erg/mm²) induce degradation of about 1.7% of the DNA within 6 hr of irradiation of unsubstituted cells; in BU-substituted cells under these conditions about 1.9% of the normal strand is degraded but 17.5% of the BU strand. After irradiation fresh bases are inserted into the BU strands at infrequent intervals throughout the DNA and this is presumed to represent repair of UV damage in the BU strands. After 1000 erg/mm² the majority (70%) of the thymidine incorporated enters the BU strand. Inhibitors of normal DNA synthesis, hydroxyurea and arabinosyl cytosine, do not appear to inhibit the repair of DNA. The increased sensitivity of mammalian cells that contain BU to irradiation may consequently be due to damage of the BU strand. A specific interference between BU and repair of DNA which leads to large amounts of DNA degradation in bacteria, does not seem to be important in the sensitization of mammalian cells with BU.     

A method for gene enrichment based on the avidin-biotin interaction. Application to the Drosophila ribosomal RNA genes.

A method of enriching, from the total DNA of an organism, for long DNA strands carrying a particular gene is described. The purified RNA corresponding to the gene is covalently attached to biotin via a cytochrome c bridge. This modified RNA is hybridized to the total DNA. Those DNA strands which hybridize are separated from all the other DNA, using the avidin-biotin interaction, by one of two methods. Avidin is covalently attached to submicroscopic polymer spheres; the complexes of avidin spheres with the DNA: RNA-biotin hybrids band in CsCl at a much lower buoyant density than does free DNA. Alternatively, the DNA:RNA-biotin hybrids are isolated by affinity chromatography on an avidin-solid support column. These methods have been used to prepare long single strands of Drosophila ribosomal DNA (rDNA) in high yield and 42 to 80% pure.     

Transformation and Deoxyribonucleic Acid Size: Extent of Degradation on Entry Varies with Size of Donor

The fate of label introduced as donor deoxyribonucleic acid (DNA) into competent cells of Diplococcus pneumoniae was determined immediately after entry at 25 C, as a function of the size of the donor DNA. Part of the label is found to be acid soluble, part has been incorporated into chromosomal DNA, apparently through reincorporation of degraded donor DNA, and part is found in single strands of length smaller than that of the input donor DNA strands. The last fraction apparently constitutes the precursor for integration of intact donor genetic markers and is referred to as the intact fraction. For large donor DNA the intact fraction contains over 80% of the total intracellular label, but the median strand length has been reduced to 2.2 x 10(6) daltons. For small donor molecules (1 x 10(5) to 6 x 10(5) daltons per strand) the fraction intact increases with donor size from 10 to 50% of the total intracellular label, and the median strand length of this fraction is half that of the donor strands. By combining these results with earlier data on the size dependence of the yield of transformants per unit of total intracellular donor label, we have calculated the probability that a marker in the intact fraction will be integrated, as a function of the length of the donor strand after entry. This probability has a linear dependence on strand length for activities below 40% of maximum, and extrapolates to zero activity at 77,000 daltons per strand.     

Evidence for a partial RNA transcript of the small circular component of kinetoplast DNA of Crithidia acanthocephali.

The major component of kinetoplast DNA (kDNA) in the protozoan Crithidia acanthocephali is an association of approximately 27,000, 0.8 micrometers (1.58 x 10(6) dalton) circular molecules apparently held together in a particular structural configuration by topological interlocking. We have carried out hybridization experiments between kDNA samples containing one or the other of the two complementary (H and L) strands of purified 0.8 micrometers molecules derived from mechanically disrupted associations and RNA samples prepared either from whole C. acanthocephali cells or from a mitochondrion-enriched fraction. The results of experiments involving cesium sulfate buoyant density centrifugation indicate that whole cell RNA contains a component(s) complementary to all kDNA H strands, but none complementary to kDNA L strands. Similar results were obtained using mitochondrion-associated RNA. Digestion of RNA/DNA hybrids and suitable controls with the single-strand-specific nuclease S1 indicated that 10% of the kDNA H strand is involved in hybrid formation. Visualization of RNA/DNA hybrids stained with bacteriophage T4 gene 32 protein revealed that hybridation involves a single region of each kDNA H strand, equal to approximately 10% of the molecule length. These data suggest that at least 10% of the small circular component of kDNA of Crithidia acanthocephali is transcribed.     

Mechanism of replication of human mitochondrial DNA. Localization of the 5' ends of nascent daughter strands

Human mitochondrial DNA contains two physically separate and distinct origins of DNA replication. The initiation of each strand (heavy and light) occurs at a unique site and elongation proceeds unidirectionally. Animal mitochondrial DNA is novel in that short nascent strands are maintained at one origin (D-loop) in a significant percentage of the molecules. In the case of human mitochondrial DNA, there are three distinct D-loop heavy strands differing in length at the 5' end. We report here the localization of the 5' ends of nascent daughter heavy strands originating from the D-loop region. Analyses of the map positions of 5' ends relative to known restriction endonuclease cleavage sites and 5' end nucleotides indicate that the points of initiation of D-loop synthesis and actual daughter strands are the same. In contrast, the second origin is located two-thirds of the way around the genome where light strand synthesis is presumably initiated on a single-stranded template. Mapping of 5' ends of daughter light strands at this origin relative to known restriction endonuclease cleavage sites reveals two distinct points of initiation separated by 37 nucleotides. This origin is in the same relative genomic position and shows a high degree of DNA sequence homology to that of mouse mitochondrial DNA. In both cases, the DNA region within and immediately flanking the origin of DNA replication contains five tightly clustered tRNA genes. A major portion of the pronounced DNA template secondary structure at this origin includes the known tDNA sequences.     

The specificity of lambda exonuclease. Interactions with single-stranded DNA.

The lambda exonuclease, an enzyme that has been implicated in genetic recombination, rapidly and processively degrades native DNA, starting at the 5' terminus. The enzyme will also degrade the 5'-terminated strand at a single-stranded branch. The experiments reported here reveal various interactions of the enzyme with single-stranded DNA. The rate of digestion is related inversely to the length of single strands. Chains of 100 nucleotides are digested at about 10% the rate of digestion of native DNA. Digestion of the single-stranded ends of lambda DNA does not appear to occur processively. The enzyme binds to circular as well as linear single strands and the affinity for single strands is also related inversely to the chain length. In an equimolar mixture of single- and double-stranded DNA the action of lambda exonuclease on the latteris about half-inhibited. At 20 degrees the initiation of digestion at the 5' terminus of duplex DNA is blocked sterically when such DNA has 3'-terminal single strands that are longer than 100 nucleotides. Information about these properties is important for the practical use of lambda exonuclease as well as for reflections on the role of the enzyme in genetic recombination.     

Synthesis of plus strands of retroviral DNA in cells infected with avian sarcoma virus and mouse mammary tumor virus

The vast majority of plus strands synthesized in quail cells acutely infected with avian sarcoma virus were subgenomic in size, generally less than 3 kilobases (kb). A series of discrete species could be identified after agarose gel electrophoresis by annealing with various complementary DNAs, indicating specificity in the initiation and termination of plus strands. The first plus strand to appear (within 2 h postinfection) was similar in length to the long redundancy at the ends of linear DNA (0.35 kb), and it annealed with complementary DNAs specific for the 3' and 5' termini of viral RNA (Varmus et al., J. Mol. Biol. 120:50-82, 1978). Several subgenomic plus-strand fragments (0.94, 1.38, 2.3, and 3.4 kb) annealed with these reagents. At least the 0.94- and 1.38-kb strands were located at the same end of linear DNA as the 0.35-kb strand, indicating that multiple specific sites for initiation were employed to generate strands which overlapped on the structural map. We were unable to detect RNA liked to plus strands isolated as early as 2.5 h postinfection; thus, the primers must be short (fewer than 50 to 100 nucleotides), rapidly removed, or not composed of RNA. To determine whether multiple priming events are a general property of retroviral DNA synthesis in vivo, we also examined plus strands of mouse mammary tumor virus DNA in chronically infected rat cells after induction of RNA and subsequent DNA synthesis with dexamethasone. In this case, multiple, discrete subgenomic DNA plus strands were not found when the same methods applied to avian sarcoma virus DNA were used; instead, the plus strands present in the linear DNA of mouse mammary tumor virus fell mainly into two classes: (i) strands of ca. 1.3 kb which appeared early in synthesis and were similar in size and genetic content to the terminally repeated sequence in linear DNA; and (ii) plus strands of the same length as linear DNA. A heterogeneous population of other strands diminished with time, was not found in completed molecules, and was probably composed of strands undergoing elongation. These two retroviruses thus appear to differ with respect to both the number of priming sites used for the synthesis of plus strands and the abundance of full-length plus strands. On the other hand the major subgenomic plus strand of mouse mammary tumor virus DNA (1.3 kb) is probably the functional homolog of a major subgenomic plus strand of avian sarcoma virus DNA (0.35 kb). The significance of this plus strand species is discussed in the context of current models which hold that it is used as a template for the completion of the minus strand, thereby generating the long terminal redundancy.     

Ligation and synthesis of chromatin deoxyribonucleic acid in vitro in neuronal, glial and liver nuclei isolated from adult guinea pig

Neuron-rich and glial nuclear preparations and liver nuclei were isolated from adult guinea pigs. These nuclei were incubated to carry out DNA-ligation and -synthesis reactions. Before and after incubation, the sizes of single-standed DNA and DNA-synthesis patterns in single strands were analysed by using alkaline sucrose-density-gradient centrifugation. Isolation of nuclei by cell-fractionation technique shortened chromatin DNA and decreased markedly the number-average molecular weight of DNA strands. Chromatin DNA in neuronal and glial nuclei was ligated at the nicks during incubation in a reaction mixture containing ATP, Mg(2+), dithiothreitol and four deoxyribonucleotides. The number-average molecular weights were estimated to increase 1.1-and 2.1-fold in neuronal and glial nuclei respectively. DNA strands in liver nuclei were shortened during incubation, but elongated under conditions that inhibit deoxyribonuclease. Since the endogenous deoxyribounuclease activity was conspicuously higher in liver nuclei than in neuronal and glial nuclei, the shortening and elongation were thought to depend on the balance between DNA ligase and deoxyribonuclease reactions. DNA synthesis occurred at the gaps in chromatin DNA and about 50% of the total synthesized DNA was found in the shorter strands having 6 to 297 bases in all species of nuclei. Based on these results, it was concluded that in nuclei isolated from non-dividing cells (neurons) and slowly dividing cells (glial and liver cells) DNA-ligation and -synthesis reactions proceeded in parallel at the breaks in single-stranded DNA, which was produced mainly by endogenous deoxyribonuclease during isolation and incubation processes.     

Molecular simulation study of assembly of DNA-grafted nanoparticles: effect of bidispersity in DNA strand length

In this paper, we use molecular dynamics simulations to study the assembly of DNA-grafted nanoparticles to demonstrate specifically the effect of bidispersity in grafted DNA strand length on the thermodynamics and structure of nanoparticle assembly at varying number of grafted single-stranded DNA (ssDNA) strands and number of guanine/cytosine (G/C) bases per strand. At constant number of grafted ssDNA strands and G/C nucleotides per strand, as bidispersity in strand lengths increases, the number of nanoparticles that assemble as well as the number of neighbours per particle in the assembled cluster increases. When the number of G/C nucleotides per strand in short and long strands is equal, the long strands hybridise with the other long strands with higher frequency than the short strands hybridise with short/long strands. This dominance of the long strands leads to bidisperse systems having similar thermodynamics to that in corresponding systems with monodisperse long strands. Structurally, however, as a result of long–long, long–short and short–short strand hybridisation, bidispersity in DNA strand length leads to a broader inter-particle distance distribution within the assembled cluster than seen in systems with monodisperse short or monodisperse long strands. The effect of increasing the number of G/C bases per strand or increasing the number of grafted DNA strands on the thermodynamics of assembly is similar for bidisperse and monodisperse systems. The effect of increasing the number of grafted ssDNA strands on the structure of the assembled cluster is dependent on the extent of strand bidispersity because the presence of significantly shorter ssDNA strands among long ssDNA strands reduces the crowding among the strands at high grafting density. This relief in crowding leads to larger number of strands hybridised and as a result larger coordination number in the assembled cluster in systems with high bidispersity in strands than in corresponding monodisperse or low bidispersity systems.     

Sequence-dependent mechanics of single DNA molecules

Atomic force microscope-based single-molecule force spectroscopy was employed to measure sequence-dependent mechanical properties of DNA by stretching individual DNA double strands attached between a gold surface and an AFM tip. We discovered that in lambda-phage DNA the previously reported B-S transition, where 'S' represents an overstretched conformation, at 65 pN is followed by a nonequilibrium melting transition at 150 pN. During this transition the DNA is split into single strands that fully recombine upon relaxation. The sequence dependence was investigated in comparative studies with poly(dG-dC) and poly(dA-dT) DNA. Both the B-S and the melting transition occur at significantly lower forces in poly(dA-dT) compared to poly(dG-dC). We made use of the melting transition to prepare single poly(dG-dC) and poly(dA-dT) DNA strands that upon relaxation reannealed into hairpins as a result of their self-complementary sequence. The unzipping of these hairpins directly revealed the base pair-unbinding forces for G-C to be 20 +/- 3 pN and for A-T to be 9 +/- 3 pN.     

Breakage of Parental DNA Strands in Haemophilus influenzae by 313 nm Radiation after Replication in the Presence of 5-Bromodeoxyuridine

Haemophilus influenzae was labeled with thymidine-³H (dThd), then grown in the presence of 5-bromodeoxyuridine (BrdUrd), and then irradiated with 313 nm light (a wavelength that selectively photolyzes DNA containing 5-bromouracil [BrUra]). Irradiation with 313 nm light induced breaks in the ³H-labeled strands in cells grown with BrdUrd at a much higher frequency than in ¹⁴C-labeled DNA of cells not exposed to BrdUrd. Breakage of the ³H-labeled strands was about 0.6% as efficient as that of fully BrUra-substituted DNA. During growth in the presence of BrdUrd, susceptibility to 313 nm-induced breakage of the ³H-labeled DNA strands increased, reaching a maximum in about one generation, and it decreased to zero during subsequent growth for one generation in medium containing dThd instead of BrdUrd. Heat denaturation of DNA extracted from dThd-³H-labeled cells grown in the presence of BrdUrd eliminated 313 nm-induced breakage of the ³H-labeled strands. It is concluded that breakage of the ³H-labeled DNA strands resulted from reaction with photoproducts in the base-paired, BrUra-containing strands, rather than from photolysis of BrdUrd incorporated into parental strands. It may be possible to utilize the phenomenon of interstrand breakage in physical studies of DNA replication.     

Identification of individual sex-factor DNA strands and their replication during conjugation in thermosensitive DNA mutants of Escherichia coli

Covalent circular sex-factor DNA has been isolated from donor and recipient cells during the conjugation of normal and temperature-sensitive DNA mutants of Escherichia coli. Single strands of sex-factor DNA were centrifuged in cesium chloride-poly(U,G) gradients to give two components that have been identified by annealing experiments as the separated complementary strands. When matings are performed with either DNA temperature-sensitive donor or recipient cells, the inhibition of vegetative DNA synthesis at the restrictive temperature does not interfere with transfer and circularization of the sex-factor DNA. If DNA is isolated from temperature-sensitive donor cells mated at the restrictive temperature, a specific stimulation of sex-factor DNA synthesis can be demonstrated. By separating the complementary strands of the sex-factor in a cesium chloride-poly (U,G) gradient, this DNA synthesis has been found to be asymmetric. The sex-factor DNA strand which is synthesized in the donor has the same polarity as the strand which is transferred to the recipient.     

Adenovirus type 12-specific RNA sequences during productive infection of KB cells.

The complementary strands of adenovirus type 12 DNA were separated, and virus-specific RNA was analyzed by saturation hybridization in solution. Late during infection whole cell RNA hybridized to 75% of the light (1) strand and 15% of the heavy (H) strand, whereas cytoplasmic RNA hybridized to 65% of the 1 strand and 15% of the h strand. Late nuclear RNA hybridized to about 90% of the 1 strand and at least 36% of the h strand. Double-stranded RNA was isolated from infected cells late after infection, which annealed to greater than 30% of each of the two complementary DNA strands. Early whole cell RNA hybridized to 45 to 50% of the 1 strand and 15% of the h strand, whereas early cytoplasmic RNA hybridized to about 15% of each of the complementary strands. All early cytoplasmic sequences were present in the cytoplasm at late times.