Sizing of single-stranded regions in double-stranded DNA by preparative benzoylated DEAE-cellulose chromatography

The concentration of caffeine required to elute wholly single-stranded DNA from benzoylated DEAE-cellulose is proportional to the polynucleotide length. The use of benzoylated DEAE-cellulose chromatography for isolating and sizing single-stranded regions in double-stranded DNA has been examined using a series of hybrid molecules. Restriction fragments of the replicating form of bacteriophage luminal diameter X174 were hybridized to the intact 'plus' strand, thereby forming hybrids having single- and/or double-stranded regions in the kilobase range. A series of such hybrid preparations were subject to caffeine concentration gradient elution from benzoylated DEAE-cellulose. After logarithmic transformation, a linear relationship (R = 0.94) could be demonstrated between eluting caffeine concentration and single-stranded length, irrespective of the length of associated double-stranded regions or the location, within a given fragment, of unpaired nucleotides. Benzoylated DEAE-cellulose chromatography may therefore be used to separate and characterize, on a preparative scale, double-stranded DNA containing single-stranded regions.     

The cyclization of Xenopus laevis 5 S DNA

DNA from Xenopus laevis containing the sequences complementary to 5 S RNA has been studied by the formation of folded rings. Maximal cyclization for fragments 1 to 2 μm in length is 45 to 55%. Thus the efficiency of folded ring formation from this tandemly-repeating DNA is about 50%, assuming that all fragments are 5 S DNA. From the ring frequency as a function of the number of nucleotides removed from the 3′ terminals of the shear-broken fragments, one may calculate that the repeating sequence is approximately 750 nucleotides long, a number that agrees with earlier partial denaturation mapping. The circumference of the folded rings confirms this repeating length since most rings correspond to modular size classes of 0.25-μm increments. Fragments 12 μm long cyclize almost as readily as 1 to 2-μm fragments do. Therefore, the length of the regions (g-regions) containing the tandemly-repeating 5 S DNA is more than 12 μm. The folded rings are about as stable to linearization by increasing concentrations of formamide as the duplex DNA is to denaturation. This indicates that the local, non-transcribed, spacer portions which represent the majority (83%) of the nucleotides in the tandemly-repeating unit, are probably homogeneous in sequence. The exonuclease-treated 5 S DNA fragments cyclize more rapidly than phage T7 DNA, and the kinetics are in accord with theoretical expectation.     

Yeast DNA topoisomerase II. An ATP-dependent type II topoisomerase that catalyzes the catenation, decatenation, unknotting, and relaxation of double-stranded DNA rings

An activity from the yeast Saccharomyces cerevisiae, initially noted for its catalysis of aggregation of covalently closed double-stranded DNA rings in the presence of ATP, has been identified as a type II DNA topoisomerase and is designated yeast DNA topoisomerase II. The formation of the DNA aggregate, which has been shown to be a network of DNA rings that are topologically interlocked, requires the presence of a yeast DNA-binding protein in addition to the topoisomerase. In the absence of the binding protein, yeast DNA topoisomerase II catalyzes decatenation and unknotting of duplex DNA rings and the relaxation of negatively or positively supercoiled DNA. All reactions are ATP-dependent and require Mg(II). Similar to other eukaryotic and phage T4-type II DNA topoisomerases, the yeast enzyme does not catalyze DNA supercoiling under the assay conditions employed. The activity is not sensitive to the gyrase inhibitor nalidixic acid, oxolinic acid, or novobiocin. Coumermycin inhibits the activity, however, at a concentration as low as 5 microgram/ml.     

Viscometric analysis of the interaction of bisphenanthridinium compounds with closed circular supercoiled and linear DNA.

The interaction with closed circular supercoiled and linear DNA of bisphenanthridinium compounds substituted through both the meta and para positions of the 6-phenyl group, along with appropriate monomer intercalators as controls, has been investigated by viscometric titration. When CPK models for the phenanthridinium rings of the three bis-compounds are oriented in a parallel manner as a model for intercalation, their ring plane to ring plane distances are approximately 7 to 8 A (SR 2430), 11 A (SR 2193), and 15 A (SR 2166). In SR 2430 the two phenanthridines are linked through the para positions of the 6-phenyl group; this chain allows intercalation of the two rings at adjacent binding sites in DNA, but is not long enough to accommodate an excluded site. The viscometric titrations with both superhelical and linear DNA clearly indicate that SR 2430 gives results close to those of the monomer control compounds while SR 2193 and SR 2166 have approximately twice the unwinding angle and DNA length increase on binding to DNA as the monomer compounds. These phenanthridinium compounds, therefore, are capable of bisintercalation only if their linking groups are of sufficient length to allow an excluded binding site between base pairs. This conclusion is supported by DNA thermal denaturation experiments in the presence of these compounds.     

Knotted single-stranded DNA rings: A novel topological isomer of circular single-stranded DNA formed by treatment with Escherichia coli ω protein

Treatment of single-stranded circular phage fd DNA with Escherichia coli ω protein yields a new species which sediments 1.2 to 1.5 times faster than the untreated DNA in an alkaline medium. The infectivity of this species in spheroplast assays, after purification of the DNA by zone sedimentation in an alkaline sucrose gradient, is only slightly lower than that of untreated fd DNA. The formation of this species requires Mg(II) and is strongly dependent on salt concentration and temperature. At 37 °C, over 85% of the input DNA can be converted to this form when incubation is carried out in media containing 0.15 to 0.25 m-salt. The yield decreases with increasing temperature or decreasing salt concentration. The increase in sedimentation coefficient of fd DNA in an alkaline medium following treatment with ω is not due to protein binding, as no change was observed upon treatment of the product with phenol or Pronase. Furthermore, neither the buoyant density of this new species in neutral CsCl nor its sedimentation coefficient in a neutral medium is significantly different from the corresponding properties of untreated fd DNA. Examination by electron microscopy shows that the new form has the appearance of a knotted ring of about the same contour length as an untreated monomeric single-stranded fd DNA. The new form can be converted to full-length linear fd DNA by treatment with pancreatic DNAase I. The rate of conversion is approximately the same as that of untreated circular fd DNA to the linear form. These results show that the new form of fd DNA is a novel topological isomer: a knotted single-stranded DNA ring. It is also found that further treatment of the knotted DNA rings with ω at low ionic strength can reverse the reaction, i.e. the knotted DNA rings can be converted back to simple DNA rings indistinguishable from fd DNA from the phage. At intermediate ionic strength the two forms are interconvertible and form an equilibrium mixture. Results similar to those obtained for fd DNA have also been observed for single-stranded circular φX174 DNA. A mechanism based on the known activity of ω protein on double-stranded DNA, the secondary structure of a single-stranded circular DNA, and the experimental results described here is proposed.     

Adeno-associated virus DNA replication in vitro: activation by a maltose binding protein/Rep 68 fusion protein.

The adeno-associated virus (AAV) nonstructural protein Rep 68 is required for viral DNA replication. An in vitro assay has been developed in which addition of Rep 68 to an extract from uninfected HeLa cells supports AAV DNA replication. In this paper, we report characterization of the replication process when a fusion of the maltose binding protein and Rep 68, expressed in Escherichia coli, was used in the assay. Replication was observed when the template was either linear double-stranded AAV DNA or a plasmid construct containing intact AAV DNA. When the recombinant plasmid construct was used as the template, there was replication of pBR322 DNA as well as the AAV DNA; however, linear pBR322 DNA was not replicated. When the plasmid construct was the template, replication appeared to initiate on the intact plasmid and led to separation of the AAV sequences from those of the vector, a process which has been termed rescue. There was no evidence that replication could initiate on the products of rescue. Rep 68 can make a site-specific nick 124 nucleotides from the 3' end of AAV DNA; the site of the nick has been called the terminal resolution site. Our data are most consistent with initiation occurring at the terminal resolution site and proceeding toward the 3' terminus. When the template was the plasmid construct, either elongation continued past the junction into pBR322 sequences or the newly synthesized sequence hairpinned, switched template strands, and replicated the AAV DNA. Replication was linear for 4 h, during which time 70% of the maximal synthesis took place. An additional finding was that the Rep fusion could resolve AAV dimer length duplex intermediates into monomer duplexes without DNA synthesis.     

Regional organization of eukaryotic DNA sequences as studied by the formation of folded rings

The observed frequency of folded rings has been determined as a function of fragment length and degree of resection for DNA from mouse and Necturus. The thermal stability of the ring closure and the kinetics of ring formation have been studied. As seen in the case of Drosophila DNA, mouse and Necturus DNA display a decreasing frequency of folded rings as fragment length increases. We interpret this to mean that repetitious sequences of a given type are clustered into many thousands of characteristic regions, called g-regions. The present paper focuses on the interior organization of g-regions. Variations of two competing models may be entertained: “tandem repetition” and “intermittent repetition”. If the g-regions were composed of exact, tandemly-repeating sequences, all observations can be easily explained. In order to maintain the idea that the g-regions contain repetitious blocks located at regular, or irregular intervals, one must suppose that such repetitious blocks are long (>200 nucleotide pairs), not internally repetitious, and represent perhaps 80% of the nucleotides in the g-region. Such a sequence can be thought of as a fractional-tandem repeat. For example: HIJXXXABC … HIJXXXABC … HIJXXX, where the X's stand for nucleotides composing sequences that are unrelated to each other, and the letters (ABC … HIJ) represent nucleotides in the non-internally-repetitive repeating sequence. We feel that debate cart now be profitably devoted to the question of whether approximately 80 or 100% of the tandemly-repetitious unit is in fact tandem.     

Maize Mitochondrial Plasmid S-1 Sequences Share Homology with Chloroplast Gene psbA

The linear, 6397-base pair (bp), mitochondrial S-1 DNA molecule from maize contains a 420-bp segment that is homologous with the chloroplast gene (psbA) that codes for the quinone binding protein of photosystem II. This is the first report of a chloroplast sequence in a naturally occurring viral-like or plasmid DNA. The complete sequence of the S-1 chloroplast segment has been compared with homologous regions of six different chloroplast genes. The S-1 segment has diverged from the other genes both by length mutation and base substitution. Several of the length mutations are exact adjacent tandem duplications of 4 and 5 bp similar to "footprints" left after excision of transposable elements in maize nuclear DNA.     

Mechanism of activation of an N-ras gene in the human fibrosarcoma cell line HT1080.

A full length N-ras gene has been cloned from both the human fibrosarcoma cell line HT1080 and from normal human DNA. N-ras isolated from HT1080 will efficiently induce morphological transformation of NIH/3T3 cells in a transfection assay, whereas N-ras isolated from normal human DNA has no effect on NIH/3T3 cells. The coding regions of the normal N-ras gene have been sequenced and the predicted amino acid sequence of the N-ras product is very similar to that of the c-Ha-ras1 and c-Ki-ras2 products. By making chimeric molecules between the two cloned genes the activating alteration in the HT1080 N-ras gene has been localised to a single base change that results in an amino acid alteration at position 61 of the p21 N-ras product.     

Ring theory

In what follows we demonstrate that the minimum requirement for the formation of a DNA ring is a pair of ordinary (ABC … ABC) or inverted (ABC … C′B′A′) repetitions. DNA fragments that are partly degraded from their ends by a 3′ (or 5′) specific exonuclease such as exonuclease III (or λ exonuelease) produce resected fragments that can only form rings by virtue of ordinary repetitions.Next we analyze how random fragments cut from DNA molecules containing ordinary repetitions would be expected to form rings. Since longer fragments (>5 to 10 μm) cyclize less efficiently than do shorter ones (2 μm), we are led to the view that the chromatid is composed of thousands of distinctive regions, called g-regions, within which characteristic repetitious sequences are clustered in an intermittent or tandem fashion. Mathematical expressions are derived that allow one to measure the length and number of these g-regions from the ring frequency, R, and its dependence on the length of the fragment.The interior organization of the g-regions is considered in terms of two models and their variants: intermittent repetition and tandem repetition. These are depicted in Figure 2. The objective of this effort is to calculate the frequency of rings that can be generated from these two models, and to explain the “shortside fall-off”, that is, the decrease in ring frequency as the fragment length becomes shorter. This could not be due to the stiffness of the DNA double helix and must reflect a distribution of spacing of the repetitious sequences within the g-regions. Mathematical expressions are obtained that allow one to estimate the average values of the repetitive or partly repetitive unit. These estimates may be obtained from the dependence of ring frequency on the extent of resection, and from the dependence of ring frequency on the length of shorter fragments.The mathematical expressions derived here are employed in the previous papers of this group, and lead to the conclusion that the g-regions are composed of tandemly repeating sequences.     

Formation of rings from segments of HeLa-cell nuclear deoxyribonucleic acid

Duplex segments of HeLa-cell nuclear DNA were generated by cleavage with DNA restriction endonuclease from Haemophilus influenzae. About 20-25% of the DNA segments produced, when partly degraded with exonuclease III and annealed, were found to form rings visible in the electron microscope. A further 5% of the DNA segments formed structures that were branched in configuration. Similar structures were generated from HeLa-cell DNA, without prior treatment with restriction endonuclease, when the complementary polynucleotide chains were exposed by exonuclease III action at single-chain nicks. After exposure of an average single-chain length of 1400 nucleotides per terminus at nicks in HeLa-cell DNA by exonuclease III, followed by annealing, the physical length of ring closures was estimated and found to be 0.02-0.1mum, or 50-300 base pairs. An almost identical distribution of lengths was recorded for the regions of complementary base sequence responsible for branch formation. It is proposed that most of the rings and branches are formed from classes of reiterated base sequence with an average length of 180 base pairs arranged intermittenly in HeLa-cell DNA. From the rate of formation of branched structures when HeLa-cell DNA segments were heat-denatured and annealed, it is estimated that the reiterated sequences are in families containing approximately 2400-24000 copies.     

Cleavage of Circular, Superhelical Simian Virus 40 DNA to a Linear Duplex by S1 Nuclease

S(1) nuclease, the single-strand specific nuclease from Aspergillus oryzae can cleave both strands of circular covalently closed, superhelical simian virus 40 (SV40) DNA to generate unit length linear duplex molecules with intact single strands. But circular, covalently closed, nonsuperhelical DNA, as well as linear duplex molecules, are relatively resistant to attack by the enzyme. These findings indicate that unpaired or weakly hydrogen-bonded regions, sensitive to the single strand-specific nuclease, occur or can be induced in superhelical DNA. Nicked, circular SV40 DNA can be cleaved on the opposite strand at or near the nick to yield linear molecules. S(1) nuclease may be a useful reagent for cleaving DNAs at regions containing single-strand nicks. Unlike the restriction endonucleases, S(1) nuclease probably does not cleave SV40 DNA at a specific nucleotide sequence. Rather, the sites of cleavage occur within regions that are readily denaturable in a topologically constrained superhelical molecule. At moderate salt concentrations (75 mM) SV40 DNA is cleaved once, most often within either one of the two following regions: the segments defined as 0.15 to 0.25 and 0.45 to 0.55 SV40 fractional length, clockwise, from the EcoR(I) restriction endonuclease cleavage site (defined as the zero position on the SV40 DNA map). In higher salt (250 mM) cleavage occurs preferentially within the 0.45 to 0.55 segment of the map.     

Primary organization of nucleosomes containing all five histories and DNA 175 and 165 base-pairs long

The sequential arrangement of histones along DNA in nucleosomes containing all five histones and DNA about 165 and 175 base-pairs in length has been determined. The data provide evidence that core histones (H2A, H2B, H3 and H4) are arranged in nucleosomes and nucleosome core particles in a largely similar way with the following differences. (1) On nucleosomal DNA about 175 basepairs long core histones are probably shifted by 20 nucleotides on one DNA strand and by 10 nucleotides on the complementary DNA strand from the 5′ end. On nucleosomal DNA 165 base-pairs long, histones appear to be shifted by 10 nucleotides from the 5′ end of DNA on both the DNA strands. (2) Histone H3 is extended beyond core DNA and is bound to the 3′ end of DNA about 175 nucleotides long. Thus, core histones span the whole length of nucleosomal DNA. (3) Histone H2A seems to be absent from the central region of nucleosomal DNA. These results indicate that during the preparation of core particles, some rearrangement of histones or some of their regions occurs.Histone H1 has been shown to be bound mainly to the ends of nucleosomal DNA and, along the whole DNA length, to the gap regions that are free of core histones.     

Mechanism of DNA degradation by the ATP-dependent DNase from Hemophilus influenzae Rd.

The rate of production of acid-soluble material during degradation of duplex DNA by Hemophilus influenzae ATP-dependent DNAse (Hind exonuclease V) has been shown to be directly dependent upon the Mg2+ concentration in the reaction mixture. At high concentrations of Mg2+ (5 to 20 mM), DNA degradation to acid-soluble products is rapid and the rate of ATP hydrolysis is slightly depressed. At low concentrations of Mg2+ (0.1 to 0.5 mM), the enzyme rapidly hydrolyzes ATP and converts up to 35% of linear duplex DNA to single-stranded material while degrading less than 0.2% of the DNA to acid-soluble products. We refer to this enzymatic production of single-stranded DNA as the "melting" activity. Under the conditions of our assay, the initial melting reaction is processive, lasting about 70s on phage T7 DNA. Using DNAs with several different lengths, we have established that the duration of the initial reaction is dependent upon DNA length, requiring approximately 1 s per 0.18 mum. The products of the initial reaction on phage T7 DNA are somewhat heterogeneous, consisting of short duplex fragments approximately 0.5 mum long, purely single-stranded products up to 7 mum long, and longer duplex fragments 3 to 11 mum in length, some of which have single-stranded tails. Nearly half of the single-stranded material remains linked to a duplex segment of DNA after the inital processive reaction. We propose that Hind exo V initiates attack at the DNA termini and then acts in a processive manner, migrating along the DNA molecule, converting some regions to single-stranded material by the combined action of the melting activity and limited phosphodiester cleavage, while leaving other regions double-stranded. At the completion of its processive movement through a single DNA molecule, it is released and then recycles onto either intact molecules or the partially degraded products, continuing in this manner until the DNA is finally reduced to oligonucleotides.     

Torsional rigidity of DNA and length dependence of the free energy of DNA supercoiling

By analyzing the Boltzmann populations of DNA topoisomers that differ only in their linking numbers, the dependence of the free energy delta G tau of DNA supercoiling on the linking number alpha has been determined for DNA rings as small as 200 base-pairs (bp) in length. All experimental data can be fitted by the relation delta G tau = K (alpha-alpha)2, where alpha is a constant for a given DNA at a given set of conditions and K is a DNA length-dependent proportionality constant. For DNA rings with length N larger than 2000 bp, K is inversely proportional to N and the product NK is nearly a constant around 1150 RT X bp. For rings smaller than 2000 bp NK increases steadily with decreasing N; for a 200 bp ring NK is 3900 RT X bp. The increase in NK when N decreases can be interpreted as a result of the decrease in the contribution of the fluctuation in the writhing number to the equilibrium distribution in alpha. Assuming that the writhing contribution approaches zero for DNA rings 200 bp in size, the torsional rigidity of the DNA double helix is calculated to be 2.9 X 10(-19) erg cm. In addition, the large value of K for the small circles allows precise calculation of the helical repeat of DNA. For the 210 bp rings, the repeat is measured to be 10.54 bp.     

Bisintercalators of DNA with a rigid linker in an extended configuration.

A new class of DNA bisintercalators is reported in which phenanthridinium or acridinium rings are connected by rigid and extended linkers of varied length. Cross-linking of DNA by bisintercalation is inferred from the unwinding and folding of linear DNA induced by the compound; after ligation and removal of the bisintercalator, superhelical circles, catenanes, and knots that bear an imprint of the bisintercalator are observed. These novel bisintercalators are of interest because they can be used to probe the organization of DNA in three-dimensional space, especially near sites of replication, recombination, or topoisomerase action, where two duplexes must be in close proximity.     

Denaturation map of the circular mitochondrial genome of Neurospora crassa.

A denaturation map of mitochondrial DNA from the wild type strain 5256 of Neurospora crassa was constructed by computer analysis of the contour length distribution of single- and double-stranded regions of nineteen circular and three full length linear molecules after partial denaturation. The data suggest that mitochondrial DNA in this strain is a homogeneous population of a circular molecule of molecular weight 41 - 10(6) with an asymmetric distribution of AT-rich regions, and that linear molecules derive from this genome by random breaks during isolation.     

Structure and sequence of the mitochondrial 20S rRNA and tRNA tyr gene of Paramecium primaurelia

The sequence and structure of the large (20s) mitochondrial (mt) rRNA gene and flanking regions from Paramecium primaurelia have been determined. The gene contains two regions of strong homology with other large mt rRNAs: one 44-base region near the 5' end and a 321-base region near the 3' end. Another region of strong homology to both ends of E. coli 23s RNA exists at loci consistent with these regions. The Paramecium gene appears to be 2204 bases in length and contains slightly more homology to E. coli rRNA than its mammalian or fungal counterparts. The gene, located about 1200 bp from the replicative terminal end of the linear mt DNA, is transcribed in the same polarity as replication. Previous R-looping studies detected no large introns within the gene. Here we describe sequences resembling degenerate rRNAs, one of which could represent a small intron. A tRNA tyr gene was found on the same DNA strand, 127 bp downstream from the large rRNA presumptive 3' end. The tRNA is flanked on both sides by short DNA regions of approximately 90% A + T content.     

Use of the single cell gel electrophoresis/comet assay for detecting DNA damage in aquatic (marine and freshwater) animals

The comet assay is a rapid, sensitive and inexpensive method for measuring DNA strand breaks. The comet assay has advantages over other DNA damage methods, such as sister chromatid exchange, alkali elution and micronucleus assay, because of its high sensitivity and that DNA strand breaks are determined in individual cells. This review describes a number of studies that used the comet assay to determine DNA strand breaks in aquatic animals exposed to genotoxicants both in vitro and in vivo, including assessment of DNA damage in aquatic animals collected from contaminated sites. One difficulty of using the comet assay in environmental work is that of comparing results from studies that used different methods, such as empirical scoring or comet tail lengths. There seems to be a consensus in more recent studies to use both the intensity of the tail and the length of the tail, i.e. DNA tail moment, percentage of DNA in the tail. The comet assay has been used to assess DNA repair and apoptosis in aquatic animals and modifications of the comet assay have allowed the detection of specific DNA lesions. There have been some recent studies to link DNA strand breaks in aquatic animals to effects on the immune system, reproduction, growth, and population dynamics. Further work is required before the comet assay can be used as a standard bio-indicator in aquatic environments, including standardization of methods (such as ASTM method E2186-02a) and measurements.