Studies of DNA organization in hydrated spermidine-condensed DNA toruses and spermidine-DNA fibres

We have investigated in vitro spermidine-condensed DNA preparations by both biochemical and freeze-etch electron microscopic approaches. These studies lead us to the conclusion that the reversibly condensed preparations, qualitatively described by Manning's counterion condensation theory, contain disk-like torus structures largely comprised of unidirectional, circumferentially wrapped DNA. Stereoscopic measurements on stereomicrographs of DNA torus and fibre objects have demonstrated the feasibility of directly measuring DNA writhe or, for that matter, mapping any secondary, tertiary or quaternary structure features of a hydrated macromolecular array in which the features can be differentially highlighted by low replica metal shadow levels.     

A Study of øX-174 DNA Torus and Lambda DNA Torus Tertiary Structure and the Implications For DNA Self-Assembly

Abstract

Hydrated torus shaped complexes were examined by transmission electron microscopy in both spermidine-condensed linear and nicked circular øX-174 DNA and lambda DNA preparations. Freeze-etch replicas of both these torus samples, produced with very low Pt metal deposition levels (9APt/C), were found to have circumferentially wound single DNA double helix size surface fibers in the range of 30A width. Measurements of torus inner and outer circumference as well as ring thickness were performed. Observed differences in the torus dimension distributions from circular øX-174 DNA and linear øX ?174 DNA may be related to the different topological constraints on DNA folding in these two samples (1). On the basis of annulus thickness measurements øX ?174 DNA toruses, in contrast to lambda DNA toruses, were observed to fall into two classes identified as being formed from monomer DNA condensation and multimer DNA condensation. All of the torus substructure and population dimensions observed here are consistent with the continuous circumferential DNA winding model of torus organization proposed by Marx and Reynolds (1) to explain the micrococcal nuclease cleavage properties of the toruses. End-on view measurements of the torus thickness were made from micrographs obtained by extensive tilting of the object replica. These direct measurements confirmed quaternary structure interpretations made from simple strand packing models. We compared the measured torus properties in this linear DNA size series (5386–48000 bp). With increasing DNA length the pattern of DNA strand self- assembly was found to be more varied producing lambda DNA toruses of varying shape. The relevance of our study to the problem of lambda bacteriophage DNA head packaging was discussed.     

A study of phi X-174 DNA torus and lambda DNA torus tertiary structure and the implications for DNA self-assembly

Hydrated torus shaped complexes were examined by transmission electron microscopy in both spermidine-condensed linear and nicked circular phi X-174 DNA and lambda DNA preparations. Freeze-etch replicas of both these torus samples, produced with very low Pt metal deposition levels (9APt/C), were found to have circumferentially wound single DNA double helix size surface fibers in the range of 30A width. Measurements of torus inner and outer circumference as well as ring thickness were performed. Observed differences in the torus dimension distributions from circular phi X-174 DNA and linear phi X-174 DNA may be related to the different topological constraints on DNA folding in these two samples (1). On the basis of annulus thickness measurements phi X-174 DNA toruses, in contrast to lambda DNA toruses, were observed to fall into two classes identified as being formed from monomer DNA condensation and multimer DNA condensation. All of the torus substructure and population dimensions observed here are consistent with the continuous circumferential DNA winding model of torus organization proposed by Marx and Reynolds (1) to explain the micrococcal nuclease cleavage properties of the toruses. End-on view measurements of the torus thickness were made from micrographs obtained by extensive tilting of the object replica. These direct measurements confirmed quaternary structure interpretations made from simple strand packing models. We compared the measured torus properties in this linear DNA size series (5386-48000 bp). With increasing DNA length the pattern of DNA strand self-assembly was found to be more varied producing lambda DNA toruses of varying shape. The relevance of our study to the problem of lambda bacteriophage DNA head packaging was discussed.     

The Absence of Cruciform Structures from pA03 Plasmid DNA In vivo

Abstract

We extracted pA03 plasmid DNA from E. coli cells, having “frozen” the transitions between cruciform and double-helical conformations in DNA. The characteristic feature of the DNA isolation procedure is that all steps were carried out at temperature between 0 and 4 C and no phenol deproteinization was used, since it has been discovered that phenol destabilizes cruciform structures in pA03 DNA. Two-dimensional gel electrophoresis has revealed no cruciform structures in the pA03 DNA preparations obtained this way, although the superhelical density of DNA was sufficient for them. Cruciform structures are absent from intracellular pA03 DNA at all growth stages of the bacterial culture: stationary and logarithmic, and under the induction of pA03 DNA replication in chloramphenicol-treated cells.     

Development of oxidovanadium and oxido-peroxido vanadium-based artificial DNA nucleases via multi spectroscopic investigations and theoretical simulation of DNA binding

Benzimidazole is a neutral ligand which is often used to synthesize bioactive compounds. Two transition metal benzimidazole-based complexes, namely, vanadium (IV) dioxido complex (complex 1) and vanadium (V) oxido-peroxido complex (complex 2) with tridentate benzimidazole ligand, 2,6-di (1H-benzo[d]imidazol-2-yl) pyridine (Byim) have been designed with the intention of developing potential DNA nuclease. Different studies involving biochemical and biophysical techniques along with molecular docking suggest that both the complexes interact with DNA, while the mode of binding is intercalation. The complexes were further used for DNA cleavage activity. Both of them were found to have substantial DNA nuclease activity, but complex 2 was more potent than complex 1 in exhibiting such activity.     

Evidence for hydrated spermidine-calf thymus DNA toruses organized by circumferential DNA wrapping

In spermidine-condensed calf thymus DNA preparations, torus-shaped condensates were shown by transmission electron microscopy to exist under the hydrated conditions of the freeze fracture experiment. Using extremely low Pt metal deposition levels (9 A Pt/C) high-contrast replicas of the spermidine-DNA toruses were obtained that showed circumferential wrapping of single DNA double helix-size surface fibres. Stereoscopic analysis of high magnification stereomicrographs established some details of the three-dimensional organization of two DNA double helix sections winding circumferentially on the inner surface of one such torus. These measurements demonstrate the usefulness of stereoscopic analysis of these high macromolecular organization magnification. Measurements on a number of torus-shaped complexes (n = 16) yielded these average dimensions: inner circumference (1840 +/- 204 A) outer circumference (2800 +/- 222 A), torus ring thickness (143 +/- 18 A). These data support a continuous circumferential DNA-winding model of torus organization proposed by Marx & Reynolds.     

MALDI-TOF Mass Spectrometry as a Powerful Tool to Study Enzymatic Processing of DNA Lesions Inserted into Oligonucleotides

Abstract

MALDI-TOF mass spectrometry measurements, coupled with either exonuclease or DNA N-glycosylases digestions of lesion-containing oligonucleotides, were used to assess biochemical features of several oxidative DNA damage. The latter analytical approach was shown to be an informative and efficient alternative technique to conventional electrophoresis and chromatographic analyses.     

MRCNN: a deep learning model for regression of genome-wide DNA methylation

Background

Determination of genome-wide DNA methylation is significant for both basic research and drug development. As a key epigenetic modification, this biochemical process can modulate gene expression to influence the cell differentiation which can possibly lead to cancer. Due to the involuted biochemical mechanism of DNA methylation, obtaining a precise prediction is a considerably tough challenge. Existing approaches have yielded good predictions, but the methods either need to combine plenty of features and prerequisites or deal with only hypermethylation and hypomethylation.

Results

In this paper, we propose a deep learning method for prediction of the genome-wide DNA methylation, in which the Methylation Regression is implemented by Convolutional Neural Networks (MRCNN). Through minimizing the continuous loss function, experiments show that our model is convergent and more precise than the state-of-art method (DeepCpG) according to results of the evaluation. MRCNN also achieves the discovery of de novo motifs by analysis of features from the training process.

Conclusions

Genome-wide DNA methylation could be evaluated based on the corresponding local DNA sequences of target CpG loci. With the autonomous learning pattern of deep learning, MRCNN enables accurate predictions of genome-wide DNA methylation status without predefined features and discovers some de novo methylation-related motifs that match known motifs by extracting sequence patterns.

     

Probing the MvaI Methyltransferase Region that Interacts with DNA: Affinity Labeling with the Dialdehyde-Containing DNA Duplexes

Abstract

Affinity labeling of methyltransferase MvaI by DNA duplexes containing oxidized 2′-O-β-D-ribofuranosylcytidine or 1-(β-D-galactopyranosyl)thymine residues was performed. Partial chemical hydrolysis of the covalently bound methylase in the conjugates with the dialdehyde-containing DNA allowed us to determine the amino acid region in the C terminus of methylase MvaI that interacts with DNA.     

AFFINITY MODIFICATION OF EcoRII DNA METHYLTRANSFERASE BY THE DIALDEHYDE-SUBSTITUTED DNA DUPLEXES: MAPPING THE ENZYME REGION THAT INTERACTS WITH DNA

ABSTRACT

Affinity modification of EcoRII DNA methyltransferase (M·EcoRII) by DNA duplexes containing oxidized 2′-O-β-D-ribofuranosylcytidine (Crib*) or 1-(β-D-galactopyranosyl)thymine (Tgal*) residues was performed. Cross-linking yields do not change irrespective of whether active Crib* replaces an outer or an inner (target) deoxycytidine within the EcoRII recognition site. Chemical hydrolysis of M·EcoRII in the covalent cross-linked complex with the Tgal*-substituted DNA indicates the region Gly²⁶⁸-Met³⁹¹ of the methylase that is likely to interact with the DNA sugar-phosphate backbone. Both specific and non-specific DNA interact with the same M·EcoRII region. Our results support the theoretically predicted DNA binding region of M·EcoRII.     

Optimization of DNA delivery by three classes of hybrid nanoparticle/DNA complexes

Plasmid DNA encoding a luciferase reporter gene was complexed with each of six different hybrid nanoparticles (NPs) synthesized from mixtures of poly (D, L-lactide-co-glycolide acid) (PLGA 50:50) and the cationic lipids DOTAP (1, 2-Dioleoyl-3-Trimethyammonium-Propane) or DC-Chol {3β-[N-(N', N'-Dimethylaminoethane)-carbamyl] Cholesterol}. Particles were 100-400 nm in diameter and the resulting complexes had DNA adsorbed on the surface (out), encapsulated (in), or DNA adsorbed and encapsulated (both). A luciferase reporter assay was used to quantify DNA expression in 293 cells for the uptake of six different NP/DNA complexes. Optimal DNA delivery occurred for 10⁵ cells over a range of 500 ng - 10 μg of NPs containing 20-30 μg DNA per 1 mg of NPs. Uptake of DNA from NP/DNA complexes was found to be 500-600 times as efficient as unbound DNA. Regression analysis was performed and lines were drawn for DNA uptake over a four week interval. NP/DNA complexes with adsorbed NPs (out) showed a large initial uptake followed by a steep slope of DNA decline and large angle of declination; lines from uptake of adsorbed and encapsulated NPs (both) also exhibited a large initial uptake but was followed by a gradual slope of DNA decline and small angle of declination, indicating longer times of luciferase expression in 293 cells. NPs with encapsulated DNA only (in), gave an intermediate activity. The latter two effects were best seen with DOTAP-NPs while the former was best seen with DC-Chol-NPs. These results provide optimal conditions for using different hybrid NP/DNA complexes in vitro and in the future, will be tested in vivo.     

Plasmid transformation in Bacillus subtilis: Fate of plasmid DNA

Summary Only multimeric, and not monomeric forms of B. subtilis plasmids can transform B. subtilis cells (Canosi et al. 1978). This finding prompted us to study the physico-chemical fate of plasmid DNA in transformation. Competent cells of B. subtilis were exposed to either unfractionated preparations or to preparations of multimeric plasmid DNA. Plasmid DNA was re-extracted from such cells and then analyzed by sedimentation and isopycnic centrifugation and also defined by its sensitivity to nuclease S1 degradation. No double-stranded plasmid DNA could be recovered from cells transformed with unfractionated plasmid preparations which contained predominantly monomeric covalently closed circular (CCC) DNA, Re-extracted plasmid DNA was single-stranded, had a molecular weight considerably smaller than monomer length DNA and had been subject to degradation to acid soluble products. However, when transformations were performed with multimeric DNA (constructed by in vitro ligation of linearized pC194 DNA), both double-stranded and partially double-stranded DNA could be recovered in addition to single-stranded DNA.We assume that plasmid DNA is converted to a single-stranded form in transformation, irrespective of its molecular structure. Double-stranded and partially double-stranded DNAs found in transformation with multimeric DNA would be the products of intramolecular annealing.Some of these results were presented at the 5th European Meeting on Bacterial Transformation and Transfection, September 1980, Florence     

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.     

A Computer Graphics Study of Sequence-Directed Bending in DNA

Abstract

We present theoretical results to account for the unusual physical properties of a 423 bp DNA restriction fragment isolated from the kinetoplast of the trypanosomatid Leishmania tarentolae. This fragment has an anomalously low electrophoretic mobility in Polyacrylamide gels and a rotational relaxation time smaller than that of normally-behaved control fragments of the same molecular weight. Our earlier work (Proc. Natl. Acad. Sci. USA 79, 7664, 1982) has attributed these anomalies to the highly periodic distribution of the dinucleotide ApA in the DNA sequence. As originally proposed by Trifonov and Sussman (Proc. Natl. Acad. Sci. USA 77, 3816,1980) local features of the DNA structure such as a small bend at ApA, if repeated with the periodicity of the helix, will cause systematic bending of the molecule.

Computer graphics representations of DNA chain trajectories are presented for different structural models. It is shown that the structural model of Calladine (J. Mol. Biol. 161, 343, 1982) which is based on crystallographic data, is unsuccessful in predicting the systematic bending of DNA in solution.     

A New Type of DNA Polymorphism Identified in the Species-Specific DNA Region Originating from the <Emphasis Type="Italic">Candida albicans</Emphasis> Mitochondrial Genome

Mitochondrial DNA of the pathogenic yeast Candida albicans contains a species-specific DNA region, designated EO3, within the duplicated region in its stem-and-loop structure, as reported in our previous paper. We have revealed that the EO3 region exhibits a DNA-size polymorphism, yielding three fragment length types: L, M, and S. This polymorphism is due to the presence (or absence) of two small nucleotide sequences of ~50 bp (designated a and b) within the EO3 region: type L possesses both a and b, type M possesses a alone, and type S possesses neither a nor b. Here we have identified a new type of EO3, which possesses b but not a, designated type M-II. Restriction fragment analyses with BglII revealed accurate discrimination of EO3 into four types: L, M-I, M-II, and S. Moreover, we show that these small nucleotide segments a and b were encompassed by the characteristic nucleotide sequence with a common inverted repeat structure. In the present report we propose a revised version of EO3 typing, suggesting epidemiological usefulness as an alternative tool for species-specific detection of C. albicans, and discuss the potentiality of EO3 for generating more variations of DNA polymorphism.     

Genetic mapping of the Saccharomyces cerevisiae DNA polymerase I gene and characterization of a pol1 temperature-sensitive mutant altered in DNA primase-polymerase complex stability

Summary The cloned DNA polymerase I gene has been used to map the POL1 locus on the left arm of chromosome XIV, between MET4 and TOP2. Temperature-sensitive mutants in POL1 have been obtained by in vitro mutagenesis of the cloned gene and in vivo replacement of the wild-type allele with the mutated copy. Physiological and biochemical characterization of one temperature-sensitive mutant (pol1-1) shows that cells shifted to the non-permissive temperature can complete one round of cell division and DNA replication before they arrest. Analysis of DNA polymerase I in crude extracts and in partially purified preparations indicates that the pol1-1 mutation results in a conformational change and affects the stability of the DNA primase-polymerase complex.     

Evaluation of Comparative DNA Amplification Fingerprinting for Rapid Species Identification within the Genus Clusia

Understanding the taxonomiy of the tropical genus Clusia (Fam. Clusiaceae, Ord. Theales) has been hampered by the difficulties inherent in studying tropical dioecious, succulent, arborescent, epi- or hemiepiphytic taxa. Species identification by morphological traits often requires the terminal inflorescences and/or the succulent capsular fruits. To allow species differentiation based exclusively on vegetative tissue, a frequent necessity during ecological field studies, a procedure has been developed for rapid isolation of genomic DNA from Clusia leaf tissue followed by DNA amplification fingerprinting with a set of single arbitrary oligomer primers (23–27 mers). Fingerprints obtained with independent DNA preparations from one individual as well as DNA preparations from several individuals of the same species were identical for the major amplification products, although minor bands were somewhat variable. Polymorphic fingerprints have been obtained with 3 different primers for 3 Clusia species (C. minor L., C. alata Pl. & Tr., C. multiflora H. B. K.), and the related Oedematopus obovatus Spruce ex. PL (Clusiaceae). The interspecific Randomly Amplified Polymorphic Markers (RAPDs) thus obtained allow a rapid identification of vegetative tissue samples collected in the field, and will assist in a revision of the controversial taxonomy of the genus Clusia.     

Mechanisms of mutagenesis induced by DNA lesions: multiple factors affect mutations in translesion DNA synthesis

Abstract

Environmental mutagens lead to mutagenesis. However, the mechanisms are very complicated and not fully understood. Environmental mutagens produce various DNA lesions, including base-damaged or sugar-modified DNA lesions, as well as epigenetically modified DNA. DNA polymerases produce mutation spectra in translesion DNA synthesis (TLS) through misincorporation of incorrect nucleotides, frameshift deletions, blockage of DNA replication, imbalance of leading- and lagging-strand DNA synthesis, and genome instability. Motif or subunit in DNA polymerases further affects the mutations in TLS. Moreover, protein interactions and accessory proteins in DNA replisome also alter mutations in TLS, demonstrated by several representative DNA replisomes. Finally, in cells, multiple DNA polymerases or cellular proteins collaborate in TLS and reduce in vivo mutagenesis. Summaries and perspectives were listed. This review shows mechanisms of mutagenesis induced by DNA lesions and the effects of multiple factors on mutations in TLS in vitro and in vivo.     

DNA Topological Context Affects Access to Eukaryotic DNA Topoisomerase I

Abstract

We have analyzed the reactivity of a 217 base pair segment of the intrinsically curved Crithidia fasciculata kinetoplast DNA towards eukaryotic DNA topoisomerase I. The substrates were open [linear fragment and nicked circle] and closed minidomains [closed relaxed circle and circles with linking differences of ?1 and ?2], We interpreted the results with the aid of a model that was used to predict the structures of the topoisomers. The modelling shows that the ΔLk(?l) form is unusually compact because of the curvature in the DNA. To determine the role of sequence-directed curvature in both the experimental and modeling studies, controls were examined in which the curved Crithidia sequence was replaced by an uncurved sequence obtained from the plasmid pBR322.

Reactivity of the Crithidia DNA [as analyzed both by the cleavage and the topoisomerization reactions] markedly varied among the DNA forms: (i) the hierarchy of overall reactivity observed is: linear fragment > nicked circular, closed circular [ΔLk(O)], interwound [ΔLk(?2)] > bent interwound [ΔLk(?l)]; (ii) the intensity of several cleavage positions differs among DNA forms.

The results show that eukaryotic DNA topoisomerase I is very sensitive to the conformation of the substrates and that its reactivity is modulated by the variation of the compactness of the DNA molecule. The C. fasciculata sequence contains a highly curved segment that determines the conformation of the closed circle in a complex way.     

The lampbrush chromosomes of Xenopus laevis: preparation,identification, and distribution of 5S DNA sequences

Details are given of a technique for making permanent preparations of the lampbrush chromosomes of Xenopus laevis. Stained preparations allow all 18 bivalent chromosomes to be identified, and a working map showing the major features has been constructed. Fifteen of the Xenopus chromosomes have one telomere conspicuously larger than the other; the two smallest chromosomes, and one other, lack large telomeres. Similar preparations, extracted with RNase and denatured, have been hybridized in situ with a ³H-labelled 5S cRNA probe. Chromosomes can be identified in the resulting autoradiographs. 5S DNA sequences are present at all the larger telomeres and at three of the smaller ones, but are absent from the telomeres at both ends of the two smallest chromosomes. There are also five interstitial sites of hybridization. At one of these, label is on the chromosome axis; at the other four, label extends well away from the axis.