Thermodynamic aspects of triplex DNA formation in crowded environments.
Explore the source record for details and available documents.
Calorimetry↗
Biomedical subjects
A Minsky
Publications and source records attributed to A Minsky.
Phase behavior, DNA ordering, and size instability of cationic lipoplexes. Relevance to optimal transfection activity.
Mechanisms of cationic lipid-based nucleic acid delivery are receiving increasing attention, but despite this the factors that determine high or low activity of lipoplexes are poorly understood. This study is focused on the fine structure of cationic lipid-DNA complexes (lipoplexes) and its relevance to transfection efficiency. Monocationic (N-(1-(2,3-dioleoyloxy)propyl),N,N,N-trimethylammonium chloride, N-(1-(2,3-dimyristyloxypropyl)-N,N-dimethyl-(2-hydroxyethyl)ammonium bromide) and polycationic (2,3-dioleyloxy-N-[2(sperminecarboxamido)ethyl]-N,N-dimethyl-1-propanammonium trifluoroacetate) lipid-based assemblies, with or without neutral lipid (1,2-dioleoyl-sn-glycero-3-phosphatidylethanolamine, 1,2-dioleoyl-sn-glycero-3-phosphatidylcholine, cholesterol) were used to prepare lipoplexes of different L(+)/DNA(-) charge ratios. Circular dichroism, cryogenic-transmission electron microscopy, and static light scattering were used for lipoplex characterization, whereas expression of human growth hormone or green fluorescent protein was used to quantify transfection efficiency. All monocationic lipids in the presence of inverted hexagonal phase-promoting helper lipids (1,2-dioleoyl-sn-glycero-3-phosphatidylethanolamine, cholesterol) induced appearance of Psi(-) DNA, a chiral tertiary DNA structure. The formation of Psi(-) DNA was also dependent on cationic lipid-DNA charge ratio. On the other hand, monocationic lipids either alone or with 1,2-dioleoyl-sn-glycero-3-phosphatidylcholine as helper lipid, or polycationic 2,3-dioleyloxy-N-[2(sperminecarboxamido)ethyl]-N,N-dimethyl-1-propanammonium trifluoroacetate-based assemblies, neither of which promotes a lipid-DNA hexagonal phase, did not induce the formation of Psi(-) DNA. Parallel transfection studies reveal that the size and phase instability of the lipoplexes, and not the formation of Psi(-) DNA structure, correlate with optimal transfection.
3T3 Cells↗
Regulated phase transitions of bacterial chromatin: a non-enzymatic pathway for generic DNA protection.
The enhanced stress resistance exhibited by starved bacteria represents a central facet of virulence, since nutrient depletion is regularly encountered by pathogens in their natural in vivo and ex vivo environments. Here we explore the notion that the regular stress responses, which are mediated by enzymatically catalyzed chemical transactions and promote endurance during the logarithmic growth phase, can no longer be effectively induced during starvation. We show that survival of bacteria in nutrient-depleted habitats is promoted by a novel strategy: finely tuned and fully reversible intracellular phase transitions. These non-enzymatic transactions, detected and studied in bacteria as well as in defined in vitro systems, result in DNA sequestration and generic protection within tightly packed and highly ordered assemblies. Since this physical mode of defense is uniquely independent of enzymatic activity or de novo protein synthesis, and consequently does not require energy consumption, it promotes virulence by enabling long-term bacterial endurance and enhancing antibiotic resistance in adverse habitats.
Bacterial Proteins↗
Contextual equilibrium effects in DNA molecules.
The thermodynamic parameters of DNA triplex formation between oligonucleotides and double-stranded DNA segments containing adenine runs (A-tracts) were investigated to explore equilibrium structural effects exerted by flanking segments upon the A-tracts. Results obtained from isothermal titration calorimetry, temperature-dependent circular dichroism (CD), and UV melting experiments indicate that A-tracts, considered as a uniquely robust and inflexible DNA motif, can be structurally perturbed by neighboring sequences in a way that significantly affects the propensity of this motif to interact with triplex-forming oligonucleotides. These contextual equilibrium effects, which depend upon the composition and location of the flanking sequences, are likely to apply not only to the interaction of A-tracts with single-stranded DNA molecules but also to interactions with drugs and proteins. As such, the current results refine the guidelines for the design of triplex-forming oligonucleotides used for antigene strategies. More generally, they substantiate the notion that significant data might be encoded by structural DNA parameters.
Base Sequence↗
Ordered intracellular RecA-DNA assemblies: a potential site of in vivo RecA-mediated activities.
The inducible SOS response increases the ability of bacteria to cope with DNA damage through various DNA repair processes in which the RecA protein plays a central role. Here we present the first study of the morphological aspects that accompany the SOS response in Escherichia coli. We find that induction of the SOS system in wild-type bacteria results in a fast and massive intracellular coaggregation of RecA and DNA into a lateral macroscopic assembly. The coaggregates comprise substantial portions of both the cellular RecA and the DNA complement. The structural features of the coaggregates and their relation to in vitro RecA-DNA networks, as well as morphological studies of strains carrying RecA mutants, are all consistent with the possibility that the intracellular assemblies represent a functional entity in which RecA-mediated DNA repair and protection activities occur.
DNA Damage↗
Chiral DNA packaging in DNA-cationic liposome assemblies.
Recent studies have indicated that the structural features of DNA-lipid assemblies, dictated by the lipid composition and cationic lipid-to-DNA ratio, critically affect the efficiency of these complexes in acting as vehicles for cellular delivery of genetic material. Using circular dichroism we find that upon binding DNA, positively-charged liposomes induce a secondary conformational transition of the DNA molecules from the native B form to the C motif. Liposomes composed of positively-charged and neutral 'helper' lipids, found to be particularly effective as transfecting agents, induce - in addition to secondary conformational changes - DNA condensation into a left-handed cholesteric-like phase. A structural model is presented according to which two distinct, yet inter-related modes of DNA packaging coexist within such assemblies. The results underline the notion that subtle changes in the components of a supramolecular assembly may substantially modulate the interplay of interactions which dictate its structure and functional properties.
Cations↗
DNA protection by stress-induced biocrystallization.
The crystalline state is considered to be incompatible with life. However, in living systems exposed to severe environmental assaults, the sequestration of vital macromolecules in intracellular crystalline assemblies may provide an efficient means for protection. Here we report a generic defence strategy found in Escherichia coli, involving co-crystallization of its DNA with the stress-induced protein Dps. We show that when purified Dps and DNA interact, extremely stable crystals form almost instantaneously, within which DNA is sequestered and effectively protected against varied assaults. Crystalline structures with similar lattice spacings are formed in E. coli in which Dps is slightly over expressed, as well as in starved wild-type bacteria. Hence, DNA-Dps co-crystallization is proposed to represent a binding mode that provides wide-range protection of DNA by sequestration. The rapid induction and large-scale production of Dps in response to stress, as well as the presence of Dps homologues in many distantly related bacteria, indicate that DNA protection by biocrystallization may be crucial and widespread in prokaryotes.
Bacterial Proteins↗
Nucleosomes: a solution to a crowded intracellular environment?
The emergence of eukaryotes was accompanied by two major events that concern their genome and are of crucial significance when considered in terms of macromolecular crowding: (i) a substantial increase in the amount of DNA, and (ii) its confinement within a defined space. The resulting highly crowded environment would have strongly promoted DNA self-assembly processes, leading to extremely condensed and thermodynamically stable DNA aggregates. Such structural transitions have indeed been observed in vitro, as well as in virtually all cellular systems in which a nucleosomal assembly is absent. In this appear we raise the hypothesis that upon transition from prokaryotic systems to eukaryotes, the nucleosomes were rendered essential in order to negate extensive DNA condensation processes that would have resulted from excluded volume effects. By suppressing such processes, the nucleosomes act to maintain and regulate the conformational space of the DNA, thus enabling conformational flexibility and reversible structural modulations.
Animals↗
Flow of structural information between four DNA conformational levels.
Closed-circular supercoiled DNA molecules have been shown to form a cholesteric assembly within bacteria as well as in vitro under physiological DNA and salt concentrations. Circular dichroism and X-ray scattering studies indicate that the macroscopic structural properties of the chiral mesophase are directly and uniquely dictated by the supercoiling parameters of the constituent molecules. Specifically, we find that the pitch of the DNA cholesteric phase derived from supercoiled DNA is determined by the superhelical density, which, in turn, is modulated by secondary conformational changes. A direct interrelationship among four DNA structural levels, namely, DNA sequence, secondary structural transitions, the tertiary superhelical conformation, and the quaternary, supramolecular organization is accordingly pointed out. Since secondary conformational changes are both sequence and environment dependent, alterations of cellular conditions may effectively modulate the properties of the packed DNA organization, through their effects on secondary structural transitions and hence on the superhelical parameters. On the basis of these results we suggest that liquid crystallinity represents an effectively regulated packaging mode of plectonemic, nucleosome-free DNA molecules in living systems.
Base Sequence↗
In vivo quantitative characterization of intermolecular interactions.
Extensive effort has been directed toward a quantitative evaluation of forces which operate between biomacromolecules since the characterization of such forces is essential to a thorough understanding of fundamental biological processes. However, all studies hitherto reported were conducted in vitro, using isolated species. Here we report the first quantitative characterization of forces operating between DNA molecules within living bacteria. Evaluation of x-ray scattering studies conducted on intact bacteria indicates that, at DNA-DNA surface separations characteristic of DNA assemblies, interactions are dominated by repulsive hydration forces which originate from the structuring of water molecules. The results support the notion that the mechanisms by means of which macromolecules function, fold, and interact with each other crucially depend upon their hydration properties.
Base Composition↗
Supercoiling-regulated liquid-crystalline packaging of topologically-constrained, nucleosome-free DNA molecules.
Electron microscopy and circular dichroism studies of cholesteric aggregates derived from topologically-constrained DNA molecules indicate that the overall morphology and structural properties of these aggregates are fundamentally different from those characterizing condensed structures of nonconstrained DNA species. Specifically, the cholesteric pitch and twist of all hitherto characterized lyotropic mesophases of biopolymers--including those obtained from linear DNA--depend predominantly upon environmental parameters such as the dielectric constant of the solvent. In contrast, the properties of aggregates derived from closed circular supercoiled DNA are found to be solely and directly dictated by the superhelical density and handedness. On the basis of these results, as well as on the demonstrated ubiquity of liquid-crystalline DNA organizations in vivo, we suggest that supercoiling-regulated liquid crystallinity represents an effective packaging mode of nucleosome-free, topologically-constrained DNA molecules in living systems.
Circular Dichroism↗
Liquid-crystalline mesophases of plasmid DNA in bacteria.
Bacterial plasmids may often reach a copy number larger than 1000 per cell, corresponding to a total amount of DNA that may exceed the amount of DNA within the bacterial chromosome. This observation highlights the problem of cellular accommodation of large amounts of closed-circular nucleic acids, whose interwound conformation offers negligible DNA compaction. As determined by x-ray scattering experiments conducted on intact bacteria, supercoiled plasmids segregate within the cells into dense clusters characterized by a long-range order. In vitro studies performed at physiological DNA concentrations indicated that interwound DNA spontaneously forms liquid crystalline phases whose macroscopic structural properties are determined by the features of the molecular supercoiling. Because these features respond to cellular factors, DNA supercoiling may provide a sensitive regulatory link between cellular parameters and the packaging modes of interwound DNA in vivo.
Birefringence↗
Relationship between functional properties and structure of ovalbumin.
The effects of ovalbumin (OVA) denaturation using urea, guanidinium chloride (GdnHCl), sodium dodecyl sulphate (SDS), cetylpyridinium chloride (CPC), 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS), and 5 different cationic detergents with various side chains, HCl, and CH3COOH were observed. Progressive unfolding in ovalbumin was measured as a function of fluorescent light intensity, peak response and shift in the maximum of emission. Kinetic measurements demonstrated that the rate of denaturation usually followed a double exponential decay pattern, but at small concentrations of urea and acids first-order reaction was indicated. The reversibility of the unfolding-folding transitions was confirmed from tryptophan fluorescence and circular dichroism (CD) measurements. Differences in secondary structure were observed and changes of alpha-helical content were calculated. Polyacrylamide gel electrophoresis (PAGE) with and without sodium dodecyl sulphate (SDS-PAGE) showed differences in the structure of native and denatured ovalbumin. Native protein samples in PAGE demonstrated smaller number and larger mobilities of subunits than denatured ones with different reductants, such as SDS and 2-mercaptoethanol (2 ME). Scanning of SDS protein patterns showed the appearance of aggregated forms in region of 45 kD.
Circular Dichroism↗
Mode of formation and structural features of DNA-cationic liposome complexes used for transfection.
Complexes formed between cationic liposomes and nucleic acids represent a highly efficient vehicle for delivery of DNA and RNA molecules into a large variety of eukaryotic cells. By using fluorescence, gel electrophoresis, and metal-shadowing electron microscopy techniques, the factors that affect the, yet unclear, interactions between DNA and cationic liposomes as well as the structural features of the resulting complexes have been elucidated. A model is suggested according to which cationic liposomes bind initially to DNA molecules to form clusters of aggregated vesicles along the nucleic acids. At a critical liposome density, two processes occur, namely, DNA-induced membrane fusion, indicated by lipid mixing studies, and liposome-induced DNA collapse, pointed out by the marked cooperativity of the encapsulation processes, by their modulations by DNA-condensing agents, and also by their conspicuous independence upon DNA length. The DNA collapse leads to the formation of condensed structures which can be completely encapsulated within the fused lipid bilayers in a fast, highly cooperative process since their exposed surface is substantially smaller than that of extended DNA molecules. The formation of the transfecting DNA-liposome complexes in which the nucleic acids are fully encapsulated within a positively-charged lipid bilayer is proposed, consequently, to be dominated by mutual effects exerted by the DNA and the cationic liposomes, leading to interrelated lipid fusion and DNA collapse.
Animals↗
Effects of adenine tracts on the B-Z transition. Fine tuning of DNA conformational transition processes.
The effects exerted by short runs of adenines (A-tracts), alternating (AT)n segments, and single-stranded DNA upon the right- to left-handed DNA transition, as well as upon the energetic and structural parameters of the B/Z junctions, were investigated by using synthetic segments in which these motifs are coupled to a potentially Z-forming core. UV, CD, and 31P NMR studies of the salt-induced B to Z transition occurring in the various segments indicate that the transition is composed of two phases: a slow rate-determining induction of an initial structural deformation followed by a cooperative propagation of this "nucleus" in the form of a left-handed Z-DNA. The first phase is found to be crucially affected by the nature of the sequences coupled to the potentially Z-forming core. Thus, a higher rigidity of the flanking segments, such as that characterizing adenine tracts, is associated with higher energy values required for the induction of the initial conformational deformation, as well as with more defined structural parameters of the ultimate B/Z junctions. The second phase is affected mainly by the composition and sequence of the Z-forming segment. The observations that DNA conformational changes can be finely tuned and modulated by parameters pertaining to both the segment which undergoes the transition and the flanking sequences support the notion that DNA secondary motifs, such as the Z form and A-tracts, might be involved in the regulation of cellular processes.
Adenine↗
On the metastability of left-handed DNA motifs.
Alternating purine-pyrimidine DNA sequences such as poly [d(C-G)] or poly[d(m5C-G)] undergo a cooperative, salt-induced structural transition from a right-handed B conformation, which prevails at relatively low ionic strength, into a left-handed Z form, generally believed to be stabilized by high salt concentrations. We report here that upon a monotonous increase of the ionic strength, the well-established B to Z transition is followed by a second, hitherto unobserved conformational change leading from Z-DNA back into a right-handed B-like form. This observation indicates that, in contrast with the current convention, the Z motif represents an unstable configuration relative to the B form at both low and high salt concentrations and that the occurrence of a left-handed DNA structure, presently depicted as a step function of the ionic strength, should rather be treated in terms of a pulse. The reported transition underscores the inherent metastability of the Z configuration, and indicates, consequently, that this motif is ideally suited to act as a structural regulatory element, as such an element should be endowed with a large susceptibility to cellular parameters.
Base Sequence↗
DNA packaging induced by micellar aggregates: a novel in vitro DNA condensation system.
Evidence for a conceptually novel DNA packaging process is presented. X-ray scattering, electron microscopy, and circular dichroism measurements indicate that in the presence of positively charged micellar aggregates and flexible anionic polymers, such as negatively charged polypeptides or single-stranded RNA species, a complex is formed in which DNA molecules are partially embedded within a micellar scaffold and partially condensed into highly packed chiral structures. Based on studies of micelle-DNA and micelle-flexible anionic polymer systems, as well as on the known effects of a high charge density upon the micellar organization, a DNA packaging model is proposed. According to this model, the DNA induces the elongation of the micelles into rodlike aggregates, forming a closely packed matrix in which the DNA molecules are immobilized. In contrast, the flexible anionic polymers stabilize clusters of spherical micelles which are proposed to effect a capping of the rodlike micelles, thus arresting their elongation and creating surfactant-free segments of the DNA that are able to converge and collapse. Thus, unlike other in vitro DNA packaging systems, in which condensation follows encounters between charge-neutralized DNA molecules, a prepackaging phase where the DNA is immobilized within a matrix is proposed in this case. Cellular and nuclear membranes have been implicated in DNA packaging processes in vivo, and negatively charged polyelectrolytes were shown to be involved in the processes. These observations, combined with the basic tenets of the DNA condensation system described here, allow for the progression to the study of more elaborate model systems and thus might lead to insights into the nature and roles of the intricate in vivo DNA-membrane complexes.
Circular Dichroism↗
Attenuation of DNA-protein interactions associated with intrinsic, sequence-dependent DNA curvature.
Inherently curved DNA segments, associated with short runs of adenines, have been identified in many gene regulatory regions, yet their physiological significance remains unknown. The observations reported in this study indicate that intrinsically bent nucleic acid fragments are characterized by substantially attenuated affinities toward DNA-binding proteins involved in structural functions, such as H1 histone and protamine, as well as toward various DNA-modifying enzymes including ligases and exo- and endonucleases. Two mechanisms might be responsible for the altered binding properties. According to the first mechanism, the attenuated binding affinities and the bending represent two independent consequences of the unique structural parameters exhibited by A-tracts. Indeed, analysis of the degradation products obtained upon exposure of the curved sequences to various chemical nucleases points toward the narrowing of the DNA minor groove, a conformational modulation known to characterize A-tracts and to run along the axially-bent motifs, as a potential determinant of the observed binding attenuation. Alternatively, the conformational constraints which result from the stable bending might act to modulate the strength of DNA-protein interactions. Although the factor directly responsible for the altered binding affinities revealed by the bent sequences cannot as yet be conclusively resolved, it is proposed that a reiteration of this specific factor, being either an A-tract or a bend, in phase with the DNA helical repeat acts to amplify the modulation of the binding.(ABSTRACT TRUNCATED AT 250 WORDS)
Adenine Nucleotides↗