Chloroviruses are unusual among viruses infecting eukaryotic organisms in that they must, like bacteriophages, penetrate a rigid cell wall to initiate infection. Chlorovirus PBCV-1 infects its host, Chlorella variabilis NC64A by specifically binding to and degrading the cell wall of the host at the point of contact by a virus-packaged enzyme(s). However, PBCV-1 does not use any of the five previously characterized virus-encoded polysaccharide degrading enzymes to digest the Chlorella host cell wall during virus entry because none of the enzymes are packaged in the virion. A search for another PBCV-1-encoded and virion-associated protein identified protein A561L. The fourth domain of A561L is a 242 amino acid C-terminal domain, named A561LD4, with cell wall degrading activity. An A561LD4 homolog was present in all 52 genomically sequenced chloroviruses, infecting four different algal hosts. A561LD4 degraded the cell walls of all four chlorovirus hosts, as well as several non-host Chlorella spp. Thus, A561LD4 was not cell-type specific. Finally, we discovered that exposure of highly purified PBCV-1 virions to A561LD4 increased the specific infectivity of PBCV-1 from about 25–30% of the particles forming plaques to almost 50%. We attribute this increase to removal of residual host receptor that attached to newly replicated viruses in the cell lysates.
Due to the heterogeneity of viruses and their hosts, a comprehensive view of viral infection is best achieved by analyzing large populations of infected cells. However, information regarding variation in infected cell populations is lost in bulk measurements. Motivated by an interest in the temporal progression of events in virally infected cells, we used image flow cytometry (IFC) to monitor changes in Acanthamoeba polyphaga cells infected with Mimivirus. This first use of IFC to study viral infection required the development of methods to preserve morphological features of adherent amoeba cells prior to detachment and analysis in suspension. It also required the identification of IFC parameters that best report on key events in the Mimivirus infection cycle. The optimized IFC protocol enabled the simultaneous monitoring of diverse processes including generation of viral factories, transport, and fusion of replication centers within the cell, accumulation of viral progeny, and changes in cell morphology for tens of thousands of cells. After obtaining the time windows for these processes, we used IFC to evaluate the effects of perturbations such as oxidative stress and cytoskeletal disruptors on viral infection. Accurate dose-response curves could be generated, and we found that mild oxidative stress delayed multiple stages of virus production, but eventually infection processes occurred with approximately the same amplitudes. We also found that functional actin cytoskeleton is required for fusion of viral replication centers and later for the production of viral progeny. Through this report, we demonstrate that IFC offers a quantitative, high-throughput, and highly robust approach to study viral infection cycles and virus-host interactions. (C) 2019 The Authors. Cytometry Part A published by Wiley Periodicals, Inc. on behalf of International Society for Advancement of Cytometry.
A fundamental stage in viral infection is the internalization of viral genomes in host cells. Although extensively studied, the mechanisms and factors responsible for the genome internalization process remain poorly understood. Here we report our observations, derived from diverse imaging methods on genome internalization of the large dsDNA Paramecium bursaria chlorella virus-1 (PBCV-1). Our studies reveal that early infection stages of this eukaryotic-infecting virus occurs by a bacteriophage-like pathway, whereby PBCV-1 generates a hole in the host cell wall and ejects its dsDNA genome in a linear, base-pair-by-base-pair process, through a membrane tunnel generated by the fusion of the virus internal membrane with the host membrane. Furthermore, our results imply that PBCV-1 DNA condensation that occurs shortly after infection probably plays a role in genome internalization, as hypothesized for the infection of some bacteriophages. The subsequent perforation of the host photosynthetic membranes presumably enables trafficking of viral genomes towards host nuclei. Previous studies established that at late infection stages PBCV-1 generates cytoplasmic organelles, termed viral factories, where viral assembly takes place, a feature characteristic of many large dsDNA viruses that infect eukaryotic organisms. PBCV-1 thus appears to combine a bacteriophage-like mechanism during early infection stages with a eukaryotic-like infection pathway in its late replication cycle.
The increasing interest in cytoplasmic factories generated by eukaryotic-infecting viruses stems from the realization that these highly ordered assemblies may contribute fundamental novel insights to the functional significance of order in cellular biology. Here, we report the formation process and structural features of the cytoplasmic factories of the large dsDNA virus Paramecium bursaria chlorella virus 1 (PBCV-1). By combining diverse imaging techniques, including scanning transmission electron microscopy tomography and focused ion beam technologies, we show that the architecture and mode of formation of PBCV-1 factories are significantly different from those generated by their evolutionary relatives Vaccinia and Mimivirus. Specifically, PBCV-1 factories consist of a network of single membrane bilayers acting as capsid templates in the central region, and viral genomes spread throughout the host cytoplasm but excluded from the membrane-containing sites. In sharp contrast, factories generated by Mimivirus have viral genomes in their core, with membrane biogenesis region located at their periphery. Yet, all viral factories appear to share structural features that are essential for their function. In addition, our studies support the notion that PBCV-1 infection, which was recently reported to result in significant pathological outcomes in humans and mice, proceeds through a bacteriophage-like infection pathway.
The discovery of giant DNA viruses and the recent realization that such viruses are diverse and abundant blurred the distinction between viruses and cells. These findings elicited lively debates on the nature and origin of viruses as well as on their potential roles in the evolution of cells. The following essay is, however, concerned with new insights into fundamental structural and physical aspects of viral replication that were derived from studies conducted on large DNA viruses. Specifically, the entirely cytoplasmic replication cycles of Mimivirus and Vaccinia are discussed in light of the highly limited trafficking of large macromolecules in the crowded cytoplasm of cells. The extensive spatiotemporal order revealed by cytoplasmic viral factories is described and contended to play an important role in promoting the efficiency of these 'nuclear-like' organelles. Generation of single-layered internal membrane sheets in Mimivirus and Vaccinia, which proceeds through a novel membrane biogenesis mechanism that enables continuous supply of lipids, is highlighted as an intriguing case study of self-assembly. Mimivirus genome encapsidation was shown to occur through a portal different from the 'stargate' portal that is used for genome release. Such a 'division of labor' is proposed to enhance the efficacy of translocation processes of very large viral genomes. Finally, open questions concerning the infection cycles of giant viruses to which future studies are likely to provide novel and exciting answers are discussed. (C) 2014 Elsevier Inc. All rights reserved.
Genome condensation is increasingly recognized as a generic stress response in bacteria. To better understand the physiological implications of this response, we used fluorescent markers to locate specific sites on Escherichia coli chromosomes following exposure to cytotoxic stress. We find that stress-induced condensation proceeds through a nonrandom, zipper-like convergence of sister chromosomes, which is proposed to rely on the recently demonstrated intrinsic ability of identical double-stranded DNA molecules to specifically identify each other. We further show that this convergence culminates in spatial proximity of homologous sites throughout chromosome arms. We suggest that the resulting apposition of homologous sites can explain how repair of double strand DNA breaks might occur in a mechanism that is independent of the widely accepted yet physiologically improbable genome-wide search for homologous templates. We claim that by inducing genome condensation and orderly convergence of sister chromosomes, diverse stress conditions prime bacteria to effectively cope with severe DNA lesions such as double strand DNA breaks.
Although extensively studied, the structure, cellular origin and assembly mechanism of internal membranes during viral infection remain unclear. By combining diverse imaging techniques, including the novel Scanning-Transmission Electron Microscopy tomography, we elucidate the structural stages of membrane biogenesis during the assembly of the giant DNA virus Mimivirus. We show that this elaborate multistage process occurs at a well-defined zone localized at the periphery of large viral factories that are generated in the host cytoplasm. Membrane biogenesis is initiated by fusion of multiple vesicles, similar to 70 nm in diameter, that apparently derive from the host ER network and enable continuous supply of lipid components to the membrane-assembly zone. The resulting multivesicular bodies subsequently rupture to form large open single-layered membrane sheets from which viral membranes are generated. Membrane generation is accompanied by the assembly of icosahedral viral capsids in a process involving the hypothetical major capsid protein L425 that acts as a scaffolding protein. The assembly model proposed here reveals how multiple Mimivirus progeny can be continuously and efficiently generated and underscores the similarity between the infection cycles of Mimivirus and Vaccinia virus. Moreover, the membrane biogenesis process indicated by our findings provides new insights into the pathways that might mediate assembly of internal viral membranes in general.
Poxviruses are considered to be unique among all DNA viruses, because their infection cycle is carried out exclusively in the host cytoplasm. Such an infection strategy is of interest, because it necessitates generation of elaborate factories in which viral replication and assembly are promoted. By using diverse imaging techniques, we show that the infection cycle of the largest virus currently identified, the Acanthamoeba polyphaga Mimivirus, similarly occurs exclusively in the host cytoplasm. We further show that newly synthesized mRNAs accumulate at discrete cytoplasmic sites that are distinct from the sites where viral replication occurs, and this is observed in vaccinia infection. By revealing substantial physiologic similarity between poxviruses and Mimivirus and thus, implying that an entirely cytoplasmic viral replication might be more common than generally considered, these findings underscore the ability of DNA viruses to generate large and elaborate replication factories.
Double-strand DNA breaks (DSBs) are the most detrimental lesion that can be sustained by the genetic complement, and their inaccurate mending can be just as damaging. According to the consensual view, precise DSB repair relies on homologous recombination. Here, we review studies on DNA repair, chromatin diffusion and chromosome confinement, which collectively imply that a genome-wide search for a homologous template, generally thought to be a pivotal stage in all homologous DSB repair pathways, is improbable. The implications of this assertion for the scope and constraints of DSB repair pathways and for the ability of diverse organisms to cope with DNA damage are discussed.
The bacterium Blastochloris viridis carries one of the simplest photosynthetic systems, which includes a single light-harvesting complex that surrounds the reaction center, membrane soluble quinones, and a soluble periplasmic protein cytochrome c(2) that shuttle between the reaction center and the bc(1) complex and act as electron carriers, as well as the ATP synthase. The close arrangement of the photosynthetic membranes in Bl. viridis, along with the extremely tight arrangement of the photosystems within these membranes, raises a fundamental question about the diffusion of the electron carriers. To address this issue, we analyzed the structure and response of the Bl. viridis photosynthetic system to various light conditions, by using a combination of electron microscopy, whole-cell cryotomography, and spectroscopic methods. We demonstrate that in response to high light intensities, the ratio of both cytochrome c(2) and bc(1) complexes to the reaction centers is increased. The shorter membrane stacks, along with the notion that the bc(1) complex is located at the highly curved edges of these stacks, result in a smaller average distance between the reaction centers and the bc(1) complexes, leading to shorter pathways of cytochrome c(2) between the two complexes. Under anaerobic conditions, the slow diffusion rate is further mitigated by keeping most of the quinone pool reduced, resulting in a concentration gradient of quinols that allows for a constant supply of theses electron carriers to the bc(1) complex.
The organization of chromatin has a major impact on cellular activities, such as gene expression. For bacteria, it was suggested that the spatial organization of the genetic material correlates with transcriptional levels, implying a specific architecture of the chromosome within the cytoplasm. Accordingly, recent technological advances have emphasized the organization of the genetic material within nucleoid structures. Gemmata obscuriglobus, a member of the phylum Planctomycetes, exhibits a distinctive nucleoid structure in which chromatin is encapsulated within a discrete membrane-bound compartment. Here, we show that this soil and freshwater bacterium tolerates high doses of UV and ionizing radiation. Cryoelectron tomography of frozen hydrated sections and electron microscopy of freeze-substituted cells have indicated a more highly ordered condensed-chromatin organization in actively dividing and stationary-phase G. obscuriglobus cells. These three-dimensional analyses revealed a complex network of double membranes that engulf the condensed DNA. Bioinformatics analysis has revealed the existence of a putative component involved in nonhomologous DNA end joining that presumably plays a role in maintaining chromatin integrity within the bacterium. Thus, our observations further support the notion that packed chromatin organization enhances radiation tolerance.
Icosahedral double-stranded DNA viruses use a single portal for genome delivery and packaging. The extensive structural similarity revealed by such portals in diverse viruses, as well as their invariable positioning at a unique icosahedral vertex, led to the consensus that a particular, highly conserved vertex-portal architecture is essential for viral DNA translocations. Here we present an exception to this paradigm by demonstrating that genome delivery and packaging in the virus Acanthamoeba polyphaga mimivirus occur through two distinct portals. By using high-resolution techniques, including electron tomography and cryo-scanning electron microscopy, we show that Mimivirus genome delivery entails a large-scale conformational change of the capsid, whereby five icosahedral faces open up. This opening, which occurs at a unique vertex of the capsid that we coined the ''stargate'', allows for the formation of a massive membrane conduit through which the viral DNA is released. A transient aperture centered at an icosahedral face distal to the DNA delivery site acts as a non-vertex DNA packaging portal. In conjunction with comparative genomic studies, our observations imply a viral packaging pathway akin to bacterial DNA segregation, which might be shared by diverse internal membrane-containing viruses.
During the photosynthetic process, highly organized membranal assemblies convert light into biochemical energy with high efficiency. We have used whole-mount cryo-electron tomography to study the intracellular architecture of the photosynthetic membranes of the anaerobic purple photosynthetic bacterium Rhodopseudomonas viridis, as well,as the organization of the photosynthetic units within the membranes. Three-dimensional reconstruction demonstrates a continuity of the plasma membrane with the photosynthetic membranes that form tunnel-like structures with an average diameter of 31 nm +/- 8 nm at the connection sites. The spacing between the photosynthetic membranes at their cytoplasmic faces was found to be 11 nm, thus enforcing a highly close packaging of the photosynthetic membranes. Analysis of successive tomographic slices allowed for derivation of the spacing between adjacent photosynthetic core complexes from a single-layered photosynthetic membrane, in situ. This analysis suggests that most, if not all, photosynthetic membranes in R. viridis are characterized by a similar two-dimensional hexagonal lattice organization. (C) 2007 Elsevier Inc. All rights reserved.
Five common assumptions about the first cells are challenged by the pre- biotic ecology model and are replaced by the following propositions: firstly, early cells were more complex, more varied and had a greater diversity of constituents than modern cells; secondly, the complexity of a cell is not related to the number of genes it contains, indeed, modern bacteria are as complex as eukaryotes; thirdly, the unit of early life was an 'ecosystem' rather than a 'cell'; fourthly, the early cell needed no genes at all; fifthly, early life depended on non-covalent associations and on catalysts that were not confined to specific reactions. We present here the outlines of a theory that connects findings about modern bacteria with speculations about their origins.
For enzymatic activities to be effectively carried out, basic prerequisites must be met. Many enzymatic tasks require continuous consumption and dissipation of energy, sometimes in massive amounts. Some activities, such as DNA replication, transcription, and repair through homologous recombination rely upon templates that provide the information required for these transactions. Yet, circumstances where intracellular energy pools are severely depleted, or where intact templates are not available, frequently occur. Moreover, the fact that in order to reach their targets, enzymes must cope with an extremely crowded and viscous cellular milieu that drastically slows down their diffusion is often neglected. These impediments are particularly evident under stress conditions such as prolonged starvation or continuous exposure to DNA-damaging agents. Here we survey recent studies, which imply that when enzymatically-mediated DNA repair pathways are hindered, alternative strategies are deployed, whose common denominator is the reorganization of bacterial nucleoids into morphologies that promote DNA repair and protection. (c) 2006 Elsevier Inc. All rights reserved.
We hypothesize that life began not with the first self-reproducing molecule or metabolic network, but as a prebiotic ecology of co-evolving populations of macromolecular aggregates (composomes). Each composome species had a particular molecular composition resulting from molecular complementarity among environmentally available prebiotic compounds. Natural selection acted on composomal species that varied in properties and functions such as stability, catalysis, fission, fusion and selective accumulation of molecules from solution. Fission permitted molecular replication based on composition rather than linear structure, while fusion created composomal variability. Catalytic functions provided additional chemical novelty resulting eventually in autocatalytic and mutually catalytic networks within composomal species. Composomal autocatalysis and interdependence allowed the Darwinian co-evolution of content and control (metabolism). The existence of chemical interfaces within complex composomes created linear templates upon which self-reproducing molecules (such as RNA) could be synthesized, permitting the evolution of informational replication by molecular templating. Mathematical and experimental tests are proposed.
Bacterial spores have long been recognized as the sturdiest known life forms on earth, revealing extraordinary resistance to a broad range of environmental assaults. A family of highly conserved spore-specific DNA-binding proteins, termed alpha/beta-type small, acid-soluble spore proteins (SASP), plays a major role in mediating spore resistance. The mechanism by which these proteins exert their protective activity remains poorly understood, in part due to the lack of structural data on the DNA-SASP complex. By using cryoelectron microscopy, we have determined the structure of the helical complex formed between DNA and SspC, a characteristic member of the alpha/beta-type SASP family. The protein is found to fully coat the DNA, forming distinct protruding domains, and to modify DNA structure such that it adopts a 3.2-nm pitch. The protruding SspC motifs allow for interdigitation of adjacent DNA-SspC filaments into a tightly packed assembly of nucleoprotein helices. By effectively sequestering DNA molecules, this dense assembly of filaments is proposed to enhance and complement DNA protection obtained by DNA saturation with the alpha/beta-type SASP.
The textbook view of the bacterial cytoplasm as an unstructured environment has been overturned recently by studies that highlighted the extent to which non-random organization and coherent motion of intracellular components are central for bacterial life-sustaining activities. Because such a dynamic order critically depends on continuous consumption of energy, it cannot be perpetuated in starved, and hence energy-depleted, stationary-state bacteria. Here, we show that, at the onset of the stationary state, bacterial chromatin undergoes a massive reorganization into ordered toroidal structures through a process that is dictated by the intrinsic properties of DNA and by the ubiquitous starvation-induced DNA-binding protein Dps. As starvation proceeds, the toroidal morphology acts as a structural template that promotes the formation of DNA-Dps crystalline assemblies through epitaxial growth. Within the resulting condensed assemblies, DNA is effectively protected by means of structural sequestration. We thus conclude that the transition from bacterial active growth to stationary phase entails a co-ordinated process, in which the energy-dependent dynamic order of the chromatin is sequentially substituted with an equilibrium crystalline order.
Nucleic acids are characterized by a vast structural variability. Secondary structural conformations include the main polymorphs A, B, and Z, cruci-forms, intrinsic curvature, and multistranded motifs. DNA secondary motifs are stabilized and regulated by the primary base sequence, contextual effects, environmental factors, as well as by high-order DNA packaging modes. The high-order modes are, in turn, affected by secondary structures and by the environment. This review is concerned with the flow of structural information among the hierarchical structural levels of DNA molecules, the intricate interplay between the various factors that affect these levels, and the regulation and physiological significance of DNA high-order structures.
DNA repair and protection processes impose arduous demands upon cellular systems. The high-fidelity recombinational repair pathway entails a rapid genome-wide search for sequence homology. The efficiency of this transaction is intriguing in light of the uniquely adverse diffusion traits of the involved species. DNA protection in cells exposed to continuous stress or prolonged starvation is equally enigmatic, because the ability of such cells to deploy energy-dependent enzymatic repair processes is hampered as a result of progressive perturbation of the intracellular energy balance. DNA repair in radio-resistant bacteria, which involves accurate chromosome reconstruction from multiple fragments, is similarly associated with apparently insurmountable logistical obstacles. The studies reviewed here imply that the mechanisms deployed to overcome these intrinsic hurdles have a basic common denominator. In all these cases, condensed and ordered chromatin assemblies are formed, within which molecular diffusion is restricted and confined. Restricted diffusion thus appears as a general strategy that is exploited by nature to facilitate homologous search, to promote energy-independent DNA protection through physical DNA sequestration and attenuated accessibility to damaging agents, and to enable error-free repair of multiple double-strand DNA breaks.
Crowding, which characterizes the interior of all living cells, has been shown to dramatically affect biochemical processes, leading to stabilization of compact morphologies, enhanced macromolecular associations, and altered reaction rates. Due to the crowding-mediated shift in binding equilibria toward association, crowding agents were proposed to act as a metabolic buffer, significantly extending the range of intracellular conditions under which interactions occur. Crowding may, however, impose a liability because, by greatly and generally enhancing macromolecular association, it can lead to irreversible interactions. To better understand the physical determinants and physiological consequences of crowding-mediated buffering, we studied the effects of crowding, or excluded volume, on DNA structures. Results obtained from isothermal titration calorimetry (ITC) and UV melting experiments indicate that crowding-induced effects are marginal under conditions that a priori favor association of DNA strands but become progressively larger when conditions deteriorate. As such, crowding exerts "genuine" buffering activity. Unexpectedly, crowding-mediated effects are found to include enthalpy terms that favorably contribute to association processes. We propose that these enthalpy terms and preferential stabilization derive from a reconfiguration of DNA hydration that occurs in dense DNA-rich phases obtained in crowded environments.
The bacterium Deinococcus radiodurans survives ionizing irradiation and other DNA-damaging assaults at doses that are lethal to all other organisms. How D. radiodurans accurately reconstructs its genome from hundreds of radiation-generated fragments in the absence of an intact template is unknown. Here we show that the D. radiodurans genome assumes an unusual toroidal morphology that may contribute to its radioresistance. We propose that, because of restricted diffusion within the tightly packed and laterally ordered DNA toroids, radiation-generated free DNA ends are held together, which may facilitate template-independent yet error-free joining of DNA breaks.
In Escherichia coli, ribosomes must interact with translocons on the membrane for the proper integration of newly synthesized membrane proteins, cotranslationally. Previous in vivo studies indicated that unlike the E coli signal recognition particle (SRP), the SRP receptor FtsY is required for membrane targeting of ribosomes. Accordingly, a putative SRP-independent, FtsY-mediated ribosomal targeting pathway has been suggested (Herskovits, A.A., E.S. Bochkareva, and E. Bibi. 2000. Mol. Microbiol. 38:927-939). However, the nature of the early contact of ribosomes with the membrane, and the involvement of FtsY in this interaction are unknown. Here we show that in cells depleted of the SRP protein, Ffh or the translocon component SecE, the ribosomal targeting pathway is blocked downstream and unprecedented, membrane-bound FtsY-ribosomal complexes are captured. Concurrently, under these conditions, novel, ribosome-loaded intracellular membrane structures are formed. We propose that in the absence of a functional SRP or translocon, ribosomes remain jammed at their primary membrane docking site, whereas FtsY-dependent ribosomal targeting to the membrane continues. The accumulation of FtsY-ribosome complexes induces the formation of intracellular membranes needed for their quantitative accommodation. Our results with E. coli, in conjunction with recent observations made with the yeast Saccharomyces cerevisiae, raise the possibility that the SRP receptor-mediated formation of intracellular membrane networks is governed by evolutionarily conserved principles.
Triple-stranded DNA structures can be formed in living cells, either by native DNA sequences or following the application of antigene strategies, in which triplex-forming oligonucleotides are targeted to the nucleus. Recent studies imply that triplex motifs may play a role in DNA transcription, recombination and condensation processes in vivo. Here we show that very short triple-stranded DNA motifs, but not double-stranded segments of a comparable length, self-assemble into highly condensed and ordered structures. The condensation process, studied by circular dichroism and polarized-light microscopy, occurs under conditions that mimic cellular environments in terms of ionic strength, ionic composition and crowding. We argue that the unique tendency of triplex DNA structures to self-assemble, a priori unexpected in light of the very short length and the large charge density of these motifs, reflects the presence of strong attractive interactions that result from enhanced ion correlations. The results provide, as such, a direct experimental link between charge density, attractive interactions between like-charge polymers and DNA packaging. Moreover, the observations strongly support the notion that triple-stranded DNA motifs may be involved in the regulation of chromosome organization in living cells.
Much of the sophisticated chemistry of life is accomplished by multicomponent complexes, which act as molecular machines. Intrinsic to their accuracy and efficiency is the energy that is supplied by hydrolysis of nucleoside triphosphates. Conditions that deplete energy sources should therefore cause decay and death. But studies on organisms that are exposed to prolonged stress indicate that this fate could be circumvented through the formation of highly ordered intracellular assemblies. In these thermodynamically stable structures, vital components are protected by a physical sequestration that is independent of energy consumption.
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.
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.
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 nonenzymatic 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.
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 coil. 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.
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 DIVA 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. (C) 1999 Federation of European Biochemical Societies.
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(1,2). 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.
Nucleic acids are characterized by a predominant right-handed helical configuration that derives from the chirality of the sugar moiety. Hitherto, only "local" effects of this helical asymmetry, exemplified by DNA interactions with small compounds, have been documented. The results described in this study indicate that an enhanced asymmetry is required for the manifestation of chiral effects in DNA self-assembly processes or for chiral discrimination upon interactions with peptides. Two cases in which the intrinsic DNA asymmetry is enhanced are reported: rod-like superhelical species derived from linear DNA molecules, and topologically constrained supercoiled DNA. In the first case, the super-helical grooves within the DNA rods allow for a stereospecific complexation with peptides, resulting in chiral discrimination. In the second case, it is shown that the properties of cholesteric assemblies derived from supercoiled DNA are strictly determined by the enhanced asymmetry associated with molecular supercoiling. The results allow for new reflections on the concept of molecular complementarity, and indicate that spontaneously obtained chiral DNA mesophases might have played a key role in determining terrestrial homochirality. (C) 1998 Wiley-Liss, Inc.
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 paper 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.
The notion that ''L-proteins interact more avidly (than D-proteins) with D-nucleic acids'' (Hegstorm, R. A.; Kondepudi, D. K. Sci. Am. 1990, 253, 98-105) represents a direct extension to the concept of stereochemical complementarity. This notion, considered as a central tenet to theories concerned with the origin of biochemical homochirality, is however completely refuted by currently available experimental data that indicate identical DNA affinities towards L- and D-peptides. Here we show that chiral discrimination in nucleic acid-peptide interactions necessitates a substantial amplification of macromolecular geometric constraints. Thus, DNA molecules are found to exhibit a higher affinity toward L-peptides-but only under conditions that enhance their chiral identity by promoting the formation of cholesteric DNA mesophases. The results allow for new reflections on the concept of molecular complementarity, and indicate that spontaneously obtained chiral DNA mesophases might have played a key role in determining the terrestrial L-homochirality of proteins. Moreover, the observations provide an intriguing example to the notion that new properties of DNA molecules emerge in their condensed state, in which a higher structural order is imposed.
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.
Keywords: Biochemistry & Molecular Biology; Biophysics; Spectroscopy
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.
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.
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 inter wound DNA in vivo.
The effects of ovalbumin (OVA) denaturation using urea, guanidinium chloride (GdnHC1), sodium dodecyl sulphate (SDS), cetylpyridinium chloride (CPC), 3-[(3-cholamidopropyl)dimethylammonio]-1-propa (CHAPS), and 5 different cationic detergents with various side chains, HC1, 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.
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.
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 P-31 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.
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.
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.
The effects of short runs of adenines (A-tracts) upon nucleic acids packaging processes and the properties of the resulting condensates were investigated by using random DNA sequences isolated from natural sources, as well as synthetic segments obtained by an extensive ligation of specific oligomers. Reiteration of short A-tracts (A(N) where N3), in which the distinct structural features that characterize this motif are fully expressed, results in a complete suppression of any chiral order in the packed particles, assigned to a significantly enhanced rigidity. DNA fragments where A-tracts are reiterated in phase, leading to a stable macroscopic curvature, are found to undergo condensation through altered pathways and to form toroidal shapes of unusually small dimensions. The results point towards the intriguing possibility that A-tracts and, in particular, the global, intrinsic curvature associated with such motifs, might be involved in the determination of nucleic acids packaging pathways, and underline the usefulness of defined sequences in the study of DNA condensation processes.
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. This suggestion is based on the findings obtained from ligated DNA fragments which are composed of hexadecamers as the repetitive unit, and in which the A-tracts and the bends are reiterated out-of-phase. The binding attenuation revealed by these DNA segments is found to be considerably lower than that exhibited by the globally curved sequences. On the basis of these observations, we suggest that sequence-dependent DNA bending associated with short, phased runs of adenine might be involved in gene regulation processes.
Circular dichroism and electron microscopy studies of various in vitro DNA packaging systems indicate that all the factors which induce and modulate the secondary conformation of DNA molecules are capable of eliciting nucleic acids condensation processes into tight, highly ordered tertiary structures as well as altering the extent of order and compactness within the resulting species. Specifically, such factors include the ionic strength, the presence of particular dehydrating agents and polyamines, as well as the pH values. It is proposed that slight alterations of these parameters induce the formation of short non-B-DNA segments that propagate as a perturbation along the B-DNA double helix. The structural fluctuations of the dsDNA molecules that result from the conformational discontinuities formed at the junction sites between the B motif and the conformationally altered segments alter the elastic response of the nucleic acids and facilitate cooperative condensation processes. Moreover, the type and frequency of the structurally modified clusters interspersed within the B conformation and determined by the environmental parameters are shown to provide a means for continuous regulation of the extent and mode of DNA packaging. The ionic strength and hydrophobic environment in the close vicinity of the DNA molecules are controlled and modulated in vivo by DNA-binding proteins such as histones and protamines; similarly, pH values and polyamine concentrations are constantly regulated in living systems. It is suggested, therefore, that the secondary structural polymorphism which characterizes the DNA molecules might display a regulatory role by acting as a functional link between cellular parameters and the extent, mode, and timing of nucleic acid packaging processes.
DNA-binding drugs used for chemotherapy originate from a rather large variety of modifications sustained by the nucleic acids upon interaction with the chemical agents. Notably, these modifications are generally considered as involving the following localized chemical or structural processes: base alkylations, frameshift mutations or strand breakages at specific sites, interstrand cross-links, and local structural transitions within the secondary configurations. We find that antitumor agents hinder or prevent altogether the long range packaging of DNA molecules into compact, ordered states. This effect, observed even at low drug to base pair ratios, is general: it is induced by DNA groove binders as well as by intercalators. Nucleoprotein complexes are found to be efficiently protected against the decondensing effect of the drugs. These observations point toward a generic mechanism for the effectiveness of DNA-binding drugs ggainst tumor cells and viruses as well as for the severe effects of chemotherapy on male fertility: actively dividing systems, such as tumor cells, are characterized by regions of chromatin which are decondensed for the purpose of replication and transcription, and therefore accessible to the drugs. Similarly, both viral infection and spermatogenesis, where histones are replaced by protamines, involve transient formation of relatively uncondensed DNA species and subsequent packaging into extremely tight structures
In biological systems nucleic acids are invariably found in highly compact forms. These rather intricate forms raise questions of basic importance which are related to the various factors involved in the condensation processes, the chemical, physical, and structural features revealed by the packed species, and the effects of the extremely tight packaging upon interactions of the DNA molecules with proteins and drugs. A means for addressing these questions on a molecular level is provided by various procedures known to induce in vitro condensation of DNA molecules into highly compact species which, in turn, may serve as a model for the in vivo physical organization of nucleic acids. A study of the optical properties of the tightly packed DNA molecules indicates that the interactions of these species with polypeptides are characterized by distinct, hitherto unobserved, chiral and structural discrimination. Specifically, the polypeptides found to be selected against are composed of those amino acids that are not normally used in protein biosynthesis, such as D-1ySine or ornithine. These findings provide new clues to long debated topics such as the specific universal chirality of amino acids in proteins or the correlation between conformational flexibility of polypeptides and their ability to form stable compact complexes with nucleic acids.