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1.
Nucleic Acids Res ; 52(1): 22-48, 2024 Jan 11.
Artículo en Inglés | MEDLINE | ID: mdl-37994702

RESUMEN

Closing each strand of a DNA duplex upon itself fixes its linking number L. This topological condition couples together the secondary and tertiary structures of the resulting ccDNA topoisomer, a constraint that is not present in otherwise identical nicked or linear DNAs. Fixing L has a range of structural, energetic and functional consequences. Here we consider how L having different integer values (that is, different superhelicities) affects ccDNA molecules. The approaches used are primarily theoretical, and are developed from a historical perspective. In brief, processes that either relax or increase superhelicity, or repartition what is there, may either release or require free energy. The energies involved can be substantial, sufficient to influence many events, directly or indirectly. Here two examples are developed. The changes of unconstrained superhelicity that occur during nucleosome attachment and release are examined. And a simple theoretical model of superhelically driven DNA structural transitions is described that calculates equilibrium distributions for populations of identical topoisomers. This model is used to examine how these distributions change with superhelicity and other factors, and applied to analyze several situations of biological interest.


Asunto(s)
ADN Superhelicoidal , ADN , ADN/química , Conformación de Ácido Nucleico , Nucleosomas , Modelos Químicos
2.
J Biol Chem ; 295(14): 4684-4695, 2020 04 03.
Artículo en Inglés | MEDLINE | ID: mdl-32107311

RESUMEN

R-loop structures are a prevalent class of alternative non-B DNA structures that form during transcription upon invasion of the DNA template by the nascent RNA. R-loops form universally in the genomes of organisms ranging from bacteriophages, bacteria, and yeasts to plants and animals, including mammals. A growing body of work has linked these structures to both physiological and pathological processes, in particular to genome instability. The rising interest in R-loops is placing new emphasis on understanding the fundamental physicochemical forces driving their formation and stability. Pioneering work in Escherichia coli revealed that DNA topology, in particular negative DNA superhelicity, plays a key role in driving R-loops. A clear role for DNA sequence was later uncovered. Here, we review and synthesize available evidence on the roles of DNA sequence and DNA topology in controlling R-loop formation and stability. Factoring in recent developments in R-loop modeling and single-molecule profiling, we propose a coherent model accounting for the interplay between DNA sequence and DNA topology in driving R-loop structure formation. This model reveals R-loops in a new light as powerful and reversible topological stress relievers, an insight that significantly expands the repertoire of R-loops' potential biological roles under both normal and aberrant conditions.


Asunto(s)
ADN Superhelicoidal/química , Estructuras R-Loop/fisiología , Animales , Replicación del ADN , ADN Superhelicoidal/metabolismo , ADN de Forma Z/química , ADN de Forma Z/metabolismo , Escherichia coli/genética , Inestabilidad Genómica , Transcripción Genética
3.
Nucleic Acids Res ; 47(12): 6360-6368, 2019 07 09.
Artículo en Inglés | MEDLINE | ID: mdl-31106378

RESUMEN

DNA unwinding is an important cellular process involved in DNA replication, transcription and repair. In cells, molecular crowding caused by the presence of organelles, proteins, and other molecules affects numerous internal cellular structures. Here, we visualize plasmid DNA unwinding and binding dynamics to an oligonucleotide probe as functions of ionic strength, crowding agent concentration, and crowding agent species using single-molecule CLiC microscopy. We demonstrate increased probe-plasmid interaction over time with increasing concentration of 8 kDa polyethylene glycol (PEG), a crowding agent. We show decreased probe-plasmid interactions as ionic strength is increased without crowding. However, when crowding is introduced via 10% 8 kDa PEG, interactions between plasmids and oligos are enhanced. This is beyond what is expected for normal in vitro conditions, and may be a critically important, but as of yet unknown, factor in DNA's proper biological function in vivo. Our results show that crowding has a strong effect on the initial concentration of unwound plasmids. In the dilute conditions used in these experiments, crowding does not impact probe-plasmid interactions once the site is unwound.


Asunto(s)
ADN Superhelicoidal/química , ADN Superhelicoidal/metabolismo , Sondas de Oligonucleótidos , Concentración Osmolar , Plásmidos/genética , Polietilenglicoles , Imagen Individual de Molécula
4.
Proc Natl Acad Sci U S A ; 116(13): 6260-6269, 2019 03 26.
Artículo en Inglés | MEDLINE | ID: mdl-30850542

RESUMEN

R-loops are abundant three-stranded nucleic-acid structures that form in cis during transcription. Experimental evidence suggests that R-loop formation is affected by DNA sequence and topology. However, the exact manner by which these factors interact to determine R-loop susceptibility is unclear. To investigate this, we developed a statistical mechanical equilibrium model of R-loop formation in superhelical DNA. In this model, the energy involved in forming an R-loop includes four terms-junctional and base-pairing energies and energies associated with superhelicity and with the torsional winding of the displaced DNA single strand around the RNA:DNA hybrid. This model shows that the significant energy barrier imposed by the formation of junctions can be overcome in two ways. First, base-pairing energy can favor RNA:DNA over DNA:DNA duplexes in favorable sequences. Second, R-loops, by absorbing negative superhelicity, partially or fully relax the rest of the DNA domain, thereby returning it to a lower energy state. In vitro transcription assays confirmed that R-loops cause plasmid relaxation and that negative superhelicity is required for R-loops to form, even in a favorable region. Single-molecule R-loop footprinting following in vitro transcription showed a strong agreement between theoretical predictions and experimental mapping of stable R-loop positions and further revealed the impact of DNA topology on the R-loop distribution landscape. Our results clarify the interplay between base sequence and DNA superhelicity in controlling R-loop stability. They also reveal R-loops as powerful and reversible topology sinks that cells may use to nonenzymatically relieve superhelical stress during transcription.


Asunto(s)
Secuencia de Bases , ADN Superhelicoidal/química , ADN/química , Conformación de Ácido Nucleico , ADN de Cadena Simple/química , Modelos Genéticos , Hibridación de Ácido Nucleico , Plásmidos/química , ARN/química , Transcripción Genética
5.
Nucleic Acids Res ; 46(9): 4622-4631, 2018 05 18.
Artículo en Inglés | MEDLINE | ID: mdl-29684182

RESUMEN

We directly visualize the topology-mediated interactions between an unwinding site on a supercoiled DNA plasmid and a specific probe molecule designed to bind to this site, as a function of DNA supercoiling and temperature. The visualization relies on containing the DNA molecules within an enclosed array of glass nanopits using the Convex Lens-induced Confinement (CLiC) imaging method. This method traps molecules within the focal plane while excluding signal from out-of-focus probes. Simultaneously, the molecules can freely diffuse within the nanopits, allowing for accurate measurements of exchange rates, unlike other methods which could introduce an artifactual bias in measurements of binding kinetics. We demonstrate that the plasmid's structure influences the binding of the fluorescent probes to the unwinding site through the presence, or lack, of other secondary structures. With this method, we observe an increase in the binding rate of the fluorescent probe to the unwinding site with increasing temperature and negative supercoiling. This increase in binding is consistent with the results of our numerical simulations of the probability of site-unwinding. The temperature dependence of the binding rate has allowed us to distinguish the effects of competing higher order DNA structures, such as Z-DNA, in modulating local site-unwinding, and therefore binding.


Asunto(s)
ADN Superhelicoidal/química , ADN-Topoisomerasas de Tipo I/metabolismo , Cinética , Microscopía Fluorescente , Sondas de Oligonucleótidos/química , Plásmidos/genética , Temperatura
6.
Cell Syst ; 4(3): 344-356.e7, 2017 03 22.
Artículo en Inglés | MEDLINE | ID: mdl-28237796

RESUMEN

DNA in cells is predominantly B-form double helix. Though certain DNA sequences in vitro may fold into other structures, such as triplex, left-handed Z form, or quadruplex DNA, the stability and prevalence of these structures in vivo are not known. Here, using computational analysis of sequence motifs, RNA polymerase II binding data, and genome-wide potassium permanganate-dependent nuclease footprinting data, we map thousands of putative non-B DNA sites at high resolution in mouse B cells. Computational analysis associates these non-B DNAs with particular structures and indicates that they form at locations compatible with an involvement in gene regulation. Further analyses support the notion that non-B DNA structure formation influences the occupancy and positioning of nucleosomes in chromatin. These results suggest that non-B DNAs contribute to the control of a variety of critical cellular and organismal processes.


Asunto(s)
Huella de ADN/métodos , ADN/química , ADN/ultraestructura , Animales , Cromatina , Biología Computacional/métodos , ADN de Cadena Simple/ultraestructura , Proteínas Fúngicas , G-Cuádruplex , Regulación de la Expresión Génica/fisiología , Genoma , Mamíferos/genética , Compuestos de Manganeso/metabolismo , Ratones , Conformación de Ácido Nucleico , Nucleosomas , Óxidos/metabolismo , Unión Proteica , Endonucleasas Específicas del ADN y ARN con un Solo Filamento
7.
Bioinformatics ; 31(3): 421-2, 2015 Feb 01.
Artículo en Inglés | MEDLINE | ID: mdl-25282644

RESUMEN

SUMMARY: Supercoiling imposes stress on a DNA molecule that can drive susceptible sequences into alternative non-B form structures. This phenomenon occurs frequently in vivo and has been implicated in biological processes, such as replication, transcription, recombination and translocation. SIST is a software package that analyzes sequence-dependent structural transitions in kilobase length superhelical DNA molecules. The numerical algorithms in SIST are based on a statistical mechanical model that calculates the equilibrium probability of transition for each base pair in the domain. They are extensions of the original stress-induced duplex destabilization (SIDD) method, which analyzes stress-driven DNA strand separation. SIST also includes algorithms to analyze B-Z transitions and cruciform extrusion. The SIST pipeline has an option to use the DZCBtrans algorithm, which analyzes the competition among these three transitions within a superhelical domain. AVAILABILITY AND IMPLEMENTATION: The package and additional documentation are freely available at https://bitbucket.org/benhamlab/sist_codes. CONTACT: dzhabinskaya@ucdavis.edu.


Asunto(s)
ADN Superhelicoidal/química , Conformación de Ácido Nucleico , Estrés Oxidativo/genética , Programas Informáticos , Algoritmos , Humanos , Modelos Estadísticos , Relación Estructura-Actividad
8.
Nucleic Acids Res ; 42(20): 12367-79, 2014 Nov 10.
Artículo en Inglés | MEDLINE | ID: mdl-25336616

RESUMEN

While individual non-B DNA structures have been shown to impact gene expression, their broad regulatory role remains elusive. We utilized genomic variants and expression quantitative trait loci (eQTL) data to analyze genome-wide variation propensities of potential non-B DNA regions and their relation to gene expression. Independent of genomic location, these regions were enriched in nucleotide variants. Our results are consistent with previously observed mutagenic properties of these regions and counter a previous study concluding that G-quadruplex regions have a reduced frequency of variants. While such mutagenicity might undermine functionality of these elements, we identified in potential non-B DNA regions a signature of negative selection. Yet, we found a depletion of eQTL-associated variants in potential non-B DNA regions, opposite to what might be expected from their proposed regulatory role. However, we also observed that genes downstream of potential non-B DNA regions showed higher expression variation between individuals. This coupling between mutagenicity and tolerance for expression variability of downstream genes may be a result of evolutionary adaptation, which allows reconciling mutagenicity of non-B DNA structures with their location in functionally important regions and their potential regulatory role.


Asunto(s)
ADN/química , Expresión Génica , Variación Genética , Genoma Humano , Tasa de Mutación , Humanos , Mutagénesis , Conformación de Ácido Nucleico , Nucleótidos/análisis , Sitios de Carácter Cuantitativo
9.
Nucleic Acids Res ; 41(21): 9610-21, 2013 Nov.
Artículo en Inglés | MEDLINE | ID: mdl-23969416

RESUMEN

A DNA molecule under negative superhelical stress becomes susceptible to transitions to alternate structures. The accessible alternate conformations depend on base sequence and compete for occupancy. We have developed a method to calculate equilibrium distributions among the states available to such systems, as well as their average thermodynamic properties. Here we extend this approach to include superhelical cruciform extrusion at both perfect and imperfect inverted repeat (IR) sequences. We find that short IRs do not extrude cruciforms, even in the absence of competition. But as the length of an IR increases, its extrusion can come to dominate both strand separation and B-Z transitions. Although many IRs are present in human genomic DNA, we find that extrusion-susceptible ones occur infrequently. Moreover, their avoidance of transcription start sites in eukaryotes suggests that cruciform formation is rarely involved in mechanisms of gene regulation. We examine a set of clinically important chromosomal translocation breakpoints that occur at long IRs, whose rearrangement has been proposed to be driven by cruciform extrusion. Our results show that the susceptibilities of these IRs to cruciform formation correspond closely with their observed translocation frequencies.


Asunto(s)
ADN Cruciforme/química , ADN Superhelicoidal/química , Algoritmos , Animales , Puntos de Rotura del Cromosoma , Cromosomas Humanos Par 1 , Biología Computacional/métodos , Humanos , Secuencias Invertidas Repetidas , Ratones , Sitio de Iniciación de la Transcripción , Translocación Genética
10.
Nucleic Acids Res ; 41(12): 5965-77, 2013 Jul.
Artículo en Inglés | MEDLINE | ID: mdl-23620297

RESUMEN

Although the right-handed double helical B-form DNA is most common under physiological conditions, DNA is dynamic and can adopt a number of alternative structures, such as the four-stranded G-quadruplex, left-handed Z-DNA, cruciform and others. Active transcription necessitates strand separation and can induce such non-canonical forms at susceptible genomic sequences. Therefore, it has been speculated that these non-B DNA motifs can play regulatory roles in gene transcription. Such conjecture has been supported in higher eukaryotes by direct studies of several individual genes, as well as a number of large-scale analyses. However, the role of non-B DNA structures in many lower organisms, in particular proteobacteria, remains poorly understood and incompletely documented. In this study, we performed the first comprehensive study of the occurrence of B DNA-non-B DNA transition-susceptible sites (non-B DNA motifs) within the context of the operon structure of the Escherichia coli genome. We compared the distributions of non-B DNA motifs in the regulatory regions of operons with those from internal regions. We found an enrichment of some non-B DNA motifs in regulatory regions, and we show that this enrichment cannot be simply explained by base composition bias in these regions. We also showed that the distribution of several non-B DNA motifs within intergenic regions separating divergently oriented operons differs from the distribution found between convergent ones. In particular, we found a strong enrichment of cruciforms in the termination region of operons; this enrichment was observed for operons with Rho-dependent, as well as Rho-independent terminators. Finally, a preference for some non-B DNA motifs was observed near transcription factor-binding sites. Overall, the conspicuous enrichment of transition-susceptible sites in these specific regulatory regions suggests that non-B DNA structures may have roles in the transcriptional regulation of specific operons within the E. coli genome.


Asunto(s)
ADN Bacteriano/química , Escherichia coli/genética , Operón , Elementos Reguladores de la Transcripción , Transcripción Genética , Sitios de Unión , ADN Bacteriano/metabolismo , ADN Cruciforme/análisis , ADN Intergénico/química , Genoma Bacteriano , Motivos de Nucleótidos , Regiones Promotoras Genéticas , Factores de Transcripción/metabolismo , Terminación de la Transcripción Genética
11.
PLoS Comput Biol ; 8(4): e1002484, 2012.
Artículo en Inglés | MEDLINE | ID: mdl-22570598

RESUMEN

We develop a statistical mechanical model to analyze the competitive behavior of transitions to multiple alternate conformations in a negatively supercoiled DNA molecule of kilobase length and specified base sequence. Since DNA superhelicity topologically couples together the transition behaviors of all base pairs, a unified model is required to analyze all the transitions to which the DNA sequence is susceptible. Here we present a first model of this type. Our numerical approach generalizes the strategy of previously developed algorithms, which studied superhelical transitions to a single alternate conformation. We apply our multi-state model to study the competition between strand separation and B-Z transitions in superhelical DNA. We show this competition to be highly sensitive to temperature and to the imposed level of supercoiling. Comparison of our results with experimental data shows that, when the energetics appropriate to the experimental conditions are used, the competition between these two transitions is accurately captured by our algorithm. We analyze the superhelical competition between B-Z transitions and denaturation around the c-myc oncogene, where both transitions are known to occur when this gene is transcribing. We apply our model to explore the correlation between stress-induced transitions and transcriptional activity in various organisms. In higher eukaryotes we find a strong enhancement of Z-forming regions immediately 5' to their transcription start sites (TSS), and a depletion of strand separating sites in a broad region around the TSS. The opposite patterns occur around transcript end locations. We also show that susceptibility to each type of transition is different in eukaryotes and prokaryotes. By analyzing a set of untranscribed pseudogenes we show that the Z-susceptibility just downstream of the TSS is not preserved, suggesting it may be under selection pressure.


Asunto(s)
ADN Superhelicoidal/química , ADN Superhelicoidal/ultraestructura , Modelos Químicos , Modelos Moleculares , Secuencia de Bases , Simulación por Computador , Datos de Secuencia Molecular , Conformación de Ácido Nucleico
12.
PLoS One ; 6(6): e20798, 2011.
Artículo en Inglés | MEDLINE | ID: mdl-21695263

RESUMEN

The susceptibility to recombination of a plasmid inserted into a chromosome varies with its genomic position. This recombination position effect is known to correlate with the average G+C content of the flanking sequences. Here we propose that this effect could be mediated by changes in the susceptibility to superhelical duplex destabilization that would occur. We use standard nonparametric statistical tests, regression analysis and principal component analysis to identify statistically significant differences in the destabilization profiles calculated for the plasmid in different contexts, and correlate the results with their measured recombination rates. We show that the flanking sequences significantly affect the free energy of denaturation at specific sites interior to the plasmid. These changes correlate well with experimentally measured variations of the recombination rates within the plasmid. This correlation of recombination rate with superhelical destabilization properties of the inserted plasmid DNA is stronger than that with average G+C content of the flanking sequences. This model suggests a possible mechanism by which flanking sequence base composition, which is not itself a context-dependent attribute, can affect recombination rates at positions within the plasmid.


Asunto(s)
Efectos de la Posición Cromosómica/genética , ADN Superhelicoidal/genética , Ácidos Nucleicos Heterodúplex/genética , Recombinación Genética/genética , Saccharomyces cerevisiae/genética , Composición de Base/genética , Análisis por Conglomerados , ADN de Hongos/genética , Genoma Fúngico/genética , Mutagénesis Insercional/genética , Plásmidos/genética , Análisis de Componente Principal
13.
Phys Biol ; 8(3): 035011, 2011 Jun.
Artículo en Inglés | MEDLINE | ID: mdl-21572180

RESUMEN

A vast literature has explored the genetic interactions among the cellular components regulating gene expression in many organisms. Early on, in the absence of any biochemical definition, regulatory modules were conceived using the strict formalism of genetics to designate the modifiers of phenotype as either cis- or trans-acting depending on whether the relevant genes were embedded in the same or separate DNA molecules. This formalism distilled gene regulation down to its essence in much the same way that consideration of an ideal gas reveals essential thermodynamic and kinetic principles. Yet just as the anomalous behavior of materials may thwart an engineer who ignores their non-ideal properties, schemes to control and manipulate the genetic and epigenetic programs of cells may falter without a fuller and more quantitative elucidation of the physical and chemical characteristics of DNA and chromatin in vivo.


Asunto(s)
Biología Computacional , ADN/química , Cromatina/química , Cromatina/genética , Simulación por Computador , ADN/genética , Regulación de la Expresión Génica , Humanos , Fenotipo , Termodinámica
14.
PLoS Comput Biol ; 7(1): e1001051, 2011 Jan 20.
Artículo en Inglés | MEDLINE | ID: mdl-21283778

RESUMEN

We present a method to calculate the propensities of regions within a DNA molecule to transition from B-form to Z-form under negative superhelical stresses. We use statistical mechanics to analyze the competition that occurs among all susceptible Z-forming regions at thermodynamic equilibrium in a superhelically stressed DNA of specified sequence. This method, which we call SIBZ, is similar to the SIDD algorithm that was previously developed to analyze superhelical duplex destabilization. A state of the system is determined by assigning to each base pair either the B- or the Z-conformation, accounting for the dinucleotide repeat unit of Z-DNA. The free energy of a state is comprised of the nucleation energy, the sequence-dependent B-Z transition energy, and the energy associated with the residual superhelicity remaining after the change of twist due to transition. Using this information, SIBZ calculates the equilibrium B-Z transition probability of each base pair in the sequence. This can be done at any physiologically reasonable level of negative superhelicity. We use SIBZ to analyze a variety of representative genomic DNA sequences. We show that the dominant Z-DNA forming regions in a sequence can compete in highly complex ways as the superhelicity level changes. Despite having no tunable parameters, the predictions of SIBZ agree precisely with experimental results, both for the onset of transition in plasmids containing introduced Z-forming sequences and for the locations of Z-forming regions in genomic sequences. We calculate the transition profiles of 5 kb regions taken from each of 12,841 mouse genes and centered on the transcription start site (TSS). We find a substantial increase in the frequency of Z-forming regions immediately upstream from the TSS. The approach developed here has the potential to illuminate the occurrence of Z-form regions in vivo, and the possible roles this transition may play in biological processes.


Asunto(s)
ADN Superhelicoidal/química , Modelos Teóricos , Algoritmos , Animales , Ratones , Termodinámica
15.
Curr Genet ; 56(4): 321-40, 2010 Aug.
Artículo en Inglés | MEDLINE | ID: mdl-20446088

RESUMEN

Although a variety of possible functions have been proposed for inverted repeat sequences (IRs), it is not known which of them might occur in vivo. We investigate this question by assessing the distributions and properties of IRs in the Saccharomyces cerevisiae (SC) genome. Using the IRFinder algorithm we detect 100,514 IRs having copy length greater than 6 bp and spacer length less than 77 bp. To assess statistical significance we also determine the IR distributions in two types of randomization of the S. cerevisiae genome. We find that the S. cerevisiae genome is significantly enriched in IRs relative to random. The S. cerevisiae IRs are significantly longer and contain fewer imperfections than those from the randomized genomes, suggesting that processes to lengthen and/or correct errors in IRs may be operative in vivo. The S. cerevisiae IRs are highly clustered in intergenic regions, while their occurrence in coding sequences is consistent with random. Clustering is stronger in the 3' flanks of genes than in their 5' flanks. However, the S. cerevisiae genome is not enriched in those IRs that would extrude cruciforms, suggesting that this is not a common event. Various explanations for these results are considered.


Asunto(s)
Genoma Fúngico/genética , Secuencias Invertidas Repetidas , Saccharomyces cerevisiae/genética , Región de Flanqueo 3' , Región de Flanqueo 5' , Algoritmos , Secuencia de Bases
16.
J Phys Chem A ; 113(16): 4213-6, 2009 Apr 23.
Artículo en Inglés | MEDLINE | ID: mdl-19371114

RESUMEN

The persistence length of DNA has been studied for decades; however, experimentally obtained values of this quantity have not been entirely consistent. We report results from Brownian dynamics simulations that address this issue, validating and demonstrating the utility of an explicitly double-stranded model for mesoscale DNA dynamics. We find that persistence lengths calculated from rotational relaxation increase with decreasing ionic strength, corroborating experimental evidence, but contradicting results obtained from wormlike coil assumptions. Further, we find that natural curvature does not significantly affect the persistence length, corroborating cyclization efficiency measurements, but contradicting results from cryo-EM.


Asunto(s)
ADN/química , Modelos Químicos , ADN/metabolismo , Reproducibilidad de los Resultados , Sales (Química)/química
17.
Stand Genomic Sci ; 1(2): 204-15, 2009 Sep 25.
Artículo en Inglés | MEDLINE | ID: mdl-21304658

RESUMEN

We present an interactive web application for visualizing genomic data of prokaryotic chromosomes. The tool (GeneWiz browser) allows users to carry out various analyses such as mapping alignments of homologous genes to other genomes, mapping of short sequencing reads to a reference chromosome, and calculating DNA properties such as curvature or stacking energy along the chromosome. The GeneWiz browser produces an interactive graphic that enables zooming from a global scale down to single nucleotides, without changing the size of the plot. Its ability to disproportionally zoom provides optimal readability and increased functionality compared to other browsers. The tool allows the user to select the display of various genomic features, color setting and data ranges. Custom numerical data can be added to the plot allowing, for example, visualization of gene expression and regulation data. Further, standard atlases are pre-generated for all prokaryotic genomes available in GenBank, providing a fast overview of all available genomes, including recently deposited genome sequences. The tool is available online from http://www.cbs.dtu.dk/services/gwBrowser. Supplemental material including interactive atlases is available online at http://www.cbs.dtu.dk/services/gwBrowser/suppl/.

18.
Phys Rev E Stat Nonlin Soft Matter Phys ; 77(3 Pt 1): 031924, 2008 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-18517439

RESUMEN

Numerical models of mesoscale DNA dynamics relevant to in vivo scenarios require methods that incorporate important features of the intracellular environment, while maintaining computational tractability. Because the explicit inclusion of ions leads to electrostatic calculations that scale as the square of the number of charged particles, such models typically handle these calculations using low-potential, mean-field approaches, rather than by considering the discrete interactions of ions. This allows approximation of the long-range, screened self-repulsion of DNA, but is unable to capture detailed electrostatic phenomena, such as short-range attractions mediated by ion-ion correlations. Here, we develop a dynamical model of explicitly double-stranded, sequence-specific DNA in a bulk environment consisting of other polyions and explicitly represented counterions and coions. DNA is represented as two interwound chains of charged Stokes spheres, and ions as free, monovalently charged Stokes spheres. Brownian dynamics simulations performed at salt concentrations of 0.1, 1, 10, and 100 mM demonstrate this model captures anticipated behaviors of the system, including increasing compaction of the polyion by the ionic atmosphere with increasing ionic strength. The decay of the distance dependence of the ion concentrations as one moves away from the polyion approaches their equilibrium values in quantitative agreement with predictions of Poisson-Boltzmann theory. The simulation results also demonstrate quantitative agreement with experimental measurements of the persistence length of B-DNA, which increases significantly at low ionic strengths. The model also captures behaviors intimating the importance of explicitly representing ionic and polyionic structure. These include penetration of the polyion interior by both coions and counterions, and counterion-mediated accumulation of coions near the surface of the polyion. Such phenomena are likely to play an important role in the formation of alternative DNA secondary structures, suggesting the present methods will prove valuable to dynamic models of superhelical stress-induced DNA structural transitions.


Asunto(s)
Biofisica/métodos , ADN/química , Sales (Química)/química , Simulación por Computador , Iones , Modelos Biológicos , Modelos Estadísticos , Modelos Teóricos , Conformación Molecular , Distribución Normal , Conformación de Ácido Nucleico , Distribución de Poisson , Polidesoxirribonucleótidos/química , Electricidad Estática
19.
PLoS Comput Biol ; 4(1): e17, 2008 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-18208321

RESUMEN

Stress-induced DNA duplex destabilization (SIDD) analysis exploits the known structural and energetic properties of DNA to predict sites that are susceptible to strand separation under negative superhelical stress. When this approach was used to calculate the SIDD profile of the entire Escherichia coli K12 genome, it was found that strongly destabilized sites occur preferentially in intergenic regions that are either known or inferred to contain promoters, but rarely occur in coding regions. Here, we investigate whether the genes grouped in different functional categories have characteristic SIDD properties in their upstream flanks. We report that strong SIDD sites in the E. coli K12 genome are statistically significantly overrepresented in the upstream regions of genes encoding transcriptional regulators. In particular, the upstream regions of genes that directly respond to physiological and environmental stimuli are more destabilized than are those regions of genes that are not involved in these responses. Moreover, if a pathway is controlled by a transcriptional regulator whose gene has a destabilized 5' flank, then the genes (operons) in that pathway also usually contain strongly destabilized SIDD sites in their 5' flanks. We observe this statistically significant association of SIDD sites with upstream regions of genes functioning in transcription in 38 of 43 genomes of free-living bacteria, but in only four of 18 genomes of endosymbionts or obligate parasitic bacteria. These results suggest that strong SIDD sites 5' to participating genes may be involved in transcriptional responses to environmental changes, which are known to transiently alter superhelicity. We propose that these SIDD sites are active and necessary participants in superhelically mediated regulatory mechanisms governing changes in the global pattern of gene expression in prokaryotes in response to physiological or environmental changes.


Asunto(s)
Daño del ADN , ADN Bacteriano/química , ADN Bacteriano/genética , Proteínas de Unión al ADN/química , Proteínas de Unión al ADN/genética , Modelos Químicos , Modelos Genéticos , Secuencias Reguladoras de Ácidos Nucleicos/genética , Factores de Transcripción/química , Factores de Transcripción/genética , Simulación por Computador , Factores de Transcripción del Choque Térmico , Modelos Moleculares , Conformación de Ácido Nucleico
20.
J Phys Chem B ; 112(51): 16788-92, 2008 Dec 25.
Artículo en Inglés | MEDLINE | ID: mdl-19368012

RESUMEN

We report results from scanning tunneling microscopy experiments in which supercoiling of the plasmid, pTNT, leads to localized duplex denaturation at a single location. Measurements of the imaged strand-separated region suggest an extent of approximately 60 base pairs (bp's), in excellent agreement with the prediction of a statistical mechanical analysis of the pTNT base sequence that accounts for the topology-mediated global coupling of melting behaviors. Notwithstanding that the sequence of pTNT includes a 30-bp run of poly-A, where thermodynamic considerations alone predict significant instability, based on the statistical result, we propose the location of denaturation is an approximately 60-bp run, spanning positions 2050-2110, that is 82% AT-rich, terminally flanks the beta-lactamase coding region, and is known to contain two sites susceptible to cleavage by the DraI restriction enzyme.


Asunto(s)
ADN/química , Conformación de Ácido Nucleico , Plásmidos , Microscopía Electrónica de Transmisión de Rastreo , Termodinámica
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