RESUMEN
DNA supercoiling plays an important role in a variety of cellular processes, including transcription, replication, and DNA compaction. To fully understand these processes, we must uncover and characterize the dynamics of supercoiled DNA. However, supercoil dynamics are difficult to access because of the wide range of relevant length and timescales. In this work, we present an algorithm to reconstruct the arrangement of identical fluorescent particles distributed around a circular DNA molecule, given their three-dimensional trajectories through time. We find that this curve reconstruction problem is analogous to solving the traveling salesman problem. We demonstrate that our approach converges to the correct arrangement with a sufficiently long observation time. In addition, we show that the time required to accurately reconstruct the fluorophore arrangement is reduced by increasing the fluorophore density or reducing the level of supercoiling. This curve reconstruction algorithm, when paired with next-generation super-resolution imaging systems, could be used to access and thereby advance our understanding of supercoil dynamics.
Asunto(s)
ADN Superhelicoidal , ADN , AlgoritmosRESUMEN
Several recent single-molecule experiments observe the response of supercoiled DNA to nicking endonucleases and topoisomerases. Typically in these experiments, indirect measurements of supercoil relaxation are obtained by observing the motion of a large micron-sized bead. The bead, which also serves to manipulate DNA, experiences significant drag and thereby obscures supercoil dynamics. Here we employ our discrete wormlike chain model to bypass experimental limitations and simulate the dynamic response of supercoiled DNA to a single strand nick. From our simulations, we make three major observations. First, extension is a poor dynamic measure of supercoil relaxation; in fact, the linking number relaxes so fast that it cannot have much impact on extension. Second, the rate of linking number relaxation depends upon its initial partitioning into twist and writhe as determined by tension. Third, the extensional response strongly depends upon the initial position of plectonemes.
Asunto(s)
Simulación por Computador , ADN Superhelicoidal , Modelos Genéticos , Algoritmos , Elasticidad , Hidrodinámica , Modelos Lineales , Temperatura , Factores de Tiempo , ViscosidadRESUMEN
DNA looping mediated by the Lac repressor is an archetypal test case for modeling protein and DNA flexibility. Understanding looping is fundamental to quantitative descriptions of gene expression. Systematic analysis of LacIâ¢DNA looping was carried out using a landscape of DNA constructs with lac operators bracketing an A-tract bend, produced by varying helical phasings between operators and the bend. Fluorophores positioned on either side of both operators allowed direct Förster resonance energy transfer (FRET) detection of parallel (P1) and antiparallel (A1, A2) DNA looping topologies anchored by V-shaped LacI. Combining fluorophore position variant landscapes allows calculation of the P1, A1 and A2 populations from FRET efficiencies and also reveals extended low-FRET loops proposed to form via LacI opening. The addition of isopropyl-ß-D-thio-galactoside (IPTG) destabilizes but does not eliminate the loops, and IPTG does not redistribute loops among high-FRET topologies. In some cases, subsequent addition of excess LacI does not reduce FRET further, suggesting that IPTG stabilizes extended or other low-FRET loops. The data align well with rod mechanics models for the energetics of DNA looping topologies. At the peaks of the predicted energy landscape for V-shaped loops, the proposed extended loops are more stable and are observed instead, showing that future models must consider protein flexibility.
Asunto(s)
ADN/química , Represoras Lac/metabolismo , Regiones Operadoras Genéticas , ADN/metabolismo , Transferencia Resonante de Energía de Fluorescencia , Colorantes Fluorescentes , Isopropil Tiogalactósido/metabolismo , Represoras Lac/química , Conformación de Ácido NucleicoRESUMEN
In the bacteriophage Ï29, DNA is packed into a preassembled capsid from which it ejects under high pressure. A recent cryo-EM reconstruction of Ï29 revealed a compact toroidal DNA structure (30-40 basepairs) lodged within the exit cavity formed by the connector-lower collar protein complex. Using multiscale models, we compute a detailed structural ensemble of intriguing DNA toroids of various lengths, all highly compatible with experimental observations. In particular, coarse-grained (elastic rod) and atomistic (molecular dynamics) models predict the formation of DNA toroids under significant compression, a largely unexplored state of DNA. Model predictions confirm that a biologically attainable compressive force of 25 pN sustains the toroid and yields DNA electron density maps highly consistent with the experimental reconstruction. The subsequent simulation of dynamic toroid ejection reveals large reactions on the connector that may signal genome release.
Asunto(s)
Fagos de Bacillus/química , ADN Viral/química , Simulación de Dinámica Molecular , Secuencia de Bases , Datos de Secuencia Molecular , Conformación de Ácido NucleicoRESUMEN
In cells, DNA is routinely subjected to significant levels of bending and twisting. In some cases, such as under physiological levels of supercoiling, DNA can be so highly strained, that it transitions into non-canonical structural conformations that are capable of relieving mechanical stress within the template. DNA minicircles offer a robust model system to study stress-induced DNA structures. Using DNA minicircles on the order of 100 bp in size, we have been able to control the bending and torsional stresses within a looped DNA construct. Through a combination of cryo-EM image reconstructions, Bal31 sensitivity assays and Brownian dynamics simulations, we have been able to analyze the effects of biologically relevant underwinding-induced kinks in DNA on the overall shape of DNA minicircles. Our results indicate that strongly underwound DNA minicircles, which mimic the physical behavior of small regulatory DNA loops, minimize their free energy by undergoing sequential, cooperative kinking at two sites that are located about 180° apart along the periphery of the minicircle. This novel form of structural cooperativity in DNA demonstrates that bending strain can localize hyperflexible kinks within the DNA template, which in turn reduces the energetic cost to tightly loop DNA.
Asunto(s)
ADN Circular/química , Microscopía por Crioelectrón , ADN Circular/ultraestructura , Endodesoxirribonucleasas , Modelos Moleculares , Conformación de Ácido Nucleico , Estrés MecánicoRESUMEN
In this study, we report what we believe to be the first multiscale simulation of the dynamic relaxation of DNA supercoils by human topoisomerase IB (topo IB). We leverage our previous molecular dynamics calculations of the free energy landscape describing the interaction between a short DNA fragment and topo IB. Herein, this landscape is used to prescribe boundary conditions for a computational, elastodynamic continuum rod model of a long length of supercoiled DNA. The rod model, which accounts for the nonlinear bending, twisting, and electrostatic interaction of the (negatively charged) DNA backbone, is extended to include the hydrodynamic drag induced by the surrounding physiological buffer. Simulations for a 200-bp-long DNA supercoil in complex with topo IB reveal a relaxation timescale of â¼0.1-1.0 µs. The relaxation follows a sequence of cascading reductions in the supercoil linking number (Lk), twist (Tw), and writhe (Wr) that follow companion cascading reductions in the supercoil elastic and electrostatic energies. The novel (to our knowledge) multiscale modeling method may enable simulations of the entire experimental setup that measures DNA supercoiling and relaxation via single molecule magnetic trapping.
Asunto(s)
ADN-Topoisomerasas de Tipo I/metabolismo , ADN Superhelicoidal/química , ADN Superhelicoidal/metabolismo , Simulación de Dinámica Molecular , Conformación de Ácido Nucleico , Biocatálisis , Humanos , TorqueRESUMEN
Protein-mediated DNA looping, such as that induced by the lactose repressor (LacI) of Escherichia coli, is a well-known gene regulation mechanism. Although researchers have given considerable attention to DNA looping by LacI, many unanswered questions about this mechanism, including the role of protein flexibility, remain. Recent single-molecule observations suggest that the two DNA-binding domains of LacI are capable of splaying open about the tetramerization domain into an extended conformation. We hypothesized that if recent experiments were able to reveal the extended conformation, it is possible that such structures occurred in previous studies as well. In this study, we tested our hypothesis by reevaluating two classic in vitro binding assays using a computational rod model of DNA. The experiments and computations evaluate the looping of both linear DNA and supercoiled DNA minicircles over a broad range of DNA interoperator lengths. The computed energetic minima align well with the experimentally observed interoperator length for optimal loop stability. Of equal importance, the model reveals that the most stable loops for linear DNA occur when LacI adopts the extended conformation. In contrast, for DNA minicircles, optimal stability may arise from either the closed or the extended protein conformation depending on the degree of supercoiling and the interoperator length.
Asunto(s)
ADN/química , ADN/metabolismo , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/metabolismo , Represoras Lac/química , Represoras Lac/metabolismo , Simulación de Dinámica Molecular , Conformación de Ácido Nucleico , Unión Proteica , Conformación ProteicaRESUMEN
Sequence-dependent intrinsic curvature of DNA influences looping by regulatory proteins such as LacI and NtrC. Curvature can enhance stability and control shape, as observed in LacI loops formed with three designed sequences with operators bracketing an A-tract bend. We explore geometric, topological, and energetic effects of curvature with an analysis of a family of highly bent sequences, using the elastic rod model from previous work. A unifying straight-helical-straight representation uses two phasing parameters to describe sequences composed of two straight segments that flank a common helically supercoiled segment. We exercise the rod model over this two-dimensional space of phasing parameters to evaluate looping behaviors. This design space is found to comprise two subspaces that prefer parallel versus anti-parallel binding topologies. The energetic cost of looping varies from 4 to 12 kT. Molecules can be designed to yield distinct binding topologies as well as hyperstable or hypostable loops and potentially loops that can switch conformations. Loop switching could be a mechanism for control of gene expression. Model predictions for linking numbers and sizes of LacI-DNA loops can be tested using multiple experimental approaches, which coupled with theory could address whether proteins or DNA provide the observed flexibility of protein-DNA loops.
Asunto(s)
Proteínas Bacterianas/metabolismo , Simulación por Computador , ADN/química , Modelos Moleculares , Conformación de Ácido Nucleico , Proteínas Represoras/metabolismo , Proteínas Bacterianas/farmacología , ADN/metabolismo , Elasticidad , Represoras Lac , Conformación de Ácido Nucleico/efectos de los fármacos , Proteínas Represoras/farmacología , Reproducibilidad de los Resultados , Rotación , TermodinámicaRESUMEN
Protein-mediated DNA looping is a common mechanism for regulating gene expression. Loops occur when a protein binds to two operators on the same DNA molecule. The probability of looping is controlled, in part, by the basepair sequence of inter-operator DNA, which influences its structural properties. One structural property is the intrinsic or stress-free curvature. In this article, we explore the influence of sequence-dependent intrinsic curvature by exercising a computational rod model for the inter-operator DNA as applied to looping of the LacR-DNA complex. Starting with known sequences for the inter-operator DNA, we first compute the intrinsic curvature of the helical axis as input to the rod model. The crystal structure of the LacR (with bound operators) then defines the requisite boundary conditions needed for the dynamic rod model that predicts the energetics and topology of the intervening DNA loop. A major contribution of this model is its ability to predict a broad range of published experimental data for highly bent (designed) sequences. The model successfully predicts the loop topologies known from fluorescence resonance energy transfer measurements, the linking number distribution known from cyclization assays with the LacR-DNA complex, the relative loop stability known from competition assays, and the relative loop size known from gel mobility assays. In addition, the computations reveal that highly curved sequences tend to lower the energetic cost of loop formation, widen the energy distribution among stable and meta-stable looped states, and substantially alter loop topology. The inclusion of sequence-dependent intrinsic curvature also leads to nonuniform twist and necessitates consideration of eight distinct binding topologies from the known crystal structure of the LacR-DNA complex.