Modeling DNA Thermodynamics under Torsional Stress

Negatively twisted DNA is essential to many biological functions. Due to torsional stress, duplex DNA can have local, sequence-dependent structural defects. In this work, a thermodynamic model of DNA was built to qualitatively predict the local sequence-dependent mechanical instabilities under torsional stress. The results were compared to both simulation of a coarse-grained model and experiment results. By using the Kirkwood superposition approximation, we built an analytical model to represent the free energy difference ΔW of a hydrogen-bonded basepair between the B-form helical state and the basepair opened (or locally melted) state, within a given sequence under torsional stress. We showed that ΔW can be well approximated by two-body interactions with its nearest-sequence-neighbor basepairs plus a free energy correction due to long-range correlations. This model is capable of rapidly predicting the position and thermodynamics of local defects in a given sequence. The result qualitatively matches with an in vitro experiment for a long DNA sequence (>4000 basepairs). The 12 parameters used in this model can be further quantitatively refined when more experimental data are available.     

Temperature Dependence of the DNA Double Helix at the Nanoscale: Structure,Elasticity, and Fluctuations

Biological organisms exist over a broad temperature range of −15°C to +120°C, where many molecular processes involving DNA depend on the nanoscale properties of the double helix. Here, we present results of extensive molecular dynamics simulations of DNA oligomers at different temperatures. We show that internal basepair conformations are strongly temperature-dependent, particularly in the stretch and opening degrees of freedom whose harmonic fluctuations can be considered the initial steps of the DNA melting pathway. The basepair step elasticity contains a weaker, but detectable, entropic contribution in the roll, tilt, and rise degrees of freedom. To extend the validity of our results to the temperature interval beyond the standard melting transition relevant to extremophiles, we estimate the effects of superhelical stress on the stability of the basepair steps, as computed from the Benham model. We predict that although the average twist decreases with temperature in vitro, the stabilizing external torque in vivo results in an increase of ∼1°/bp (or a superhelical density of Δσ ?  + 0.03) in the interval 0–100°C. In the final step, we show that the experimentally observed apparent bending persistence length of torsionally unconstrained DNA can be calculated from a hybrid model that accounts for the softening of the double helix and the presence of transient denaturation bubbles. Although the latter dominate the behavior close to the melting transition, the inclusion of helix softening is important around standard physiological temperatures.     

Calibration of Optical Tweezers for In?Vivo Force Measurements: How do Different Approaches Compare?

There is significant interest in quantifying force production inside cells, but since conditions in vivo are less well controlled than those in vitro, in vivo measurements are challenging. In particular, the in vivo environment may vary locally as far as its optical properties, and the organelles manipulated by the optical trap frequently vary in size and shape. Several methods have been proposed to overcome these difficulties. We evaluate the relative merits of these methods and directly compare two of them, a refractive index matching method, and a light-momentum-change method. Since in vivo forces are frequently relatively high (e.g., can exceed 15 pN for lipid droplets), a high-power laser is employed. We discover that this high-powered trap induces local temperature changes, and we develop an approach to compensate for uncertainties in the magnitude of applied force due to such temperature variations.     

Applying the stochastic difference equation to DNA conformational transitions: a study of B-Z and B-A DNA transitions

Despite the existence of numerous models to account for the B-Z DNA transition, experimenters have not yet arrived at a conclusive answer to the structural and dynamical features of the B-Z transition. By applying the stochastic difference equation to simulate the B-Z DNA transition, we have shown that the stretched intermediate model of the B-Z transition is more probable than other B-Z transition models such as the Harvey model. This is accomplished by comparing potential energy profiles of various B-Z DNA transition models and calculating relative probabilities based on the stochastic difference equation with respect to length (SDEL) formalism. The results garnered in this article allow for new approaches in determining the structural transition of B-DNA to Z-DNA experimentally. We have also simulated the B-A DNA transition using the stochastic difference equation. Unlike the B-Z DNA transition, the mechanism for the B-A DNA transition is well established. The variation in the pseudorotation angle during the transition is in good agreement with experimental results. Qualitative features of the simulated B-A transition also agree well with experimental data. The SDEL approach is thus a suitable numerical technique to compute long-time molecular dynamics trajectory for DNA molecules.     

High‐efficiency delivery of CRISPR‐Cas9 by engineered probiotics enables precise microbiome editing

Antibiotic resistance threatens our ability to treat infectious diseases, spurring interest in alternative antimicrobial technologies. The use of bacterial conjugation to deliver CRISPR‐cas systems programmed to precisely eliminate antibiotic‐resistant bacteria represents a promising approach but requires high in situ DNA transfer rates. We have optimized the transfer efficiency of conjugative plasmid TP114 using accelerated laboratory evolution. We hence generated a potent conjugative delivery vehicle for CRISPR‐cas9 that can eliminate > 99.9% of targeted antibiotic‐resistant Escherichia coli in the mouse gut microbiota using a single dose. We then applied this system to a Citrobacter rodentium infection model, achieving full clearance within four consecutive days of treatment.     

A Model of the Strain-induced B-Z Transition

Abstract

The B-to-Z transition in supercoiled circular DNA is modeled as a strain-induced nonlinear excitation process. Using a model, in which DNA is regarded as a chain of units with a bistable energy function along the twisting coordinate together with a harmonic inter-unit interaction, we show that a Z region and the accompanying two B-Z junctions of finite width appear naturally as a solution of nonlinear equations, when the strain exceeds a critical value. We examine the B-Z transition behaviour as a function of twist under various situations. We also analyse available experimental results on B-Z transition in supercoiled plasmid with G-C insertions by this mechanistic model in order to estimate the magnitude of model parameters. The energy barrier of the B-Z transition is estimated to be of the order of 1 kcal/mole per base pair. The analysis shows that if the length of the insertion is less than a certain value, the entire insertion converts to Z form at a transition point, but if the insertion is much longer, the B-Z transition exhibits a different behavior, in which part of the insertion flips to Z form and the Z region expands linearly upon changing linking number.     

Torsion Profiling of Proteins Using Magnetic Particles

We report a method to profile the torsional spring properties of proteins as a function of the angle of rotation. The torque is applied by superparamagnetic particles and has been calibrated while taking account of the magnetization dynamics of the particles. We record and compare the torsional profiles of single Protein G-Immunoglobulin G (IgG) and IgG-IgG complexes, sandwiched between a substrate and a superparamagnetic particle, for torques in the range between 0.5 × 10³ and 5 × 10³ pN·nm. Both molecular systems show torsional stiffening for increasing rotation angle, but the elastic and inelastic torsion stiffnesses are remarkably different. We interpret the results in terms of the structural properties of the molecules. The torsion profiling technique opens new dimensions for research on biomolecular characterization and for research on bio-nanomechanical structure-function relationships.     

A model of the strain-induced B-Z transition

The B-to-Z transition in supercoiled circular DNA is modeled as a strain-induced nonlinear excitation process. Using a model, in which DNA is regarded as a chain of units with a bistable energy function along the twisting coordinate together with a harmonic inter-unit interaction, we show that a Z region and the accompanying two B-Z junctions of finite width appear naturally as a solution of nonlinear equations, when the strain exceeds a critical value. We examine the B-Z transition behaviour as a function of twist under various situations. We also analyse available experimental results on B-Z transition in supercoiled plasmid with G-C insertions by this mechanistic model in order to estimate the magnitude of model parameters. The energy barrier of the B-Z transition is estimated to be of the order of 1 kcal/mole per base pair. The analysis shows that if the length of the insertion is less than a certain value, the entire insertion converts to Z form at a transition point, but if the insertion is much longer, the B-Z transition exhibits a different behavior, in which part of the insertion flips to Z form and the Z region expands linearly upon changing linking number.     

Energetics of B-Z junction formation in a sixteen base-pair duplex DNA

We report analysis of the NaCl-induced B-Z transition in a 16 base-pair duplex DNA with sequence designed such that when NaCl is increased the left half of the molecule undergoes the B-Z transition while the right half remains in the B-form. An equilibrium thermodynamic model based on the body of available published experimental data and the recent theoretical work of Soumpasis, which indicate, in the salt range above approximately 0.9 M-NaCl, the transition free-energy of B-Z conversion in DNA is a linear function of the NaCl concentration, is employed. Analysis of the B-Z transition of the junction-containing molecule indicates the B-Z junction formed in this 16 base-pair DNA is composed of approximately three base-pairs and has a free energy of formation of approximately 4.7 kcal/mol junction. These values for the junction are in excellent agreement with published estimates of B-Z junction size and energy derived from much longer DNA pieces.     

The stretched intermediate model of B-Z DNA transition

There have been numerous attempts to describe the mechanism of B-Z transition. Our simulations based on the stochastic difference equation with length algorithm show that a short DNA oligomer will tend to unwind and overstretch during the transition. The overstretching of DNA is then understood from the Zhou, Zhang, and Ou-Yang model. Unlike the Harvey model, the stretched intermediate model does not pose any steric dilemma; we are able to show that the chain sense reversal progresses spontaneously using the stretched intermediate model. A nonlinear DNA model is used to describe the origins and mechanism of base rotation in the stretched intermediate state of DNA. We also propose an experiment that can verify the existence of a stretched intermediate state during B-Z transition, thus opening up fresh grounds for experimentation in this field.     

Bone morphogenetic protein 6–mediated crosstalk between endothelial cells and hepatocytes recapitulates the iron-sensing pathway in vitro

Liver sinusoidal endothelial cell–derived bone morphogenetic protein 6 (BMP6) and the BMP6–small mothers against decapentaplegic homolog (SMAD) signaling pathway are essential for the expression of hepcidin, the secretion of which is considered the systemic master switch of iron homeostasis. However, there are continued controversies related to the strong and direct suppressive effect of iron on hepatocellular hepcidin in vitro in contrast to in vivo conditions. Here, we directly studied the crosstalk between endothelial cells (ECs) and hepatocytes using in vitro coculture models that mimic hepcidin signaling in vivo. Huh7 cells were directly cocultured with ECs, and EC conditioned media (CM) were also used to culture Huh7 cells and primary mouse hepatocytes. To explore the reactions of ECs to surrounding iron, they were grown in the presence of ferric ammonium citrate and heme, two iron-containing molecules. We found that both direct coculture with ECs and EC-CM significantly increased hepcidin expression in Huh7 cells. The upstream SMAD pathway, including phosphorylated SMAD1/5/8, SMAD1, and inhibitor of DNA binding 1, was induced by EC-CM, promoting hepcidin expression. Efficient blockage of this EC-mediated hepcidin upregulation by an inhibitor of the BMP6 receptor ALK receptor tyrosine kinase 2/3 or BMP6 siRNA identified BMP6 as a major hepcidin regulator in this coculture system, which highly fits the model of hepcidin regulation by iron in vivo. In addition, EC-derived BMP6 and hepcidin were highly sensitive to levels of not only ferric iron but also heme as low as 500 nM. We here establish a hepatocyte–endothelial coculture system to fully recapitulate iron regulation by hepcidin using EC-derived BMP6.     

Metformin inhibits MAPK signaling and rescues pancreatic aquaporin 7 expression to induce insulin secretion in type 2 diabetes mellitus

Metformin is the first-line antidiabetic agent for type 2 diabetes mellitus (T2DM) treatment. Although accumulated evidence has shed light on the consequences of metformin action, the precise mechanisms of its action, especially in the pancreas, are not fully understood. Aquaporin 7 (AQP7) acts as a critical regulator of intraislet glycerol content, which is necessary for insulin production and secretion. The aim of this study was to investigate the effects of different doses of metformin on AQP7 expression and explore the possible mechanism of its protective effects in the pancreatic islets. We used an in vivo model of high-fat diet in streptozocin-induced diabetic rats and an in vitro model of rat pancreatic β-cells (INS-1 cells) damaged by hyperglycemia and hyperlipidemia. Our data showed that AQP7 expression levels were decreased, whereas p38 and JNK mitogen-activated protein kinases (MAPKs) were activated in vivo and in vitro in response to hyperglycemia and hyperlipidemia. T2DM rats treated with metformin demonstrated a reduction in blood glucose levels and increased regeneration of pancreatic β-cells. In addition, metformin upregulated AQP7 expression as well as inhibited activation of p38 and JNK MAPKs both in vivo and in vitro. Overexpression of AQP7 increased glycerol influx into INS-1 cells, whereas inhibition of AQP7 reduced glycerol influx, thereby decreasing subsequent insulin secretion. Our findings demonstrate a new mechanism by which metformin suppresses the p38 and JNK pathways, thereby upregulating pancreatic AQP7 expression and promoting glycerol influx into pancreatic β-cells and subsequent insulin secretion in T2DM.     

Crossover-site sequence and DNA torsional stress control strand interchanges by the Bxb1 site-specific serine recombinase

DNA segment exchange by site-specific serine recombinases (SRs) is thought to proceed by rigid-body rotation of the two halves of the synaptic complex, following the cleavages that create the two pairs of exchangeable ends. It remains unresolved how the amount of rotation occurring between cleavage and religation is controlled. We report single-DNA experiments for Bxb1 integrase, a model SR, where dynamics of individual synapses were observed, using relaxation of supercoiling to report on cleavage and rotation events. Relaxation events often consist of multiple rotations, with the number of rotations per relaxation event and rotation velocity sensitive to DNA sequence at the center of the recombination crossover site, torsional stress and salt concentration. Bulk and single-DNA experiments indicate that the thermodynamic stability of the annealed, but cleaved, crossover sites controls ligation efficiency of recombinant and parental synaptic complexes, regulating the number of rotations during a breakage-religation cycle. The outcome is consistent with a ‘controlled rotation’ model analogous to that observed for type IB topoisomerases, with religation probability varying in accord with DNA base-pairing free energies at the crossover site. Significantly, we find no evidence for a special regulatory mechanism favoring ligation and product release after a single 180° rotation.     

In vitro eradication of abasic site-mediated DNA–peptide/protein cross-links by Escherichia coli long-patch base excision repair

Apurinic/apyrimidinic (AP or abasic) sites are among the most abundant DNA lesions. Numerous proteins within different organisms ranging from bacteria to human have been demonstrated to react with AP sites to form covalent Schiff base DNA–protein cross-links (DPCs). These DPCs are unstable due to their spontaneous hydrolysis, but the half-lives of these cross-links can be as long as several hours. Such long-lived DPCs are extremely toxic due to their large sizes, which physically block DNA replication. Therefore, these adducts must be promptly eradicated to maintain genome integrity. Herein, we used in vitro reconstitution experiments with chemically synthesized, stable, and site-specific Schiff base AP-peptide/protein cross-link analogs to demonstrate for the first time that this type of DPC can be repaired by Escherichia coli (E. coli) long-patch base excision repair. We demonstrated that the repair process requires a minimum of three enzymes and five consecutive steps, including: (1) 5′-DNA strand incision of the DPC by endonuclease IV; (2 to 4) strand-displacement DNA synthesis, removal of the 5′-deoxyribose phosphate-peptide/protein adduct-containing flap, and gap-filling DNA synthesis by DNA polymerase I; and (5) strand ligation by a ligase. We further demonstrated that endonuclease IV plays a major role in incising an AP-peptide cross-link within E. coli cell extracts. We also report that eradicating model AP-protein (11.2–36.1 kDa) DPCs is less efficient than that of an AP-peptide_10mer cross-link, supporting the emerging model that proteolysis is likely required for efficient DPC repair.     

The Energetics of the B-Z Transition in DNA

Abstract

The paper deals with the energetics of the transition to left-handed Z form in DNA with an arbitrary base sequence. There is a brief outline of the statistical-mechanical model of the B-Z transition allowing for three possible states of each base pair. The parameters of the model can be determined by comparing the theory with experimental data for the B-Z transition in inserts with given sequences in circular DNA The model contains six energy parameters, most of which have been determined before. In order to find the remaining parameters of the model and test its adequacy, a number of oligonucleotide sequences were synthesized and inserted into the pUC 19 plasmid. Two-dimensional gel electrophoresis was used to determine the superhelical density at which the inserts adopt the Z form. A statistical-mechanical treatment of these data yielded a complete set of six energy parameters for the B-Z transition. The theoretical assumption that the free energy of Z-form pairs does not depend on the type of adjacent pairs proved to be in agreement with the experimental data.