DNA Twist Research Articles

Gene repression by minimal lac loops in vivo.

The inflexibility of double-stranded DNA with respect to bending and twisting is well established in vitro. Understanding apparent DNA physical properties in vivo is a greater challenge. Here, we exploit repression looping with components of the Escherichia coli lac operon to monitor DNA flexibility in living cells. We create a minimal system for testing the shortest possible DNA repression loops that contain an E. coli promoter, and compare the results to prior experiments. Our data reveal that loop-independent repression occurs for certain tight operator/promoter spacings. When only loop-dependent repression is considered, fits to a thermodynamic model show that DNA twisting limits looping in vivo, although the apparent DNA twist flexibility is 2- to 4-fold higher than in vitro. In contrast, length-dependent resistance to DNA bending is not observed in these experiments, even for the shortest loops constraining <0.4 persistence lengths of DNA. As observed previously for other looping configurations, loss of the nucleoid protein heat unstable (HU) markedly disables DNA looping in vivo. Length-independent DNA bending energy may reflect the activities of architectural proteins and the structure of the DNA topological domain. We suggest that the shortest loops are formed in apical loops rather than along the DNA plectonemic superhelix.

Torsional Mechanics of DNA Are Regulated by Small-Molecule Intercalation

Whether the bend and twist mechanics of DNA molecules are coupled is unclear. Here, we report the direct measurement of the resistive torque of single DNA molecules to study the effect of ethidium bromide (EtBr) intercalation and pulling force on DNA twist mechanics. DNA molecules were overwound and unwound using recently developed magnetic tweezers where the molecular resistive torque was obtained from Brownian angular fluctuations. The effect of EtBr intercalation on the twist stiffness was found to be significantly different from the effect on the bend persistence length. The twist stiffness of DNA was dramatically reduced at low intercalator concentration (<10 nM); however, it did not decrease further when the intercalator concentration was increased by 3 orders of magnitude. We also determined the dependence of EtBr intercalation on the torque applied to DNA. We propose a model for the elasticity of DNA base pairs with intercalated EtBr molecules to explain the abrupt decrease of twist stiffness at low EtBr concentration. These results indicate that the bend and twist stiffnesses of DNA are independent and can be differently affected by small-molecule binding.

Effect of Spontaneous Twist on DNA Minicircles

Monte Carlo simulations are used to study the effect of spontaneous (intrinsic) twist on the conformation of topologically equilibrated minicircles of dsDNA. The twist, writhe, and radius of gyration distributions and their moments are calculated for different spontaneous twist angles and DNA lengths. The average writhe and twist deviate in an oscillatory fashion (with the period of the double helix) from their spontaneous values, as one spans the range between two neighboring integer values of intrinsic twist. Such deviations vanish in the limit of long DNA plasmids.

Torsional sensing of small-molecule binding using magnetic tweezers

DNA-binding small molecules are widespread in the cell and heavily used in biological applications. Here, we use magnetic tweezers, which control the force and torque applied to single DNAs, to study three small molecules: ethidium bromide (EtBr), a well-known intercalator; netropsin, a minor-groove binding anti-microbial drug; and topotecan, a clinically used anti-tumor drug. In the low-force limit in which biologically relevant torques can be accessed (<10 pN), we show that ethidium intercalation lengthens DNA ∼1.5-fold and decreases the persistence length, from which we extract binding constants. Using our control of supercoiling, we measure the decrease in DNA twist per intercalation to be 27.3 ± 1° and demonstrate that ethidium binding delays the accumulation of torsional stress in DNA, likely via direct reduction of the torsional modulus and torque-dependent binding. Furthermore, we observe that EtBr stabilizes the DNA duplex in regimes where bare DNA undergoes structural transitions. In contrast, minor groove binding by netropsin affects neither the contour nor persistence length significantly, yet increases the twist per base of DNA. Finally, we show that topotecan binding has consequences similar to those of EtBr, providing evidence for an intercalative binding mode. These insights into the torsional consequences of ligand binding can help elucidate the effects of small-molecule drugs in the cellular environment.

Anharmonic Torsional Stiffness of DNA Revealed under Small External Torques

DNA supercoiling plays an important role in a variety of cellular processes. The torsional stress related to supercoiling may also be involved in gene regulation through the local structure and dynamics of the double helix. To check this possibility, steady torsional stress was applied in the course of all-atom molecular dynamics simulations of two DNA fragments with different base pair sequences. For one fragment, the torsional stiffness significantly varied with small twisting. The effect is traced to sequence-specific asymmetry of local torsional fluctuations, and it should be small in long random DNA due to compensation. In contrast, the stiffness of special short sequences can change significantly, which gives a simple possibility of gene regulation via probabilities of strong fluctuations. These results have important implications for the role of DNA twisting in complexes with transcription factors.

Positional dependence of transcriptional inhibition by DNA torsional stress in yeast chromosomes

How DNA helical tension is constrained along the linear chromosomes of eukaryotic cells is poorly understood. In this study, we induced the accumulation of DNA (+) helical tension in Saccharomyces cerevisiae cells and examined how DNA transcription was affected along yeast chromosomes. The results revealed that, whereas the overwinding of DNA produced a general impairment of transcription initiation, genes situated at <100 kb from the chromosomal ends gradually escaped from the transcription stall. This novel positional effect seemed to be a simple function of the gene distance to the telomere: It occurred evenly in all 32 chromosome extremities and was independent of the atypical structure and transcription activity of subtelomeric chromatin. These results suggest that DNA helical tension dissipates at chromosomal ends and, therefore, provides a functional indication that yeast chromosome extremities are topologically open. The gradual escape from the transcription stall along the chromosomal flanks also indicates that friction restrictions to DNA twist diffusion, rather than tight topological boundaries, might suffice to confine DNA helical tension along eukaryotic chromatin.

Direct Observation of Twisting Steps During Rad51 Polymerization on DNA

The human recombinase hRad51 is a key protein for the maintenance of genome integrity and for cancer development. This protein plays a central role is the DNA strand exchange occurring during homologous recombination. Here we report the polymerization and depolymerization of hRad51 on duplex DNA observed with a new generation of magnetic tweezers, allowing the measurement of DNA twist with a resolution of 5° in real time. At odds with earlier claims, we show that, after initial deposition of a multimeric nucleus, nucleoprotein filament growth occurs by addition of single proteins, involving DNA twisting steps of 65±5°.

A Direct Observation of Highly Bent and Twisted DNA at the Single Molecule Level

Many DNA-binding proteins interact with twisted or bent DNA. To characterize the activity of these proteins as a function of the torsional and bending stresses, we must first understand how these mechanical stresses affect the DNA tertiary structure (topology). To experimentally define this relationship on scales that are biologically relevant to DNA-binding proteins requires DNA molecules which stably maintain high degrees of stress and deformation on a length scale appreciably below the persistence length.

Single Molecule Analysis of Substeps in the Mechanochemical Cycle of DNA Gyrase

DNA gyrase is a molecular motor that harnesses the free energy of ATP hydrolysis to introduce negative supercoils into DNA. We have characterized the structural dynamics of processive supercoiling using a real-time single molecule assay in which DNA gyrase activity drives the directional, stepwise rotation of a submicron rotor bead attached to the side of a stretched DNA molecule. We are able to directly observe rotational pauses corresponding to rate-limiting kinetic steps under varying [ATP], and have used simultaneous measurements of DNA twist and extension in order to characterize transient supercoil trapping and DNA compaction during the reaction cycle. We have mapped out structural intermediates of the DNA:gyrase complex on a twist-extension plane, and have characterized transitions between these states driven by chemical events such as the cooperative binding of ATP. These measurements motivate several revisions to previous models based on lower resolution assays [1], and we will present our results in the context of a new branched kinetic model for the mechanochemical cycle. We are now using theoretical calculations together with measurements of force-dependent changes in extension in order to test specific geometric models for structural intermediates, and we have begun to analyze structure-function relationships using single-molecule analysis of gyrase fragments.[1] Jeff Gore, Zev Bryant, Michael D. Stone, Marcelo Nollmann, Nicholas R. Cozzarelli, Carlos Bustamante, “Mechanochemical analysis of DNA gyrase using rotor bead tracking”, Nature, 439 (2006)

Two perspectives on the twist of DNA.

Because of the double-helical structure of DNA, in which two strands of complementary nucleotides intertwine around each other, a covalently closed DNA molecule with no interruptions in either strand can be viewed as two interlocked single-stranded rings. Two closed space curves have long been known by mathematicians to exhibit a property called the linking number, a topologically invariant integer, expressible as the sum of two other quantities, the twist of one of the curves about the other, and the writhing number, or writhe, a measure of the chiral distortion from planarity of one of the two closed curves. We here derive expressions for the twist of supercoiled DNA and the writhe of a closed molecule consistent with the modern view of DNA as a sequence of base-pair steps. Structural biologists commonly characterize the spatial disposition of each step in terms of six rigid-body parameters, one of which, coincidentally, is also called the twist. Of interest is the difference in the mathematical properties between this step-parameter twist and the twist of supercoiling associated with a given base-pair step. For example, it turns out that the latter twist, unlike the former, is sensitive to certain translational shearing distortions of the molecule that are chiral in nature. Thus, by comparing the values for the two twists for each step of a high-resolution structure of a protein-DNA complex, we may be able to determine how the binding of various proteins contributes to chiral structural changes of the DNA.

Direct observation of twisting steps during Rad51 polymerization on DNA

The human recombinase hRad51 is a key protein for the maintenance of genome integrity and for cancer development. Polymerization and depolymerization of hRad51 on duplex DNA were studied here using a new generation of magnetic tweezers, measuring DNA twist in real time with a resolution of 5 degrees . Our results combined with earlier structural information suggest that DNA is somewhat less extended by hRad51 than by RecA (4.5 vs. 5.1 A per base pair) and untwisted by 18.2 degrees per base pair. They also confirm a stoichiometry of 3-4 bp per protein in the hRad51-dsDNA nucleoprotein filament. At odds with earlier claims, we show that after initial deposition of a multimeric nucleus, nucleoprotein filament growth occurs by addition/release of single proteins, involving DNA twisting steps of 65 degrees +/- 5 degrees. Simple numeric simulations show that this mechanism is an efficient way to minimize nucleoprotein filament defects. Nucleoprotein filament growth from a preformed nucleus was observed at hRad51 concentrations down to 10 nM, whereas nucleation was never observed below 100 nM in the same buffer. This behavior can be associated with the different stoichiometries of nucleation and growth. It may be instrumental in vivo to permit efficient continuation of strand exchange by hRad51 alone while requiring additional proteins such as Rad52 for its initiation, thus keeping the latter under the strict control of regulatory pathways.

The single polypeptide restriction–modification enzyme LlaGI is a self-contained molecular motor that translocates DNA loops

To cleave DNA, the single polypeptide restriction–modification enzyme LlaGI must communicate between a pair of indirectly repeated recognition sites. We demonstrate that this communication occurs by a 1-dimensional route, namely unidirectional dsDNA loop translocation rightward of the specific recognition sequence 5′-CTnGAyG-3′ as written (where n is either A, G, C or T and y is either C or T). Motion across thousands of base pairs is catalysed by the helicase domain and requires the hydrolysis of 1.5-2 ATP per base pair. DNA loop extrusion is accompanied by changes in DNA twist consistent with the motor following the helical pitch of the polynucleotide track. LlaGI is therefore an example of a polypeptide that is a completely self-contained, multi-functional molecular machine.

An iterative strategy combining biophysical criteria and duration hidden Markov models for structural predictions of Chlamydia trachomatis σ66 promoters

BackgroundPromoter identification is a first step in the quest to explain gene regulation in bacteria. It has been demonstrated that the initiation of bacterial transcription depends upon the stability and topology of DNA in the promoter region as well as the binding affinity between the RNA polymerase σ-factor and promoter. However, promoter prediction algorithms to date have not explicitly used an ensemble of these factors as predictors. In addition, most promoter models have been trained on data from Escherichia coli. Although it has been shown that transcriptional mechanisms are similar among various bacteria, it is quite possible that the differences between Escherichia coli and Chlamydia trachomatis are large enough to recommend an organism-specific modeling effort.ResultsHere we present an iterative stochastic model building procedure that combines such biophysical metrics as DNA stability, curvature, twist and stress-induced DNA duplex destabilization along with duration hidden Markov model parameters to model Chlamydia trachomatis σ66 promoters from 29 experimentally verified sequences. Initially, iterative duration hidden Markov modeling of the training set sequences provides a scoring algorithm for Chlamydia trachomatis RNA polymerase σ66/DNA binding. Subsequently, an iterative application of Stepwise Binary Logistic Regression selects multiple promoter predictors and deletes/replaces training set sequences to determine an optimal training set. The resulting model predicts the final training set with a high degree of accuracy and provides insights into the structure of the promoter region. Model based genome-wide predictions are provided so that optimal promoter candidates can be experimentally evaluated, and refined models developed. Co-predictions with three other algorithms are also supplied to enhance reliability.ConclusionThis strategy and resulting model support the conjecture that DNA biophysical properties, along with RNA polymerase σ-factor/DNA binding collaboratively, contribute to a sequence's ability to promote transcription. This work provides a baseline model that can evolve as new Chlamydia trachomatis σ66 promoters are identified with assistance from the provided genome-wide predictions. The proposed methodology is ideal for organisms with few identified promoters and relatively small genomes.

Stacking Interactions and DNA Intercalation

The relationship between stacking interactions and the intercalation of proflavine and ellipticine within DNA is investigated using a nonempirical van der Waals density functional for the correlation energy. Our results, employing a binary stack model, highlight fundamental, qualitative differences between base-pair-base-pair interactions and that of the stacked intercalator-base-pair system. The most notable result is the paucity of torque, which so distinctively defines the twist of DNA. Surprisingly, this model, when combined with a constraint on the twist of the surrounding base-pair steps to match the observed unwinding of the sugar-phosphate backbone, was sufficient for explaining the experimentally observed proflavine intercalator configuration. Our extensive mapping of the potential energy surface of base-pair-intercalator interactions can provide valuable information for future nonempirical studies of DNA intercalation dynamics.

Twist- and Tension-Mediated Elastic Coupling between DNA-Binding Proteins

We study the effective interaction between DNA-binding proteins that arises from elastic stresses in the DNA when tension is applied. Using the wormlike chain model, we calculate the free energy cost of introducing multiple nearby bends in the DNA. We find that the bend deformation energy promotes aggregation to straighten the linker DNA, while twist resistance of the linker leads to damped oscillations in the coupling free energy between two proteins. We calculate the mean first encounter time for proteins sliding along DNA, indicating, in some cases, an optimal applied tension for protein assembly. Our results highlight the need to consider DNA twist even when no torsion is applied and the DNA ends are free to rotate. The variable-range oscillatory coupling between DNA-binding proteins may provide a versatile mechanism for tension-mediated gene regulation.

DNA Nanomechanics in the Nucleosome

The sequence-dependent mechanics of DNA-histone binding are essential for nucleosome positioning and mobility. Here we reanalyze nucleosome crystal structures in terms of the well-characterized base-pair scale DNA elasticity, extracting the forces and torques acting on all bound DNA base pairs. We find that the strongest forces follow a characteristic repeating pattern that recovers the 12 known DNA backbone-histone contact sites. DNA twist defects and histone point mutations modify this pattern in interpretable ways. Additional, irregular forces between contact sites reveal histone tail-DNA interactions, whereas requiring the absence of external forces leads to a structural refinement of linker DNA. Based on these observations, we propose a simple, structure-based mechanical model of the nucleosome that is able to explain the placement of DNA twist defects in 146 base-pair nucleosomes and allows an estimate of the elastic energy spectrum of nucleosome twist defect states.

Small-Molecule Binding to DNA under Tension and Twist Studied by Magnetic Tweezers

DNA-binding small molecules are present in the cellular environment and are ubiquitously used in biochemistry and biotechnology. Here, we use single molecule magnetic tweezers experiments to study the effect of small-molecule ligands on DNA mechanical properties.Using the magnetic tweezers ability to control both the applied stretching force and torque, we have systematically characterized the mechanical properties of DNA in the presence of Ethidium Bromide (EtBr), a well-known intercalator, and Netropsin, an anti-microbial drug and known minor groove binder. In addition, we have characterized the interactions of Topotecan, a clinically used anti-tumor drug, with bare DNA.Our results show a lengthening of DNA upon EtBr intercalation to an extension of ∼1.5 times the initial contour length and a decrease in DNA twist by 29 +- 3 degrees per intercalation event, in agreement with previous estimates from bulk experiments. Further effects of intercalation are a stabilization of the double strand under high forces and negative torques and a decrease in both the bending and twisting persistence lengths. In contrast, minor groove binding by Netropsin does not change the contour or persistence length significantly, but increases the twist per base of the DNA.For Topotecan, both intercalative and minor-groove binding have been postulated. Our magnetic tweezers results indicate that drug binding at > 50 micromolar concentrations has consequences qualitatively similar to the changes observed for EtBr, in support of a intercalative binding mode for Topotecan.These results point to important consequences of intercalative ligand binding for DNA torsional behavior, which we characterized at the single-molecule level using magnetic tweezers. Ultimately, such insights can help elucidate the effects of small molecule drugs in the cellular environment and their interference with DNA transcription and replication.

Twist and Tension-mediated Elastic Coupling between DNA-Bending Proteins

We study the effective interaction between DNA-binding proteins that arises from elastic stresses in the DNA when tension is applied. Using the wormlike chain model, we calculate the free energy cost of introducing multiple nearby bends in the DNA. We find that the bend deformation energy promotes aggregation to straighten the linker DNA, while twist resistance of the linker leads to damped oscillations in the coupling free energy between two proteins. We calculate the mean first encounter time for proteins sliding along DNA, indicating, in some cases, an optimal applied tension for protein assembly. Our results highlight the need to consider DNA twist even when no torsion is applied and the DNA ends are free to rotate. The variable-range oscillatory coupling between DNA-binding proteins may provide a versatile mechanism for tension-mediated gene regulation.

1TP5-10 Rad51の重合によるDNAねじり運動の実時間計測(核酸結合蛋白質,第47回日本生物物理学会年会)

1TP5-10 Rad51の重合によるDNAねじり運動の実時間計測(核酸結合蛋白質,第47回日本生物物理学会年会)

Twist-stretch correlation of DNA

We present an elastic model for B-form DNA with variable radius to study the elastic twist-stretch coupling of stretched DNA. In this model, only two strain variables as well as the changes in the energy of the hydrogen and covalent bonds of DNA during the deformation are considered. It is found that, depending on the elastic constants, the correlation between twisting and stretching of a helical molecule can be positive or negative. It is shown that for the suitable elastic constants in the model, the twist-stretch coupling of DNA behaves nonmonotonically, and contrary to intuition, the DNA twisting and stretching are positively correlated for small distortions. This result is entirely consistent with recent experimental results.