High-fidelity DNA sensing by protein binding fluctuations

One of the major functions of RecA protein in the cell is to bind single-stranded DNA exposed upon damage, thereby triggering the SOS repair response. We present fluorescence anisotropy measurements at the binding onset, showing enhanced DNA length discrimination induced by adenosine triphosphate consumption. Our model explains the observed DNA length sensing as an outcome of out-of-equilibrium binding fluctuations, reminiscent of microtubule dynamic instability. The cascade architecture of the binding fluctuations is a generalization of the kinetic proofreading mechanism. Enhancement of precision by an irreversible multistage pathway is a possible design principle in the noisy biological environment.     

Biophysical characterization of DNA binding from single molecule force measurements

Single molecule force spectroscopy is a powerful method that uses the mechanical properties of DNA to explore DNA interactions. Here we describe how DNA stretching experiments quantitatively characterize the DNA binding of small molecules and proteins. Small molecules exhibit diverse DNA binding modes, including binding into the major and minor grooves and intercalation between base pairs of double-stranded DNA (dsDNA). Histones bind and package dsDNA, while other nuclear proteins such as high mobility group proteins bind to the backbone and bend dsDNA. Single-stranded DNA (ssDNA) binding proteins slide along dsDNA to locate and stabilize ssDNA during replication. Other proteins exhibit binding to both dsDNA and ssDNA. Nucleic acid chaperone proteins can switch rapidly between dsDNA and ssDNA binding modes, while DNA polymerases bind both forms of DNA with high affinity at distinct binding sites at the replication fork. Single molecule force measurements quantitatively characterize these DNA binding mechanisms, elucidating small molecule interactions and protein function.     

C60 fullerene binding to DNA

Fullerenes have attracted considerable attention in various areas of science and technology. Owing to their exceptional physical, chemical, and biological properties, they have many applications, particularly in cosmetic and medical products. Using the Lennard-Jones 6-12 potential function and the continuum approximation, which assumes that intermolecular interactions can be approximated by average atomic surface densities, we determine the binding energies of a C₆₀ fullerene with respect to both single-strand and double-strand DNA molecules. We assume that all configurations are in a vacuum and that the C₆₀ fullerene is initially at rest. Double integrals are performed to determine the interaction energy of the system. We find that the C₆₀ fullerene binds to the double-strand DNA molecule, at either the major or minor grooves, with binding energies of ?4.7 eV or ?2.3 eV, respectively, and that the C₆₀ molecule binds to the single-strand DNA molecule with a binding energy of ?1.6 eV. Our results suggest that the C₆₀ molecule is most likely to be linked to the major groove of the dsDNA molecule.     

Cutting DNA: Mechanics of the topoisomerase

The framework of relativistic self-consistent mean-field models is extended to include correlations related to restoration of broken symmetries and to fluctuations of collective variables. The generator coordinate method is used to perform configuration mixing of angular-momentum and particle-number projected relativistic wave functions. The model, currently restricted to axially symmetric shapes, employs a relativistic point-coupling (contact) nucleon-nucleon effective interaction in the particle-hole channel, and a δ-interaction in the pairing channel. Both bulk and spectroscopic nuclear properties are explored.     

The elastic theory of a single DNA molecule

We study the elastic responses of double-(ds) and single-stranded (ss) DNA at external force fields. A double-strand-polymer elastic model is constructed and solved by path integral methods and Monte Carlo simulations to understand the entropic elasticity, cooperative extensibility, and supercoiling property of dsDNA. The good agreement with experiments indicates that short-ranged base-pair stacking interaction is crucial for the stability and the high deformability of dsDNA. Hairpin-coil transition in ssDNA is studied with generating function method. A threshold force is needed to pull the ssDNA hairpin patterns, stabilized by base pairing and base-pair stacking, into random coils. This phase transition is predicted to be of first order for stacking potential higher than some critical level, in accordance with experimental observations.     

从经典力学到现代力学

力学是研究物质机械运动规律的科学．自然界物质有多种层次，从宇观的宇宙体系、宏观的天体和通常物体，到微观的分子、原子、基本粒子；通常力学以研究宏观对象为主．但由于学科的相互渗透，力学有时也涉及宇观乃至微观对象的有关运动规律．机械运动是物质运动的最基本的形式，包括移动、转动、流动、变形、振动、波动、扩散等．     

Stretching single stranded DNA,a model polyelectrolyte

The elastic properties of single stranded (ss)DNA, studied by pulling on an isolated molecule, are shown to agree with a recent model of ssDNA that takes into account base pairings and screened electrostatic repulsion of the phosphodiester backbone. By an appropriate physicochemical treatment, the pairing interactions were suppressed and ssDNA used as an experimental model for a generic polyelectrolyte. The elastic behavior of such an altered ssDNA deviates strongly from the behavior of an ideal polymer. This deviation is shown to result from the elasticity of the chain and its electrostatic self-avoiding interactions.     

Direct determination of vibrational density of states change on ligand binding to a protein

The change in the vibrational density of states of a protein (dihydrofolate reductase) on binding a ligand (methotrexate) is determined using inelastic neutron scattering. The vibrations of the complex soften significantly relative to the unbound protein. The resulting free-energy change, which is directly determined by the density of states change, is found to contribute significantly to the binding equilibrium.     

Pseudoelectrostatic effects in the thermodynamics of dye binding to DNA

Summary The interaction of DNA with the dye ethidium bromide (EB) has been investigated under the presence of methanol at varying temperatures. The Gibbs free energy associated with the DNA-EB binding is found to be well described by its linear dependence upon the dielectric constant of the solution, a property also found for other alcohols. When the enthalpy and entropy associated with the binding are considered, it is found that their role becomes rather simple if their evaluation is performed isodielectrically (ε=const). According to this procedure, the varying properties of the solvent can be separated from those of the interacting DNA and EB and the presence of hydrophobic interactions becomes then well established. Partially supported by Gruppo Nazionale Struttura della Materia, C.N.R.     

Methylene blue as a DNA probe for a comparative study of Cd, Pb and Cr ions binding to calf thymus DNA

Methylene blue (MB) was developed as a sensitive DNA probe for a comparative study of Cd²⁺, Pb²⁺ and Cr³⁺ ions binding with calf thymus DNA (ctDNA). The fluorescence intensity of the MB-ctDNA system increased dramatically when heavy metal ions (Cd²⁺, Pb²⁺ and Cr³⁺ ions) were added, which indicated that some of the bound MB molecules were released from the ctDNA base pairs. To compare the binding affinity of these three different heavy metal ions with ctDNA, the relationships between the fluorescence intensity of the MB-ctDNA-M (Metal ions) system and the concentration ratio of [M]/[DNA(p)] were investigated. The results showed that the order of the binding affinity of heavy metal ions with ctDNA had the following sequence: Cr³⁺> Cd²⁺>Pb²⁺. This order was further proved by the effects of heavy metal ions on the number of MB bound to ctDNA, the measurements of binding constants of these heavy metal ions to ctDNA, and the effects of heavy metal ions on the absorption of the MB-ctDNA system. In addition, the interaction mechanisms of Cd²⁺, Pb²⁺ and Cr³⁺ ions with ctDNA were also discussed in detail. These results indicated that their interaction mechanisms are related to the concentration ratios of heavy metal ions to DNA.     

Rotational drag on DNA: a single molecule experiment

Within a single-molecule configuration, we have studied rotational drag on double stranded linear DNA by measuring the force during mechanical opening and closing of the double helix at different rates. The molecule is cranked at one end by the effect of unzipping and is free to rotate at the other end. In this configuration the rotational friction torque tau on double-stranded DNA leads to an additional contribution to the opening force. It is shown that the effect of rotational drag increases with the length of the molecule, is approximately proportional to the angular velocity of cranking, and we estimate that the torque tau is of the order of 1k(B)T for 10 000 base pairs of DNA cranked at 2000 turns per second.     

MALDI-MS detection of noncovalent interactions of single stranded DNA with Escherichia coli single-stranded DNA-binding protein

The Escherichia coli single-stranded DNA binding protein (SSB) selectively binds single-stranded (ss) DNA and participates in the process of DNA replication, recombination and repair. Different binding modes have previously been observed in SSB?ssDNA complexes, due to the four potential binding sites of SSB. Here, chemical cross-linking, combined with high-mass matrix-assisted laser desorption/ionization (MALDI) mass spectrometry (MS), is used to determine the stoichiometry of the SSB?ssDNA complex. SSB forms a stable homotetramer in solution, but only the monomeric species (m/z 19,100) can be detected with standard MALDI-MS. With chemical cross-linking, the quaternary structure of SSB is conserved, and the tetramer (m/z 79,500) was observed. We found that ssDNA also functions as a stabilizer to conserve the quaternary structure of SSB, as evidenced by the detection of a SSB?ssDNA complex at m/z 94,200 even in the absence of chemical cross-linking. The stability of the SSB?ssDNA complex with MALDI strongly depends on the length and strand of oligonucleotides and the stoichiometry of the SSB?ssDNA complex, which could be attributed to electrostatic interactions that are enhanced in the gas phase. The key factor affecting the stoichiometry of the SSB?ssDNA complex is how ssDNA binds to SSB, rather than the protein-to-DNA ratio. This further suggests that detection of the complex by MALDI is a result of specific binding, and not due to non-specific aggregation in the MALDI plume.     

Fluorescence enhancement of radix angelica dahurica by binding to single silver sphere

We present a theoretical study of the influence of a single silver sphere on the fluorescence of radix angelica dahurica,which is a kind of traditional Chinese medicine. The enhancement factors of the excitation and the relaxation processes are deduced. The excitation can be enhanced more than 100 times at 315 nm. The enhancement factor of the emission can reach up to 9 at a center wavelength of 400 nm.     

Mechanics of single pass equal channel angular extrusion of powder in tubes

In the current study powder in tubes (PITs) are processed through single pass equal channel angular extrusion (ECAE), for two different powders by using three different tube materials. Studies were conducted for the first time to understand the processing mechanism of ECAE of PITs. In the case of hard brittle intermetallic magnesium boride (MgB₂) powder, the process was found to primarily involve compaction and shear-sliding of the powder, and localized-deformation of the tube. Reasons for localized-deformation occurring during ECAE were discussed in detail. Compaction efficiency was understood to depend not only on the material of the tube but also on the homogeneity of stress and strain in the composite PIT. Various frictional stresses and mechanisms of localized-deformation were found to be the reasons for stress-strain inhomogeneity. In the case of copper powder, even though localized-deformation occurred, higher inter-particle friction and low yield strength of the powder helped in the complete densification of the powders. PACS 81.20.Ev; 83.50.-V; 07.10.Pz; 62.20.Fe; 74.25.Ld     

DNA单分子弹性理论

随着单分子操纵技术的发明与发展，人们已经可以对单个生物大分子施以力或力矩，并测量它们的物理性质，DNA单分子的力学实验表明，在分子尺度上理解生物大分子的生化过程，力与能量是同等重要的结构与功能参数。一个梯子模型被用来描述双链DNA的外力拉伸曲线，在这个模型中，DNA是由许多碱基对(梯子的横杆，横杆之间存在吸引势)连接两条聚核苷酸虫链(梯子的两侧)形成的高分子，利用路径积分法得出的理论曲线与实验曲线吻合得很好，对于单链DNA，用分立的杂化高分子链统计理论的母函数方法来计算其弹性行为，得出与实验相符合的外力引起的解链相变结果。此外，对于抑瘤蛋白p53识别序列DNA微环弹性进行分析，发现其弹性模量只是通常随机序列的三分之一。     

Elastic response of single DNA molecules exhibits a reentrant collapsing transition

We measured the elastic response of single DNA molecules at various concentrations of the trivalent cation, spermidine. When added spermidine caused the DNA to collapse, the force-extension curves showed either plateaus or stick-release patterns depending on the concentration. The periodic stick-release response determines a characteristic length, which may reflect toroidal supercoiling. At high concentrations of spermidine, we observed the reelongation of single molecules of collapsed DNA. Thus condensation occurs between lower and upper critical concentrations, verifying that the transition is reentrant as theoretically predicted.     

Confinement-induced entropic recoil of single DNA molecules in a nanofluidic structure

The behavior of DNA molecules is observed in a nanofluidic device near the interface of two regions that produce different configuration entropies. An electric field is applied to drive the molecules partway across the interface. Upon removal of the field, the molecules recoil to the higher-entropy region with a profile characteristic of a force localized to the interface and independent of length. This is consistent with a confinement-mediated entropic force, distinct from the well-known entropic elasticity common to all polymers. An estimate of the hydrodynamic drag is used to produce a lower bound for the force. The phenomenon can be exploited to separate long-strand polyelectrolytes according to length.     

Statistical Mechanics Analysis of ATP Binding to a Multisubunit Enzyme

Due to inter-subunit communication, multisubunit enzymes usually hydrolyze ATP in a concerted fashion. However, so far the principle of this process remains poorly understood. In this study, from the viewpoint of statistical mechanics, a simple model is presented. In this model, we assume that the binding of ATP will change the potential of the corresponding enzyme subunit, and the degree of this change depends on the state of its adjacent subunits. The probability of enzyme in a given state satisfies the Boltzmann's distribution. Although it looks much simple, this model can fit the recent experimental data of chaperonin TRiC/CCT well. From this model, the dominant state of TRiC/CCT can be obtained. This study provide a new way to understand biophysical processe by statistical mechanics analysis.