Elastic Properties Of DNA Research Articles

Elastic Properties of DNA as the Entropic Driving Force for Dehybridization Transitions

Through the use of a coarse-grained lattice model informed from all-atomic simulations near equilibrium, we obtain a theoretical estimate of the sequence-dependent slow melting rates (> nanoseconds) of DNA duplexes with activation free energies accurate within 3kT. We show that the change in elastic properties of short DNAs is the key entropic driving force for DNA melting and hybridization. Using a vibration and torsional continuum model, we relate this entropy to the bending and torsional persistence lengths captured from the phonon spectra in all-atomic simulations, near equilibrium, by capturing both the momentum and configuration degrees of freedom simultaneously. A key to the theory is the cooperative melting of all but one base pair at the transition state. This hinge transition state allows us to replace the hydrogen bond vibration modes with a set of out/of-phase bending modes with a much higher vibration entropy (lower phonon frequencies). A multidimensional version of Kramers transition state theory allows us to assign this change to an effective vibrational entropy term that counters the enthalpic gain required to break the hydrogen bonds, leading to an elastic theory for the melting rate of short DNAs. As the hydrogen bond enthalpies are thermodynamic quantities, we estimate them from the classical nearest-neighbor theory. The vibration entropy of the hinge transition state is strictly kinetic but, as it relates only to the persistence lengths, we are able to capture it from near-equilibrium simulations of the duplex. This theory allows us both to make quantitative estimates of the melting rates of short DNAs as well as to differentiate between unzipping and other reaction pathways that may be followed in the presence of secondary structures or mismatches. It represents the first theory for DNA melting with quantitative accuracy.

Measuring the elasticity of ribonucleotide(s)-containing DNA molecules using AFM.

Ribonucleotides, ribonucleoside monophosphates (rNMPs), have been revealed as possibly the most noncanonical nucleotides in genomic DNA. rNMPs, either not removed from Okazaki fragments during DNA replication or incorporated and scattered throughout the genome, pose a perturbation to the structure and a threat to the stability of DNA. The instability of DNA is mainly due to the extra 2'-hydroxyl (OH) group of rNMPs which give rise to local structural effects, which may disturb various molecular interactions in cells. As a result of these structural perturbations by rNMPs, the elastic properties of DNA are also affected. Here, we show the approach to test whether the presence of rNMPs in DNA duplexes could alter the elasticity of DNA by implementing atomic force microscopy (AFM)-based single molecule force-measurements of short rNMP(s)-containing oligonucleotides (oligos).

Structural and Thermodynamic Principles of Viral Packaging

Packaging of genetic material inside a capsid is one of the major processes in the lifecycle of bacteriophages. To establish the basic principles of packing double-stranded DNA into a phage, we present a low-resolution model of bacteriophage varphi29 and report simulations of DNA packaging. The simulations show excellent agreement with available experimental data, including the forces of packaging and the average structures seen in cryo-electron microscopy. The conformation of DNA inside the bacteriophage is primarily determined by the shape of the capsid and the elastic properties of DNA, but the energetics of packaging are dominated by electrostatic repulsions and the large entropic penalty associated with DNA confinement. In this slightly elongated capsid, the DNA assumes a folded toroidal conformation, rather than a coaxial spool. The model can be used to study packaging of other bacteriophages with different shapes under a range of environmental conditions.

Role of inter and intramolecular interactions in protein–DNA recognition

Protein–DNA recognition plays an essential role in the regulation of gene expression. Regulatory proteins are known to recognize specific DNA sequences directly through atomic contacts between protein and DNA, and/or indirectly through the conformational properties of the DNA. In this work, we have analyzed the specificity of intermolecular interactions by statistical analysis of base–amino acid interactions within protein–DNA complexes as well as the computer simulations of base–amino acid interactions. The specificity of the intramolecular interactions was studied by statistical analysis of the sequence-dependent DNA conformational parameters and the elastic properties of DNA. Systematic comparison of these specificities in a large number of protein–DNA complexes revealed that both intermolecular and intramolecular interactions contribute to the specificity of protein–DNA recognition, and their relative contributions vary depending upon the protein–DNA complex. We demonstrated that combination of the intermolecular and intramolecular energies leads to enhanced specificity and the combined energy could explain experimental data on binding affinity changes caused by base mutations. These results provided new insight into the relationship between specificity and structure in the process of protein–DNA recognition, which would lead to prediction of specific protein–DNA binding sites.

Mechanisms for nucleosome mobilization

Nucleosomes are the ubiquitous and fundamental packaging for eukaryotic genomes, and are the substrate for many processes in the nucleus. Nucleosomes are not static entities but can readily be moved by thermal energy and ATP-dependent chromatin remodeling complexes in a process known as sliding or shifting. We summarize from a mechanical perspective the twist defect and bulge diffusion mechanisms proposed as the most likely pathway for nucleosome mobilization. We then consider the elastic properties of DNA and how this affects the potential for each mechanism, concentrating on kinetic aspects of twist diffusion and possible planar bulge sizes and summarize the experimental evidence reflecting on each. Either, or both, mechanisms could occur, and careful experimentation focusing on their uniquely distinguishing features will be required to determine their relative contributions to chromatin dynamics.

Mechanical Model of the Nucleosome and Chromatin

A theoretical framework for evaluating the approximate energy and dynamic properties associated with the folding of DNA into nucleosomes and chromatin is presented. Experimentally determined elastic constants of linear DNA and a simple fold geometry are assumed in order to derive elastic constants for extended and condensed chromatin. The model predicts the Young's modulus of extended and condensed chromatin to within an order of magnitude of experimentally determined values. Thus we demonstrate that the elastic properties of DNA are a primary determinant of the elastic properties of the higher order folded states. The derived elastic constants are used to predict the speed of propagation of small amplitude waves that excite an extension(sound), twist, bend or shear motion in each folded state. Taken together the results demonstrate that folding creates a hierarchy of time, length and energy scales.

The mechanical advantages of DNA

The elastic properties of DNA and the contractile activities of enzymes involved in transcription, translation and supercoiling may have contributed to the ability of early cells (protocells) to withstand turgor pressure. In the hypothesis proposed here, resistance to turgor resulted from (1) the elastic properties of DNA which was connected to the membrane by association with positively charged lipids and with membrane peptides, (2) the coupled transcription–translation–insertion of peptides into membrane—transertion—which connected membranes with phase-condensed DNA, and (3) the action of topoisomerases which supercoiled and shortened DNA. The existence of a negative feedback system is also proposed to explain how weakened regions of membrane were preferentially strengthened. It may prove possible to test this hypothesis by studying transertion using optical tweezers and by studying wall-less l-form bacteria.

Direct mechanical measurement of single DNA molecules through an optical microscope

Understanding the mechanical properties of DNA such as their bending and torsional rigidity can provide a great deal of insight about the factors that determine the folding of DNA during the cell cycle. These quantities have been obtained from bulk experiments using light scattering, equilibrium sedimentation, DNA cyclization, etc. Determination of the elastic properties of DNA with these methods is indirect, via a theoretical framework relating the observable quantities to the elastic parameters of the molecules. Moreover, the results represent an ensemble average over all accessible configurations of the molecule, with small contribution from stretched un-likely states.We have carried a systematic study of the elastic properties of single DNA molecules. Single DNA molecules were chemically attached by one end to a glass surface and by their other end to a magnetic bead. Equilibrium positions of the beads are observed in an optical microscope while the beads are acted on by known magnetic and hydrodynamic forces.

Importance of DNA stiffness in protein-DNA binding specificity.

From the first high-resolution structure of a repressor bound specifically to its DNA recognition sequence it has been shown that the phage 434 repressor protein binds as a dimer to the helix. Tight, local interactions are made at the ends of the binding site, causing the central four base pairs (bp) to become bent and overtwisted. The centre of the operator is not in contact with protein but repressor binding affinity can be reduced at least 50-fold in response to a sequence change there. This observation might be explained should the structure of the intervening DNA segment vary with its sequence, or if DNA at the centre of the operator resists the torsional and bending deformation necessary for complex formation in a sequence dependent fashion. We have considered the second hypothesis by demonstrating that DNA stiffness is sequence dependent. A method is formulated for calculating the stiffness of any particular DNA sequence, and we show that this predicted relationship between sequence and stiffness can explain the repressor binding data in a quantitative manner. We propose that the elastic properties of DNA may be of general importance to an understanding of protein-DNA binding specificity.

Investigation of DNA dynamics and drug-DNA interaction by steady state fluorescence anisotropy.

We have used steady-state fluorescence polarization anisotropy (FPA) of ethidium probe molecules bound to DNA to investigate DNA-DNA interactions and the effect of high densities of intercalating drugs on the internal motions of DNA responsible for depolarization of the ethidium fluorescence. To calibrate the method, we examined the effect of DNA length on (FPA) using DNA varying in size from 10-150 base pair. The association of approximately 30 base pair DNA at high concentrations was then detected by its effect on (FPA). With sample concentrations approaching those commonly used in various physical experiments (NMR, Raman) significant DNA-DNA interactions are observed. With high molecular weight DNA (greater than 500 base pair), the limiting value of the (FPA) (0.23) is due to internal motions of the DNA (and bound chromophores). The (FPA) of ethidium probe molecules (1 drug/200 base pair) is unaffected by the addition of high levels (1 drug/2 base pair) proflavine. This indicates that either the elastic properties of DNA are unaffected by high densities of intercalated drug or that the depolarization of the ethidium fluorescence is due to highly localized motions of the base pairs that are unperturbed by binding of drugs at neighboring sites.