Quasi-continuum models of twist-like and accordion-like low-frequency motions in DNA.

Formulae for calculating low-frequency twist-like and accordion-like modes of DNA molecules have been derived using a quasi-continuum model. The formulae can be employed in essentially all (viz. A, B, C, D, E, and Z) forms of DNA. Calculated results indicate that the experimentally observed low-frequency modes at 22 cm-1 for the A-form octanucleotide (d[CCCCGGGG]) and at 18 cm-1 for the B-form dodecanucleotide (d[CGCAA ATTTGCG]) may result from accordion-like motions, while those observed at 12 cm-1 and 15 cm-1 may result from combinations of twist-like oscillations excited in the intact segments of B- and A-DNA's, respectively. Frequency shifts in the low-frequency modes observed when DNA molecules undergo conformational changes among different forms are also discussed in terms of the current model.     

Low-frequency motions in protein molecules. Beta-sheet and beta-barrel.

Low-frequency internal motions in protein molecules play a key role in biological functions. Based on previous work with alpha-helical structure, the quasi-continuum model is extended to the beta-structure, another fundamental element in protein molecules. In terms of the equations derived here, one can easily calculate the low-frequency wave number of a beta-sheet in an accordionlike motion, and the low-frequency wave number of a beta-barrel in a breathing motion. The calculated results for immunoglobulin G and concanavalin A agree well with the observations. These findings further verify that the observed low-frequency motion (or the so-called dominant low-frequency mode) in a protein molecule is essentially governed by the collective fluctuations of its weak bonds, especially hydrogen bonds, and the internal displacement of the massive atoms therein, as described by the quasi-continuum model.     

Conformation oscillations of DNA

A conformational mobility model is proposed to describe the low-frequency DNA structure dynamics. The transverse oscillations of structure elements in a double-stranded chain are studied. The frequency of DNA long-wave conformational oscillations are estimated and the theory is shown to agree with experiment. DNA low-frequency Raman spectra are interpreted.     

Identification of low-frequency modes in protein molecules.

It is demonstrated that the observed low-frequency motions with wave numbers of 22 cm-1 and 25 cm-1 for insulin and lysozyme respectively originate from the accordion-like motions of the principal helices therein. The calculated results based on such a model are in good agreement with the observed values. During calculations the role of the internal microenvironment upon the low-frequency motion is naturally revealed, so as to elucidate as well why this kind of low-frequency motion is so sensitive to the conformations of proteins observed.     

Theory of low-frequency vibrations in DNA macromolecules

To describe low-frequency dynamics of DNA macromolecules a model is developed taking into account the hydrogen bond stretching in base pairs, the backbone flexibility and intranucleoside mobility. For double-stranded DNA the normal vibrations are found and the structure of low-frequency spectrum is determined. The agreement between theory and Raman spectroscopy data for B-DNA is demonstrated. Conformational dependences of vibration spectrum during the B----A and helix----coil DNA transitions are studied. The contribution coming from low-frequency mobility to the nucleic-protein recognition processes is discussed.     

Low-frequency Raman spectra of lysozyme crystals and oriented DNA films: dynamics of crystal water.

We observed low-frequency Raman spectra of tetragonal lysozyme crystals and DNA films, with varying water content of the samples. The spectra are fitted well by sums of relaxation modes and damped harmonic oscillators in the region from approximately 1 cm(-1) to 250 cm(-1). The relaxation modes are due to crystal water, and the distribution of relaxation times is determined. In wet samples, the relaxation time of a small part of the water molecules is a little longer than that of bulk water. The relaxation time of a considerable part of the crystal water, which belongs mainly to the secondary hydration shell, is an order of magnitude longer than that of bulk water. Furthermore, the relaxation time of some water molecules in the primary hydration shell of semidry samples is shorter than we expected. Thus we have shown that low-frequency Raman measurements combined with properly oriented samples can give specific information on the dynamics of hydration water in the ps range. On the other hand, we concluded, based on polarized Raman spectra of lysozyme crystals, that the damped oscillators correspond to essentially intramolecular vibrational modes.     

Prediction of a low-frequency mode in bovine pancreatic trypsin inhibitor molecule

One of the frontiers today in molecular biology is to measure, identify and go further to predict the low-frequency internal motion of biological macromolecules, which is crucially important for understanding the dynamic mechanism of various biological functions occurring in such molecules. Based on the theory of continuity model developed recently for dealing with the internal low-frequency motion of a biological macromolecule, it is predicted that the low-frequency phonons with wave number of about 23 cm^?1 might be excited in BPTI molecule.     

Electrophoretic charge density and persistence length of DNA as measured by fluorescence microscopy

Individual ethidium-stained DNA molecules, embedded in an agarose gel made with electrophoresis buffer (0.05 molar salt), are observed using a fluorescence microscope. In the first experiment, open circular 66 kilobase pair (kbp) plasmids, immobilized by agarose fibers threaded through their centers, display entropic "rubber" elasticity. The charged molecules extend in an electric field of several volts per centimeter and contract to a compact random coil when the field is removed. The extension of the plasmids as a function of field strength is consistent with the freely jointed chain model when the effective electrophoretic charge density is set at 15 e-per persistence length. In a second experiment, stained linear 48.5 kbp DNA molecules are observed as random coils immobilized in agarose. A measure of their size, here named the "maximal-X-extent," is taken for 100 molecules and found to average 1.47 mu. A Monte Carlo computer simulation of random coils (freely jointed chain model) gives the same maximal-X-extent value when the persistence length is set at 0.08 mu.     

Gel electrophoretic technique for separating crosslinked RNAs. Application to improved electron microscopic analysis of psoralen crosslinked 16 S ribosomal RNA

We have developed a gel electrophoresis technique for separating crosslinked RNA molecules into a series of discrete fractions. The gel used is polyacrylamide made in formamide and low salt designed to denature the RNA during electrophoresis. The mobility depends upon the position of crosslinking within each molecule, as demonstrated by electron microscopy of RNA eluted from the gel. In general, molecules with large loops electrophorese more slowly than molecules with small loops or uncrosslinked molecules. We have used this technique to re-examine the psoralen crosslinking pattern of Escherichia coli 16 S ribosomal RNA in inactivated 30 S ribosomal subunits. To determine the correct orientation of each type of crosslink, we have covalently attached DNA restriction fragments to the RNA so that the polarity of the RNA in the microscope would be known. Our previous major conclusions are confirmed: the predominant long-distance crosslink detected by gel electrophoresis involves a residue close to the 3′ end and a residue approximately 600 nucleotides away: the formamide/polyacrylamide gel is able to separate two closely spaced 1100-nucleotide interactions beginning close to the 3′ end, which were reported as one interaction before: and an interaction joining the ends is detected as before. However, one low-frequency crosslinked interaction, between positions 950 and 1400, and possibly another low-frequency interaction, between positions 550 and 870, are determined to be in the opposite polarity to that described previously.     

Single molecule DNA sequencing in submicrometer channels: state of the art and future prospects

We demonstrate a new method for single molecule DNA sequencing which is based upon detection and identification of single fluorescently labeled mononucleotide molecules degraded from DNA-strands in a cone shaped microcapillary with an inner diameter of 0.5 microm. The DNA was attached at an optical fiber via streptavidin/biotin binding and placed approximately 50 microm in front of the detection area inside of the microcapillary. The 5'-biotinylated 218-mer model DNA sequence used in the experiments contained 6 fluorescently labeled cytosine and uridine residues, respectively, at well defined positions. The negatively charged mononucleotide molecules were released by addition of exonuclease I and moved towards the detection area by electrokinetic forces. Adsorption of mononucleotide molecules onto the capillary walls as well as the electroosmotic (EOF) flow was prevented by the use of a 3% polyvinyl pyrrolidone (PVP) matrix containing 0.1% Tween 20. For efficient excitation of the labeled mononucleotide molecules a short-pulse diode laser emitting at 638 nm with a repetition rate of 57 MHz was applied. We report on experiments where single-stranded model DNA molecules each containing 6 fluorescently labeled dCTP and dUTP residues were attached at the tip of a fiber, transferred into the microcapillary and degraded by addition of exonuclease I solution. In one experiment, the exonucleolytic cleavage of 5-6 model DNA molecules was observed. 86 photon bursts were detected (43 Cy5-dCMP and 43 MR121-dUMP) during 400 s and identified due to the characteristic fluorescence decay time of the labels of 1.43+/-0.19 ns (Cy5-dCMP), and 2.35+/-0.29 ns (MR121-dUMP). The cleavage rate of exonuclease I on single-stranded labeled DNA molecules was determined to 3-24 Hz under the applied experimental conditions. In addition, the observed burst count rate (signals/s) indicates nonprocessive behavior of exonuclease I on single-stranded labeled DNA.     

Long-range DNA-water interactions

DNA functions only in aqueous environments and adopts different conformations depending on the hydration level. The dynamics of hydration water and hydrated DNA leads to rotating and oscillating dipoles that, in turn, give rise to a strong megahertz to terahertz absorption. Investigating the impact of hydration on DNA dynamics and the spectral features of water molecules influenced by DNA, however, is extremely challenging because of the strong absorption of water in the megahertz to terahertz frequency range. In response, we have employed a high-precision megahertz to terahertz dielectric spectrometer, assisted by molecular dynamics simulations, to investigate the dynamics of water molecules within the hydration shells of DNA as well as the collective vibrational motions of hydrated DNA, which are vital to DNA conformation and functionality. Our results reveal that the dynamics of water molecules in a DNA solution is heterogeneous, exhibiting a hierarchy of four distinct relaxation times ranging from ∼8 ps to 1 ns, and the hydration structure of a DNA chain can extend to as far as ∼18 Å from its surface. The low-frequency collective vibrational modes of hydrated DNA have been identified and found to be sensitive to environmental conditions including temperature and hydration level. The results reveal critical information on hydrated DNA dynamics and DNA-water interfaces, which impact the biochemical functions and reactivity of DNA.     

DNA toroids: stages in condensation.

The effects of polylysine (PLL) and PLL-asialoorosomucoid (AsOR) on DNA condensation have been analyzed by AFM. Different types of condensed DNA structures were observed, which show a sequence of conformational changes as circular plasmid DNA molecules condense progressively. The structures range from circular molecules with the length of the plasmid DNA to small toroids and short rods with approximately 1/6 to 1/8 the contour length of the uncondensed circular DNA. Single plasmid molecules of 6800 base pairs (bp) condense into single toroids of approximately 110 nm diameter, measured center-to-center. The results are consistent with a model for DNA condensation in which circular DNA molecules fold several times into progressively shorter rods. Structures intermediate between toroids and rods suggest that at least some toroids may form by the opening up of rods as proposed by Dunlap et al. [(1997) Nucleic Acids Res. 25, 3095]. Toroids and rods formed at lysine:nucleotide ratios of 5:1 and 6:1. This high lysine:nucleotide ratio is discussed in relation to entropic considerations and the overcharging of macroions. PLL-AsOR is much more effective than PLL alone for condensing DNA, because several PLL molecules are attached to a single AsOR molecule, resulting in an increased cation density.     

S-DNA, over-supercoiled DNA with a 1.94-to 2.19-Å rise per base pair

Supercoiled 3993-bp pGEMEX DNA immobilized on four substrates (freshly cleaved mica, standard amino mica, and modified amino mica with an increased or decreased surface charge density in comparison to standard amino mica) has been visualized by atomic force microscopy in the air. Plectonomically supercoiled DNA molecules, as well as single molecules with an extremely high compaction level (i.e., with a significantly higher superhelix density compared to those previously observed experimentally or estimated theoretically), have been visualized on modified amino mica with an increased surface charge density. The distance between nucleotide pairs along the duplex axis has been determined by measuring the contour length of individual oversupercoiled DNA molecules. The estimated rise per base pair varies from 1.94 to 2.19 Å. These supercoiled DNA molecules, which are compressed like a spring and have a decreased rise per base pair compared to previously known DNA forms are considered to be a new form of DNA, S-DNA. A model of S-DNA has been constructed. Molecules of S-DNA may be an intermediate in the course of the compaction of single supercoiled DNA molecules into spheroids and minitoroids. The DNA oversupercoiling, followed by the compression of the supercoiled molecules, has been shown to be accounted for by a high surface charge density of amino mica on which DNA molecules are immobilized.     

Theory of Low-Frequency Vibrations in DNA Macromolecules

Abstract

To describe low-frequency dynamics of DNA macromolecules a model is developed taking into account the hydrogen bond stretching in base pairs, the backbone flexibility and intranucleoside mobility. For double-stranded DNA the normal vibrations are found and the structure of low- frequency spectrum is determined. The agreement between theory and Raman spectroscopy data for B-DNA is demonstrated. Conformational dependences of vibration spectrum during the B→A and helix→coil DNA transitions are studied. The contribution coming from low-frequency mobility to the nucleic-protein recognition processes is discussed.     

On the mechanism of dielectric relaxation in aqueous DNA solutions.

The complex dielectric response of calf thymus DNA in aqueous saline solutions has been measured from 1 MHz to 1 GHz. The results are presented in terms of the relaxation of the incremental contributions to the permittivity and conductivity from the condensed counterions surrounding the DNA molecules. Measurements of the low-frequency conductivity of the samples also lends support to the condensed counterion interpretation.     

The optical birefringence of DNA solutions induced by oscillatory electric and hydrodynamic fields

The optical birefringence induced in DNA solutions by both oscillating hydrodynamic fields (flow birefringence) and oscillating electric fields (Kerr effect) is measured over a wide frequency range. The observed frequency response of the birefrigence is compared with theories for rigid ellipsoidal particles and for Gaussian chains. DNA at 6 × 10⁵ molecular weight is found to exhibit rigid particle hydrodynamic behavior, while DNA at 5 × 10⁶ molecular weight behaves like a flexible chain. Characterization of the hydrodynamic relaxation spectra for the DNA's by oscillatory flow birefringence allows precise comparison between theory and the experimental Kerr effect response. The dielectric model for DNA contains both permanent and dispersionless induced dipole moments. The dielectric behavior of DNA has the character of a permanent dipole but with anomalous low-frequency dispersion in the Kerr effect. The existing theories do not adequately describe this dispersion. A fluctuation dipole mechanism with relaxation times comparable to those associated with the hydrodynamic motion could possibly demonstrate the observed polar behavior.     

Visualization of novel simian virus 40 DNA recombination intermediates induced by ultraviolet light irradiation.

Electron microscopic technique was used to examine the structures of SV40 DNA recombination intermediates induced by ultraviolet irradiation as an approach for understanding recombination mechanisms in animal cells. Putative recombination intermediate with the characteristic Holliday junction was observed in both SV40 and CV-1 monkey kidney cell DNA. These results suggest that Holliday recombination intermediate is a common intermediate in eukaryotic as well as prokaryotic recombination pathways. In UV irradiated cells, putative SV40 DNA recombination intermediates with multiple recombining partners were observed. In addition, UV irradiation induced two types of novel joint molecules of SV40 DNA. The first type contains replication intermediates as one of the joint molecules with the putative recombination junction located in the newly replicated DNA arms. The second type of novel joint molecules is represented by of the 'dumbbell' structures with two circular SV40 DNA linked by a linear DNA of varying lengths. The structures of these novel recombination intermediates suggest a strand-invasion mechanism for UV-induced DNA recombination.     

Replication of simian virus 40 DNA after UV irradiation: evidence of growing fork blockage and single-stranded gaps in daughter strands.

The molecular mechanisms of in vivo inhibition of mammalian DNA replication by exposure to UV light (at 254 nm) was studied in monkey and human cells infected with simian virus 40. Analysis of viral DNA by electron microscopy and sucrose gradients confirmed that the presence of UV-induced lesions severely blocks DNA synthesis, and thus the conversion of replicative intermediates (RIs) into fully replicated form I DNA is inhibited by UV irradiation. These blocked RI molecules present several special features when visualized by electron microscopy. (i) In excision repair-proficient monkey and human cells they are composed of a double-stranded circular DNA with a double-stranded tail whose size corresponds to the average interpyrimidine dimer distance, as determined by the dimer-specific T4 endonuclease V. (ii) In excision repair-deficient human cells from patients with xeroderma pigmentosum, UV-irradiated RIs present a Cairns-like structure similar to that observed for replicating molecules obtained from unirradiated infected cells. (iii) Single-stranded gaps are visualized in the replicated portions of UV-irradiated RI molecules; such regions are detected and clearly distinguishable from double-stranded DNA when probed by a specific single-stranded DNA-binding protein such as the bacteriophage T4 gene 32 product. Consistent with the presence of gaps in UV-irradiated RI molecules, single-strand-specific S1 nuclease digestion causes a shift in their sedimentation properties when analyzed in neutral sucrose gradients compared with undamaged molecules. These results are in agreement with and reinforce the model in which UV lesions are a barrier to the replication fork movement when present in the template for the leading strand; when lesions are in the template for the lagging strand they inhibit synthesis or completion of Okazaki fragments, leaving gaps opposite the lesion. Moreover, cellular DNA repair-linked endonucleolytic activity may induce double-stranded breaks in the blocked region of the replication forks, resulting in the tailed structures observed in viral DNA molecules obtained from excision repair-proficient cell lines.     

Single-molecule studies reveal dynamics of DNA unwinding by the ring-shaped T7 helicase

Helicases are molecular motors that separate DNA strands for efficient replication of genomes. We probed the kinetics of individual ring-shaped T7 helicase molecules as they unwound double-stranded DNA (dsDNA) or translocated on single-stranded DNA (ssDNA). A distinctive DNA sequence dependence was observed in the unwinding rate that correlated with the local DNA unzipping energy landscape. The unwinding rate increased approximately 10-fold (approaching the ssDNA translocation rate) when a destabilizing force on the DNA fork junction was increased from 5 to 11 pN. These observations reveal a fundamental difference between the mechanisms of ring-shaped and nonring-shaped helicases. The observed force-velocity and sequence dependence are not consistent with a simple passive unwinding model. However, an active unwinding model fully supports the data even though the helicase on its own does not unwind at its optimal rate. This work offers insights into possible ways helicase activity is enhanced by associated proteins.     

Reaction–diffusion wave model for self-assembled network formation of poly(dA)·poly(dT) DNA on mica and HOPG surfaces

Deoxyribonucleic acid (DNA) is a vital molecule for life since it contains genetic information. However, DNA has recently been reported to have unique properties that make it suitable for bionanoelectronic applications, such as the possibility of electrical conductivity and self-organisation. Self-assembled DNA network structures have been observed on several substrates, but the detailed self-assembly mechanism has yet to be determined. The present study investigates self-assembled structures of DNA both theoretically and experimentally. We developed a reaction–diffusion model and used it to investigate pattern formations observed by atomic force microscopy. The computational results qualitatively replicate the network patterns of DNA molecules based on a quantitative agreement with the surface size and timescale. The model can account for the effect of the DNA concentration on pattern formation. Furthermore, peculiar geometric patterns are simulated for mica and highly oriented pyrolytic graphite surfaces.