Toroidal globular state of circular DNA

The influence of torsional elasticity of the double helix on compactization and structure of circular DNA in a compact form is studied in the case when the compact (globular) particle has a torus shape. For closed circular DNA the topological invariant, linking number of two strains, yields strict connection between conformation of double helix, considered as a unifilar homopolymer, and elastic energy of torsional twisting. The contribution of torsional elasticity to free energy of the toruslike globule is calculated. This contribution is shown to be proportional to the square of superturn's density. Torsional elasticity decreases the equilibrium radius of the toruslike globule formed by circular DNA in comparison with the case of linear DNA. Closure of linear DNA into a ring widens the stability range of the relatively short DNA compact form and tightens it for long DNA.     

Mechanics of single kinesin molecules measured by optical trapping nanometry.

We have analyzed the mechanics of individual kinesin molecules by optical trapping nanometry. A kinesin molecule was adsorbed onto a latex bead, which was captured by an optical trap and brought into contact with an axoneme that was bound to a glass surface. The displacement of kinesin during force generation was determined by measuring the position of the beads with nanometer accuracy. As the displacement of kinesin was attenuated because of the compliance of the kinesin-to-bead and kinesin-to-microtubule linkages, the compliance was monitored during force generation and was used to correct the displacement of kinesin. Thus the velocity and the unitary steps could be obtained accurately over a wide force range. The force-velocity curves were linear from 0 to a maximum force at 10 microM and 1 mM ATP, and the maximum force was approximately 7 pN, which is larger by approximately 30% than values previously reported. Kinesin exhibited forward and occasionally backward stepwise displacements with a size of approximately 8 nm. The histograms of step dwell time show a monotonic decrease with time. Model calculations indicate that each kinesin head steps by 16-nm, whereas kinesin molecule steps by 8-nm.     

Direct visualization of supercoiled DNA molecules in solution.

The shape of supercoiled DNA molecules in solution is directly visualized by cryo-electron microscopy of vitrified samples. We observe that: (i) supercoiled DNA molecules in solution adopt an interwound rather than a toroidal form, (ii) the diameter of the interwound superhelix changes from about 12 nm to 4 nm upon addition of magnesium salt to the solution and (iii) the partition of the linking deficit between twist and writhe can be quantitatively determined for individual molecules.     

Sequencing single molecules of DNA

In 2004, the NIH set a remarkable challenge: the 1000 dollars genome. Roughly speaking, success would provide, by 2015, the ability to sequence the complete genome of an individual human, quickly and at an accessible price. An intermediate goal of a 100,000 dollars genome was set for 2010. While the cost of Sanger sequencing has dropped dramatically over the past two decades, it is unlikely that the 100,000 dollars genome will be achieved by this means. New massively parallel technologies will push the cost of sequencing towards this mark, but it is doubtful whether these efforts will match the 1000 dollars goal. The best bets for ultrarapid, low-cost sequencing are single-molecule approaches.     

Selective trapping of circular double-stranded DNA molecules in solidifying agarose

When a solution containing agarose and DNA at 65 °C is allowed to solidify in the well of a preformed gel, it is found that circular DNAs become “trapped” in the newly formed matrix and resist electrophoretic migration. This finding provides an independent method for the characterization of circular DNA. The trapping phenomenon is dependent on the size, conformation, and concentration of circular DNAs as well as on the concentration of agarose. It is demonstrated that this technique can be used as a sensitive assay for detecting circular DNA.     

Mechanical stability of single DNA molecules

Using a modified atomic force microscope (AFM), individual double-stranded (ds) DNA molecules attached to an AFM tip and a gold surface were overstretched, and the mechanical stability of the DNA double helix was investigated. In lambda-phage DNA the previously reported B-S transition at 65 piconewtons (pN) is followed by a second conformational transition, during which the DNA double helix melts into two single strands. Unlike the B-S transition, the melting transition exhibits a pronounced force-loading-rate dependence and a marked hysteresis, characteristic of a nonequilibrium conformational transition. The kinetics of force-induced melting of the double helix, its reannealing kinetics, as well as the influence of ionic strength, temperature, and DNA sequence on the mechanical stability of the double helix were investigated. As expected, the DNA double helix is considerably destabilized under low salt buffer conditions (相似文献   

Stretching of single collapsed DNA molecules

The elastic response of single plasmid and lambda phage DNA molecules was probed using optical tweezers at concentrations of trivalent cations that provoked DNA condensation in bulk. For uncondensed plasmids, the persistence length, P, decreased with increasing spermidine concentration before reaching a limiting value 40 nm. When condensed plasmids were stretched, two types of behavior were observed: a stick-release pattern and a plateau at approximately 20 pN. These behaviors are attributed to unpacking from a condensed structure, such as coiled DNA. Similarly, condensing concentrations of hexaammine cobalt(III) (CoHex) and spermidine induced extensive changes in the low and high force elasticity of lambda DNA. The high force (5-15 pN) entropic elasticity showed worm-like chain (WLC) behavior, with P two- to fivefold lower than in low monovalent salt. At lower forces, a 14-pN plateau abruptly appeared. This corresponds to an intramolecular attraction of 0.083-0.33 kT/bp, consistent with osmotic stress measurements in bulk condensed DNA. The intramolecular attractive force with CoHex is larger than with spermidine, consistent with the greater efficiency with which CoHex condenses DNA in bulk. The transition from WLC behavior to condensation occurs at an extension about 85% of the contour length, permitting looping and nucleation of condensation. Approximately half as many base pairs are required to nucleate collapse in a stretched chain when CoHex is the condensing agent.     

Sequence-dependent mechanics of single DNA molecules

Atomic force microscope-based single-molecule force spectroscopy was employed to measure sequence-dependent mechanical properties of DNA by stretching individual DNA double strands attached between a gold surface and an AFM tip. We discovered that in lambda-phage DNA the previously reported B-S transition, where 'S' represents an overstretched conformation, at 65 pN is followed by a nonequilibrium melting transition at 150 pN. During this transition the DNA is split into single strands that fully recombine upon relaxation. The sequence dependence was investigated in comparative studies with poly(dG-dC) and poly(dA-dT) DNA. Both the B-S and the melting transition occur at significantly lower forces in poly(dA-dT) compared to poly(dG-dC). We made use of the melting transition to prepare single poly(dG-dC) and poly(dA-dT) DNA strands that upon relaxation reannealed into hairpins as a result of their self-complementary sequence. The unzipping of these hairpins directly revealed the base pair-unbinding forces for G-C to be 20 +/- 3 pN and for A-T to be 9 +/- 3 pN.     

Twisting and stretching single DNA molecules

The elastic properties of DNA are essential for its biological function. They control its bending and twisting as well as the induction of structural modifications in the molecule. These can affect its interaction with the cell machinery. The response of a single DNA molecule to a mechanical stress can be precisely determined in single-molecule experiments which give access to an accurate measurement of the elastic parameters of DNA.     

Real-time imaging of single DNA molecules with fluorescence microscopy.

A fluorescence microscopy technique was used to image the dynamics of individual DNA molecules. Lambda, calf thymus, cosmid (circular), and T4 DNA were studied with the fluorescent dye acridine orange. Experiments with DNAase I were conducted, and the results indicate that these observations correspond to DNA molecules. The results of experiments with circular DNA provide strong evidence that these were single DNA molecules. Molecules were observed free in solution or attached to a glass or copper surface at one or several points. The Brownian motion of these molecules was observed, indicating that DNA in solution exists in a partially supercoiled state. Some molecules appeared stretched and were attached to the surface by their termini; the lengths of these molecules were measured. Such molecules also exhibited elastic behavior upon breaking. The power of this technique is demonstrated in images of cosmid DNA molecules, catenanes, and DNA extending from T4 phage particles. These results suggest immediate applications to molecular biology, such as examining the dynamics of protein-DNA interactions. Areas of ongoing research are discussed.     

Direct transfer of Ku between DNA molecules with nonhomologous ends.

The Ku protein is an essential protein for DNA double-strand-break repair by the pathway of nonhomologous DNA end-joining (NHEJ). A previous study showed that Ku bound to one DNA molecule could transfer directly to another DNA molecule without being released into the solution first. Direct transfer requires the two DNA molecules having homologous cohesive ends with a minimum of four complementary bases. Results of this study reveal that direct transfer activity of Ku is regulated by NaCl and MgCl2. Increasing either one of the two cations can decrease the required amount of the other. However, the DNA end-binding activity of Ku is not affected by changing the concentration of the cations, indicating that the two activities are regulated independently. Most importantly, the results also show that Ku can transfer directly from one DNA molecule to another one with nonhomologous ends under the condition of 200 mM NaCl and 5mM MgCl2. The ability of direct transfer between DNAs with nonhomologous ends suggests that Ku can align or juxtapose two DNA ends during NHEJ.     

Estimating friction coefficients of mixed globular/chain molecules, such as protein/DNA complexes.

Existing methods for predicting translational friction properties of complex molecules start by explicitly building up their three-dimensional shape with spherical subunits. This treatment has been used especially for two types of systems: rigid assemblies and flexible chain molecules. However, many protein/DNA complexes such as chromatin consist of a small number of globular, relatively rigid, bound protein interspersed by long stretches of flexible DNA chain. I present a higher level of treatment of such macromolecules that avoids explicit subunit modeling as much as possible. An existing analytical formulation of the hydrodynamics equations is shown to be accurate when used with the present treatment. Thus the approach is fast and can be applied to hydrodynamic studies of highly degenerate multiple equilibria, such as those encountered in problems of the regulation of chromatin structure. I demonstrate the approach by predicting the effect of a hypothetical unwinding process in dinucleosomes and by simulating the distribution of sedimentation coefficients for cooperative and random models for a chromatin saturation process.     

Mechanical denaturation of globular protein in the solid state

A method for taking stress-strain diagrams in microsamples prepared from glutaraldehyde-treated monocrystals and amorphous films of hen egg-white lysozyme has been developed. Analysis of the diagrams has shown that the deformation obeys Hooke's law within 0-2%. Upon further deformation of a crystalline sample (up to 6-10%), when a critical stress, sigma(cr), is reached, the protein molecules in the sample denature and become greatly extended. Depending on the crystal type and crystallographic direction, the sample length increases 2-4 times. The critical stress is essentially dependent on the factors affecting intra- and intermolecular interactions: temperature, hydration level and urea concentration. Mechanisms for mechanical denaturation are proposed.     

Detection of single DNA molecules by multicolor quantum-dot end-labeling

Observation of DNA–protein interactions by single molecule fluorescence microscopy is usually performed by using fluorescent DNA binding agents. However, such dyes have been shown to induce cleavage of the DNA molecule and perturb its interactions with proteins. A new method for the detection of surface-attached DNA molecules by fluorescence microscopy is introduced in this paper. Biotin- and/or digoxigenin-modified DNA fragments are covalently linked at both extremities of a DNA molecule via sequence-specific hybridization and ligation. After the modified DNA molecules have been stretched on a glass surface, their ends are visualized by multicolor fluorescence microscopy using conjugated quantum dots (QD). We demonstrate that under carefully selected conditions, the position and orientation of individual DNA molecules can be inferred with good efficiency from the QD fluorescence signals alone. This is achieved by selecting QD pairs that have the distance and direction expected for the combed DNA molecules. Direct observation of single DNA molecules in the absence of DNA staining agent opens new possibilities in the fundamental study of DNA–protein interactions. This work also documents new possibilities regarding the use of QD for nucleic acid detection and analysis.     

Direct, genome-wide assessment of DNA mutations in single cells

DNA mutations are the inevitable consequences of errors that arise during replication and repair of DNA damage. Because of their random and infrequent occurrence, quantification and characterization of DNA mutations in the genome of somatic cells has been difficult. Random, low-abundance mutations are currently inaccessible by standard high-throughput sequencing approaches because they cannot be distinguished from sequencing errors. One way to circumvent this problem and simultaneously account for the mutational heterogeneity within tissues is whole genome sequencing of a representative number of single cells. Here, we show elevated mutation levels in single cells from Drosophila melanogaster S2 and mouse embryonic fibroblast populations after treatment with the powerful mutagen N-ethyl-N-nitrosourea. This method can be applied as a direct measure of exposure to mutagenic agents and for assessing genotypic heterogeneity within tissues or cell populations.     

Dynamics of protamine 1 binding to single DNA molecules

Protamine molecules bind to and condense DNA in the sperm of most vertebrates, packaging the sperm genome in an inactive state until it can be reactivated following fertilization. By using methods that enable the analysis of protamine binding to individual DNA molecules, we have monitored the kinetics of DNA condensation and decondensation by protamine 1 (P1) and synthetic peptides corresponding to specific segments of the bull P1 DNA binding domain. Our results show that the number of clustered arginine residues present in the DNA binding domain is the most important factor affecting the condensation and stability of the DNA-protamine complex prior to the formation of inter-protamine disulfide cross-links. The high affinity of P1 for DNA is achieved by the coordinated binding of three anchoring domains, which together in bull P1 contain 19 Arg residues. The single DNA molecule experiments show that sequences containing two or more anchoring domains have an off-rate that is at least 3 orders of magnitude slower than those containing a single domain. The use of Arg, rather than Lys residues, and the inclusion of Tyr or Phe residues in the hinge regions between anchoring domains provide additional stability to the complex.     

An integrative approach for the optical sequencing of single DNA molecules

A new approach for optically sequencing ensembles of single DNA molecules using DNA polymerase to mediate the consecutive incorporation of fluorochrome-labeled nucleotides into an array of large single DNA molecules is presented. The approach utilizes cycles of labeled fluorochrome addition, detection to count incorporations, and bleaching to reset the counter. These additions are imaged and analyzed to estimate the number of labeled additions and to correlate them on a per-locus basis along DNA backbones. Initial studies used precisely labeled polymerase chain reaction products to aid the development and validation of simple models of fluorochrome point spread functions within the imaging system. In complementary studies, nucleotides labeled with the fluorochrome R110 were incorporated into surface-elongated lambda DNA, and fluorescent signals corresponding to the addition of R110-dUTP were counted and assigned precise loci along DNA backbones. The labeled DNAs were then subjected to photobleaching and to a second cycle of addition of R110-labeled nucleotides-a second round of additions was correlated with the first to establish strings of addition histories among the ensemble of largely double-stranded templates. These results confirm the basic operational validity of this approach and point the way to the development of a practical system for optical sequencing.