Bent DNA Molecules Research Articles

Mechanical-energy-based amplifiers for probing interactions of DNA with metal ions

We report our development of a simple and cost-effective method to amplify and probe the interactions of DNA with metal ions, which are important for various fundamental processes in living systems. This method is based on perturbing energy landscapes using mechanical energy stored in bent DNA molecules. In this proof-of-principle study, the mechanical-energy-based amplifiers were applied to examine the interactions between DNA and ${\mathrm{Mg}}^{2+}$ ions or ${\mathrm{Ag}}^{+}$ ions. We demonstrated that interactions between DNA and ${\mathrm{Mg}}^{2+}$ or ${\mathrm{Ag}}^{+}$ ions, which are not detectable using gel electrophoresis without amplification, can be easily measured using our molecular amplifiers. In addition, we showed that quantitative details about the DNA-metal interactions can be estimated using our method. Our method is simple, sensitive, and cost-effective. We expect that the developed method will be useful for various applications.

Recent progress on the mechanics of sharply bent DNA

Despite extensive studies on the mechanics of DNA under external constrains, such as tension, torsion, and bending, several important aspects have remained poorly understood. One biologically important example is the mechanics of DNA under sharp bending conditions, which has been debated for a decade without thorough comprehension. The debate is about the interesting phenomenon raised from a series of different experiments: sharply bent DNA has a surprisingly high apparent bending flexibility that deviates from the canonical bending elasticity of DNA. This finding has motivated various theoretical models, which mainly incorporate the excitation of mechanical defects inside severely bent DNA molecules. Here, we review the recent progress on the understanding of the mechanics of sharply bent DNA and provide our view on this important question by interrogating the theoretical foundation of these experimental measurements.

Target Capture during Mos1 Transposition

DNA transposition contributes to genomic plasticity. Target capture is a key step in the transposition process, because it contributes to the selection of new insertion sites. Nothing or little is known about how eukaryotic mariner DNA transposons trigger this step. In the case of Mos1, biochemistry and crystallography have deciphered several inverted terminal repeat-transposase complexes that are intermediates during transposition. However, the target capture complex is still unknown. Here, we show that the preintegration complex (i.e., the excised transposon) is the only complex able to capture a target DNA. Mos1 transposase does not support target commitment, which has been proposed to explain Mos1 random genomic integrations within host genomes. We demonstrate that the TA dinucleotide used as the target is crucial both to target recognition and in the chemistry of the strand transfer reaction. Bent DNA molecules are better targets for the capture when the target DNA is nicked two nucleotides apart from the TA. They improve strand transfer when the target DNA contains a mismatch near the TA dinucleotide.

Winding single-molecule double-stranded DNA on a nanometer-sized reel

A molecular system of a nanometer-sized reel was developed from F1–ATPase, a rotary motor protein. By combination with magnetic tweezers and optical tweezers, single-molecule double-stranded DNA (dsDNA) was wound around the molecular reel. The bending stiffness of dsDNA was determined from the winding tension (0.9–6.0 pN) and the diameter of the wound loop (21.4–8.5 nm). Our results were in good agreement with the conventional worm-like chain model and a persistence length of 54 ± 9 nm was estimated. This molecular reel system offers a new platform for single-molecule study of micromechanics of sharply bent DNA molecules and is expected to be applicable to the elucidation of the molecular mechanism of DNA-associating proteins on sharply bent DNA strands.

Dissection of the DNA Mimicry of the Bacteriophage T7 Ocr Protein using Chemical Modification

The homodimeric Ocr (overcome classical restriction) protein of bacteriophage T7 is a molecular mimic of double-stranded DNA and a highly effective competitive inhibitor of the bacterial type I restriction/modification system. The surface of Ocr is replete with acidic residues that mimic the phosphate backbone of DNA. In addition, Ocr also mimics the overall dimensions of a bent 24-bp DNA molecule. In this study, we attempted to delineate these two mechanisms of DNA mimicry by chemically modifying the negative charges on the Ocr surface. Our analysis reveals that removal of about 46% of the carboxylate groups per Ocr monomer results in an ∼50-fold reduction in binding affinity for a methyltransferase from a model type I restriction/modification system. The reduced affinity between Ocr with this degree of modification and the methyltransferase is comparable with the affinity of DNA for the methyltransferase. Additional modification to remove ∼86% of the carboxylate groups further reduces its binding affinity, although the modified Ocr still binds to the methyltransferase via a mechanism attributable to the shape mimicry of a bent DNA molecule. Our results show that the electrostatic mimicry of Ocr increases the binding affinity for its target enzyme by up to ∼800-fold.

Defect Excitation In Sharply Bent Dna And Its Micromechanical Consequences

Kinkable defect excitation in sharply bent DNA molecules have drawn attentions in recent years. Such excitations were recently observed in molecular dynamics simulations, and were experimentally observed in circular DNAs of 64 - 65 base pairs. In addition, it was shown that rare excitation of flexible defect provided an explanation to the unusually large cyclization probability of 94 bp DNA reported recently. In this presentation, we show that defect excitation also explains a few other experiments where unusual DNA mechanical responses were observed, and we present our predictions of other measurable DNA mechanical responses for future experiments. In addition, we provide a new experimental evidence demonstrating the breakdown of the traditional WLC model when DNA is sharply bent. Finally, we discuss the molecular details of the possible defects based on our molecular dynamics simulations.

Dynamics of Bent Molecules in Gels

A quantitative predictive model of the electrophoretic mobility of a bent molecule of cylindrical symmetry relative to a straight one in gel is described. The force acting on the surface of the molecule is shown to be the minimum value of a variational functional of the fluid velocities whose minimizer satisfies the large viscosity solution of the incompressible Brinkman−Navier−Stokes equations. By exploiting the variational approach, our model readily predicts, in agreement with experimental data, that the electrophoretic mobility of the molecule bent at the middle is smaller than that of the same molecule with a bend at the end. We apply the methodology to describe the electrophoretic mobility of intrinsically bent DNA molecules in polyacrylamide gels. The predictions are compared with available experimental data as well as a semiempirical relation between the relative electrophoretic mobility and the bend angle by Thompson and Landy.

Gapped DNA is anisotropically bent.

Ionizing radiation damages DNA in several ways, including through formation of a single-nucleoside gap in one DNA strand. We have developed a two-dimensional gel electrophoresis method to investigate the effect of a strand gap on DNA structure. We generate a library of gapped DNA molecules by treating a DNA restriction fragment with the hydroxyl radical, generated by the reaction of Fe(II) EDTA with hydrogen peroxide. The DNA molecule studied contains a fixed bend produced by a set of phased adenine tracts. The A-tract bend serves as a reference bend for investigating the conformational nature of a strand gap. In the first electrophoretic dimension, a bent DNA molecule that has been treated with the hydroxyl radical is electrophoresed on a native gel. Smearing of the band on the native gel indicates that the library of gapped DNA molecules contains a variety of DNA conformations. In the second electrophoretic dimension, gapped DNA molecules having different native gel mobilities are electrophoresed on separate lanes of a denaturing gel to reveal how each strand gap affects the native gel mobility (and thus shape) of the DNA. Our results demonstrate that a single-nucleoside gap in a DNA duplex leads to an anisotropic, directional bend in the DNA helix axis. The implications of our findings for recognition of this lesion by DNA repair proteins are discussed.

Plasmid promiscuity: meeting the challenge of DNA immigration control.

Plasmid promiscuity: meeting the challenge of DNA immigration control.

Structure of Ocr from Bacteriophage T7, a Protein that Mimics B-Form DNA

We have solved, by X-ray crystallography to a resolution of 1.8 Å, the structure of a protein capable of mimicking approximately 20 base pairs of B-form DNA. This ocr protein, encoded by gene 0.3 of bacteriophage T7, mimics the size and shape of a bent DNA molecule and the arrangement of negative charges along the phosphate backbone of B-form DNA. We also demonstrate that ocr is an efficient inhibitor in vivo of all known families of the complex type I DNA restriction enzymes. Using atomic force microscopy, we have also observed that type I enzymes induce a bend in DNA of similar magnitude to the bend in the ocr molecule. This first structure of an antirestriction protein demonstrates the construction of structural mimetics of long segments of B-form DNA.

Sequence-specific High Mobility Group Box Factors Recognize 10–12-Base Pair Minor Groove Motifs

Sequence-specific High Mobility Group Box Factors Recognize 10–12-Base Pair Minor Groove Motifs

Cloning of a regulatory gene fromStreptomyces cattleya and study on itscis-acting element

Primers were designed from the highly conserved protein regions of luxl, one of the two component regulatory genes in prokaryotic, and an about 180 bp DNA from Streptomyces cattleya was amplified by PCR. Using this fragment as a probe, Southern hybridization was accomplished with chromosome DNA of S. cattleya, leading to the cloning of the foreign DNA into pBluescript (SK-). DNA sequencing and frame analysis showed an intact ORF consisting of 1800 bp; the G+C composition was 69.3%. Upstream from the ORF was a 249 bp AT rich region, downstream of the ORF several complicated secondary structures were found. DNA-binding assay by gel retardation demonstrated the presence of a protein that specifically bound to the 249 bp AT rich region. The region also showed anomalous electrophoretic mobility at low temperature, like that of a bent DNA molecule, indicating that the region contains a cis-acting element.

Mapping a Protein-Binding Site on Straightened DNA by Atomic Force Microscopy

We have developed an Atomic Force Microscopy (AFM)-based method for mapping protein-binding sites on individual, long DNA molecules (>5 kb) at nanometer resolution. The protein is clearly detected at the apex of the bent DNA molecules. Randomly coiled DNA molecules or protein:DNA complexes were extended by a motor-controlled moving meniscus on an atomically flat surface. The immobilized molecules were detected by AFM. The straightened DNA displayed a sharp bend at the site of bound protein with the two DNA segments linearly extending from the protein-binding site. Using GAL4, a yeast transcription factor, we demonstrate good agreement of the position of the observed binding site on straightened DNA templates to the predicted binding site. The technique is expected to have significant implications in elucidating DNA and protein interactions in general, and specifically, for the measurement of promoter occupancy with unlabeled regulatory proteins at the single-molecule level.

1] DNA bending, flexibility, and helical repeat by cyclization kinetics

Publisher Summary This chapter discusses experimental approaches to the measurement of ring closure probability ( J factor or Jacobson–Stockmayer factor) and the theory used to analyze ring closure, and thereby obtain fundamental physical information from the J factor. It illustrates these procedures with examples from the cyclization of bent DNA molecules (A tract multimers) and recent work on the application of cyclization methods to protein–DNA complexes. Cyclization kinetics is unusual among molecular biological techniques in its requirement for computation of the properties of models to interpret the experimental results in anything more than a highly qualitative way. The theory of cyclization kinetics can also be applied to other systems where DNA assumes conformations and where its ends are fixed in a defined spatial orientation. In vivo and in vitro results on lac repressor-mediated DNA loops have been interpreted using the cyclization paradigm, and the enhancement of cyclization has been used to provide evidence for looping. The chapter also highlights the analysis of topoisomer distributions established by ligation under various conditions that can provide complementary information to that available from cyclization kinetics.

Intrinsically bent DNA.

Intrinsically bent or curved DNA molecules result when special base sequences or structural motifs are repeated in phase with the DNA helical repeat (~10.5 bp’/turn). This places the recurrent elements along the same side of the double helix so that small bends associated with them add constructively to generate a large global curvature. Many base sequences can impart systematic curvature to DNA, but most such bends are small compared with the special effect produced by runs of homopolymeric dA.dT base pairs (“Atracts”), each tract about half a helical turn long and repeated at lo-11-bp intervals. Bent DNA was discovered by the laboratories of Englund and Crothers (1) during study of a minicircle DNA from the kinetoplast body of Leishmania tarentolae, in which appropriately phased A-tracts occur prominently (1, 2). The anomalous gel mobilities shown by bent DNA molecules have provided crucial experimental insight into the origin and nature of DNA bending. Application of early theories describing gel mobilities for molecules undergoing reptational migration (3,4) leads one to expect slower motion for bent molecules because they have a shortened end-to-end distance. This criterion has proved to be a reliable guide to experimental properties, even though it ignores significant theoretical issues 6). A critical experiment for diagnosing the presence and location of DNA bends has been comparison of the electrophoretie properties of circularly permuted DNA molecules, all of the same length but each having the bend at a different position. In the first application of this concept, Wu and Crothers (6) were able to identify a locus containing phased dA.dT tracts which provided the primary source of bending in Leishmaniu kinetoplast minicircle DNA. Since that time, phased dA.dT tracts have been identified in various gene regulatory regions (7-lo), but the biological function of intrinsically bent DNA remains uncertain.

Presence of female-specific bent-repetitive DNA sequences in the genomes of turkey and pheasant and their interactions with W-protein of chicken.

Two female-specific repetitive DNA units, the 0.4 kb PstI and 0.5 kb TaqI sequences, were detected in the genomic DNA of turkey and pheasant, respectively, by Southern blot hybridization under non-stringent conditions with the W chromosome-specific 0.7 kb XhoI repetitive unit of chicken as a probe. Cloning and sequencing of these two repetitive units revealed that they shared features with the XhoI family repetitive unit of chicken although the overall similarities of the nucleotide sequences were less than 60%. In common with the chicken XhoI family they consisted of tandem repeats of about 21 bp, the majority of which contained (A)3-5 and (T)3-5 clusters separated by six or seven relatively G + C-rich sequences, and they behaved as bent DNA molecules on polyacrylamide gel electrophoresis at room temperature. W-protein, purified from chicken liver nuclei and shown to bind with high affinity to the XhoI family repetitive unit, also bound with the cloned repetitive units from turkey and pheasant DNase I footprint analysis suggested that the mode of interaction of W-protein with these units was similar to that with the 0.7 kb XhoI sequence. On the other hand, W-protein did not bind to the female-specific 0.4 kb BamHI repetitive unit from the Bobwhite quail. The 0.4 kb BamHI sequence contained some A and T clusters but these clusters did not appear in phase with the pitch of DNA helix and the repetitive unit did not show DNA bending.

Gel electrophoretic analysis of the geometry of a DNA four-way junction

Branched DNA molecules (Holliday structures) are believed to be key intermediates in the process of homologous genetic recombination. However, despite the importance of such structures, their transient nature makes it difficult to analyze their physical properties. In an effort to evaluate several models for the geometry of such branched molecules, a stable, synthetic DNA four-way junction has been constructed. The geometry of the synthetic junction has been probed by gel electrophoresis, utilizing the fact that bent DNA molecules demonstrate reduced mobilities on polyacrylamide gels to an extent that varies with the degree of the bend angle. From the synthetic four-way junction, we have produced a set of molecules in which all combinations of two junction arms have been extended by 105 base-pairs. The electrophoretic mobilities of the extended junctions differ in a manner which indicates that the junction is not a completely flexible structure; nor is it tetrahedral or planar-tetragonal. Instead, the four strands that comprise the DNA four-way junction are structurally non-equivalent. The significance of these observations with regard to previous models for four-way junction geometry is discussed.

Ring closure probabilities for DNA fragments by Monte Carlo simulation

The rate of ligation of DNA molecules into circular forms depends on the ring closure probability, commonly called the j-factor, which is a sensitive measure of the extent to which thermal fluctuations contribute to bending and twisting of DNA molecules in solution. We present a theoretical treatment of the cyclization equilibria of DNA that employs a special Monte Carlo method for generating large ensembles of model DNA chains. Using this method, the chain length dependence of the j-factor was calculated for molecules in the size range 250 to 2000 base-pairs. The Monte Carlo results are compared with recent analytical theory and experimental data. We show that a value of 475 Å for the persistence length of DNA, close to values measured by a number of other methods, is in excellent agreement with the cyclization results. Preliminary applications of the Monte Carlo method to the problem of systematically bent DNA molecules are presented. The calculated j-factor is shown to be very sensitive to the amount of bending in these fragments. This fact suggests that ligase closure measurements of systematically bent DNA molecules should be a useful method for studying sequence-directed bending in DNA.

Possibility of nonkinked packing of DNA in chromatin.

The idea that DNA in chromatin can be packed smoothly, without breaking of base-stacking interactions, has been explored by both energetical estimations and stereochemical model building. A model of deformed DNA is built that fits reasonably to the known dimensions of a nucleosome. The model has slightly changed torsion angles in the sugarphosphate moieties relative to B DNA, varying gradually along the chains. The angle between planes of adjacent base pairs varies between 1 and 7 degrees. This model of deformed DNA does not have any unusually close nonbonded contacts and is evidently not the only possible model of smooth packing of DNA in chromatin. An energetical estimation of the critical radius of curvature of a smoothly bent DNA molecule is made using approximate potential functions for different van der Waals contracts in the B DNA structure. The critical radius of curvature of the deformed DNA-axis is close to the radius of a nucleosome (similar to 50 A). The smooth packing is a good alternative to models of kinked folding of DNA in nucleosomes.