Changes in DNA superhelical density monitored by polarized light scattering.

Linear and circular lambda-DNA at different ethidium bromide concentrations have been studied by means of polarized light scattering, namely the S14, S34, S33 and S13 elements of Mueller matrix. While S33 at low angle appears well correlated with the total light scattering evaluated by optical density measurements at 632.8 nm for linear and circular DNA of the same mass, the magnitude and slope of the S14, S34 and S13 signals display significant changes for the circular lambda-DNA depending on the degree of negative superhelical density as induced by the different ethidium bromide concentrations. At the same time, for linear lambda-DNA the signal remains invariant, making explicit for the differential scattering of polarized light the possibility to obtain additional information by its angular dependence. Strikingly also the effect of 0.2% glutaraldehyde versus ethanol fixation on the native lambda-DNA structural properties appears to confirm earlier findings by other well-established probes. Results are discussed in terms of first physical principles and of their potential bearings towards our understanding of the mechanism controlling gene expression.     

The bacteriophage lambda int gene product. A filter assay for genetic recombination, purification of int, and specific binding to DNA.

Bacteriophage lambda int gene is required for the integration of viral DNA into the chromosome of Escherichia coli. We have extensively purified the product of the int gene (Int) from a lysogen of E. coli that constitutively expresses this gene. Int was assayed by its ability to promote integrative recombination of supertwisted substrate DNA in vitro using a new method based on filter trapping of a recombinant product DNA. In order to catalyze integrative recombination, Int must be supplemented by other factors that can be extracted from bacterial host cells. By itself, purified Int does not demonstrate detectable endonuclease, exonuclease, or nicking-closing activities. However, Int does make stable complexes with double-stranded lambda-DNA containing an attachment site, the region at which recombination takes place. No stable complexes are observed between Int and lambda-DNA without an attachment site or between Int and DNA containing the bacterial site of integration. Int, therefore, appears to be a specificity element that relies on additional factor(s) to provide or activate the catalytic functions required for recombination.     

Rapid DNA mapping by fluorescent single molecule detection

DNA mapping is an important analytical tool in genomic sequencing, medical diagnostics and pathogen identification. Here we report an optical DNA mapping strategy based on direct imaging of individual DNA molecules and localization of multiple sequence motifs on the molecules. Individual genomic DNA molecules were labeled with fluorescent dyes at specific sequence motifs by the action of nicking endonuclease followed by the incorporation of dye terminators with DNA polymerase. The labeled DNA molecules were then stretched into linear form on a modified glass surface and imaged using total internal reflection fluorescence (TIRF) microscopy. By determining the positions of the fluorescent labels with respect to the DNA backbone, the distribution of the sequence motif recognized by the nicking endonuclease can be established with good accuracy, in a manner similar to reading a barcode. With this approach, we constructed a specific sequence motif map of lambda-DNA. We further demonstrated the capability of this approach to rapidly type a human adenovirus and several strains of human rhinovirus.     

Interactive measurement and characterization of DNA molecules by analysis of AFM images.

BACKGROUND: In the past few years, computer-based analysis of atomic-force microscopic images has acquired increasing importance for studying biomolecules such as DNA. On the one hand, fully automated methods do not allow analysis of complex shapes; on the other hand, manual methods are usually time consuming and inaccurate. The semiautomated approach presented in this report overcomes the drawbacks of both methods. METHODS: Two kinds of images were analyzed: computer-generated filaments that modeled circular DNA molecules on a surface and real atomic-force microscopic images of DNA molecules adsorbed on an appropriate substrate surface. RESULTS: The algorithm was tested on a group of 140 simulated and 189 real plasmids with a nominal length of 913 nm. The accuracy of the length measurement was statistically evaluated on the ensemble of molecules, with particular attention to the influence of the noise. Mean contour lengths of 912 +/- 5 nm and 910 +/- 47 nm were found for simulated and real plasmids, respectively. The measured end-to-end distance of lambda-DNA molecules as a function of their contour length is reported, from which it is possible to estimate the stiffness of the DNA molecules adsorbed onto a surface; the value obtained for the DNA persistence length (42 +/- 5 nm) is consistent with values measured by other imaging techniques. CONCLUSIONS: An interactive algorithm for DNA molecule measurements based on the detection of the filament ridge line in a digitized image is presented. The simulation of artificial filaments combined with the experimental data demonstrates that the proposed method can be a valuable tool for the DNA contour length evaluation, especially in the case of complex shapes where the use of automatic methods is not possible.     

Bacterial conjugative transfer: visualization of successful mating pairs and plasmid establishment in live Escherichia coli

We used the LacO/GFP-LacI system to label and visualize the IncP beta plasmid R751 fluorescently during conjugative transfer between live donor and recipient bacteria. Comparisons of R751 in conjugative and non-conjugative conditions have allowed us to identify key localizations and movements associated with the initiation of conjugative transfer in the donor and the establishment of R751 in the recipient. A survey of successful mating pairs demonstrates that close physical contact between donor and recipient bacteria is required for DNA transfer and that regions of intimate contact can occur at any location on the donor or recipient cell membrane. The transferred DNA is positioned at the characteristic centre or quarter-cell position after conversion to a double-stranded molecule in the recipient cell. Initial duplication of plasmids often results in an asymmetric distribution of plasmid foci. Symmetric localization (either at centre or at 1/4 and 3/4 cell lengths) occurs only after a significant lag, presumably reflecting the time required to synthesize the plasmid-encoded partitioning proteins.     

Physicochemical properties of polymers: An important system to overcome the cell barriers in gene transfection

Delivery of the macromolecules including DNA, miRNA, and antisense oligonucleotides is typically mediated by carriers due to the large size and negative charge. Different physical (e.g., gene gun or electroporation), and chemical (e.g., cationic polymer or lipid) vectors have been already used to improve the efficiency of gene transfer. Polymer‐based DNA delivery systems have attracted special interest, in particular via intravenous injection with many intra‐ and extracellular barriers. The recent progress has shown that stimuli‐responsive polymers entitled as multifunctional nucleic acid vehicles can act to target specific cells. These nonviral carriers are classified by the type of stimulus including reduction potential, pH, and temperature. Generally, the physicochemical characterization of DNA‐polymer complexes is critical to enhance the transfection potency via protection of DNA from nuclease digestion, endosomal escape, and nuclear localization. The successful clinical applications will depend on an exact insight of barriers in gene delivery and development of carriers overcoming these barriers. Consequently, improvement of novel cationic polymers with low toxicity and effective for biomedical use has attracted a great attention in gene therapy. This article summarizes the main physicochemical and biological properties of polyplexes describing their gene transfection behavior, in vitro and in vivo. In this line, the relative efficiencies of various cationic polymers are compared. © 2015 Wiley Periodicals, Inc. Biopolymers 103: 363–375, 2015.     

Prevalence of quadruplexes in the human genome

Guanine-rich DNA sequences of a particular form have the ability to fold into four-stranded structures called G-quadruplexes. In this paper, we present a working rule to predict which primary sequences can form this structure, and describe a search algorithm to identify such sequences in genomic DNA. We count the number of quadruplexes found in the human genome and compare that with the figure predicted by modelling DNA as a Bernoulli stream or as a Markov chain, using windows of various sizes. We demonstrate that the distribution of loop lengths is significantly different from what would be expected in a random case, providing an indication of the number of potentially relevant quadruplex-forming sequences. In particular, we show that there is a significant repression of quadruplexes in the coding strand of exonic regions, which suggests that quadruplex-forming patterns are disfavoured in sequences that will form RNA.     

A sequence-independent study of the influence of short loop lengths on the stability and topology of intramolecular DNA G-quadruplexes

G-Rich sequences found within biologically important regions of the genome have been shown to form intramolecular G-quadruplexes with varied loop lengths and sequences. Many of these quadruplexes will be distinguishable from each other on the basis of their thermodynamic stabilities and folded conformations. It has been proposed that loop lengths can strongly influence the topology and stability of intramolecular G-quadruplexes. Previous studies have been limited to the analysis of quadruplex sequences with particular loop sequences, making it difficult to make generalizations. Here, we describe an original study that aimed to elucidate the effect of loop length on the biophysical properties of G-quadruplexes in a sequence-independent context. We employed UV melting and circular dichroism spectroscopy to examine and compare the properties of 21 DNA quadruplex libraries, each comprising partially randomized loop sequences with lengths ranging from one to three nucleotides. Our work supports a number of general predictions that can be made solely on the basis of loop lengths. In particular, the results emphasize the strong influence of single-nucleotide loops on quadruplex properties. This study provides a predictive framework that may help identify or classify biologically relevant G-quadruplex-forming sequences.     

Single-molecule DNA digestion by lambda-exonuclease.

We used a bead displacement sensor to determine the enzymatic shortening of individual molecules of unstained lambda-DNA attached to optically trapped beads. The setup has been described previously (Dapprich and Nicklaus: Bioimaging 6:25-32, 1998) and works by observing the change in position of a trapped bead depending on its viscous drag force during motion. The drag force of a naked bead increases with each attached DNA molecule to a characteristic level that depends on the length and the number of DNAs per bead. A single undigested DNA molecule on a bead will remain stable for extended periods and exhibit a constant drag force in flow. If lambda-exonuclease is added, the drag force decreases from the level for one strand of DNA on a bead to that of a naked bead in about 45 min. This result indicates that the digestion of native lambda-DNA by lambda-exonuclease occurs at an average rate of approximately 15-20 Hz.     

Pathways and timescales of primary charge separation in the photosystem II reaction center as revealed by a simultaneous fit of time-resolved fluorescence and transient absorption

We model the dynamics of energy transfer and primary charge separation in isolated photosystem II (PSII) reaction centers. Different exciton models with specific site energies of the six core pigments and two peripheral chlorophylls (Chls) in combination with different charge transfer schemes have been compared using a simultaneous fit of the absorption, linear dichroism, circular dichroism, steady-state fluorescence, transient absorption upon different excitation wavelengths, and time-resolved fluorescence. To obtain a quantitative fit of the data we use the modified Redfield theory, with the experimental spectral density including coupling to low-frequency phonons and 48 high-frequency vibrations. The best fit has been obtained with a model implying that the final charge separation occurs via an intermediate state with charge separation within the special pair (RP(1)). This state is weakly dipole-allowed, due to mixing with the exciton states, and can be populated directly or via 100-fs energy transfer from the core-pigments. The RP(1) and next two radical pairs with the electron transfer to the accessory Chl (RP(2)) and to the pheophytin (RP(3)) are characterized by increased electron-phonon coupling and energetic disorder. In the RP(3) state, the hole is delocalized within the special pair, with a predominant localization at the inactive-branch Chl. The intrinsic time constants of electron transfer between the three radical pairs vary from subpicoseconds to several picoseconds (depending on the realization of the disorder). The equilibration between RP(1) and RP(2) is reached within 5 ps at room temperature. During the 5-100-ps period the equilibrated core pigments and radical pairs RP(1) and RP(2) are slowly populated from peripheral chlorophylls and depopulated due to the formation of the third radical pair, RP(3). The effective time constant of the RP(3) formation is 7.5 ps. The calculated dynamics of the pheophytin absorption at 545 nm displays an instantaneous bleach (30% of the total amplitude) followed by a slow increase of the bleaching amplitude with time constants of 15 and 12 ps for blue (662 nm) and red (695 nm) excitation, respectively.     

Genetic map of Salmonella typhimurium, edition VIII.

We present edition VIII of the genetic map of Salmonella typhimurium LT2. We list a total of 1,159 genes, 1,080 of which have been located on the circular chromosome and 29 of which are on pSLT, the 90-kb plasmid usually found in LT2 lines. The remaining 50 genes are not yet mapped. The coordinate system used in this edition is neither minutes of transfer time in conjugation crosses nor units representing "phage lengths" of DNA of the transducing phage P22, as used in earlier editions, but centisomes and kilobases based on physical analysis of the lengths of DNA segments between genes. Some of these lengths have been determined by digestion of DNA by rare-cutting endonucleases and separation of fragments by pulsed-field gel electrophoresis. Other lengths have been determined by analysis of DNA sequences in GenBank. We have constructed StySeq1, which incorporates all Salmonella DNA sequence data known to us. StySeq1 comprises over 548 kb of nonredundant chromosomal genomic sequences, representing 11.4% of the chromosome, which is estimated to be just over 4,800 kb in length. Most of these sequences were assigned locations on the chromosome, in some cases by analogy with mapped Escherichia coli sequences.     

Factors influencing the ability of nuclear localization sequence peptides to enhance nonviral gene delivery

Nonviral gene delivery is limited by inefficient transfer of DNA from the cytoplasm to the nucleus. Nuclear localization sequence (NLS) peptides have been widely used to exploit intracellular transport mechanisms and promote nuclear uptake of DNA. However, the exact conditions to successfully utilize the properties of NLS peptides are still unclear. In the present study a panel of NLS peptides that bind different transport receptors were compared for their ability to enhance nonviral gene transfer. Several factors such as method of incorporating the NLS peptide, type of NLS peptide, DNA morphology, and proper characterization of NLS peptide/DNA conjugates were identified as important considerations in utilizing NLS peptides to enhance gene transfer. In particular, it was shown that a peptide derived from human T cell leukaemia virus type 1 (HTLV) was able to effectively condense DNA into discrete particles and mediate levels of transgene expression up to 32-fold greater than polylysine-based polyplexes. This is the first study to demonstrate efficient transfection mediated by an importin beta-binding peptide based on the HTLV sequence. Promising results were also achieved with a 7-fold increase in gene expression using a NLS peptide/DNA conjugate formed by site-specific linkage of an extended SV40 peptide via a peptide nucleic acid (PNA) clamp. Altogether, the results from this study should help to define the requirements for successful NLS-enhanced transfection.     

The effect of intrinsic curvature on conformational properties of circular DNA.

Both thermal fluctuations and the intrinsic curvature of DNA contribute to conformations of the DNA axis. We looked for a way to estimate the relative contributions of these two components of the double-helix curvature for DNA with a typical sequence. We developed a model and Monte Carlo procedure to simulate the Boltzmann distribution of DNA conformations with a specific intrinsic curvature. Two steps were used to construct the equilibrium conformation of the model chain. We first specified the equilibrium DNA conformation at the base pair level of resolution, using a set of the equilibrium dinucleotide angles and DNA sequence. This conformation was then approximated by the conformation of the model chain consisting of a reduced number of longer, straight cylindrical segments. Each segment of the chain corresponded to a certain number of DNA base pairs. We simulated conformational properties of nicked circular DNA for different sets of equilibrium dinucleotide angles, different random DNA sequences, and lengths. Only random sequences of DNA generated with equal probability of appearance for all types of bases at any site of the sequence were used. The results showed that for a broad range of intrinsic curvature parameters, the radius of gyration of DNA circles should be nearly independent of DNA sequence for all DNA lengths studied. We found, however, a DNA properly that should strongly depend on DNA sequence if the double helix has essential intrinsic curvature. This property is the equilibrium distribution of the linking number for DNA circles that are 300-1000 bp in length. We found that a large fraction of the distributions corresponding to random DNA sequences should have two separate maxima. The physical nature of this unexpected effect is discussed. This finding opens new opportunities for joined experimental and theoretical studies of DNA intrinsic curvature.     

Transient orientation of linear DNA molecules during pulsed-field gel electrophoresis.

The transient orientation of lambda DNA and lambda-DNA oligomers has been measured during pulsed field gel electrophoresis. The DNA becomes substantially aligned parallel to the electric field E. In response to a single rectangular pulse, orientation shows an overshoot with a peak at 1 second, then a small undershoot, and finally a plateau. When the field is turned off, the orientation dissipates in two distinct exponential phases. Field inversion leads to periods of orientation with intervening periods of reduced orientation as the chains reverse direction. Field inversion pulses applied to linear oligomers of lambda-DNA show that orientation responses slow down but increase in amplitude as molecular weight increases, for a given field. Because DNA stretching and alignment parallel to E are expected to correlate with DNA velocity, the velocity in response to a pulsed field is also expected to exhibit an overshoot.     

The Escherichia coli dam DNA methyltransferase modifies DNA in a highly processive reaction

The Escherichia coli dam adenine-N6 methyltransferase modifies DNA at GATC sequences. It is involved in post-replicative mismatch repair, control of DNA replication and gene regulation. We show that E. coli dam acts as a functional monomer and methylates only one strand of the DNA in each binding event. The preferred way of ternary complex assembly is that the enzyme first binds to DNA and then to S-adenosylmethionine. The enzyme methylates an oligonucleotide containing two dam sites and a 879 bp PCR product with four sites in a fully processive reaction. On lambda-DNA comprising 48,502 bp and 116 dam sites, E. coli dam scans 3000 dam sites per binding event in a random walk, that on average leads to a processive methylation of 55 sites. Processive methylation of DNA considerably accelerates DNA methylation. The highly processive mechanism of E. coli dam could explain why small amounts of E. coli dam are able to maintain the methylation state of dam sites during DNA replication. Furthermore, our data support the general rule that solitary DNA methyltransferase modify DNA processively whereas methyltransferases belonging to a restriction-modification system show a distributive mechanism, because processive methylation of DNA would interfere with the biological function of restriction-modification systems.     

Dynamics of charge carrier transfer in a DNA molecule

An equivalent electric circuit has been developed which describes the charge transfer in DNA molecule. A computer simulation of the charge carrier transfer dynamics in the molecule has been performed based on this circuit. It was found that the switching time of a molecular junction lies in the femtosecond range and depends on the frequency of the input electric signal. An increase in the frequency of the input signal in the range from 1 GHz to 4 THz and a reduction of temperature lead to a decrease in the current passing through the DNA molecule. It has been shown that the sequence of the DNA base pairs defines the rate of localization and delocalization of holes and controls the signal propagation rate in the DNA molecule.     

Langevin dynamics simulation of DNA ejection from a phage

We have performed Langevin dynamics simulations of a coarse-grained model of ejection of dsDNA from Φ29 phage. Our simulation results show significant variations in the local ejection speed, consistent with experimental observations reported in the literature for both in vivo and in vitro systems. In efforts to understand the origin of such variations in the local speed of ejection, we have investigated the correlations between the local ejection kinetics and the packaged structures created at various motor forces and chain flexibility. At lower motor forces, the packaged DNA length is shorter with better organization. On the other hand, at higher motor forces typical of realistic situations, the DNA organization inside the capsid suffers from significant orientational disorder, but yet with long orientational correlation times. This in turn leads to lack of registry between the direction of the DNA segments just to be ejected and the direction of exit. As a result, a significant amount of momentum transfer is required locally for successful exit. Consequently, the DNA ejection temporarily slows down exhibiting pauses. This slowing down occurs at random times during the ejection process, completely determined by the particular starting conformation created by prescribed motor forces. In order to augment our inference, we have additionally investigated the ejection of chains with deliberately changed persistence length. For less inflexible chains, the demand on the occurrence of large momentum transfer for successful ejection is weaker, resulting in more uniform ejection kinetics. While being consistent with experimental observations, our results show the nonergodic nature of the ejection kinetics and call for better theoretical models to portray the kinetics of genome ejection from phages.     

Formation of nucleus-like structure in the cytoplasm of lambda-DNA-injected fertilized eggs and its partition into blastomeres during early embryogenesis in Xenopus laevis

The fate of bacteriophage lambda-DNA was examined after injection into the fertilized eggs of Xenopus laevis. Injection of a large amount of lambda-DNA (ca. 24 ng) into a fertilized Xenopus egg induced the formation around the injected DNA of a giant nucleus-like structure which was surrounded by an apparently normal bilayered nuclear membrane with nuclear pore complexes. Southern blot analysis revealed the persistence of injected lambda-DNA until the blastula stage. The nucleus-like structure was partitioned into blastomeres during cleavage through a process of nuclear fission, and was maintained in a group of extraordinarily large blastomeres until the blastula stage.