Escherichia coli low-copy-number plasmid R1 centromere parC forms a U-shaped complex with its binding protein ParR

The Escherichia coli low-copy-number plasmid R1 contains a segregation machinery composed of parC, ParR and parM. The R1 centromere-like site parC contains two separate sets of repeats. By atomic force microscopy (AFM) we show here that ParR molecules bind to each of the 5-fold repeated iterons separately with the intervening sequence unbound by ParR. The two ParR protein complexes on parC do not complex with each other. ParR binds with a stoichiometry of about one ParR dimer per each single iteron. The measured DNA fragment lengths agreed with B-form DNA and each of the two parC 5-fold interon DNA stretches adopts a linear path in its complex with ParR. However, the overall parC/ParR complex with both iteron repeats bound by ParR forms an overall U-shaped structure: the DNA folds back on itself nearly completely, including an angle of ∼150°. Analysing linear DNA fragments, we never observed dimerized ParR complexes on one parC DNA molecule (intramolecular) nor a dimerization between ParR complexes bound to two different parC DNA molecules (intermolecular). This bacterial segrosome is compared to other bacterial segregation complexes. We speculate that partition complexes might have a similar overall structural organization and, at least in part, common functional properties

Organisation of nucleosomal arrays reconstituted with repetitive African green monkey α-satellite DNA as analysed by atomic force microscopy

Alpha-satellite DNA (AS) is part of centromeric DNA and could be relevant for centromeric chromatin structure: its repetitive character may generate a specifically ordered nucleosomal arrangement and thereby facilitate kinetochore protein binding and chromatin condensation. Although nucleosomal positioning on some satellite sequences had been shown, including AS from African green monkey (AGM), the sequence-dependent nucleosomal organisation of repetitive AS of this species has so far not been analysed. We therefore studied the positioning of reconstituted nucleosomes on AGM AS tandemly repeated DNA. Enzymatic analysis of nucleosome arrays formed on an AS heptamer as well as the localisation of mononucleosomes on an AS dimer by atomic force microscopy (AFM) showed one major positioning frame, in agreement with earlier results. The occupancy of this site was in the range of 45–50%, in quite good agreement with published in vivo observations. AFM measurements of internucleosomal distances formed on the heptamer indicated that the nucleosomal arrangement is governed by sequence-specific DNA-histone interactions yielding defined internucleosomal distances, which, nevertheless, are not compatible with a uniform phasing of the nucleosomes with the AGM AS repeats

Filamentous Biopolymers on Surfaces: Atomic Force Microscopy Images Compared with Brownian Dynamics Simulation of Filament Deposition

Nanomechanical properties of filamentous biopolymers, such as the persistence length, may be determined from two-dimensional images of molecules immobilized on surfaces. For a single filament in solution, two principal adsorption scenarios are possible. Both scenarios depend primarly on the interaction strength between the filament and the support: i) For interactions in the range of the thermal energy, the filament can freely equilibrate on the surface during adsorption; ii) For interactions much stronger than the thermal energy, the filament will be captured by the surface without having equilibrated. Such a ‘trapping’ mechanism leads to more condensed filament images and hence to a smaller value for the apparent persistence length. To understand the capture mechanism in more detail we have performed Brownian dynamics simulations of relatively short filaments by taking the two extreme scenarios into account. We then compared these ‘ideal’ adsorption scenarios with observed images of immobilized vimentin intermediate filaments on different surfaces. We found a good agreement between the contours of the deposited vimentin filaments on mica (‘ideal’ trapping) and on glass (‘ideal’ equilibrated) with our simulations. Based on these data, we have developed a strategy to reliably extract the persistence length of short worm-like chain fragments or network forming filaments with unknown polymer-surface interactions

Enzymatic Digestion of Single DNA Molecules Anchored on Nanogold-Modified Surfaces

To study enzyme–DNA interactions at single molecular level, both the attachment points and the immediate surroundings of surfaces must be carefully considered such that they do not compromise the structural information and biological properties of the sample under investigation. The present work demonstrates the feasibility of enzymatic digestion of single DNA molecules attached to nanoparticle-modified surfaces. With Nanogold linking DNA to the mica surface by electrostatic interactions, advantageous conditions with fewer effects on the length and topography of DNA are obtained, and an appropriate environment for the activities of DNA is created. We demonstrate that by using Dip-Pen Nanolithography, individual DNA molecules attached to modified mica surfaces can be efficiently digested by DNase I

Mechanics of the IL2RA Gene Activation Revealed by Modeling and Atomic Force Microscopy

Transcription implies recruitment of RNA polymerase II and transcription factors (TFs) by DNA melting near transcription start site (TSS). Combining atomic force microscopy and computer modeling, we investigate the structural and dynamical properties of the IL2RA promoter and identify an intrinsically negative supercoil in the PRRII region (containing Elf-1 and HMGA1 binding sites), located upstream of a curved DNA region encompassing TSS. Conformational changes, evidenced by time-lapse studies, result in the progressive positioning of curvature apex towards the TSS, likely facilitating local DNA melting. In vitro assays confirm specific binding of the General Transcription Factors (GTFs) TBP and TFIIB over TATA-TSS position, where an inhibitory nucleosome prevented preinitiation complex (PIC) formation and uncontrolled DNA melting. These findings represent a substantial advance showing, first, that the structural properties of the IL2RA promoter are encoded in the DNA sequence and second, that during the initiation process DNA conformation is dynamic and not static

The Role of Histone H4 Biotinylation in the Structure of Nucleosomes

Background: Post-translational modifications of histones play important roles in regulating nucleosome structure and gene transcription. It has been shown that biotinylation of histone H4 at lysine-12 in histone H4 (K12Bio-H4) is associated with repression of a number of genes. We hypothesized that biotinylation modifies the physical structure of nucleosomes, and that biotin-induced conformational changes contribute to gene silencing associated with histone biotinylation. Methodology/Principal Findings: To test this hypothesis we used atomic force microscopy to directly analyze structures of nucleosomes formed with biotin-modified and non-modified H4. The analysis of the AFM images revealed a 13% increase in the length of DNA wrapped around the histone core in nucleosomes with biotinylated H4. This statistically significant (p,0.001) difference between native and biotinylated nucleosomes corresponds to adding approximately 20 bp to the classical 147 bp length of nucleosomal DNA. Conclusions/Significance: The increase in nucleosomal DNA length is predicted to stabilize the association of DNA with histones and therefore to prevent nucleosomes from unwrapping. This provides a mechanistic explanation for the gene silencing associated with K12Bio-H4. The proposed single-molecule AFM approach will be instrumental for studying the effects of various epigenetic modifications of nucleosomes, in addition to biotinylation

Protein/DNA interactions in complex DNA topologies: expect the unexpected

DNA supercoiling results in compacted DNA structures that can bring distal sites into close proximity. It also changes the local structure of the DNA, which can in turn influence the way it is recognised by drugs, other nucleic acids and proteins. Here, we discuss how DNA supercoiling and the formation of complex DNA topologies can affect the thermodynamics of DNA recognition. We then speculate on the implications for transcriptional control and the three-dimensional organisation of the genetic material, using examples from our own simulations and from the literature. We introduce and discuss the concept of coupling between the multiple length-scales associated with hierarchical nuclear structural organisation through DNA supercoiling and topology

Do Femtonewton Forces Affect Genetic Function? A Review

Protein-Mediated DNA looping is intricately related to gene expression. Therefore any mechanical constraint that disrupts loop formation can play a significant role in gene regulation. Polymer physics models predict that less than a piconewton of force may be sufficient to prevent the formation of DNA loops. Thus, it appears that tension can act as a molecular switch that controls the much larger forces associated with the processive motion of RNA polymerase. Since RNAP can exert forces over 20 pN before it stalls, a ‘substrate tension switch’ could offer a force advantage of two orders of magnitude. Evidence for such a mechanism is seen in recent in vitro micromanipulation experiments. In this article we provide new perspective on existing theory and experimental data on DNA looping in vitro and in vivo . We elaborate on the connection between tension and a variety of other intracellular mechanical constraints including sequence specific curvature and supercoiling. In the process, we emphasize that the richness and versatility of DNA mechanics opens up a whole new paradigm of gene regulation to explore.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/41816/1/10867_2005_Article_9002.pd

Sequence-specific physical properties of African green monkey alpha-satellite DNA contribute to centromeric heterochromatin formation

Satellite DNA, a major component of eukaryotic centromeric heterochromatin, is potentially associated with the processes ensuring the faithful segregation of the genetic material during cell division. Structural properties of alpha-satellite DNA (AS) from African green monkey (AGM) were studied. Atomic force microscopy imaging showed smaller end-to-end distances of AS fragments than would be expected for the persistence length of random sequence DNA. The apparent persistence length of the AS was determined as 35 nm. Gel-electrophoresis indicated only a weak contribution of intrinsic curvature to the DNA conformations suggesting an additional contribution of an elevated bending flexibility to the reduced end-to-end distances. Next, the force-extension behavior of the naked AS and in complex with nucleosomes was studied using optical tweezers. The naked AS showed a reduced overstretching transition force (−18% the value determined for random DNA) and higher forces required to straighten the DNA. Finally, reconstituted AS nucleosomes disrupted at significantly higher forces as compared with random DNA nucleosomes which is probably due to structural properties of the AS which stabilize the nucleosomes. The data support that the AS plays a role in the formation of centromeric heterochromatin due to specific structural properties and suggest that a relatively higher mechanical stability of nucleosomes is important in AGM–AS chromatin