125 research outputs found
Modeling Bacterial DNA: Simulation of Self-avoiding Supercoiled Worm-Like Chains Including Structural Transitions of the Helix
Under supercoiling constraints, naked DNA, such as a large part of bacterial
DNA, folds into braided structures called plectonemes. The double-helix can
also undergo local structural transitions, leading to the formation of
denaturation bubbles and other alternative structures. Various polymer models
have been developed to capture these properties, with Monte-Carlo (MC)
approaches dedicated to the inference of thermodynamic properties. In this
chapter, we explain how to perform such Monte-Carlo simulations, following two
objectives. On one hand, we present the self-avoiding supercoiled Worm-Like
Chain (ssWLC) model, which is known to capture the folding properties of
supercoiled DNA, and provide a detailed explanation of a standard MC simulation
method. On the other hand, we explain how to extend this ssWLC model to include
structural transitions of the helix.Comment: Book chapter to appear in The Bacterial Nucleoid, Methods and
Protocols, Springer serie
Force spectroscopy in studying infection
Biophysical force spectroscopy tools - for example optical tweezers, magnetic
tweezers, atomic force microscopy, - have been used to study elastic,
mechanical, conformational and dynamic properties of single biological
specimens from single proteins to whole cells to reveal information not
accessible by ensemble average methods such as X-ray crystallography, mass
spectroscopy, gel electrophoresis and so on. Here we review the application of
these tools on a range of infection-related questions from antibody-inhibited
protein processivity to virus-cell adhesion. In each case we focus on how the
instrumental design tailored to the biological system in question translates
into the functionality suitable for that particular study. The unique insights
that force spectroscopy has gained to complement knowledge learned through
population averaging techniques in interrogating biomolecular details prove to
be instrumental in therapeutic innovations such as those in structure-based
drug design
Direct Observation of Strand Passage by DNA-Topoisomerase and Its Limited Processivity
Type-II DNA topoisomerases resolve DNA entanglements such as supercoils, knots and catenanes by passing one segment of DNA duplex through a transient enzyme-bridged double-stranded break in another segment. The ATP-dependent passage reaction has previously been demonstrated at the single-molecule level, showing apparent processivity at saturating ATP. Here we directly observed the strand passage by human topoisomerase IIα, after winding a pair of fluorescently stained DNA molecules with optical tweezers for 30 turns into an X-shaped braid. On average 0.51±0.33 µm (11±6 turns) of a braid was unlinked in a burst of reactions taking 8±4 s, the unlinked length being essentially independent of the enzyme concentration between 0.25–37 pM. The time elapsed before the start of processive unlinking decreased with the enzyme concentration, being ∼100 s at 3.7 pM. These results are consistent with a scenario where the enzyme binds to one DNA for a period of ∼10 s, waiting for multiple diffusional encounters with the other DNA to transport it across the break ∼10 times, and then dissociates from the binding site without waiting for the exhaustion of transportable DNA segments
Reconstruction of bacterial transcription-coupled repair at single-molecule resolution
International audienc
Spatial and topological organization of DNA chains induced by gene co-localization
Transcriptional activity has been shown to relate to the organization of
chromosomes in the eukaryotic nucleus and in the bacterial nucleoid. In
particular, highly transcribed genes, RNA polymerases and transcription factors
gather into discrete spatial foci called transcription factories. However, the
mechanisms underlying the formation of these foci and the resulting topological
order of the chromosome remain to be elucidated. Here we consider a
thermodynamic framework based on a worm-like chain model of chromosomes where
sparse designated sites along the DNA are able to interact whenever they are
spatially close-by. This is motivated by recurrent evidence that there exists
physical interactions between genes that operate together. Three important
results come out of this simple framework. First, the resulting formation of
transcription foci can be viewed as a micro-phase separation of the interacting
sites from the rest of the DNA. In this respect, a thermodynamic analysis
suggests transcription factors to be appropriate candidates for mediating the
physical interactions between genes. Next, numerical simulations of the polymer
reveal a rich variety of phases that are associated with different topological
orderings, each providing a way to increase the local concentrations of the
interacting sites. Finally, the numerical results show that both
one-dimensional clustering and periodic location of the binding sites along the
DNA, which have been observed in several organisms, make the spatial
co-localization of multiple families of genes particularly efficient.Comment: Figures and Supplementary Material freely available on
http://dx.doi.org/10.1371/journal.pcbi.100067
Helical Chirality: a Link between Local Interactions and Global Topology in DNA
DNA supercoiling plays a major role in many cellular functions. The global DNA conformation is however intimately linked to local DNA-DNA interactions influencing both the physical properties and the biological functions of the supercoiled molecule. Juxtaposition of DNA double helices in ubiquitous crossover arrangements participates in multiple functions such as recombination, gene regulation and DNA packaging. However, little is currently known about how the structure and stability of direct DNA-DNA interactions influence the topological state of DNA. Here, a crystallographic analysis shows that due to the intrinsic helical chirality of DNA, crossovers of opposite handedness exhibit markedly different geometries. While right-handed crossovers are self-fitted by sequence-specific groove-backbone interaction and bridging Mg2+ sites, left-handed crossovers are juxtaposed by groove-groove interaction. Our previous calculations have shown that the different geometries result in differential stabilisation in solution, in the presence of divalent cations. The present study reveals that the various topological states of the cell are associated with different inter-segmental interactions. While the unstable left-handed crossovers are exclusively formed in negatively supercoiled DNA, stable right-handed crossovers constitute the local signature of an unusual topological state in the cell, such as the positively supercoiled or relaxed DNA. These findings not only provide a simple mechanism for locally sensing the DNA topology but also lead to the prediction that, due to their different tertiary intra-molecular interactions, supercoiled molecules of opposite signs must display markedly different physical properties. Sticky inter-segmental interactions in positively supercoiled or relaxed DNA are expected to greatly slow down the slithering dynamics of DNA. We therefore suggest that the intrinsic helical chirality of DNA may have oriented the early evolutionary choices for DNA topology
A Geometrical Model for DNA Organization in Bacteria
Recent experimental studies have revealed that bacteria, such as C. crescentus, show a remarkable spatial ordering of their chromosome. A strong linear correlation has been found between the position of genes on the chromosomal map and their spatial position in the cellular volume. We show that this correlation can be explained by a purely geometrical model. Namely, self-avoidance of DNA, specific positioning of one or few DNA loci (such as origin or terminus) together with the action of DNA compaction proteins (that organize the chromosome into topological domains) are sufficient to get a linear arrangement of the chromosome along the cell axis. We develop a Monte-Carlo method that allows us to test our model numerically and to analyze the dependence of the spatial ordering on various physiologically relevant parameters. We show that the proposed geometrical ordering mechanism is robust and universal (i.e. does not depend on specific bacterial details). The geometrical mechanism should work in all bacteria that have compacted chromosomes with spatially fixed regions. We use our model to make specific and experimentally testable predictions about the spatial arrangement of the chromosome in mutants of C. crescentus and the growth-stage dependent ordering in E. coli
Malaria pigment crystals as magnetic micro-rotors: Key for high-sensitivity diagnosis
The need to develop new methods for the high-sensitivity
diagnosis of malaria has initiated a global activity in medical
and interdisciplinary sciences. Most of the diverse variety of
emerging techniques are based on research-grade instruments,
sophisticated reagent-based assays or rely on expertise. Here,
we suggest an alternative optical methodology with an easy-to-
use and cost-effective instrumentation based on unique
properties of malaria pigment reported previously and determined
quantitatively in the present study. Malaria pigment, also
called hemozoin, is an insoluble microcrystalline form of heme.
These crystallites show remarkable magnetic and optical
anisotropy distinctly from any other components of blood. As a
consequence, they can simultaneously act as magnetically driven
micro-rotors and spinning polarizers in suspensions. These
properties can gain importance not only in malaria diagnosis and
therapies, where hemozoin is considered as drug target or immune
modulator, but also in the magnetic manipulation of cells and
tissues on the microscopic scale
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