Lorentz Center - Physics goes DNA: from base-pairs to chromatin from 7 Sep 2009 through 11 Sep 2009
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    Physics goes DNA: from base-pairs to chromatin
    from 7 Sep 2009 through 11 Sep 2009






Probing DNA hybridization parameters from microarray experiments


Enrico Carlon

(Institute for Theoretical Physics, KULeuven, Belgium)


Quantifying interactions in DNA microarrays is of central importance for a better understanding of their functioning. Hybridization thermodynamics for nucleic acid strands in aqueous solution can be described by the so-called nearest neighbor model, which estimates the hybridization free energy of a given sequence as a sum of dinucleotide terms. Compared with its solution counterparts, hybridization in DNA microarrays may be hindered due to the presence of a solid surface and of a high density of DNA strands. We present a study aimed at the determination of hybridization free energies in DNA microarrays. Experiments are performed on custom Agilent slides. The solution contains a single oligonucleotide. The microarray contains spots with a perfect matching (PM) complementary sequence and other spots with one or two mismatches (MM) : in total 1006 different probe spots, each replicated 15 times per microarray. The free energy parameters are directly fitted from microarray data. The experiments demonstrate a clear correlation between hybridization free energies in the microarray and in solution. The experiments are fully consistent with the Langmuir model at low intensities, but show a clear deviation at intermediate (non-saturating) intensities. The origins of these deviations are discussed. These results provide new interesting insights for the quantification of molecular interactions in DNA microarrays.


J. Hooyberghs, P. Van Hummelen and E. Carlon, The effects of mismatches on hybridization in DNA microarrays: determination of nearest neighbor parameters. Nucl Acid Res 37, e53 (2009)






Unravelling the organization of bacterial chromatin


Remus T. Dame

(Leiden Institute of Chemistry, Gorlaeus Laboratories, Laboratory of Molecular Genetics, Leiden University &

Division of Physics and Astronomy, VU University, Amsterdam, Netherlands)


The bacterial genome is folded and compacted into a body referred to as the nucleoid due to the activity of nucleoid-associated proteins (NAP?s). As a consequence of their role in global genome organization these proteins also act as pleiotropic regulators of transcription. One of the key players in these processes is H-NS. This is an abundant, multimeric protein with a binding preference for A/T rich regions along the genome. Its binding to these regions is associated with transcriptional silencing and has been suggested to be a mechanism to specifically target and silence newly acquired foreign DNA and protect the host against its potentially harmful effects.
A lot of progress in the understanding of H-NS action has been booked in recent years. Our main aim has been to establish the structure, kinetics, mode of binding and the role of H-NS in global genome organization. To this purpose we used a combination of scanning force microscopy imaging, single-molecule micromanipulation and theoretical modeling of H-NS-DNA complexes. We demonstrated that H-NS organizes DNA by bridging two DNA duplexes and put forward evidence that this mode of binding is also key to the role of H-NS as repressor. In recent follow up studies, we determined the quaternary structure of H-NS, the organization within H-NS bridged regions and the forces required to open up these regions. Combining the in vitro structural observations and genome-wide binding studies of H-NS, we can now explain the higher order organization of the genome in the long known topologically isolated domains.






Translocations of single RNA molecules through solid state nanopores


M. van den Hout, G.M. Skinner, O.D. Broekmans, C. Dekker, and N.H. Dekker


Solid-state nanopores offer a promising method for rapidly probing the structural properties of biopolymers such as DNA and RNA.  We have for the first time translocated RNA molecules through solid-state nanopores, comparing the signatures of translocating double-stranded RNA molecules and of single-stranded homopolymers poly(A), poly(U), poly(C).  Based on their differential blockade currents, we can rapidly discriminate between both single- and double-stranded nucleic-acid molecules, as well as separate purine-based homopolymers from pyrimidine-based homopolymers.  Molecule identification is facilitated through the application of high voltages (~600 mV), which contribute to the entropic stretching of these highly flexible molecules.  This striking sensitivity to relatively small differences in the underlying polymer structure greatly improves the prospects for using nanopore-based devices for DNA or RNA mapping.






Transcriptional proofreading and the competing demands of fidelity and velocity


Martin Depken


The RNA polymerase is a protein molecular machine that transcribes genetic information from DNA into RNA with remarkably high fidelity. The growth of the RNA transcript is frequently interrupted by pauses, the detailed nature of which has remained controversial. Based on theoretical modeling and comparison to single molecule data, we argue that the majority of such pauses are due to a diffusive state where the polymerase slides backward along the transcript. We further show how these pause states can be used for proofreading the nascent RNA transcript, and discuss the inherent competition between velocity, efficiency, and fidelity during transcription.






Scale Dependent Statistical Properties of DNA in 2 and 3 Dimensions


Giovanni Dietler

(EPFL, Lausanne, Switzerland)


Single and double-stranded DNA molecules of different topologies (linear, circular and knotted) in 2 D and 3 D conformation were imaged using Atomic Force Microscopy (AFM). After tracing the trajectory of the molecules, it was possible to calculate different scale dependent statistical quantities like the radius of gyration, the end-to-end distance, the distribution of the end-to-end distance and the shape (asphericity). These quantities in turn permit to determine the critical exponents describing the divergence of the radius of gyration with the length of the DNA molecule and their distributions. It was possible to compare the experimental distributions for the above-mentioned quantities with the theoretical one. Additionally, because of the fact that DNA has a rather large persistence length (50 nm), theories for semi-flexible polymers could be tested. The above experiments were carried out with topologically constrained DNA molecules: namely linear, circular, and knotted DNA.  






Dynamic chromatin folding inside the interphase nucleus: biological function and constraints


Roel van Driel

(University of Amsterdam and Netherlands Inst. for Systems Biology)


The folding of the chromatin fibre inside the interphase nucleus of higher eukaryotes is tightly related to local functional properties of the genome. Although our insight into underlying physical principles is still limited, certain folding principles, both static and dynamic, are being uncovered. I will give a brief overview of the present status of our knowledge in this field and discuss key questions.



Mateos-Langerak, et al. (2009) Spatially confined folding of chromatin in the interphase nucleus. Proc Natl Acad Sci USA. 106:3812-3817.






Structure and Dynamics of Interphase Chromosomes


Angelo Rosa1, Ralf Everaers2

(1 Institute for Biocomputation and Physics of Complex Systems (BIFI), Zaragoza, Spain,

2 Université de Lyon, Laboratoire de Physique et Centre Blaise Pascal de l’Ecole Normale Supérieure de Lyon, CNRS UMR 5672, Lyon, France)


During interphase chromosomes decondense, but fluorescent in situ hybridization experiments reveal the existence of distinct territories occupied by individual chromosomes inside the nuclei of most eukaryotic cells. We have used computer simulations to show that entanglement effects cause sufficiently long chromosomes to remain segregated during interphase and to form ‘‘territories.’’ Our parameter-free minimal model of decondensing chromosomes (1) reproduces currently available experimental results for the existence and shape of territories as well as for the internal chromosome structure and dynamics in interphase nuclei and (2) explains why entanglement effects do not interfere with the reverse process of chromosome condensation at the end of interphase.


Rosa A, Everaers R (2008) Structure and Dynamics of Interphase Chromosomes. PLoS Comput Biol 4(8): e1000153. doi:10.1371/journal.pcbi.1000153






Duplex DNA sequence-specific targeting for single-molecule experiments


Maxim D. Frank-Kamenetskii

(Department of Biomedical Engineering and Center for Advanced Biotechnology, Boston University, Boston, USA)


In recent years a remarkable variety of micro and nanotechnology devices and tools have emerged, which carry an enormous potential for the development of totally new approaches for single-molecule interrogation of DNA and RNA. Among them, there are microfluidic devices with laser excitation and fluorescent detection; micro and nanoelectromechanical systems (MEMS and NEMS); various nanopores, etc. Nucleic acids (NAs) in duplex form, double-stranded DNA (dsDNA), the DNA/RNA hybrid and double-stranded RNA (dsRNA), are most compatible with micro and nanotechnology tools since they are extremely stable with respect to large shear forces common at the nanoscale.  For various applications of single molecule DNA technologies it is crucial to be able to target dsDNA in a strictly sequence specific manner. Such specifically tagged dsDNA is subjected to single-molecule interrogation using one or another micro or nanotechnology tool. 

Since in dsDNA nucleobases are involved in complementary pairing and buried inside the double helix, sequence-specific tagging of dsDNA (as well as any other duplex NA) presents a great challenge. The situation became especially challenging after realization that triplex forming oligonucleotides, which had long been known to be capable of sequence specific targeting certain sites on dsDNA, could not be used in combination with micro and nanotechnology tools for a variety of reasons. Still, we have succeeded in developing approaches compatible with the single-molecule detection grounding on two biochemical tools: peptide nucleic acid (PNA), an artificial analog of DNA, and nicking enzymes, which sequence specifically produce single-strand breaks in dsDNA. PNAs make it possible the sequence-specifically assembly of dsDNA nanostructures, P-loops and PD-loops, which are used for exceedingly specific targeting of dsDNA. The nicking enzyme approach makes it possible to prepare dsDNA carrying tags covalently attached to DNA in extremely sequence-specific manner.  In all these cases the dsDNA tagging is very stable making the approaches suitable for single-molecule applications. The feasibility of single-molecule DNA experiments based on the developed methods of sequence-specific dsDNA tagging has been demonstrated.






DNA capture into a nanopore


Alexander Grosberg


While DNA translocation is widely regarded as one of the most sensitive single molecule experimental techniques, the statistical mechanics of DNA capture into a nanopore had not been properly understood. In this work, a rather detailed view of DNA capture is developed which is found in good agreement with experiment.






Entropy and the large scale organisation of chromosomes: from bacteria to plants


Bela Mulder


Irrespective of the local packing effects there is a length scale beyond which genomic DNA behaves like a generic volume-excluding polymer. In this regime the entropy associated with the polymeric conformations becomes a major determinant of chromosome structure, especially in circumstances where spatial confinement and/or interactions with other chromosomes are relevant. We will discuss two examples where entropy effects lead to non-trivial process and spatial structuring effects. The first is chromosome segregation during bacterial cell division. The second is the architecture of the nucleus of the model plant organism Arabidopsis thaliana.



Suckjoon Jun and Bela Mulder, Entropy-driven spatial organization of highly confined polymers: Lessons for the bacterial chromosome, Proc. Natl. Acad. Sci. 103:12388–12393 (2006)


Silvester de Nooijer, Joan Wellink, Bela Mulder and Ton Bisseling, Non-specific interactions are sufficient to explain the position of heterochromatic chromocenters and nucleoli in interphase nuclei, Nucl. Acids. Res. 37:3558-3568 (2009)






Single molecule force spectroscopy on chromatin


John van Noort


The compaction of eukaryotic DNA into chromatin has been implicated in the regulation of all cellular processes whose substrate is DNA. To understand this regulation, it is essential to reveal the structure and mechanism by which chromatin fibers fold and unfold. I will discuss how we used magnetic tweezers to probe the mechanical properties of chromatin fibers consisting of a single, well-defined array of 25 nucleosomes and compare the results with data obtained when pulling on a single nucleosome. It appears that neighboring nucleosomes stabilize DNA folding into a nucleosome.

When an array of nucleosomes is folded into a 30 nm fiber, representing the first level of chromatin condensation, the fiber stretched like a Hookian spring at forces up to 4 pN. Together with a nucleosome-nucleosome stacking energy of 14 kT, four times larger than previously reported, this points to a solenoid as the underlying topology of the 30 nm fiber. Surprisingly, linker histones do not affect the length or stiffness of the fibers, but stabilize fiber folding up to forces of 7 pN. Fibers with a nucleosome repeat length of 167 bp instead of 197 bp are significantly stiffer, consistent with a two-start helical arrangement. The extensive thermal breathing of the chromatin fiber that is a consequence of the observed high compliance provides a structural basis for understanding the balance between chromatin condensation and transparency for DNA transactions.






Base Sequence and the Architecture of Nucleosomal DNA


Wilma K. Olson

(Rutgers, the State University of New Jersey)



In order to understand the mechanisms by which DNA base sequence and tightly bound proteins control the biophysical properties of the long, threadlike molecule, we have developed a coarse-grained model, in which the DNA base pairs are treated as rigid bodies subject to realistic, knowledge-based energy constraints, and computational techniques to determine the configuration-dependent propensities of these molecules. The presentation will highlight some of the unique, sequence-dependent spatial information that has been gleaned from analyses of the high-resolution structures of DNA and its complexes with other molecules, including nucleosomes, and illustrate how this information can be used to gain new insights into the positioning of non-specific proteins on DNA and how the positions of nucleosomes and the local structural and deformation properties of free and protein-bound DNA base-pair steps affect the configurational properties of fluctuating chromatin fibers.






Hierarchical Modeling of DNA – Flow, Confinement, and Hybridization


Juan de Pablo


Over the past several years we have developed a hierarchical modeling approach that has enabled description of DNA over scales ranging from nanometers to millimeters. This talk will describe this approach in the context of three particular examples. In the first, we show how our coarse grain representation of DNA can describe the behavior of long molecules in a variety of flows, both in the bulk and in the vicinity of surfaces. The emphasis is on the proper description of hydrodynamic interactions and their effects on the structure and dynamics of DNA. The second example is concerned with development of a model that permits calculation of the thermodynamic and mechanical properties of DNA at the level of individual base pairs. We focus on description of melting temperatures, persistence lengths, structure, and the effect of sequence on such properties. In the third example we examine in detail the actual process of hybridization at molecular length scales. We present a detailed analysis from transition path simulations of the transition state ensembles corresponding to hybridization of different sequences. That analysis reveals intriguing new insights into the process of re-hybridization that might enable design of effective experimental strategies






DNA nanotechnology: Nanodevices and supramolecular assemblies made from DNA


Friedrich C. Simmel

(Biomolecular Systems and Bionanotechnology, Physics Department, Technische Universität München, Garching, Germany)


DNA base-pairing interactions have been utilized for the realization of a large variety of artificial nanoscale structures. Among them are small switchable devices based on aptamers which may be used as biosensors or for the controlled binding and release of substances. We will discuss the design principles and operation cycles of several of such devices. Another field of application for DNA in nanotechnology is the construction of precisely addressable supramolecular assemblies. Due to the predictability of base-pairing interactions between complementary sequences of DNA, programmable and even automatable nanoassembly becomes feasible. In particular, the recent invention of the origami technique has revolutionized this field and we will demonstrate several potential applications for origami structures.  






The Genomic Code for Nucleosome Positioning


Jonathan Widom

 (Dept. of Biochemistry, Molecular Biology and Cell Biology, and Dept. of Chemistry, Northwestern University, Evanston, IL, USA)



Eukaryotic genomes are packaged into nucleosome particles that occlude the DNA from interacting with most DNA binding proteins.  We have discovered that genomes care where their nucleosomes are located on average, and that genomes manifest this care by encoding an additional layer of genetic information, superimposed on top of other kinds of regulatory and coding information that were previously recognized.  We have developed a partial ability to read this nucleosome positioning code and predict the in vivo locations of nucleosomes.  Most recently, we showed that the distribution of nucleosomes reconstituted on yeast genomic DNA in a purified in vitro system closely resembles that in vivo, implying that much of the in vivo nucleosome organization is explicitly encoded in the genomic DNA sequence itself, through the nucleosomes’ DNA sequence preferences.  A statistical model based only on the in vitro nucleosome DNA sequence data is significantly predictive of the detailed distribution of nucleosome locations in yeast, C. elegans, and human, suggesting that there may exist a universal genomic code for nucleosome positioning.  Our results suggest that genomes utilize the nucleosome positioning code to facilitate specific chromosome functions, including to delineate functional versus nonfunctional binding sites for key gene regulatory proteins, and to define the next higher level of chromosome structure.   The physical basis of the nucleosome DNA sequences preferences lies in the sequence-dependent mechanics of DNA itself.






Force-induced DNA interactions: From small molecules to viral replication


Mark Williams


I will discuss experiments in which single DNA molecules are stretched in the presence of DNA binding ligands using an optical tweezers instrument. From these measurements, we obtain the force required to extend the DNA molecule and to convert double-stranded DNA into single-stranded DNA, which we refer to as force-induced melting. Depending on the nature of the ligand binding mode, several different effects are observed. For example, intercalators such as ethidium increase the contour length of double-stranded DNA as well as the melting force. In contrast, single-stranded DNA binding proteins, such as gp32 from bacteriophage T4 and gp2.5 from bacteriophage T7, strongly destabilize double-stranded DNA, resulting in a decrease in melting force. The observed decrease in melting force as a function of protein concentration allows us to quantify the protein binding free energy. We then analyze the salt dependence of protein binding to characterize the interactions that regulate protein-DNA binding in these systems.






Quantifying the physics inside the genome one molecule at a time


Gijs Wuite


The genetic information of an organism is encoded in the base pair sequence of its DNA. Many specialized proteins are involved in organizing, preserving and processing the vast amounts of information on the DNA. In order to do this swiftly and correctly these proteins have to move quickly and accurately along and/or around the DNA constantly rearranging it. In order to elucidate these kind of processes we perform single-molecule experiments on model systems such as restriction enzymes, DNA polymerases and repair proteins. The data we use to extract forces, energies and mechanochemistry driving these dynamic transactions. The results obtained from these model systems are then generalized and thought to be applicable to many DNA-protein interactions. In particular, I will report experiments that use a combination of fluorescence microscopy and optical tweezers to:


(i) directly visualize the DNA overstretching transition and demonstrate that its origin is the cooperative melting of the two DNA strands. In the experiments we use intercalating dyes and fluorescently labeled single-stranded binding proteins to specifically visualize double- and single-stranded segments in DNA molecules undergoing the transition. Our data unambiguously show that the overstretching transition comprises a gradual conversion from double-stranded to single-stranded DNA, in agreement with the force-induced melting model. Interestingly and in contrast to either model, we found that melting is nucleation-limited, initiating exclusively from DNA extremities and nicks.


(ii) image single Rad51 monomer disassembling from the edges of nucleoprotein filaments. Applying external tension to the DNA, we found that disassembly slows down and can even be stalled. We quantified the fluorescence of RAD51 patches and found that disassembly occurs in bursts interspersed by long pauses. Upon relaxation of a stalled complex, pauses were suppressed resulting in a large burst. These results imply that tension-dependent disassembly takes place only from filament ends, after tension-independent ATP hydrolysis.






Helix-Coil models

Applications and extensions in Algorithmics, Bioinformatics, Physics and Genomics


Édouard Yeramian


Coming back to basics, the original helix-coil model (describing the opening of the double-helix) can serve as a fruitful template for applications and extensions in different directions:


1) Algorithmics.

Powerful ideas in the original Fixman-Freire method for the helix-coil model can be generalized and extended to increasingly more complex biophysical models. Such generalizations (in the context of the SIMEX formulation) allow to handle large-scale realistic models (with long-ranged constraints) in reduced computation times (calculations reduced up to million folds).


2) Bioinformatics.

The helix-coil model represents perhaps the very first bioinformatics elaboration proper, but the original tight links between biophysics and bioinformatics have progressively loosened. Coming back to strict isomorphisms between models it is possible to apply in bioinformatics (dynamic programming) methodological ideas from biophysics, for the handling of realistic models with efficient algorithms. Such a situation is illustrated with the basic problem of sequence alignments, with realistic representations of gaps (corresponding to loops in helix-coil models).


3) Physics.

In a ‘DNA goes physics’ approach, helix-coil models and methods provide interesting potentials for the exploration of certain properties of ‘disordered systems’ (with sequence-specificity as a natural instance of disorder in Ising models).


4) Genomics.

One basic motivation in the elaboration of the helix-coil model was the analysis of DNA sequences in terms of genetic properties. More specifically it was asked if there were correspondences between the genetic (coding/non-coding) and physical (helix/coil) segmentations of DNA sequences. This original question progressively faded with no clear answers, notably in the background of dogmatic debates (between oversimplified generalizations and complete rejections based on argument such as ‘DNA does not melt at physiological temperatures’). Beyond dogmatisms, when resorting to helix-coil properties as powerful sequence descriptors, a very contrasted landscape emerges following genomes and chromosome regions. The correspondence can be so sharp as to allow ab initio gene identifications (notably for very short exons, such as in Plasmodium falciparum, with experimental validations), but completely non-significant in other cases. These contrasted results raise a large series of interesting evolutionary questions in terms of competing drives shaping the structure of genomes. 






Controlling the Structural Transition of giant DNA and Chromatin


Kenichi Yoshikawa

(Department of Physics, Kyoto University, Kyoto 606-8502, Japan)


The persistence length of double stranded DNA is on the order of 200 base pairs (bp), indicating that giant DNA molecules above the order of several tens kbp exhibits the characteristic to undergo large discrete transition between disordered coil and ordered compact states. Among the ordered structure, morphologies such as toroid, rod, spherical and spool structures are generated reflecting the free energy difference and also kinetic pathway. Interestingly, reconstituted chromatin with giant DNA above the size of tens kbp also exhibits large discrete transition between dispersed polynucleosome structure and tightly packed structure.

Biological significance of the discrete nature of the folding transition of DNA and chromatin will be discussed in relation to the epigenetic character of genomic DNA.






Higher order structure of DNA and its environmental response


Yuko Yoshikawa

(Research Organization of Science and Engineering, Ritsumeikan University, Kusatsu, Shiga 525-8577, Japan)


Genomic DNA is packaged to various degrees in response to cellular environments, including cell cycle, and the manner of packaging is involved in biological functions of DNA, such as replication and transcription. During the last decade, we have performed direct observation of the higher order structure of DNA in a single-molecule level using fluorescence microscopy. We have found that giant DNA molecules undergo a large discrete transition from an elongated state to a compact state upon the addition of various condensing agents including polyamines, cationic proteins and neutral polymers. In addition, we have shown that the higher-order structure of DNA can be controlled by optical tweezers without any structural modification of DNA

In the present, I will focus my talk on the application of single-molecule observation for the study of photo- and radiation-induced double-strand breaks on giant DNA. Double-strand break is the most significant DNA lesion, leading to the fatal cell damage.

It will be shown that the double-strand break of DNA is markedly protected accompanying by the folding transition into compact conformation. I will argue that a single DNA observation with fluorescence microscopy is applicable to the quantitative evaluation on DNA damage.