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    Nuclear Architecture and Dynamics
    Nuclear Architecture and Dynamics
    Nuclear Architecture and Dynamics
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    Nuclear Architecture and Dynamics

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    Nuclear Architecture and Dynamics provides a definitive resource for (bio)physicists and molecular and cellular biologists whose research involves an understanding of the organization of the genome and the mechanisms of its proper reading, maintenance, and replication by the cell. This book brings together the biochemical and physical characteristics of genome organization, providing a relevant framework in which to interpret the control of gene expression and cell differentiation. It includes work from a group of international experts, including biologists, physicists, mathematicians, and bioinformaticians who have come together for a comprehensive presentation of the current developments in the nuclear dynamics and architecture field.

    The book provides the uninitiated with an entry point to a highly dynamic, but complex issue, and the expert with an opportunity to have a fresh look at the viewpoints advocated by researchers from different disciplines.

    • Highlights the link between the (bio)chemistry and the (bio)physics of chromatin
    • Deciphers the complex interplay between numerous biochemical factors at task in the nucleus and the physical state of chromatin
    • Provides a collective view of the field by a large, diverse group of authors with both physics and biology backgrounds
    LanguageEnglish
    PublisherAcademic Press
    Release dateOct 27, 2017
    ISBN9780128035030
    Nuclear Architecture and Dynamics

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      Nuclear Architecture and Dynamics - Christophe Lavelle

      France

      Preface

      Christophe Lavelle¹,² and Jean-Marc Victor²,³, ¹National Museum of Natural History, CNRS UMR7196/INSERM U1154, Paris, France, ²UPMC University, CNRS UMR7600, Paris, France, ³Nuclear Architecture and Dynamics, CNRS GDR3536, Paris, France

      In an eukaryotic cell, much of the genome and the machinery for its replication, maintenance, and expression is hosted in a membrane-enclosed organelle that occupies about 10% of the cell interior: the nucleus. Each chromosome, which bears a part of the genome, consists of a single molecule of DNA compacted by various proteins into a nucleoprotein substance known as chromatin. The precise 3D organization and dynamics of chromatin in the nucleus is thought to be a key feature in transcriptional regulation, hence also one of the determiners of differential gene expression during development. As such, it pertains to epigenetics in its original definition by Conrad Waddington as the mechanisms by which the genes of the genotypes bring about phenotypic effects.

      Indeed, evidence has accumulated pointing out chromatin dynamics as a critical means of control of genome accessibility in time and space, regulating the smooth progress of fundamental processes such as DNA replication, repair, and transcription. Moreover, there is a growing body of evidence that mechanical constraints experienced by DNA (e.g., supercoiling) control the affinity of transcription factors to DNA. As such the nucleus cannot be seen as a mere genome container, but rather a complex cellular entity which structural and chemical compositions change during development and exhibit cell- and tissue-specific differences. To understand how these phenomena take place, one needs to decipher the complex interplay between numerous biochemical factors at task in the nucleus and the physical state of chromatin.

      This book is dedicated to these biochemical and physical characteristics of genome organization, with the aim of providing a relevant framework in which to interpret the control of gene expression and cell differentiation. For this, we brought together a select group of international experts, including biologists, physicists, mathematicians, and bioinformaticians, for a comprehensive presentation of the current developments in the nuclear dynamics and architecture field and their relevance to nuclear functions and physiological processes. We hope that this collection will provide both the uninitiated with an entry point to a highly dynamic but complex issue and the expert with an opportunity to have a fresh look at the viewpoints advocated by the researchers from the different disciplines that have contributed.

      I

      Chromatin Organization and Dynamics

      Outline

      1 DNA Mechanics

      2 The Role of Nucleosome Positioning in Genome Function and Evolution

      3 DNA Supercoiling(omics)

      4 Dynamic Chromatin Folding in the Cell

      5 Mesoscale Modeling of Chromatin Fibers

      6 A Polymer Physics View on Universal and Sequence-Specific Aspects of Chromosome Folding

      7 Persistence of Long-Range Contacts at Insulators

      8 Long-Range Intranuclear Interactions

      9 The Multiple Effects of Molecular Crowding in the Cell Nucleus

      1

      DNA Mechanics

      John F. Marko,    Northwestern University, Evanston, IL, United States

      Abstract

      This chapter discusses the mechanical and statistical–mechanical properties of the DNA double helix. Basic DNA polymer elasticity will be reviewed, with a focus on the bending stiffness of the double helix. The twisting stiffness, which is a consequence of the double-helical structure of DNA will also be discussed, along with the phenomenon of supercoiling, which is associated with situations where the linking number of the two strands inside the double helix is held fixed. The mechanics of strand separation and of protein–DNA interactions will also be reviewed.

      Keywords

      DNA; persistence length; supercoiling; topology; twist modulus; strand separation; ssDNA; protein–DNA interactions; facilitated dissociation

      To understand the biophysics of chromatin and chromosomes, we need to understand the physical properties of the long DNA polymers that underlie them. The mechanical properties of DNA are central to many aspects of chromatin biology, from DNA transcription, replication and repair, to the removal of DNA entanglements and physical segregation of chromosomes that must occur in order for cell division to be successful (Marko and Siggia, 1997a; Bloom, 2008). Over the past 20 years, our understanding of DNA mechanics has advanced tremendously, thanks in part to the development of single-molecule nanomechanics experiments which allow the direct study of the mechanical response of individual DNA molecules. A major lesson has been that thermal fluctuations and statistical mechanics are central to understanding DNA mechanics.

      This chapter introduces the DNA double helix, with an emphasis on the basic statistical physics which is inextricably intertwined with its mechanical properties. The basic polymer elasticity of DNA will be discussed, which for forces (≈ piconewtons) is controlled by the bending stiffness of the double helix, a general feature of semiflexible polymers. The double helix also has an internal linking number of its two strands which gives rise to twisting stiffness, which in turn has strong physical consequences for DNA conformation. The nonlinear elasticity of the double helix associated with separation of the two strands within the double helix will also be described. Finally, the chapter ends with a brief introduction to protein–DNA interactions, which are essential to the processing of DNA molecules inside the cell and of course to the properties of chromatin which are the subject of the remainder of the book.

      1.1 Basic Properties of DNA

      1.1.1 The DNA Double Helix

      DNA molecules in cells are found in double-helix form, consisting of two long polymer chains wrapped around one another, with complementary chemical structures. The double helix encodes genetic information through the sequence of chemical groups—the bases adenine, thymine, guanine and cytosine (A, T, G, and C). Corresponding bases on the two chains in a double helix bind one another according to the complementary base-pairing rules A=T and G≡C. These rules follow from the chemical structures of the bases, which permit two hydrogen bonds to form between A and T (indicated by =), versus three that form between G and C (indicated by ≡). Each base pair has a chemical weight of about 600 Daltons (Da).

      The presence of the two complementary copies along the two polynucleotide chains in the double helix provides redundant storage of genetic information and also facilitates DNA replication, via the use of each chain as a template for assembly of a new complementary polynucleotide chain.

      1.1.2 Physical Properties of the DNA Double Helix

      The basic physical properties of DNA molecules found inside cells are key to thinking about how cellular machinery reads, replicates, repairs, and stores them.

      1.1.2.1 Length

      Double-helix DNAs in vivo are long polymers: the chromosome of the λ bacteriophage (a virus that infects Escherichia coli bacteria) is 48,502 base pairs (bp) or about 16 µm in length; the E. coli bacterial chromosome is 4.6 × 10⁶ bp (4.6 Mb) or about 1.5 mm long; small E. coli plasmid DNA molecules used in genetic engineering are typically 2–10 kb (0.7–3 µm) in length; and the larger chromosomal DNAs in human cell nuclei are roughly 200 Mb or a few cm in length. Each base pair contributes about 0.34 nm of length (or rise) to a double-helix DNA.

      1.1.2.2 Electrical Charge

      The environment in the cell is essentially aqueous solution, in which DNA molecules are ionized, so as to carry essentially one electron charge per base (2 e−/bp≈6e−/nm, each negative charge coming from an ionized phosphate on the DNA backbone, see Fig. 1.1A). The high electric charge density along the double helix makes it a strong polyelectrolyte, and gives it strong electrostatic interactions with other electrically charged molecules. Notably, in cells, the univalent salt concentration is 100–200 mM, making the Debye length shorter than 1 nm (λD, where M is the concentration of 1:1 salt in mol/liter=M): thus electrostatic interactions with DNA, while strong, are essentially short-ranged. Electrostatic repulsions give rise to an effective hard-core diameter of dsDNA of ≈3.5 nm under physiological salt conditions (Rybenkov et al., 1993).

      Figure 1.1 DNA double-helix structure. (A) Chemical structure of one DNA chain, showing the deoxyribose sugars (note numbered carbons) and charged phosphates along the backbone, and the attached bases (A, T, G, and C following the 5′ to 3′ direction from top to bottom). (B) Space-filling diagram of the double helix. Two complementary-sequence strands as in (A) noncovalently bind together via base-pairing and stacking interactions, and coil around one another to form a regular helix. The two strands can be seen to have directed chemical structures and are oppositely directed. Note the different sizes of the major (M) and minor (m) grooves, and the negatively charged phosphates along the backbones (dark groups). The helix repeat is 3.6 nm, and the DNA cross-sectional diameter is 2 nm. Image reproduced from Goodsell, D.S., 1992. The Machinery of Life. Springer-Verlag, New York, NY.

      1.1.2.3 Bending Stiffness

      Although the structure of the DNA double helix is sometimes presented in books as if it is static, at room temperature and in solution the double helix undergoes continual thermally excited changes in shape. The DNA double helix is a semiflexible polymer, with a persistence length of A≈50 nm (≈150 bp). Roughly speaking, this length is the distance along a double-helix DNA that thermally excited bends of about a radian in total angle occur. The thickness of the double helix is about 2 nm (Fig. 1.1B), so a persistence length of double-helix DNA is long and thin. These long lengths combined with the stiffness of the double helix mean that by themselves, genomic-length DNAs (more than a few kilobases long) will behave as flexible, random-coil polymers.

      It is important to keep in mind that the origin of the bending flexibility of the double helix, and indeed all elastic properties of biological molecules, arise from the intrinsic molecular flexibility of the backbones and the bases (Fig. 1.1). The fact that the double helix can be easily bent by a few degrees at each base pair means that thermal fluctuations will give rise to appreciable local distortions of the double helix. Fig. 1.2 shows a snapshot of a typical conformation of a state-of-the-art molecular dynamics simulation of a short, 10-bp segment of DNA: the fluctuations make the zero-temperature classical double-helix structure (Fig. 1.1A) nearly unrecognizable. These distortions and the flexibility of the double helix are key to its interactions with other molecules in the cell, notably DNA-interacting proteins.

      Figure 1.2 Molecular dynamics snapshot gives an idea of the nature of a typical double-helix DNA conformation for a short 10 bp molecule in solution at room temperature. Reproduced from Feig, M., Marko, J.F., Pettitt, M., 2003. Microscopic DNA fluctuations are in accord with macroscopic DNA stretching elasticity without strong dependence on force field choice. In: Russo, N. (Ed.), NATO ASI Series: Metal Ligand Interactions, vol. 193. Kluwer Academic Press.

      Per base pair, the fluctuations are usually small displacements (about 4 degrees of bend, 0.03 nm average separations of the bases) but over long stretches of double helix, they build up to significant, thermally excited random bends. One can estimate what length of double helix one needs to have a random 1-radian net bend; given randomly signed bends of about 0.08 radian per base pair, a net bend of a radius will build up over about 1/(0.08)²≈150 bp, or about 50 nm, giving a microscopic picture of the persistence length.

      1.1.2.4 Helical Structure and Twisting Stiffness

      The DNA double helix is really two polymers wrapped around one another, with one right-handed turn every ≈10.5 bp, or about 0.6 radian/bp (Fig. 1.1B). This double-helix structure gives rise to twist rigidity, which is a quite unique polymer property. The two-strand structure also has the possibility of trapping a fixed linking number of the two strands when a DNA is closed into a loop. Constraint of strand linking number—a topological property of DNA—gives rise to a rich array of biologically relevant phenomena. Finally, if we consider the moderate strength of the base-pairing interactions holding the two strands together (about 2.5 kBT per base pair when averaged over base-pair sequence (SantaLucia, 1998)) indicates the possibility of stress-driven structural defects (bubbles of locally base-unpaired single-strands) or transitions (stress-driven strand separation).

      1.1.3 Physical Scales Relevant to DNA

      Given some basic idea of the physical properties of DNA, we can identify a number of characteristic physical scales relevant to DNA biophysics, as well as to many aspects of molecular biophysics.

      1.1.3.1 Length

      The basic length scale relevant to molecular biology is the nanometer (nm=10−9 m). DNA bases, amino acids, simple sugars, energy-transferring molecules such as ATP, and many of the other basic molecular units used by living things are all roughly 1 nm in size. Therefore, the nm is the scale of modularity of biochemical structure, and also the scale of information granularity in a living cell. It is good to keep in mind that the length of a small bacterial cell or a small fraction of a eukaryote cell is ≈10−6 m or µm, 1000 nm in length.

      1.1.3.2 Concentration

      Another scale to remember is the concentration of one molecule per cubic micron, which is 10¹⁵ molecules per liter, or about 1.6×10−9 mol/liter=1.6 nM (the number of molecules in a mol is NA≈6×10²³). This is roughly the concentration of a transcription factor protein one might find in a bacterial or eukaryote cell.

      1.1.3.3 Energy

      There are two main energy scales of interest to us. First, there is the thermal energy per degree of freedom, kBT≈4×10−21 J at room temperature (T=300 K; for living cells, T is never too far from this so we will regard kBT as essentially fixed). The binding energies of the noncovalent bonds that hold biological molecules in their folded conformations (folded proteins, double-helix structure of DNA) are naturally measured in kBT units, e.g., base-pair binding energies along the DNA double helix range from ≈1 to 4kBT per base pair under normal physiological solution conditions.

      The second energy scale of relevance here is that of a covalent chemical bond, which is much larger, comparable to 1 eV≈40kBT. This level of energy stabilizes the polymer backbones of protein and nucleic acid chains, and allows biological molecules to have their secondary (folded) structure changed, without breaking their primary (backbone) structure.

      1.1.3.4 Force

      The force scale most relevant to molecular biology is kBT/nm≈4×10−12 N=4 piconewtons (pN). The pN force scale appears as the characteristic force scale associated with biomolecule conformational change, since it corresponds to the breaking of noncovalent bonds of a few kBT binding energy, by stretching them a fraction of a nm.

      The few pN force scale should be contrasted with the much larger force scale of ≈eV/Å≈10−9 N=1 nN, the characteristic force one might expect to break a covalent bond (Grandbois et al., 1999).

      We can quickly estimate the force scale to tear a protein off a DNA molecule (or to tear apart a dimeric protein complex). To do this one can expect to have to do a few kBT of work over a reaction distance of a nm or so (the size of the binding site), indicating a rupture force of ≈10 pN in accord with experimental data, e.g., Krasnoslobodtsev et al. (2007).

      apart. In this case, the work done against the applied force f is≈f(Marko and Siggia, 1997b). If looping is to occur by thermal fluctuation against that force, we can expect a strong suppression of the rate of loop formation relative to the zero-force case when f> 10kBT, or for f > 10kBT(for more detailed calculations including effects of DNA bending, see Sankararaman and Marko, 2005; Blumberg et al., 2005; Yan et al., 2005; note that DNA bending elastic energy controls loop formation rate at zero tension and is an additional free energy cost of loop formation, adding to the force–extension free energy).

      =100 nm (300 bp, a rather typical distance for loop formation by site-specific DNA-looping proteins as occurs during gene regulation) we have strong suppression of loop formation when f > 0.1kBT/nm≈0.4 pN. In this situation we expect the loop formation rate to be suppressed relative to the zero-force case by a factor≈e−10 < 10−4. Strong suppression of DNA looping by roughly piconewton forces has been observed (Skoko et al., 2005; Gemmen et al., 2006; Chen et al., 2010).

      A larger force is associated with the stalling of molecular machines which burn ATP or similar energy-rich molecules, transferring ≈10kBT from chemical to mechanical energy per step of nm dimensions. This stall force scale is roughly ≈10kBT/nm≈40 pN. This is comparable to the stall forces observed for RNA and DNA polymerases (Wang et al., 1998; Maier et al., 2000).

      1.1.3.5 Time

      At molecular scales, all dynamics is driven by thermal motion, and is highly overdamped: we don’t need to worry about inertia for nm or even µm-sized objects. All the motion we will worry about is diffusive, controlled by diffusion constants of the form D=kBT/(6πηR), where R is the scale size of the object in question (the formula is the Einstein diffusion constant for a sphere of radius R) and where η is the fluid viscosity (η≈10−3 Pa s for water, η≈5×10−3 Pa s for cytoplasm). This gives rise to a self-diffusion time τ≈6πηR³/(kBT) which is on the order of 10−9 s for R=1 nm, and about 1 s for R=1 µm. The strong R dependence of this diffusive relaxation time makes it change from a molecular timescale for nm-sized small molecules, to human-observable timescales for µm-scale objects.

      1.2 The Double Helix Is a Semiflexible Polymer

      The starting point for thinking about double-helix DNA conformation is the semiflexible polymer, which coarse-grains microscopic fluctuations (see Fig. 1.2) to model the double helix as having bending stiffness (Hagerman, 1988). If we consider the double helix to have a fixed length L (not a bad approximation to start with although we will discuss elastic stretching of DNA length later), then we can describe it with a space curve r(s), where s is contour distance along the polymer, running from s=0 to s=L. The polymer conformation is controlled by bending energy:

      (1.1)

      which is zero for the straight configuration κ=0. This energy function is just that describing weak bending of an elastic rod, as studied in the theory of elasticity (Landau and Lifshitz, 1986). The bending stiffness is controlled by the constant A, which has dimensions of length; this is the persistence length mentioned earlier. The flexible polymer limit is obtained for L A; for L A, the polymer will be essentially unbent by thermal fluctuations. For double-helix DNA, A≈50 nm, or about 150 bp.

      One should keep in mind that the persistence length is a mesoscopic property of the double helix, and that the bending energy (1.1) is really a free energy describing bending deformations obtained by coarse-graining over smaller-scale thermal fluctuations.

      1.2.1 Bending DNA Into Small Loops

      Before we move to consider statistical mechanics of Eq. (1.1), let’s think about the static energy of a few configurations. A circle of radius R , and therefore a piece of DNA of length L bent along such a circular arc has energy Ecirc/(kBT)=AL/(2R²). So, if we ask what length of DNA is bent into a 1-radian bend—for which R=L—by a thermal fluctuation—of energy kBT/2—we find that length as Ebend/kBT=AL/(2L²)=1/2, i.e., L=A. Thus, each persistence length (A) worth of double helix gets bent by about 1 radian in a random direction.

      Now, it happens that ≈50 nm segments of DNA are bent into nearly circular arcs of radius R=5 nm to form nucleosomes. Nucleosomes are the main DNA-packaging scheme used in eukaryote cells and will be discussed in detail later in this book. The elastic energy associated with bending DNA to form a nucleosome can be estimated to be (A=50 nm, L=50 nm, R=5 nm) Ebend≈50kBT. While this seems like quite severe bending, it is actually a mere (0.34 nm)/(5 nm)≈0.07 radians per base pair (4 degrees per base pair) and gives a reasonable idea of the energy scales involved in DNA packaging. This bending energy is only about 0.3kBT per base pair, and the double-helix structure is only moderately disrupted by bending in the nucleosome (Luger et al., 1997).

      Along similar lines, if we take a piece of DNA of length L and form it into a circle (R=L/[2π]), there is an energy cost of Ecirc/(kBT)=2π²A/L≈19.7 A/L. This is not far from the minimal energy for any shape that brings the ends together; for the optimal teardrop-shaped configuration, Eteardrop/(kBT)≈14.1A/L (Yamakawa and Stockmayer, 1972). This kind of description works quite well down to ≈200 bp lengths, and some experiments suggest that the simple elastic description of double-helix bending may be applicable down to bending radii of ≈4 nm (corresponding to a 75 bp circle) (Schopflin et al., 2012; Du et al., 2008; Le and Kim, 2014).

      1.2.1.1 Permanent Sequence-Driven Bends

      The model described earlier has a perfectly straight configuration as its lowest-energy state. In reality, the average shape of any DNA molecule depends on its sequence: different sequences have slightly different average distortions. However, for most sequences in most situations, the crude model described earlier is sufficient. It is possible, however, by phasing sequences that generate kinks, to obtain DNAs with strong permanent bends along them (Crothers et al., 1990). Some of these strong permanent bends are implicated in biological processes, e.g., facilitation of the binding of proteins that bend or wrap DNA (Lowary and Widom, 1998). In this way, DNA sequence can play a role in positioning nucleosomes (Kaplan et al., 2012).

      1.2.2 Statistical Mechanics of the Semiflexible Polymer

      Now to statistical mechanics of double-helix conformations. Thermal fluctuations give rise to bending and are described by the partition function

      (1.2)

      and β=(kBT)−1. This free polymer model (no applied force or self-interactions) can be solved in closed form (. Since the end-to-end vector R , the mean square end-to-end distance can be computed from the tangent vector correlation function. This approaches the limit, for L AR[r(L)−r=2ALR=2AL[1−(1−e−L/A)A/L) and has the small-L R=L², see Ref. Marko (2005).] The correspondence between A and the statistical segment length b R=Nb²) is b=2A and N=L/(2A)=L/b.

      1.2.2.1 Stretching the Semiflexible Polymer by Small Forces (<kBT/A)

      Rxyz, where x, y, and z are the Cartesian components of the end-to-end vector RRx. This zero-force fluctuation tells us the spring constant for linear response of a force applied to separate the ends, namely k=kBTx=3kBT/(2AL, with the linear response regime essentially holding for f<kBT/A. For double-helix DNA, this characteristic force is quite low since A=50 nm; kBT/A≈0.1 pN (recall kBT/(1 nm)≈4 pN).

      As DNA length L is increased, the linear low-force behavior will eventually be replaced by the nonlinear scaling law for a self-avoiding polymer, for sufficiently large L (de Gennes, 1979). However, for double-helix DNA, the narrow effective thickness (≈3.5 nm at 100 mM univalent salt including electrostatic effects (Rybenkov et al., 1993)) of the double helix compared to its effective segment length b=2A≈100 nm leads to quite weak self-avoidance, and makes dsDNA elasticity quite close to that of an ideal polymer for DNA lengths (<50 kb≈16 µm) routinely studied experimentally (Marko and Siggia, 1995b).

      We note that for single-stranded nucleic acid molecules (e.g., one of the polynucleotide chains in the double helix) the far shorter persistence length ≈1 nm leads to much stronger self-avoidance effects (Saleh et al., 2009; McIntosh et al., 2009), especially for low-salt conditions.

      1.2.2.2 External Force on a Polymer

      For any polymer model, to go beyond linear force response, we need to include force in the energy function:

      (1.3)

      Force is added as a field coupled to the end-to-end vector, so that averages of end-to-end extension are generated by derivatives of the partition function Z with respect to force, as expected for identification of kBT ln Z as a free energy in the fluctuating-extension, constant-force ensemble (the ensemble relevant to magnetic tweezers experiments, which apply a constant force to a paramagnetic particle attached to one end of a DNA (Neuman et al., 2007)).

      There are a number of general consequences for this form of statistical weight. For nonzero force along the z z =∂kBT ln Z/(∂βfzz ²=∂² ln Z/∂(βf)². Components of R transverse x y

      An important feature of any model of the form of Eq. (1.3) where there is no preferred orientation other than that of the force f is that the free energy only depends on the magnitude of force f, ln Z=ln Z(|f|). For nonzero force f along z, if we imagine applying small transverse forces fx and fy, and expand the partition function as

      (1.4)

      A simple relationship between longitudinal and transverse derivatives of ln Z follows:

      (1.5)

      where both sides are evaluated for fx=fy=xz /(βf). Therefore, if we measure thermally averaged transverse fluctuations and average extension we can infer the applied force:

      (1.6)

      This exact relationship holds for any polymer model with a rotationally symmetric conformational energy (essentially any model without a preferred direction in space other than the applied force, notably including models with polymer self-interactions) and is a powerful tool used for force calibration in magnetic tweezers experiments. This relation is model-independent and not limited to the case of small fluctuations (Yan et al., 2005).

      1.2.2.3 Higher-Force Behavior of the Semiflexible Polymer and DNA

      , with force applied in the z :

      (1.7)

      , the energy is one-dimensional and local, which allows one to solve for the partition function using continuum transfer matrix (Schrodinger-like equation) methods (Marko and Siggia, 1995b).

      , with u in the xy , so u is small; to Gaussian order we have

      (1.8)

      , for q=2πn/L, n=0, ±1, ±2,···) we have

      (1.9)

      Using the equipartition theorem,

      (1.10)

      where the factor of 2 in the numerator reflects the two (x and y) components of uui(s)uj(s∝ δije−|s−s′|/ξ.

      Fourier transforming (the result shown is the long-polymer limit L→∞, where Σq → L ∫dq/(2π).) Eq. (1.10) gives the fluctuation of u,

      (1.11)

      This allows us to compute the average extension

      (1.12)

      This characteristic reciprocal square-root dependence of extension on force for a semiflexible polymer in the regime f kBT/A is observed in single-molecule experiments on double-helix DNA for forces from about 0.1 up to 10 pN (Fig. 1.3).

      Figure 1.3 Force versus extension data for 97 kb dsDNA (L≈33 µm) of and shows a linear dependence on extension as expected for the semiflexible polymer. Note that 1kBT/nm=4.1 pN. Figure adapted from Marko, J.F., Siggia, E.D. 1995b. Macromolecules 28, 8759.

      The main force scales for DNA molecules encountered so far, then, are that required to start to stretch out the random-coil fluctuations, about kBT/A≈0.08 pN, and the force range from 0.1 to about 10 pN where the force is a strongly nonlinear function of the extension.

      1.2.2.4 High-Force Elastic Deformation of the Double Helix

      For forces in the range 10–40 pN, the double helix starts to stretch elastically. This stretching can be described by adding a term to the result above (Marko and Siggia, 1995b; Odijk, 1995):

      (1.13)

      The constant f0 has dimensions of a force and represents the stretching elastic constant of the double helix (f0 is the force at which the extrapolated linear elasticity would double the length of the double helix). Experimental data have determined f0≈1000 pN (Cluzel et al., 1996; Smith et al., 1996).

      We can understand this large force scale by going back to the microscopic fluctuations in length per base pair (see Fig. 1.2, which is about 10% of the equilibrium length (or rise) of the double helix per base pair (Feig et al., 2003)). Given that the root-mean-squared fluctuation in length is about δ=0.035 nm per base pair, we can estimate a double-helix spring constant of about kBT/δ²; multiplying this by the equilibrium rise of Δ=0.34 nm (to find the force at which this elastic model predicts doubling of helix length) gives f0=kBTΔ/δ²≈1000 pN.

      1.2.2.5 Effective Young’s Modulus of the Double Helix

      We can also think about f0 in terms of an effective Young’s modulus for DNA. If we suppose the double helix to be a circular rod of cross-sectional radius r0, then the relation between f(Landau and Lifshitz, 1986). Using the DNA radius r0=1 nm gives us Y≈3×10⁸ Pa, comparable to noncovalently bonded materials like plexiglas (filaments made of folded proteins have been found to have slightly higher moduli in the GPa range (Gittes et al., 1993), presumably because of their more three-dimensionally bonded structure).

      We can link Y back to the persistence length, again using the classical formula relating modulus to bending stiffness (. Using double-helix values for Y and r0 gives B=2.4×10−28 J m; recalling that persistence length is just B in kBT units gives A=B/(kBT)≈50 nm. DNA microscopic fluctuations, bending properties, and stretching properties are thus consistently related (Feig et al., 2003). We now turn to twisting properties of the double helix, but we will return to discuss strongly nonlinear stretching response near the end of the chapter.

      1.3 Double-Helix Topology and Twisting Stiffness

      We now turn to the twisting properties of DNA, a topic naturally connected to the internal linking number of double-stranded DNA double helix, or the wrapping of one strand around the other in the double helix. When DNA is formed into a loop or a circular DNA with both strands constrained, as often occurs in vivo, the topology of the looped DNA becomes fixed. All cells contain topoisomerase enzymes which catalyze topological changes of the double helix, and maintenance of DNA topology is a hallmark of all living cells. Interference with cellular control of DNA topology can easily be lethal, and drugs that target topoisomerases are used in many cancer chemotherapies and in antibiotics used to counter bacterial infections.

      Topology of polymers generally refers to linking or entanglement properties, which are invariant under smooth geometrical deformations, and which can only change when one polymer passes through another. A simple example is the linking of two rings; they can be unlinked, or linked together; one cannot pass from the unlinked to a linked state without breaking one of the rings (Fig. 1.4).

      Figure 1.4 Simple links of oriented loops. Lk for each pair is computed by adding up the signs of the crossings and dividing the sum by 2. (A) Unlinked rings; the signs of the crossings cancel, so Lk=0. (B) The Hopf link; the signs of the crossings add, so Lk=+1 (Lk would be −1 if the orientation of one of the loops were reversed). (C) For this link (sometimes called Solomon’s knot) the signs of the crossings again add, making Lk=+2. (D) the Whitehead link has canceling signs of its crossings, and has Lk=0 despite being a nontrivial link.

      1.3.1 Linking Number of Two Curves

      We can compute the linking number of two oriented closed curves by just counting up all of their mutual signed crossings, according to the rules shown in Fig. 1.5. Dividing the total crossing number by two gives an integer, the linking number Lk of the two curves (Fig. 1.4). This quantity can only change when one curve is passed through another. (Linking topology is perfectly well defined only for closed curves or polymers. However, it is sometimes useful to define linkage of open curves, using suitably defined closure boundary conditions, e.g., closing chains at infinity by extending them with long straight paths. This introduces small corrections to the properties of entanglement of interest here, primarily estimates of linking number. Qualitatively this can be understood by considering the definition of linking number in terms of signed crossings (Fig. 1.5). If we imagine deforming part of one of the links of Fig. 1.4 so that it closes far from the other crossings (not introducing any new crossings in the process) the topology and linking number of the polymer will be unchanged. This will be true for all closure paths that do not introduce additional strand crossings, indicating a rather weak dependence of linking number on closure boundary conditions, and further allowing us to talk about the topology of the region of the polymers not including the closure in a reasonably well-defined way. This is particularly true for linking of stretched polymers as will be discussed later; see, for example, Vologodskii and Marko (1997).)

      Figure 1.5 Sign convention for computation of linking number using crossings. Left: left-handed (−1) crossing. Right: right-handed (+1) crossing.

      The Gauss invariant computes the same quantity, but determines it from the geometry of the two curves:

      (1.14)

      For DNA, we can distinguish between external linking of two double-helix molecules together, and the internal linking property of the double helix itself.

      1.3.2 Internal Double-Helix Linking Number Lk

      We consider the internal linking of the two strands inside one double helix. The double helix contains two single-stranded DNAs (ssDNAs) which are wrapped around one another in a right-handed fashion, with a preferred twist rate of one turn every nh≈10.5 bp, or every h≈3.6 nm of contour length. For helical wrapping, we can associate a linking number, which is just the number of times one strand crosses over the other. (For internal DNA linking the Gauss invariant is computed using orientation of the two backbones in the same direction along the double helix, giving positive crossings to right-handed wrapping.) For a double helix of length L and Nbp base pairs, Lk≈Lk0=L/h=Nbp/nh. However, Lk is an integer for a closed double helix and is not in general equal to Lk0.

      The difference between double-helix linking number and the preferred linking number, ΔLk=Lk − Lk0, is often expressed as a fraction of the preferred linking number (linking number density), σ ≡ ΔLk/Lk0 (the excess linking number per DNA length is ΔLk/L=σ/h). In E. coli and many other species of bacteria, circular DNA molecules are maintained in a state of appreciably perturbed Lk, with σ≈−0.05. This is a sufficient perturbation to drive the DNA to supercoil, or wrap around itself in the manner of a twisted extension cord, due to competition between bending and twisting elasticity of the double helix.

      1.3.3 DNA Twist Stiffness

      If Lk is sufficiently different from Lk0, then there will be a buildup of twist in the DNA, leading to a response in the form of chiral bending. This response is often a wrapping of the double helix around itself, a phenomenon known as supercoiling. One can observe this by taking a stiff cord and twisting it. This behavior arises from a competition between the bending energy (Eq. (1.1)) and the elastic twist energy, the latter being

      (1.15)

      where Θ is the net twist angle along the double helix. This is just the form of the twisting energy for a uniform elastic rod (Landau and Lifshitz, 1986). Experimentally, this simple linear model has been observed to have a surprisingly wide range of validity for DNA, for C≈100 nm (Bryant et al., 2003).

      In the absence of other constraints, thermal fluctuations of twist give rise to a fluctuation

      (1.16)

      suggesting the interpretation of C as a characteristic length for twist fluctuations. For the double helix, this twist persistence length is C≈100 nm. Note that the derivative of Etwist with respect to Θ is the torque or torsional stress in the DNA:

      (1.17)

      If there is no bending, then any excess linking number ΔLk goes entirely into twisting the double helix: Θ=2πΔLk (or σ=Θ/[2πL/h]). The mechanical torque in DNA will be τ=2πkBTCΔLk/L=(2πkBTC/h)σ. The parameter 2πC/h≈175 sets the scale for when the linking number density will start to appreciably perturb DNA conformation, i.e., when |τ|≈kBT. This level of torque occurs for |σ|≈0.005.

      The twist persistence length C≈100 nm can be connected to the shear modulus µ (Landau and Lifshitz, 1986), giving µ≈2.5×10⁸ Pa (Landau and Lifshitz, 1986). Given that µ is expected to be comparable to Y, the value of C observed for DNA is consistent with use of the elastic deformation model.

      1.3.4 Decomposition of Double-Helix Lk Into Twist Tw and Writhe Wr

      The previous computation supposed that there was no bending, in which case all of the ΔLk is put into twisting the double helix. This DNA twisting can be quantified through the twist angle Θ, or equivalently through the twisting number. [The total twist of a DNA molecule is often written as the excess twist ΔTw plus the intrinsic twist, or Tw=ΔTw+Lk0=ΔTw+L/h, where ΔTw=Θ/(2π).]

      If DNA bending occurs, there may be nonlocal crossings of the double helix over itself. These nonlocal crossings contribute to double-helix linking number, and the separation of length scales between DNA thickness and the longer scale of DNA self-crossing (controlled by the persistence length A) allows linking number to be decomposed into local (twist) and nonlocal (writhe) crossing contributions:

      (1.18)

      or equivalently, ΔLk=ΔTw+Wr.

      One can demonstrate this with a thin strip of paper (30 cm by 1 cm works well). Put one twist into the strip, closing it in a ring. The two edges of the strip are linked together once. Now without opening the ring, let it assume a figure-8 shape; you will see that you can make the twist go away: in this state there is only writhe (Fig. 1.6).

      Figure 1.6 Left: a ribbon with Tw≈−1 and Wr≈0. Right: deforming the ribbon allows the twist to be transferred to writhe, so that Tw≈0 and Wr≈−1. The linking number is fixed at Lk=−1 as long as the strip is not broken.

      For elastic ribbon models of DNA, suitable definition of the twist allows Wr to be expressed by the analytical formula (White, 1969; Fuller, 1971):

      (1.19)

      where r1 and r2 are the two edges of the ribbon. The similarity of this equation to the Gauss invariant, Eq. (1.14), arises from the partitioning of the double integral into contributions from local wrapping of the strands in the double helix (Tw), and from nonlocal contributions (Wr) arising from nonlocal crossings of the centerline of the molecule. Eq. (1.19) is the sum of the signed nonlocal crossings for one curve (following the rule of Fig. 1.5), averaged over all orientations (Fuller, 1971). While Lk is a topological property and is quantized for a covalently closed double helix, Wr and Tw are geometrical, and change value smoothly as the molecule is distorted.

      1.3.5 DNA Supercoiling

      The ability to transfer Tw to Wr suggests that when there is appreciable torsional stress in a flexible filament, it can be relaxed by wrapping the filament around itself. For DNA we should also include the entropic cost of bringing the filament close to itself. A type of model widely used to describe the plectonemic wrapping of DNA around itself (Fig. 1.7) is based on treating the wrapping as helical, and by writing down a variational free energy (Marko and Siggia, 1994b, 1995a; Marko and Neukirch, 2012, 2013):

      (1.20)

      where Θ=2πΔTw is the DNA twisting (which costs twist elastic energy), κ is the bending curvature, which is κ=r/[r²+p²] for a regular helix of radius r and pitch p =πp, Fig. 1.7). The final two terms describe the entropic confinement free energy for a semiflexible polymer in a tube (Marko and Siggia, 1995a; Helfrich and Harbich, 1985; Burkhardt, 1997) and direct electrostatic and hard-core interactions per molecule length, v(r).

      Figure 1.7 Geometry of plectonemic supercoil, based on consideration of the shape as two interwound regular helices of radius r and the helix pitch p= /π.

      The confinement entropy is based on estimation of the correlation length for bending fluctuations for a confined chain, which will have curvature fluctuations ≈r/ξ². The fluctuations in the bending free energy per length (in kBT units) will be ≈Ar²/ξ⁴, and over a correlation length the fluctuation free energy is therefore ≈Ar²/ξ³. But the fluctuation free energy over a correlation length in kBT units should be ≈1, giving us ξ³≈Ar², and the confinement free energy per length (still kBT units) of ≈1/ξ=1/(Ar²)¹/³.

      The important final ingredient is Eq. (1.18) which allows the twist to be expressed in terms of linking number and the writhe: Θ=2πΔTw=2πLp/(2π[r²+p²]), where the upper/lower signs are for right/left-handed plectonemic wrapping (Marko and Siggia, 1995a).

      Putting this together gives the free energy per length

      (1.21)

      where the sign of the writhe has been chosen to provide the lower twist energy for positive ΔLk, which is the case of a left-handed superhelix (note that left-handed plectonemes form for ΔLk >0 while right-handed ones form for ΔLk<0).

      The free energy (1.21) can be optimized numerically to determine r and p (Marko and Siggia, 1995a; Marko and Neukirch, 2012, 2013; Neukirch and Marko, 2011). However, it is instructive to consider an approximate computation for the case of a slender superhelix (r p), for which the curvature is κ≈r/p², and the writhe per length is Wr/L≈1/(2πpof DNA. Dropping the molecular interaction potential v(r) gives

      (1.22)

      Now, this expression can be minimized to determine optimal values of r and p. Up to numerical constants, minimization over r sets Ar² ∝ p³ (setting the correlation length for bending fluctuations in the plectoneme to ξ≈p), and reduces the final two terms of Eq. (1.22) to k/p where k constant. Subsequent minimization with respect to 1/p gives −2π²C(ΔLk/L − 1/[2πp])+k=0, or 1/(2πp)=ΔLk/L − k/(2π²C). There is a threshold ΔLk*=kl/(2π²C) for appearance of a valid minimum (p > 0), introduced by the entropic cost of confinement; in terms of superhelix density σ=ΔLk/(L/h) this is σ*=k/(2π²C/h), which is small compared to unity due to the ratio of length scales C/h≈(100 nm)/(3.6 nm). Beyond this characteristic value of linking number, the plectoneme becomes stable (see Fig. 1.8 which displays the transition from open to supercoiled structures with increasing linking number), and has a free energy below the essentially unwrithed, twisted molecule. This provides a rough idea of the behavior of the full plectoneme model Eq. (1.21) (Marko and Siggia, 1994b, 1995a; Marko and Neukirch, 2013; Neukirch and Marko, 2011). For sufficient ΔLk, screening of the twist energy Eq. (1.15) by the writhe becomes favorable, which has little bending free energy cost if the superhelix radius r is kept relatively small.

      Figure 1.8 Electron micrographs of supercoiled DNA at a few different σ values. Scale bar is 100 nm (300 bp); molecules are all 7 kb (2300 nm) in length. Reproduced from Boles, T.C., White, J.H., Cozzarelli, N.R., 1990. J. Mol. Biol. 213.

      Given that the main result for the free energy of the plectoneme is a free energy that rises from zero and eventually becomes superlinear, a useful approximate form to use for the free energy per length of the plectoneme is βF(σ)/L=(2π²Cp/h²)σ², where Cp≈25 nm, Cp < C reflecting the twist-energy-screening effect (Marko, 2007). An important feature of plectonemically supercoiled DNA is its branched structure. Branch points can be thought of as defects in the plectonemic supercoil structure: like the ends, there is some energy cost associated with them. However, there is an entropy gain≈kB ln L/A of having a branch point, since it can be placed anywhere in the molecule. Balance of branch point energy and entropy determines the observed density of a Y-shaped branch point for every 2 kb along a supercoil with σ=−0.05 (Marko and Siggia, 1995a). Branching is also important to the internal sliding of DNA sequence around in the interior of a plectonemically supercoiled DNA, a process important to some enzymes which bind to two sequences simultaneously across a plectnomemic superhelix (Marko et al., 1997).

      1.3.6 Twisting Stretched DNA

      In single-molecule DNA stretching experiments, if a force in the pN range is applied the double helix will be nearly straight. If it is then slightly twisted while under≈pN forces, the molecule will tend to coil chirally, leading to a slight contraction. For larger amounts of twisting, the torque in the DNA will build up to a point where the molecule will buckle, and plectonemic supercoils will start to form along it.

      For small twisting, a small-fluctuation-amplitude computation can be done (, where u are the components of t ). We begin with the energy for a DNA under tension and twist:

      (1.23)

      just Eq. (1.8) with the addition of the twist energy. For a single-DNA experiment, ΔLk is just the number of full turns made of the end of the molecule (in a magnetic tweezers experiment, the number of times the magnet and therefore the bead at the end of the DNA is rotated (Strick et al., 1996)).

      The challenge is how to include the linking number constraint in Eq. (1.23). The solution is to use an alternative representation of the writhe which takes the form of a single integral over contour length s (Fuller, 1978):

      (1.24)

      The appearance of the mod 1 in Eq. (1.24) reflects the fact that this expression for Wr (unlike the double integral Eq. (1.19)) is not sensitive to antipodal points, essentially nonlocal crossings which contribute ±1 to the total writhe (for a detailed discussion, see Neukirch and Starostin (2008)). The huge advantage of Eq. (1.24) over Eq. (1.19) is the presence of only a single integral, permitting expansion in powers of u for small deformations away from a straight configuration:

      (1.25)

      This quantity is quadratic in u since the writhe of a straight line configuration is zero.

      Using this in the twisting energy Eq. (1.23) and expanding to quadratic order in u gives:

      (1.26)

      which when Fourier transformed is

      (1.27)

      For the untwisted case σ=0 this reduces to the fluctuation free energy of the untwisted chain, Eq. (1.9).

      In terms of Cartesian components of u, nonzero twisting leads to an off-diagonal coupling, which can lead to a zero eigenvalue and an elastic instability. The stability condition is the requirement of a positive determinant (Aq²+βf)²−(2πC/h)²σ²q²>0. The eigenvalue vanishing condition occurs for σc . This instability places a hard limit on the maximum value of σ* for which this type of model can be applied; for f=0.5 pN, this is σc≈0.028. By expressing this in terms of the DNA torque for a straight molecule τ=kBT(2πC/h)σ , which is the classical buckling instability of a rod subject to tension and torque (Love, 1944). The same instability can be observed in dynamical models of twisted and stretched DNA (Banigan and Marko, 2014).

      Diagonalization (the eigenvectors of the matrix in Eq. (1.27) are proportional to the circularly polarized states [1,±iu, or

      (1.28)

      where the neglected terms are of higher order in 1/f. Changing σ from zero leads to additional shrinkage over the untwisted case, due to chiral bending fluctuations.

      Either integration of the extension with force or direct computation of the partition function gives the free energy per length in a similar 1/f expansion:

      (1.29)

      The last term shows that the effect of the chiral fluctuations is to, as for DNA supercoiling, partially screen the twist energy, generating a reduction in the effective twist modulus C → Cf=C[1−(C/2 A)(kBT/4Af)¹/²]. This effect was used by Moroz and Nelson (1997) to estimate the twist elastic constant C from single-molecule data of Strick et al. (1996) and led to a substantial revision in the accepted value of C from 75 nm up to the range 100–125 nm.

      1.3.7 Coexistence of Supercoiled and Twisted Stretched DNA

      For fixed force and sufficient ΔLk, one has phase coexistence of domains of plectonemic supercoiling and extended DNA (sketched in Fig. 1.9) (Marko and Siggia, 1994b, 1995a; Marko and Neukirch, 2013; Marko, 2007). These pure states can be described by free energies per B-DNA length dependent on applied force f and the linking number density σfor plectonemic DNA (the free energies per length discussed in the prior two sections, i.e., up to a factor of kBT, Eqs. (1.29) and (1.21)). For these pure states, the rate that work is done injecting linking number is proportional to torque, for example:

      (1.30)

      Figure 1.9 Sketch of a DNA molecule under tension f, and with linking number fixed so as to put the double helix under torsional stress. Over a range of applied tension, the molecule breaks up into domains of extended and plectonemically supercoiled DNA. Only a single domain of plectonemic DNA is shown for clarity.

      The prefactor ω0=2π/h=2π/(3.6 nm) is the angle of twist per molecule length for relaxed B-DNA, which converts the σ derivatives to ones with respect to angle.

      Along a molecule which is a fraction xand fraction xp=1 − xs , the free energy per base pair of the mixed phase is

      (1.31)

      The equilibrium length fraction xs and the free energy are determined by minimization of this free energy subject to the constraint σ=xsσs+xpσp: linking number is considered as being partitioned between the two states. In the case of interest here, the plectonemic regions are essentially closed loops (see Fig. 1.9). By pinching of those loops off to form circular plectonemic supercoils separated from the extended DNA, the calculation of writhe can be decoupled into separate writhes for extended and plectonemic regions.

      If the pure state free energy densities plotted as a function of linking number density never cross, then one pure state or the other will be the equilibrium state, i.e., one of the two extreme cases xs=0 or xs=1 will always minimize Eq. (1.31). If the two free energy densities cross, then there will be a range of σ over which there will be coexisting domains of the two states. Fig. 1.10 shows this situation, sketched to correspond to the case of main interest here, where at low values of σ the stretched state is stable (lower in free energy) relative to the plectoneme state, but where at large σ the stability reverses due to screening of the twist energy by the plectonemic state’s writhe (Marko and Siggia, 1994b, 1995b; Marko and Neukirch, 2013).

      Figure 1.10 ) DNA states as a function of linking number σ. For σ < σsor any mixture of the two. Similarly, for σ > σpis the lowest free energy configuration. On the other hand, for σ between σs and σp , is the lowest free energy state. Note that the gap between the two states near σ; this difference grows with applied force and is due to the term −β·f in the extended state free energy Eq. (1.29).

      Minimization of Eq. (1.31) leads to a double-tangent construction familiar from other examples of phase coexistence (e.g., liquid–gas); in this case the conserved density is that of linking number (Fig. 1.10). The two coexisting states of linking number densities σs and σp , i.e., they have equal torques. They mix in proportions xs and xp, so the free energy in the coexistence region is

      (1.32)

      In the coexistence region, the fractions of the two states in the mixed state depend linearly on σ, as

      (1.33)

      The coexistence construction guarantees that the free energy is a convex function of linking number, and therefore that the torque is a monotonic function of linking number, as required for mechanical stability. In the coexistence region (σ between the limits σs and σp) the torques in the two types of domains are equal and σ-independent; i.e., the σ-derivative of Eq. (1.32) is constant. This is quite useful for experiments on topoisomerases, since measurements carried out in the rather broad plectoneme-extended coexistence regions (along the linear portions of the hat curves of Fig. 1.11) are done at fixed torque. The value of torque in the coexistence regions is controlled by the constant force, varying from about 7 pN nm at 0.5 pN (approximately the torque in a plasmid with physiological supercoiling σ≈0.06 (Marko, 2007; Mosconi et al., 2009), to a little more than 25 pN nm at 3 pN (Marko, 2007; Forth et al., 2008) (note that there is an appreciable torque decrease with increased salt (Mosconi et al., 2009), since DNA hard-core diameter drops and therefore plectoneme tightness increases (Neukirch and Marko, 2011) with increased salt concentration).

      Figure 1.11 Extension versus linking number curves for fixed forces. (A) Experimental data of G. Charvin for 48 kb segment of λ), 0.74 pN (◊), 1.31 pN (+), and 2.95 pN (×). As force is increased, the extension increases, and the contracting effect of torsional stress (linking number) is reduced. For low values of force below 0.5 pN, the curves are symmetric around σ=0, but for larger forces asymmetry appears due to stress-driven DNA melting. (B) The parabolic peak of each extension curve occurs when the DNA is purely in the extended state; extended and plectonemic DNA are in coexistence on the steep linear parts of each extension curve. The beginning of the steep black linear segments for positive supercoiling for 0.2, 0.5, and 1.5 pN, and for negative supercoiling for 0.2 and 0.5 pN indicates σs, and their intercepts with the σ axis indicates σp. For 10 pN and positive supercoiling, as well as for 2, 5, and 10 pN for negative supercoiling, formation of plectonemic DNA is preempted by DNA strand separation (the torque exceeds the critical torque for melting) with the result that a much shallower coexistence line is obtained, corresponding to coexistence of the extended state and torque-melted DNA. For details of the model and the entire force-σ and force–torque phase diagrams, see Marko and Neukirch (2013). (A) Data from Neukirch, S., 2004. Phys. Rev. Lett 93, 198107. (B) Theoretical curves from Marko, J.F., 2007. Phys. Rev. E 76,

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