Mechanically Controlled DNA Extrusion from a Palindromic Sequence by Single Molecule Micromanipulation

A magnetic tweezers setup is used to control both the stretching force and the relative linking number $\Delta \mathrm{Lk}$ of a palindromic DNA molecule. We show here, in absence of divalent ions, that twisting negatively the molecule while stretching it at $\sim 1\mathrm{pN}$ induces the formation of a cruciform DNA structure. Furthermore, once the cruciform DNA structure is formed, the extrusion of several kilo-base pairs of palindromic DNA sequence is directly and reversibly controlled by varying $\Delta \mathrm{Lk}$. Indeed the branch point behaves as a nanomechanical gear that links rotation with translation, a feature related to the helicity of DNA. We obtain experimentally a very good linear relationship between the extension of the molecule and $\Delta \mathrm{Lk}$. We use then this experiment to obtain a precise measurement of the pitch of B-DNA in solution : $3.61\pm 0.03$ nm/turn.

Figure 1

DNA extrusion from of a palindromic molecule. Example of measurement of the extension (Ext) vs the relative linking number ($\Delta \mathrm{Lk}$) of a palindromic DNA molecule, for two levels of force, 0.3 pN and 1.5 pN, respectively. The curves are obtained by averaging the extension every 10 turns for a period of 1 s to 4 min depending on the force level (arrows indicate the direction of rotations). Buffer conditions: 25 mM Tris-Acetate pH 8, 0.5 mM EDTA, 0.1% BSA, 0.01% ${\mathrm{NaN}}_3$, $T=27°\mathrm{C}$. The slope of the curve segments 2, 3, and 4 is $\sim 3.1\mathrm{nm}/\mathrm{\text{turn}}$.

Figure 2

Coupling between torsion and extrusion (arrows) for a palindromic DNA sequence. The gray and black double strands are related by a two-fold symmetry. (a) Cruciform DNA formation. (b) Strand exchange or branch point migration.

Figure 3

Helical pitch measurement. (a) Migration curves (Ext vs $\Delta \mathrm{Lk}$) at forces from 0.08 pN to 4.7 pN (buffer as in Fig. 1, $T=37°\mathrm{C}$). Each curve is obtained by averaging together a forward and reverse extrusion. The same DNA molecule is used for all data points in this figure. Identical results are found on other molecules. gray lines: linear fits with a common intercept imposed. (b) Force vs slope ($\mathrm{\text{slope}}=d\mathrm{Ext}/d\Delta \mathrm{Lk}$). Filled diamonds: results from the linear fits of the spindle in (a). Open diamonds: same experiment on another molecule in the presence of $0.1\mu \mathrm{M}$ of ethidium bromide (EtBr). Solid lines: fits using the WLC model (see text). Results: black line: $p=3.59\pm 0.01\mathrm{nm}/\mathrm{\text{turn}}$, $L_P=50\pm 3\mathrm{nm}$; gray line: $p^{0.1\mathrm{EB}}=4.12\pm 0.05\mathrm{nm}/\mathrm{\text{turn}}$, $L_P^{0.1\mathrm{EB}}=52\pm 5\mathrm{nm}$. Inset: pitch distribution obtained from 12 different DNA molecules in the absence of EtBr.