Integrating single-molecule visualization and DNA micromanipulation

MacKintosh, F.C. [Promotor]Peterman, E.J.G. [Copromotor]Wuite, G.J.L. [Copromotor

Calibrating bead displacements in optical tweezers using acousto-optic deflectors

Displacements of optically trapped particles are often recorded using back-focal-plane interferometry. In order to calibrate the detector signals to displacements of the trapped object, several approaches are available. One often relies either on scanning a fixed bead across the waist of the laser beam or on analyzing the power spectrum of movements of the trapped bead. Here, we introduce an alternative method to perform this calibration. The method consists of very rapidly scanning the laser beam across the solvent-immersed, trapped bead using acousto-optic deflectors while recording the detector signals. It does not require any knowledge of solvent viscosity and bead diameter, and works in all types of samples, viscous or viscoelastic. Moreover, it is performed with the same bead as that used in the actual experiment. This represents marked advantages over established methods. © 2006 American Institute of Physics

Unraveling DNA tori under tension

Motivated by recent experiments, we develop a model for DNA toroids under external tension. We find that tori are the equilibrium states for our model up to a critical tension, above which they become only metstable. Above this tension, we find a cascade of transitions between discrete toroid states that successively lowers the winding number, until the ground state (rod) is reached. In this process, this model predicts a nearly constant force plateau as a function of extension, in agreement with experiment.Comment: 9 pages, 11 figure

Mesoscopic models for DNA stretching under force: new results and comparison to experiments

Single molecule experiments on B-DNA stretching have revealed one or two structural transitions, when increasing the external force. They are characterized by a sudden increase of DNA contour length and a decrease of the bending rigidity. It has been proposed that the first transition, at forces of 60--80 pN, is a transition from B to S-DNA, viewed as a stretched duplex DNA, while the second one, at stronger forces, is a strand peeling resulting in single stranded DNAs (ssDNA), similar to thermal denaturation. But due to experimental conditions these two transitions can overlap, for instance for poly(dA-dT). We derive analytical formula using a coupled discrete worm like chain-Ising model. Our model takes into account bending rigidity, discreteness of the chain, linear and non-linear (for ssDNA) bond stretching. In the limit of zero force, this model simplifies into a coupled model already developed by us for studying thermal DNA melting, establishing a connexion with previous fitting parameter values for denaturation profiles. We find that: (i) ssDNA is fitted, using an analytical formula, over a nanoNewton range with only three free parameters, the contour length, the bending modulus and the monomer size; (ii) a surprisingly good fit on this force range is possible only by choosing a monomer size of 0.2 nm, almost 4 times smaller than the ssDNA nucleobase length; (iii) mesoscopic models are not able to fit B to ssDNA (or S to ss) transitions; (iv) an analytical formula for fitting B to S transitions is derived in the strong force approximation and for long DNAs, which is in excellent agreement with exact transfer matrix calculations; (v) this formula fits perfectly well poly(dG-dC) and $\lambda$-DNA force-extension curves with consistent parameter values; (vi) a coherent picture, where S to ssDNA transitions are much more sensitive to base-pair sequence than the B to S one, emerges.Comment: 14 pages, 9 figure

Effect of the BRCA2 CTRD domain on RAD51 filaments analyzed by an ensemble of single molecule techniques

Homologous recombination is essential for the preservation of genome stability, thereby preventing cancer. The recombination protein RAD51 drives DNA strand exchange, which requires the assembly, rearrangement and disassembly of a RAD51 filament on DNA, coupled to ATP binding and hydrolysis. This process is facilitated and controlled by recombination mediators and accessory factors. Here, we have employed a range of single molecule techniques to determine the influence of the C-terminal RAD51 interaction domain (CTRD) of the breast cancer tumor suppressor BRCA2 on intrinsic aspects of RAD51-DNA interactions. We show that at high concentration the CTRD entangles RAD51 filaments and reduces RAD51 filament formation in a concentration dependent manner. It does not affect the rate of filament disassembly measured as the loss of fluorescent signal due to intrinsic RAD51 protein dissociation from double-stranded DNA (dsDNA). We conclude that, outside the context of the full-length protein, the CTRD does not reduce RAD51 dissociation kinetics, but instead hinders filament formation on dsDNA. The CTRDs mode of action is most likely sequestration of multiple RAD51 molecules thereby rendering them inactive for filament formation on dsDNA