Biomedical subjects

Timothée Lionnet

Publications and source records attributed to Timothée Lionnet.

4 recordsLinked to original sources

PubMed2006

Sequence-dependent twist-stretch coupling in DNA.

Recent single-molecule micromanipulation experiments on DNA subject to small distortion revealed positive coupling between DNA stretching and twisting--for instance, DNA elongates when overtwisted. Here we propose a method to calculate the twist-stretch coupling constant specific to a DNA fragment of a given sequence. The method employs a sequence-dependent dinucleotide force field and is based on constrained minimization of the fragment's deformation energy. Using a force field inferred from atomistic molecular dynamics simulations, we obtain the twist-stretch coupling for random sequence to be 0.30 nm/turn, close to experimental values. An exhaustive calculation for all oligomers of nine basepairs yields values between 0.14 and 0.45 nm/turn, positively correlated with the contents of pyrimidine-purine steps in the sequence. Our method is simple to use and allows one to explore the hypothesis that some sequences may be optimized for twist-stretch coupling.

Base Sequence↗

PubMed2006

DNA mechanics as a tool to probe helicase and translocase activity.

Helicases and translocases are proteins that use the energy derived from ATP hydrolysis to move along or pump nucleic acid substrates. Single molecule manipulation has proved to be a powerful tool to investigate the mechanochemistry of these motors. Here we first describe the basic mechanical properties of DNA unraveled by single molecule manipulation techniques. Then we demonstrate how the knowledge of these properties has been used to design single molecule assays to address the enzymatic mechanisms of different translocases. We report on four single molecule manipulation systems addressing the mechanism of different helicases using specifically designed DNA substrates: UvrD enzyme activity detection on a stretched nicked DNA molecule, HCV NS3 helicase unwinding of a RNA hairpin under tension, the observation of RecBCD helicase/nuclease forward and backward motion, and T7 gp4 helicase mediated opening of a synthetic DNA replication fork. We then discuss experiments on two dsDNA translocases: the RuvAB motor studied on its natural substrate, the Holliday junction, and the chromosome-segregation motor FtsK, showing its unusual coupling to DNA supercoiling.

DNA↗

PubMed2006

Wringing out DNA.

The chiral nature of DNA plays a crucial role in cellular processes. Here we use magnetic tweezers to explore one of the signatures of this chirality, the coupling between stretch and twist deformations. We show that the extension of a stretched DNA molecule increases linearly by 0.42 nm per excess turn applied to the double helix. This result contradicts the intuition that DNA should lengthen as it is unwound and get shorter with overwinding. We then present numerical results of energy minimizations of torsionally restrained DNA that display a behavior similar to the experimental data and shed light on the molecular details of this surprising effect.

Base Pairing↗

PubMed2004

Single-molecule assay reveals strand switching and enhanced processivity of UvrD.

DNA helicases are enzymes capable of unwinding double-stranded DNA (dsDNA) to provide the single-stranded DNA template required in many biological processes. Among these, UvrD, an essential DNA repair enzyme, has been shown to unwind dsDNA while moving 3'-5' on one strand. Here, we use a single-molecule manipulation technique to monitor real-time changes in extension of a single, stretched, nicked dsDNA substrate as it is unwound by a single enzyme. This technique offers a means for measuring the rate, lifetime, and processivity of the enzymatic complex as a function of ATP, and for estimating the helicase step size. Strikingly, we observe a feature not seen in bulk assays: unwinding is preferentially followed by a slow, enzyme-translocation-limited rezipping of the separated strands rather than by dissociation of the enzymatic complex followed by quick rehybridization of the DNA strands. We address the mechanism underlying this phenomenon and propose a fully characterized model in which UvrD switches strands and translocates backwards on the other strand, allowing the DNA to reanneal in its wake.

Adenosine Triphosphate↗