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Recombinaison Génétique à l'Échelle de la Molécule Unique : Micromécanique des Jonctions de Holliday et Activité du Complexe RuvAB

Abstract : This work is a study, at the single molecule level, of the recombination intermediate produced
when two homologous DNA molecules exchange their single-strands : the Holliday junction.
First, we show that a negative torque applied on a DNA molecule with an entirely palindromic
sequence leads to the formation of a Holliday junction. The strand exchange can then be directly driven
by mechanical torsion. Using this technique we accessed experimentally, and for the first time with such
a precision, to the value in solution of the helical pitch of DNA : 3.61 ± 0.03 nm/tr.
We also studied the kinetics of the strand exchange process under the influence of mechanical
constraints and in the presence of magnesium ions. We then developed a simple model of the mechanical
behavior of the Holliday junction under mechanical constraint in order to interpret the experimental data.
The single-strand exchange can also be catalyzed by specific enzymes. The mechanical work
produced during this activity defines these proteins as molecular motors. The second part of this work
concerns the study, at the single molecule level, of one of these enzymes : the RuvAB complex of the
bacteria Escherichia coli.
Firstly, we characterized the RuvAB migration activity on single Holliday junctions. In particular,
we found that the complex is highly processive and we estimated its speed at 37◦C and in presence
of 1 mM ATP : ∼ 43 base pairs exchanged per second.
Furthermore, we have shown that the RuvA subunit alone catalyzes the exchange of base-pairs
that occurs at the branch point of the structure.
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https://tel.archives-ouvertes.fr/tel-00012063
Contributor : Philippe Thomen <>
Submitted on : Thursday, March 30, 2006 - 2:41:18 PM
Last modification on : Thursday, December 10, 2020 - 12:37:22 PM
Long-term archiving on: : Saturday, April 3, 2010 - 11:04:22 PM

Identifiers

  • HAL Id : tel-00012063, version 1

Citation

Alexandre Dawid. Recombinaison Génétique à l'Échelle de la Molécule Unique : Micromécanique des Jonctions de Holliday et Activité du Complexe RuvAB. Biophysique [physics.bio-ph]. Université Pierre et Marie Curie - Paris VI, 2005. Français. ⟨tel-00012063⟩

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