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Proprietes elastiques d'une molécule d'ADN simple brin, et interactions ADN hélicases à l'échelle de la molécule unique

Abstract : In the first part of this thesis, we studied the elasticity of a single-stranded DNA molecule using a magnetic trap technique. We have experimentally verified that a complete theoretical description of single-stranded DNA should take into account both the formation of secondary structures at low forces, and excluded volume effects. Indeed, single-stranded DNA is so flexible that the electrostatic repulsions due to the phosphates groups along the chain can no longer be neglected. We characterized the elasticity of the single-stranded DNA molecule under various salt conditions, i.e. under various charge screening conditions and under conditions permitting complete inhibition of secondary structures. Numerical simulations that take into account both effects were in excellent agreement with our experimental observations. In the second part of this thesis, we studied the activity of two DNA helicases at the single molecule level. Helicases are motor proteins responsible for the unwinding of the double helix of DNA into separated strands of single-stranded DNA, thus permitting access to the individual bases forming the genetic code. In our experiment a single molecule of double-stranded DNA was trapped using magnetic tweezers, and its extension was measured with 10 nm accuracy. This allowed us to follow the unwinding activity of helicases, as this latter yielded a measurable change in extension as the double-stranded DNA is transformed into single-stranded DNA. This method has the advantage of tracking the activity of one single protein in real time. Statistical analysis of the data yielded access to the complete distributions of enzyme velocity and processivity. We thus characterized the dynamical behaviour of single helicases (their velocity, processivity, cooperativity and elementary step size on DNA). As we followed the activity of helicases in real time, we observed a dynamic instability in their mode of action, which could be used to explain velocity measurements obtained in bulk experiments. These helicases appeared to be more efficient than generally estimated: the hydrolysis of ATP seemed to serve principally to the unwinding of the double helix.
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https://tel.archives-ouvertes.fr/tel-00001765
Contributor : Marie-Noelle Dessinges <>
Submitted on : Wednesday, October 16, 2002 - 2:25:07 PM
Last modification on : Thursday, December 10, 2020 - 12:36:21 PM
Long-term archiving on: : Monday, September 17, 2012 - 3:10:46 PM

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  • HAL Id : tel-00001765, version 1

Citation

Marie Noelle Dessinges. Proprietes elastiques d'une molécule d'ADN simple brin, et interactions ADN hélicases à l'échelle de la molécule unique. Biophysique [physics.bio-ph]. Université Pierre et Marie Curie - Paris VI, 2002. Français. ⟨tel-00001765⟩

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