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Diffusion de l'enzyme de restriction EcoRV sur des molécules d'ADN etirées

Abstract : Many proteins have to localize short target sequences on long DNA molecules. The time necessary for the localization is often much shorter than the upper limit estimated for a 3D diffusion process. Several mechanisms have been proposed to account for this extraordinary efficiency (1D diffusion along DNA, jumps...). However, in spite of the large number of studies performed by biochemists in the last twenty years, especially on EcoRI and EcoRV enzymes, a clear conclusion regarding the involved mechanisms has not yet been reached.

In this work, we used fluorescence microscopy to observe the interaction between a single EcoRV enzyme and a DNA molecule. To achieve this aim, we developed a new technique to stretch DNA on a surface, and we labeled EcoRV enzymes with quantum dots, which are very bright dyes that are devoid of photobleaching. The labeled enzymes were still active and could be detected with a spatial resolution close to 10 nm and a temporal resolution of 20 ms.

We observed many association/dissociation events of enzymes on stretched DNA molecules. The analysis of these events allowed us to measure the dissociation rate of the labeled enzymes, and provided evidence for a facilitated diffusion of EcoRV along DNA, whose characteristics could be obtained.
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https://tel.archives-ouvertes.fr/tel-00011594
Contributor : Aurelien Crut <>
Submitted on : Monday, February 13, 2006 - 2:06:27 PM
Last modification on : Monday, December 14, 2020 - 9:49:18 AM
Long-term archiving on: : Monday, September 17, 2012 - 11:51:09 AM

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  • HAL Id : tel-00011594, version 1

Citation

Aurélien Crut. Diffusion de l'enzyme de restriction EcoRV sur des molécules d'ADN etirées. Biophysique [physics.bio-ph]. Université Pierre et Marie Curie - Paris VI, 2005. Français. ⟨tel-00011594⟩

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