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NOUVELLES SONDES NUCLEIQUES POUR LA MESURE D'ACTIVITES ENZYMATIQUES DE REPARATION DES DOMMAGES DE L'ADN PAR UN TEST FRET

Alexia Chollat-Namy 1
1 LAN - Laboratoire Lésions des Acides Nucléiques
SCIB - UMR E3 - Service de Chimie Inorganique et Biologique : DSM/INAC/SCIB
Abstract : CLASSICAL ANALYSIS METHODS FOR ENZYMATIC DNA REPAIR ACTIVITY MONITORING ARE TEDIOUS AND TIME-CONSUMING (GEL ELECTROPHORESIS COUPLED TO ISOTOPE LABELING OR HIGH PERFORMANCE LIQUID CHROMATOGRAPHY). HENCE, WE DEVELOPED A NEW DNA REPAIR ASSAY BASED ON FRET (FLUORESCENT RESONANCE ENERGY TRANSFER) DETECTION. THIS HAS REQUIRED THE DESIGN OF AN ORIGINAL DNA PROBE: A HAIRPIN STRUCTURE CONTAINING SPECIFIC LESIONS LOCATED AT DEFINED SITES WITHIN THE STEM SEQUENCE, CLOSE TO THE CHROMOPHORE-LABELED EXTREMITIES. THE LESION EXCISION BY DNA N-GLYCOSYLASES LEADS TO THE COMPLEMENTARY STRAND SEPARATION, CAUSING A DROP IN FLUORESCENCE QUENCHING. THUS, THE CLEAVAGE EXTENT IS REFLECTED BY THE FLUOROPHORE EMISSION INTENSITY INCREASE AND STABILIZATION OVER THE REPAIR REACTION TIME-COURSE. AFTER PROVING THE LINEARITY OF THE ASSAY RESPONSE, WE DEMONSTRATED THE ABILITY OF THE CURRENT METHOD TO GIVE ACCESS TO REPAIR ENZYME KINETIC PARAMETERS. THE RELEVANCE OF THESE PARAMETERS WAS CHECKED BY COMPARISON TO VALUES, OBTAINED BY PAGE ANALYSES. THEN, WE INVESTIGATED THE CAPABILITY OF OUR TEST TO BE USED AS A HIGH-THROUGHPUT SCREENING TOOL TO QUANTIFY ENZYMATIC REPAIR ACTIVITIES AND CHECK INHIBITION EFFECTS (BY USING PURIFIED ENZYMES OR CELL EXTRACTS). FINALLY, A MINIATURIZATION PROJECT WAS CARRIED OUT TO IMPROVE THE SENSITIVITY OF THIS NEW DETECTION METHOD. THE PERFORMANCE OF THIS HOMOGENEOUS LIQUID-PHASE ASSAY SHOULD LEAD TO APPLICATIONS IN THE BIOANALYTICAL FIELD RELATED TO FUNDAMENTAL, BIOMEDICAL AND PHARMACEUTICAL RESEARCH.
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https://tel.archives-ouvertes.fr/tel-00106970
Contributor : Alexia Chollat-Namy <>
Submitted on : Monday, October 16, 2006 - 6:25:24 PM
Last modification on : Wednesday, November 4, 2020 - 1:49:30 PM
Long-term archiving on: : Tuesday, April 6, 2010 - 7:55:42 PM

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  • HAL Id : tel-00106970, version 1

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Alexia Chollat-Namy. NOUVELLES SONDES NUCLEIQUES POUR LA MESURE D'ACTIVITES ENZYMATIQUES DE REPARATION DES DOMMAGES DE L'ADN PAR UN TEST FRET. Ingénierie biomédicale. Université Joseph-Fourier - Grenoble I, 2006. Français. ⟨tel-00106970⟩

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