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A homogeneous resonance energy transfer-based assay to monitor MutS/DNA interactions.
Lopez-Crapez, Evelyne; Malinge, Jean-Marc; Gatchitch, François; Casano, Laetitia; Langlois, Thierry; Pugnière, Martine; Roquet, Françoise; Mathis, Gérard; Bazin, Hervé.
Afiliación
  • Lopez-Crapez E; Department of Oncobiology, Cancer Research Center, CRLC Val d'Aurelle-Paul Lamarque, 34298 Montpellier Cedex 5, France. ecrapez@valdorel.fnclcc.fr
Anal Biochem ; 383(2): 301-6, 2008 Dec 15.
Article en En | MEDLINE | ID: mdl-18824153
ABSTRACT
Probing the interactions of the DNA mismatch repair protein MutS with altered and damaged DNA is of great interest both for the understanding of the mismatch repair system function and for the development of tools to detect mutations. Here we describe a homogeneous time-resolved fluorescence (HTRF) assay to study the interactions of Escherichia coli MutS protein with various DNA substrates. First, we designed an indirect HTRF assay on a microtiter plate format and demonstrated its general applicability through the analysis of the interactions between MutS and mismatched DNA or DNA containing the most common lesion of the anticancer drug cisplatin. Then we directly labeled MutS with the long-lived fluorescent donor molecule europium tris-bipyridine cryptate ([TBP(Eu(3+))]) and demonstrated by electrophoretic mobility shift assay that this chemically labeled protein retained DNA mismatch binding property. Consequently, we used [TBP(Eu(3+))]-MutS to develop a faster and simpler semidirect HTRF assay.
Asunto(s)

Texto completo: 1 Banco de datos: MEDLINE Asunto principal: ADN / Proteínas de Escherichia coli / Transferencia de Energía / Proteína MutS de Unión a los Apareamientos Incorrectos del ADN Idioma: En Revista: Anal Biochem Año: 2008 Tipo del documento: Article País de afiliación: Francia

Texto completo: 1 Banco de datos: MEDLINE Asunto principal: ADN / Proteínas de Escherichia coli / Transferencia de Energía / Proteína MutS de Unión a los Apareamientos Incorrectos del ADN Idioma: En Revista: Anal Biochem Año: 2008 Tipo del documento: Article País de afiliación: Francia