A homogeneous resonance energy transfer-based assay to monitor MutS/DNA interactions.
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.
Lopez-Crapez
Evelyne
E
Department of Oncobiology, Cancer Research Center, CRLC Val d'Aurelle-Paul Lamarque, 34298 Montpellier Cedex 5, France. ecrapez@valdorel.fnclcc.fr
Malinge
Jean-Marc
JM
Gatchitch
François
F
Casano
Laetitia
L
Langlois
Thierry
T
Pugnière
Martine
M
Roquet
Françoise
F
Mathis
Gérard
G
Bazin
Hervé
H
D020137:Base Pair Mismatch; D001483:Base Sequence; D004247:DNA / Q000235:genetics / Q000378:metabolism*; D004735:Energy Transfer; D004926:Escherichia coli; D029968:Escherichia coli Proteins / Q000378:metabolism*; D051719:MutS DNA Mismatch-Binding Protein / Q000378:metabolism*; D009838:Oligodeoxyribonucleotides / Q000235:genetics / Q000378:metabolism; D011485:Protein Binding; D013997:Time Factors
D029968:Escherichia coli Proteins; D009838:Oligodeoxyribonucleotides; D004247:DNA; D051719:MutS DNA Mismatch-Binding Protein