Recombinases and Related Proteins in the Context of Homologous Recombination Analyzed by Molecular Microscopy.
Fiche publication
Date publication
janvier 2018
Journal
Methods in molecular biology (Clifton, N.J.)
Auteurs
Membres identifiés du Cancéropôle Est :
Dr PIETREMENT Olivier
Tous les auteurs :
Dupaigne P, Tavares EM, Piétrement O, Le Cam E
Lien Pubmed
Résumé
Transmission electron microscopy (TEM) and atomic force microscopy (AFM) are powerful tools to study the behavior of various actors in homologous recombination including molecular motors such as recombinases and helicases/translocases. Here we present specific approaches developed in terms of sample preparation and imaging methods to contribute to the understanding of homologous recombination process and its regulation focusing on the interplay between recombinases and other related proteins such as mediators or antirecombinase actors.Homologous recombination (HR) is a high-fidelity DNA repair pathway since it uses a homologous DNA as template. Recombinases such as RecA in bacteria, RadA in archaea, and Rad51 in eukaryotes are key proteins in the HR pathway: HR is initiated with formation of an ssDNA overhang on which recombinases polymerize and form a dynamic active nucleoprotein filament able to search for homology and to exchange DNA strand in an ATP-dependent manner. We provide practical methods to analyze presynaptic filament formation on ssDNA, its composition and regulation in presence of mediator partners, antirecombinase activity of translocase, and chromatin remodeling events.
Mots clés
AFM, Chromatin, DNA, Electron microscopy, Helicases, Homologous recombination, Rad51, Recombinases
Référence
Methods Mol. Biol.. 2018 ;1805:251-270