title: Visualizing the Nonhomogeneous Structure of RAD51 Filaments Using Nanofluidic Channels.
abstract: RAD51 is the key component of the homologous recombination pathway in eukaryotic cells and performs its task by forming filaments on DNA. In this study we investigate the physical properties of RAD51 filaments formed on DNA using nanofluidic channels and fluorescence microscopy. Contrary to the bacterial ortholog RecA, RAD51 forms inhomogeneous filaments on long DNA in vitro, consisting of several protein patches. We demonstrate that a permanent "kink" in the filament is formed where two patches meet if the stretch of naked DNA between the patches is short. The kinks are readily seen in the present microscopy approach but would be hard to identify using conventional single DNA molecule techniques where the DNA is more stretched. We also demonstrate that protein patches separated by longer stretches of bare DNA roll up on each other and this is visualized as transiently overlapping filaments. RAD51 filaments can be formed at several different conditions, varying the cation (Mg(2+) or Ca(2+)), the DNA substrate (single-stranded or double-stranded), and the RAD51 concentration during filament nucleation, and we compare the properties of the different filaments formed. The results provide important information regarding the physical properties of RAD51 filaments but also demonstrate that nanofluidic channels are perfectly suited to study protein-DNA complexes.
authors.forename: Louise H
authors.initials: LH
authors.lastname: Fornander
authors.forename: Karolin
authors.initials: K
authors.lastname: Frykholm
authors.forename: Joachim
authors.initials: J
authors.lastname: Fritzsche
authors.affiliation: Department of Chemistry and Chemical Biology, Northeastern University , Boston, Massachusetts 02115, United States .
authors.forename: Joshua
authors.initials: J
authors.lastname: Araya
authors.affiliation: Department of Chemistry and Chemical Biology, Northeastern University , Boston, Massachusetts 02115, United States .
authors.forename: Philip
authors.initials: P
authors.lastname: Nevin
authors.affiliation: Department of Physics, University of Gothenburg , 412 96 Gothenburg, Sweden.
authors.forename: Erik
authors.initials: E
authors.lastname: Werner
authors.affiliation: Department of Physics, University of Gothenburg , 412 96 Gothenburg, Sweden.
authors.forename: Ali
authors.initials: A
authors.lastname: Çakır
authors.affiliation: Department for Cell and Molecular Biology, Science for Life Laboratory, Uppsala University , 751 24 Uppsala, Sweden.
authors.forename: Fredrik
authors.initials: F
authors.lastname: Persson
authors.affiliation: Cancer Research Center of Marseille, CNRS UMR7258, Inserm U1068, Institut Paoli-Calmettes, Aix-Marseille Université , 13273 Marseille, France.
authors.forename: Edwige B
authors.initials: EB
authors.lastname: Garcin
authors.affiliation: Department of Chemistry and Chemical Biology, Northeastern University , Boston, Massachusetts 02115, United States .
authors.forename: Penny J
authors.initials: PJ
authors.lastname: Beuning
authors.affiliation: Department of Physics, University of Gothenburg , 412 96 Gothenburg, Sweden.
authors.forename: Bernhard
authors.initials: B
authors.lastname: Mehlig
authors.affiliation: Cancer Research Center of Marseille, CNRS UMR7258, Inserm U1068, Institut Paoli-Calmettes, Aix-Marseille Université , 13273 Marseille, France.
authors.forename: Mauro
authors.initials: M
authors.lastname: Modesti
authors.forename: Fredrik
authors.initials: F
authors.lastname: Westerlund