Visualizing the Nonhomogeneous Structure of RAD51 Filaments Using Nanofluidic Channels
Journal article, 2016

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 (Mg2+ or Ca2+), 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.

Inserm

nucleoprotein filaments

U1068

polymerization

binding

single dna-molecules

reca filaments

F-13273

RS

UMR7258

double-stranded-dna

dynamics

protein

spectroscopy

homologous recombination

Author

Louise Fornander

Chalmers, Chemistry and Chemical Engineering, Chemistry and Biochemistry, Physical Chemistry

Karolin Frykholm

Chalmers, Biology and Biological Engineering, Chemical Biology

Joachim Fritzsche

Chalmers, Physics, Chemical Physics

J. Araya

Northeastern University

P. Nevin

Northeastern University

Erik Werner

University of Gothenburg

Ali Cakir

University of Gothenburg

F. Persson

Uppsala University

E. B. Garcin

Centre de Recherche en Cancerologie de Marseille

P. J. Beuning

Northeastern University

Bernhard Mehlig

University of Gothenburg

M. Modesti

Centre de Recherche en Cancerologie de Marseille

Fredrik Westerlund

Chalmers, Biology and Biological Engineering, Chemical Biology

Langmuir

0743-7463 (ISSN) 1520-5827 (eISSN)

Vol. 32 33 8403-8412

Subject Categories

Physical Sciences

DOI

10.1021/acs.langmuir.6b01877

PubMed

27479732

More information

Latest update

2/28/2018