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Visualizing the Nonhomogeneous Structure of RAD51 Filaments Using Nanofluidic Channels

Louise Fornander (Institutionen för kemi och kemiteknik, Fysikalisk kemi) ; Karolin Frykholm (Institutionen för biologi och bioteknik, Kemisk biologi) ; Joachim Fritzsche (Institutionen för fysik, Kemisk fysik (Chalmers)) ; J. Araya ; P. Nevin ; Erik Werner ; Ali Cakir ; F. Persson ; E. B. Garcin ; P. J. Beuning ; Bernhard Mehlig ; M. Modesti ; Fredrik Westerlund (Institutionen för biologi och bioteknik, Kemisk biologi)
Langmuir (0743-7463). Vol. 32 (2016), 33, p. 8403-8412.
[Artikel, refereegranskad vetenskaplig]

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.

Nyckelord: single dna-molecules, double-stranded-dna, nucleoprotein filaments, homologous recombination, reca filaments, protein, dynamics, binding, polymerization, spectroscopy, RS,UMR7258,Inserm,U1068, F-13273



Denna post skapades 2016-09-19. Senast ändrad 2016-10-21.
CPL Pubid: 241980

 

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