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Visualizing the Nonhomogeneous Structure of RAD51 Filaments Using Nanofluidic Channels

Författare och institution:
L. H. Fornander (-); K. Frykholm (-); J. Fritzsche (-); J. Araya (-); P. Nevin (-); Erik Werner (Institutionen för fysik (GU)); Ali Cakir (Institutionen för fysik (GU)); F. Persson (-); E. B. Garcin (-); P. J. Beuning (-); Bernhard Mehlig (Institutionen för fysik (GU)); M. Modesti (-); F. Westerlund (-)
Publicerad i:
Langmuir, 32 ( 33 ) s. 8403-8412
ISSN:
0743-7463
Publikationstyp:
Artikel, refereegranskad vetenskaplig
Publiceringsår:
2016
Språk:
engelska
Fulltextlänk:
Sammanfattning (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 (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.
Ämne (baseras på Högskoleverkets indelning av forskningsämnen):
NATURVETENSKAP ->
Fysik
Nyckelord:
single dna-molecules, double-stranded-dna, nucleoprotein filaments, homologous recombination, reca filaments, protein, dynamics, binding, polymerization, spectroscopy, RS,UMR7258,Inserm,U1068, F-13273
Postens nummer:
241980
Posten skapad:
2016-09-19 15:47

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