Visualizing the Nonhomogeneous Structure of RAD51 Filaments Using Nanofluidic Channels

Louise H. Fornander, Karolin Frykholm, Joachim Fritzsche, Joshua Araya, Philp Nevin, Erik Werner, Ali Cakir, Fredrik Persson, Edwige B. Garcin, Penny J. Beuning, Bernhard Mehlig, Mauro Modesti, Fredrik Westerlund

Research output: Contribution to journalArticlepeer-review

10 Scopus citations

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.
Original languageEnglish (US)
Pages (from-to)8403-8412
Number of pages10
JournalLANGMUIR
Volume32
Issue number33
DOIs
StatePublished - Aug 1 2016
Externally publishedYes

Bibliographical note

KAUST Repository Item: Exported on 2022-06-02
Acknowledged KAUST grant number(s): KUK-11-008-23
Acknowledgements: This project was supported by the Swedish Research Council Grant [2011-4324 to F.W. and 2013-3992 to B.M.], the Chalmers Area of Advance in Nanoscience and Nano technology [to F.W.], King Abdullah University of Science and Technology Grant [KUK-11-008-23 to L.F.], Investissements d'Avenir French Government program French National Research Agency A*MIDEX project [ANR-11-IDEX-0001-02 to M.M.], grant ANR Blanc 1521 01, project RADORDER, the ARC Foundation and OPTITEC to M.M., Ligue contre le Cancer [to E.B.G.], and American Cancer Society Research Scholar Grant [RSG-12-161-01-DMC to P.J.B.].
This publication acknowledges KAUST support, but has no KAUST affiliated authors.

ASJC Scopus subject areas

  • Spectroscopy
  • General Materials Science
  • Surfaces and Interfaces
  • Electrochemistry
  • Condensed Matter Physics

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