Journal
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
Volume 111, Issue 42, Pages 15090-15095Publisher
NATL ACAD SCIENCES
DOI: 10.1073/pnas.1307824111
Keywords
homologous recombination; optical tweezers; BRCA2; single-molecule fluorescence; RAD51
Categories
Funding
- Netherlands Organisation for Scientific Research (NWO)
- NWO TopTalent grant
- NWO Vici grant
- Physics of the Genome program of Foundation for Fundamental Research on Matter, NWO
- European Research Council starting grant
- Agence Nationale pour la Recherche (Project RADORDER) [ARN Blanc 1521 01]
- Optitec
- La Ligue Contre le Cancer
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During recombinational repair of double-stranded DNA breaks, RAD51 recombinase assembles as a nucleoprotein filament around single-stranded DNA to form a catalytically proficient structure able to promote homology recognition and strand exchange. Mediators and accessory factors guide the action and control the dynamics of RAD51 filaments. Elucidation of these control mechanisms necessitates development of approaches to quantitatively probe transient aspects of RAD51 filament dynamics. Here, we combine fluorescence microscopy, optical tweezers, and microfluidics to visualize the assembly of RAD51 filaments on bare single-stranded DNA and quantify the process with single-monomer sensitivity. We show that filaments are seeded from RAD51 nuclei that are heterogeneous in size. This heterogeneity appears to arise from the energetic balance between RAD51 self-assembly in solution and the size-dependent interaction time of the nuclei with DNA. We show that nucleation intrinsically is substrate selective, strongly favoring filament formation on bare single-stranded DNA. Furthermore, we devised a single-molecule fluorescence recovery after photobleaching assay to independently observe filament nucleation and growth, permitting direct measurement of their contributions to filament formation. Our findings yield a comprehensive, quantitative understanding of RAD51 filament formation on bare single-stranded DNA that will serve as a basis to elucidate how mediators help RAD51 filament assembly and accessory factors control filament dynamics.
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