title: Visualization and quantification of nascent RAD51 filament formation at single-monomer resolution.
abstract: 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.
authors.affiliation: LaserLaB Amsterdam and Department of Physics and Astronomy, VU University, NL-1081HV, Amsterdam, The Netherlands;
authors.forename: Andrea
authors.initials: A
authors.lastname: Candelli
authors.affiliation: Department of Genetics, Cancer Genomics Center, Erasmus University Medical Center, NL-3015CN, Rotterdam, The Netherlands;
authors.forename: Jan Thomas
authors.initials: JT
authors.lastname: Holthausen
authors.affiliation: Department of Bionanoscience, Kavli Institute of Nanoscience, Delft University of Technology, NL-2600GA, Delft, The Netherlands;
authors.forename: Martin
authors.initials: M
authors.lastname: Depken
authors.affiliation: LaserLaB Amsterdam and Department of Physics and Astronomy, VU University, NL-1081HV, Amsterdam, The Netherlands;
authors.forename: Ineke
authors.initials: I
authors.lastname: Brouwer
authors.affiliation: LaserLaB Amsterdam and Department of Physics and Astronomy, VU University, NL-1081HV, Amsterdam, The Netherlands;
authors.forename: Mariëlla A M
authors.initials: MA
authors.lastname: Franker
authors.affiliation: LaserLaB Amsterdam and Department of Physics and Astronomy, VU University, NL-1081HV, Amsterdam, The Netherlands;
authors.forename: Margherita
authors.initials: M
authors.lastname: Marchetti
authors.affiliation: LaserLaB Amsterdam and Department of Physics and Astronomy, VU University, NL-1081HV, Amsterdam, The Netherlands;
authors.forename: Iddo
authors.initials: I
authors.lastname: Heller
authors.affiliation: Centre de Recherche en Cancérologie de Marseille, CNRS UMR 7258, F-13009 Marseille, France; Institut National de la Santé et de la Recherche Médicale U1068, F-13009 Marseille, France; Institut Paoli-Calmettes, F-13009 Marseille, France; Aix-Marseille Université, F-13284 Marseille, France; and.
authors.forename: Stéphanie
authors.initials: S
authors.lastname: Bernard
authors.affiliation: Centre de Recherche en Cancérologie de Marseille, CNRS UMR 7258, F-13009 Marseille, France; Institut National de la Santé et de la Recherche Médicale U1068, F-13009 Marseille, France; Institut Paoli-Calmettes, F-13009 Marseille, France; Aix-Marseille Université, F-13284 Marseille, France; and.
authors.forename: Edwige B
authors.initials: EB
authors.lastname: Garcin
authors.affiliation: Centre de Recherche en Cancérologie de Marseille, CNRS UMR 7258, F-13009 Marseille, France; Institut National de la Santé et de la Recherche Médicale U1068, F-13009 Marseille, France; Institut Paoli-Calmettes, F-13009 Marseille, France; Aix-Marseille Université, F-13284 Marseille, France; and.
authors.forename: Mauro
authors.initials: M
authors.lastname: Modesti
authors.affiliation: Department of Genetics, Cancer Genomics Center, Erasmus University Medical Center, NL-3015CN, Rotterdam, The Netherlands; Department of Radiation Oncology, Erasmus University Medical Center, NL-3015CE, Rotterdam, The Netherlands.
authors.forename: Claire
authors.initials: C
authors.lastname: Wyman
authors.affiliation: LaserLaB Amsterdam and Department of Physics and Astronomy, VU University, NL-1081HV, Amsterdam, The Netherlands;
authors.forename: Gijs J L
authors.initials: GJ
authors.lastname: Wuite
authors.affiliation: LaserLaB Amsterdam and Department of Physics and Astronomy, VU University, NL-1081HV, Amsterdam, The Netherlands; e.j.g.peterman@vu.nl.
authors.forename: Erwin J G
authors.initials: EJ
authors.lastname: Peterman
mesh_terms: D002467:Cell Nucleus / Q000378:metabolism; D004277:DNA, Single-Stranded / Q000737:chemistry*; D005456:Fluorescent Dyes / Q000737:chemistry; D006801:Humans; D016013:Likelihood Functions; D044085:Microfluidics; D008856:Microscopy, Fluorescence; D052898:Optical Tweezers; D034741:RNA, Small Interfering / Q000378:metabolism; D051135:Rad51 Recombinase / Q000737:chemistry*; D011995:Recombination, Genetic; D015203:Reproducibility of Results; D013269:Stochastic Processes; D013379:Substrate Specificity
chemical_list: D004277:DNA, Single-Stranded; D005456:Fluorescent Dyes; D034741:RNA, Small Interfering; C495153:RAD51 protein, human; D051135:Rad51 Recombinase
keywords: BRCA2; RAD51; homologous recombination; optical tweezers; single-molecule fluorescence