title: Fluorescent human RAD51 reveals multiple nucleation sites and filament segments tightly associated along a single DNA molecule.
abstract: The DNA strand-exchange reactions defining homologous recombination involve transient, nonuniform allosteric interactions between recombinase proteins and their DNA substrates. To study these mechanistic aspects of homologous recombination, we produced functional fluorescent human RAD51 recombinase and visualized recombinase interactions with single DNA molecules in both static and dynamic conditions. We observe that RAD51 nucleates filament formation at multiple sites on double-stranded DNA. This avid nucleation results in multiple RAD51 filament segments along a DNA molecule. Analysis of fluorescent filament patch size and filament kinks from scanning force microscopy (SFM) images indicate nucleation occurs minimally once every 500 bp. Filament segments did not rearrange along DNA, indicating tight association of the ATP-bound protein. The kinetics of filament disassembly was defined by activating ATP hydrolysis and following individual filaments in real time.
authors.affiliation: Department of Cell Biology & Genetics, Erasmus Medical Center, PO Box 2040, 3000 CA Rotterdam, The Netherlands. m.modesti@erasmusmc.nl
authors.forename: Mauro
authors.initials: M
authors.lastname: Modesti
authors.forename: Dejan
authors.initials: D
authors.lastname: Ristic
authors.forename: Thijn
authors.initials: T
authors.lastname: van der Heijden
authors.forename: Cees
authors.initials: C
authors.lastname: Dekker
authors.forename: Joost
authors.initials: J
authors.lastname: van Mameren
authors.forename: Erwin J G
authors.initials: EJ
authors.lastname: Peterman
authors.forename: Gijs J L
authors.initials: GJ
authors.lastname: Wuite
authors.forename: Roland
authors.initials: R
authors.lastname: Kanaar
authors.forename: Claire
authors.initials: C
authors.lastname: Wyman
mesh_terms: D001665:Binding Sites / Q000502:physiology; D004247:DNA / Q000378:metabolism*; D005456:Fluorescent Dyes; D006801:Humans; D011485:Protein Binding / Q000502:physiology; D051135:Rad51 Recombinase / Q000737:chemistry* / Q000378:metabolism*
chemical_list: D005456:Fluorescent Dyes; D004247:DNA; C495153:RAD51 protein, human; D051135:Rad51 Recombinase