title: Direct imaging of human Rad51 nucleoprotein dynamics on individual DNA molecules.
abstract: Rad51 protein (Rad51) is central to recombinational repair of double-strand DNA breaks. It polymerizes onto DNA and promotes strand exchange between homologous chromosomes. We visualized the real-time assembly and disassembly of human Rad51 nucleoprotein filaments on double-stranded DNA by single-molecule fluorescence microscopy. Rad51 assembly extends the DNA by approximately 65%. Nucleoprotein filament formation occurs via rapid nucleation followed by growth from these nuclei. Growth does not continue indefinitely, however, and nucleoprotein filaments terminate when approximately 2 mum in length. The dependence of nascent filament formation on Rad51 concentration suggests that 2-3 Rad51 monomers are involved in nucleation. Rad51 nucleoprotein filaments are stable and remain extended when ATP hydrolysis is prevented; however, when permitted, filaments decrease in length as a result of conversion to ADP-bound nucleoprotein complexes and partial protein dissociation. Dissociation of Rad51 from dsDNA is slow and incomplete, thereby rationalizing the need for other proteins that facilitate disassembly.
authors.affiliation: Departments of Microbiology and Molecular and Cellular Biology, University of California, Davis, CA 95616-8665, USA.
authors.forename: Jovencio
authors.initials: J
authors.lastname: Hilario
authors.forename: Ichiro
authors.initials: I
authors.lastname: Amitani
authors.forename: Ronald J
authors.initials: RJ
authors.lastname: Baskin
authors.forename: Stephen C
authors.initials: SC
authors.lastname: Kowalczykowski
mesh_terms: D000244:Adenosine Diphosphate / Q000378:metabolism; D004247:DNA / Q000378:metabolism*; D003952:Diagnostic Imaging; D006801:Humans; D007381:Intermediate Filament Proteins / Q000378:metabolism; D007700:Kinetics; D008856:Microscopy, Fluorescence / Q000379:methods*; D009698:Nucleoproteins; D052898:Optical Tweezers; D011485:Protein Binding; D051135:Rad51 Recombinase / Q000378:metabolism*
chemical_list: D007381:Intermediate Filament Proteins; D009698:Nucleoproteins; D000244:Adenosine Diphosphate; D004247:DNA; D051135:Rad51 Recombinase