Real-Time Visualization of Assembly and Disassembly of S. Cerevisiae Rad51 on Duplex DNA

DNA double-strand break (DSB) is a lethal type of DNA damage. Homologous recombination is the most accurate method to repair broken DNA. The process of recombinational DNA repair consists of three phases. First, the ends of broken DNA are processed to generate a 3′-ended single-strand DNA (ssDNA) tail on which the DNA strand exchange protein self-assembles. Second, the protein-DNA complex searches for homology on a donor double-strand DNA (dsDNA), and catalyzes the pairing and exchange of DNA strands. Finally, the heteroduplex DNA product is resolved.In S. cerevisiae, DNA strand exchange is catalyzed by Rad51 protein. Rad51 forms a right-handed helical nucleoprotein filament in an ATP-dependent manner. In the filament, Rad51 occupies 3-4 nucleotides and stretches DNA to ∼150% of its B-form length. Interestingly, Rad51 binds to dsDNA with a similar affinity as for ssDNA, even though the functional form of Rad51 requires assembly on ssDNA. Also, since Rad51 filament formation on dsDNA inhibits DNA strand exchange, the turnover of Rad51 from dsDNA is stimulated by a dsDNA translocase, Rad54.We used single-molecule fluorescence microscopy to visualize both the kinetics of assembly and disassembly of Rad51 nucleoprotein filaments. Visualization was achieved either by directly imaging filament formation using fluorescent Rad51 protein, or by measuring the DNA length change due to Rad51 binding by fluorescently tagging the DNA end. We show that the nucleation of Rad51 on dsDNA requires about 3 monomers; that Rad51 assembly occurs through frequent nucleation; and that the Rad51 filament is stabilized by maintaining ATP-bound state.