Visualizing the Assembly of Human Rad51 Filaments on Double-stranded DNA

摘要: Abstract Rad51 is the core component of eukaryotic homologous recombination machinery and assembles into extended nucleoprotein filaments on DNA. To study dynamic behavior we have developed a single-molecule assay that relies combination hydrodynamic force microscale diffusion barriers to align individual DNA molecules surface microfluidic sample chamber coated with lipid bilayer. When visualized total internal reflection fluorescence microscopy (TIRFM), these “molecular curtains” allow for direct visualization hundreds molecules. Using this approach, analyzed binding human single double-stranded under variety different reaction conditions by monitoring extension fluorescently labeled DNA, which coincides assembly filament. We also generated several mutants in conserved regions implicated binding, tested them their ability assemble filaments. show proteins mutations within DNA-binding located N-terminal domain still retain form Mutations L1 loop, projects towards central axis filament, completely abolish In contrast, most or near L2 do not affect protein (ds)DNA. Taken together, results demonstrate L1-loop plays crucial role dsDNA, but loop significantly less impact process. The presented here provide an important initial framework beginning biochemical behaviors using our novel experimental system.