RAD51-catalyzed homology recognition and DNA strand exchange: monomer order and dynamics in nucleoprotein filaments assembled on single DNA molecules and during filament interaction with a duplex DNA molecule. – RADORDER

Homologous Recombination is a universal life process essential for maintenance of genome integrity, cell survival and protection against tumorigenesis. Accurate genome duplication, chromosome segregation and repair of DNA double-strand breaks depend on the ability of cells to perform DNA strand exchange reactions between homologous DNA molecules.

The catalytic core of homologous recombination resides in a complex molecular structure formed by an ATP-dependent recombinase protein polymerized as a right-handed helical nucleoprotein filament around single-stranded DNA (ssDNA). Despite several decades of research on the prototype bacterial RecA recombinase, the mechanisms by which such a filament promotes homology recognition in a duplex DNA molecule and ultimately DNA strand exchange at a homologous locus have remained undetermined.

The main reason that makes the study of these mechanisms refractory to experimentation and interpretation is that one must be able to probe recombinase monomers and DNA substrate conformational dynamics within nucleoprotein filaments. Due to reaction asynchrony, structural heterogeneity and population averaging, study of filament conformational dynamics by conventional ensemble biochemistry escapes precise and quantitative analysis. Structural and static visualization techniques such as X-ray crystallography, Atomic Force Microscopy and Electron Microscopy provide relative high spatial resolution information on recombinase filament structure. However, these techniques offer limited information on conformational dynamics since they report on a small number of possible spatial configurations and temporal resolution is typically missing.

For these reasons several research groups embarked on the analysis of recombinase filament conformational dynamics and action via so-called “single molecule” approaches, or perhaps more correctly said “single reaction level”. Two main trends for analyzing the behavior of recombinases on individual DNA molecules have emerged. One school uses magnetic tweezers to manipulate single DNA molecules, attaching one end of the molecule to the surface of a flow chamber and the other to a magnetic micro-sphere whose spatial location and rotation can be controlled with high precision. This instrumentation allows monitoring of changes in length, in torque and tension exerted on the DNA molecule during interaction with a protein but without direct visualization of the protein during the course of the reaction. The other school, however, aims at directly visualizing the fluorescently labeled protein (in some experiments DNA can also be visualized) during its interaction with a single DNA molecule held and manipulated with optical tweezers inside a flow chamber; or attached via one end to the surface and stretched by applying flow. Wide field fluorescence microscopy or total internal reflection fluorescence microscopy (when using the surface as anchor point and stretching the construct by applying flow) is used to monitor protein dynamic behavior in “real-time” using fast and sensitive EM-CCD cameras. Optical traps further allow concomitant measurements of the force exerted on the DNA molecule.

Here, we propose to probe the conformational dynamics of human RAD51 nucleoprotein filaments using the direct visualization single molecule/reaction approach but with the added innovation of exploiting light polarization-anisotropy imaging to gain new insights into the mechanisms of homology recognition and DNA strand exchange by ATP-dependent recombinases.