The dynamics of RAD51 foci formation and elongation in living human cells

Homologous recombination is a DNA repair process that requires binding of RAD51 to ssDNA at the break site. This facilitates the search for a homologous repair template on the sister chromatid, or on the homologous chromosome. How broken DNA ends loaded with RAD51 filaments are brought toward their repair template in the crowded 3D genome is currently poorly understood. This is largely due to a lack of tools to visualize homology search in living human cells. Here, we show that RAD51 and MND1, two proteins operating in homology search, become visible in long, extended structures several hours after double-stranded break formation. Using GFP-MND1 we capture these elongated foci in living human cells and reveal their highly dynamic nature as they traverse the nuclear space and gradually disassemble. We show that resolution of these structures depends on RAD54L, known for its role in RAD51-driven homology search. In addition, we find that loss of cohesin inhibits their resolution, in accordance with a role for cohesin in homology search. Thus, our data suggest that these elongated foci are visible intermediates of an active DNA repair process, and that GFP-MND1 is a powerful tool to study the dynamics of homology search in living human cells.