Two three-strand intermediates are processed during Rad51-driven DNA strand exchange

During homologous recombination, Rad51 forms a nucleoprotein filament with single-stranded DNA (ssDNA) that undergoes strand exchange with homologous double-stranded DNA (dsDNA). Here, we use real-time analysis to show that strand exchange by fission yeast Rad51 proceeds via two distinct three-strand intermediates, C1 and C2. Both intermediates contain Rad51, but whereas the donor duplex remains intact in C1, the ssDNA strand is intertwined with the complementary strand of the donor duplex in C2. Swi5–Sfr1, an evolutionarily conserved recombination activator, facilitates the C1–C2 transition and subsequent ssDNA release from C2 to complete strand exchange in an ATP-hydrolysis-dependent manner. In contrast, Ca 2+ , which activates the Rad51 filament by curbing ATP hydrolysis, facilitates the C1–C2 transition but does not promote strand exchange. These results reveal that Swi5–Sfr1 and Ca 2+ have different activation modes in the late synaptic phase, despite their common function in stabilizing the presynaptic filament. Real-time FRET analyses and biochemical assays reveal that Rad51 recombinase promotes DNA strand exchange via two distinct three-strand intermediate states.