DNA strand exchange assay and the role of DNA strand exchange in double-strand break repair. Chromosome breakage is followed by nucleolytic resection to generate 3' ssDNA tails on the broken ends. The exposed ssDNA tails are the substrates for DNA strand exchange catalyzed by recombinases of the RecA/Rad51/UvsX family in collaboration with SSB, RMP, and other recombination proteins. The invasion of a homologous duplex (blue) by one of the 3' ssDNA tails generates a heteroduplex D-loop intermediate in which the 3' end of the invading strand is annealed to a template strand and can serve as a primer for recombination-dependent DNA replication (red). Strand displacement DNA synthesis in the forward direction (left to right as drawn) expands the D-loop until the displaced strand can anneal to the exposed ssDNA on the remaining DNA end. This 3' end can now prime DNA synthesis in the reverse direction (right to left as drawn). Ligation generates Holliday junctions that can branch migrate and ultimately are resolved by structure-specific endonucleses to generate recombinant products (not shown). (B) Classic in vitro assay for DNA strand exchange activity of RecA/Rads51/UvsX family recombinases. Homologous circular ssDNA and linear dsDNA substrates derived from bacteriophage M13 are incubated with recombinase and accessory proteins in the presence of ATP. Recombinase-catalyzed homologous pairing generates partially heteroduplex D-loop intermediates. Polar branch migration driven by the recombinase and/or helicases extends the heteroduplex to generated nicked circular dsDNA and linear ssDNA products.