Fig. 3.

Hop2-Mnd1 is not an efficient stabilizer of the Dmc1 presynaptic filament. (A) Dmc1-driven strand exchange reaction with RPA-precoated ssDNA. css was precoated with RPA, then mixed with Dmc1, lds, and the indicated auxiliary factor. (B) Schematic of the RPA chase assay. (C) RPA chase assay. css annealed to a biotinylated oligo was initially precoated with Dmc1, then incubated in the presence of the indicated auxiliary factor, followed by the addition of RPA. css was precipitated with streptavidin-coated magnetic beads and associated proteins were examined (css-bound), along with proteins left in the supernatant (unbound), by SDS-PAGE. css, 10 µM; Dmc1, 5 µM; Hop2-Mnd1, 0.25 or 0.5 µM; RPA, 1 µM; Swi5-Sfr1, 0.5 or 1 µM. (D) Presynaptic filament stability assayed by fluorescence anisotropy. Dmc1 presynaptic filaments were formed by mixing Dmc1 with fluorescently labeled oligo-dT (72-mer), then the mixture was incubated with the indicated auxiliary factor. Stability of the formed filaments was examined by diluting the mixture (40-fold) and monitoring the change in fluorescence anisotropy in real time. (E) Synergistic activation of Dmc1-driven strand exchange by Hop2-Mnd1 and Swi5-Sfr1 with RPA-precoated ssDNA. css precoated with RPA was mixed with Dmc1, the indicated auxiliary factor(s), and lds prepared with StuI. The reaction was then incubated at 30 °C for 60 min. css, 10 µM; Dmc1, 5 µM; Hop2-Mnd1, 1 µM; lds, 10 µM; RPA, 2 µM; Swi5-Sfr1, 4 µM. For the graphs in A, C, and E, mean values ± SD from three independent experiments are shown. Representative kinetics of the reactions are shown in D.