Fig. 2: DNA strand exchange in real time.

a FRET-based real-time assays for the Rad51-mediated DNA strand pairing reaction. (upper) Schematic diagram of the pairing reaction. Yellow circles represent Rad51 monomers. Green and brown stars represent fluorescein and rhodamine, respectively. (lower) Time course of the DNA strand pairing reaction. Reactions containing fluorescein-labeled presynaptic filaments (83 nt) consisting of wild-type Rad51, Rad51-L1 (L1), Rad51-L2 (L2), or Rad51-S2 (S2) (1.5 µM each) with/without Swi5-Sfr1 (0.15 µM) were started (time = 0) by addition of donor dsDNA (40 bp), which was labeled with rhodamine at the end of the complementary strand of the presynaptic filament. Fluorescence emission was monitored at 525 nm upon excitation at 493 nm. Wild-type Rad51; light blue, wild-type Rad51 with Swi5-Sfr1; red, Rad51-L1; green, Rad51-L1 with Swi5-Sfr1; purple, Rad51-L2; yellow, Rad51-L2 with Swi5-Sfr1; black, Rad51-S2; orange, Rad51-S2 with Swi5-Sfr1; dark blue. b FRET-based real-time assays of the Rad51-mediated DNA strand displacement reaction. (upper) Schematic diagram of the displacement reaction. (lower) Time course of the displacement reaction. Reaction conditions were the same as in (a), except that the two ends of the donor dsDNA were labeled with fluorescein and rhodamine. c The upper panel shows the schematics of the three-step reaction. In the reaction formula, [A], [B], [C1], and [C2] correspond to the concentrations of presynaptic filament, donor dsDNA, C1 intermediate, and C2 intermediate, respectively. [D] and [E] correspond to the two products of the strand exchange reaction, heteroduplex DNA, and released ssDNA, respectively. The lower panel shows rate constants for DNA strand pairing derived from the DynaFit-based fitting procedure applied to the data obtained in (a). Data are expressed as the mean ± s.d. (n = 3 independent experiments). See also Supplementary Table 2. Source data are provided as a Source data file.