Fig. 6: Rad51-L1 and -S2 are defecting in dsDNA binding, whereas Rad51-L2 binds but does not completely elongate dsDNA.
From: Real-time tracking reveals catalytic roles for the two DNA binding sites of Rad51
a Formation of Rad51-dsDNA filaments by wild-type Rad51 (blue), Rad51-L1 (red), -L2 (green), or -S2 (purple) was observed by measuring fluorescence anisotropy. Rad51 protein was titrated into the reaction mixture containing dsDNA (3 µM bp). b Gel shift assay. Various concentrations of wild-type and mutant Rad51 proteins (1.25, 2.5, 5.0, and 10 µM) were mixed with dsDNA (10 µM nucleotide). After incubation at 37 °C for 15 min, Rad51-dsDNA filaments were cross-linked with glutaraldehyde. c Elongation of Rad51-dsDNA filament. (upper) Schematic diagram depicting observation of the elongation of Rad51-dsDNA filament using FRET. Emission spectra of fluorescein (green) and rhodamine (red), which are separated by 11 nucleotides on the same strand, were collected by excitation at 493 nm. Yellow balls represent Rad51 monomers. (lower) Emission spectra of fluorescein and rhodamine. Wild-type Rad51 (lower left) or Rad51-L2 (lower center) (8 µM each) and Swi5-Sfr1 (0.8 µM) were mixed with the double-labeled dsDNA (3 µM bp) and the emission spectra of fluorescein and rhodamine were collected after excitation at 493 nm, as described in the “Methods”. (lower right) FRET efficiency (EFRET) of each reaction condition containing wild-type Rad51 or Rad51-L2 in the presence of ATP or AMP-PNP was calculated from the emission spectra shown here and in Supplementary Fig. 5. Orange dot-lines in d, e show the distances between two fluorophores compared with B-form DNA. *p = 1.32 × 10−5, **p = 4.17 × 10−13 and ***p = 1.17 × 10−5 by two-tailed Student′s t test. Data (a, c) are expressed as the mean ± s.d. (n = 3 independent experiments, except n = 6 independent experiments for DNA only in c). Source data are provided as a Source data file.
