Fig. 1: Rad51 DNA binding site mutants are defective in DNA strand exchange.

a Domain structures of S. pombe Rad51. DNA binding site I contains two loops, L1 and L2. Site II is located C-terminal to L2. Amino acid alignments of these regions are shown. Sp: Schizosaccharomyces pombe; Sc: Saccharomyces cerevisiae; Hs: Homo sapiens. Rad51 mutants with mutations R257A in L1, V295A in L2, and R324A K334A in Site II are referred to as Rad51-L1, Rad51-L2, and Rad51-S2, respectively. b A model of the SpRad51-ssDNA filament constructed by homology modeling using the HsRad51-ssDNA filament structure (PDBID:5H1B). The initial ssDNA is shown in red. L1 and L2 loops are in green and cyan, respectively. R257 in the L1 loop is shown in orange. V295 in the L2 loop is shown in yellow. R324 and K334 in site ll are shown in magenta. c A model of the SpRad51-dsDNA filament constructed by homology modeling using the HsRad51-dsDNA filament structure (PDBID:5H1C). The initial ssDNA, L1 loop, L2 loop, R257 in L1 loop, V295 in L2 loop, and R324 and K334 in site ll have the same colors as those in Fig. 1b. The complementary strand of the initial ssDNA is in blue. d DNA three-strand exchange assay using long DNA substrates. (upper left) Schematic of the assay. (lower left) Gel image of the three-strand exchange assay. Wild-type Rad51 (WT), Rad51-L1 (L1), Rad51-L2 (L2), or Rad51-S2 (S2) (5 µM each) and Swi5-Sfr1 (0.1, 0.5, and 2.5 µM) were used in the assay. (right) Quantified yields of nicked-circular dsDNA (NC, one of the final reaction products) and joint molecules (reaction intermediates). e K_D values of wild-type and mutant Rad51 proteins for trinitrophenyl-ATP. WT, 0.646 ± 0.032; Rad51-L1, 0.729 ± 0.031; Rad51-L2, 0.448 ± 0.030; Rad51-S2, 0.627 ± 0.12. f k_cat values of ATP hydrolysis by wild-type and mutant Rad51 proteins. Blue bars, Rad51 only. Red bars, Rad51 with Swi5-Sfr1. Data (d–f) are expressed as the mean ± s.d. (n = 3 independent experiments). Source data are provided as a Source data file.