Fig. 4: A pairing assay with 2AP indicates that Rad51-L2 is defective in forming the C2 intermediate, which contains heteroduplex DNA.
From: Real-time tracking reveals catalytic roles for the two DNA binding sites of Rad51
a Schematic diagram of DNA strand exchange reaction using ssDNA containing 2AP. b Time course of the DNA strand pairing reaction using wild-type Rad51. (left) A pairing reaction by Rad51 with 2AP-containing ssDNA and dsDNA with homologous (blue) or heterologous (red) sequences. (center) A pairing reaction by Rad51 + Swi5-Sfr1 with 2AP-containing ssDNA and dsDNA with homologous (blue) or heterologous (red) sequences. (right) Time course of the FRET-based DNA strand pairing reaction using wild type Rad51, fluorescein-labeled ssDNA, and rhodamine-labeled dsDNA with homologous (blue) or heterologous (red) sequences. c Time course of the DNA strand pairing reaction using Rad51-L2 mutant. (left) A pairing reaction by Rad51-L2 with 2AP-containing ssDNA and dsDNA with homologous (blue) or heterologous (red) sequences. (center) A pairing reaction by Rad51-L2 + Swi5-Sfr1 with 2AP-containing ssDNA and dsDNA with homologous (blue) or heterologous (red) sequences. (right) Time course of the FRET-based DNA strand pairing reaction using Rad51-L2, fluorescein-labeled ssDNA, and rhodamine-labeled dsDNA with homologous (blue) or heterologous (red) sequences. d Change in fluorescence intensity of 2AP 25 min after the pairing reaction started, calculated from (b). The results of reactions with homologous dsDNA and completely heterologous dsDNA are displayed as blue and red bars, respectively. Data are expressed as the mean ± s.d. (n = 3 independent experiments). Source data are provided as a Source data file.
