Fig. 4
FIGNL1 dismantles RAD51 from ssDNA. a Schematics of RAD51 disassembly assay from ssDNA immobilized on magnetic beads. b RAD51 disassembly assay in the presence of FIGNL1ΔN. ssDNA-prebound to RAD51 was incubated with an increased concentrations of purified FIGNL1ΔN. After 30 min, supernatants and bound fractions were recovered. Top, a representative SDS-PAGE gel for supernatants and bound fractions stained with CBB. Bottom, Quantification of dissociated RAD51 (Supernatant) and ssDNA-bound RAD51. Intensity of each band of RAD51 was quantified by Imager. The values of RAD51 bands in the supernatant or ssDNA-bound fractions were divided by the total value of RAD51 bands (both in supernatant and bound fractions). Data are mean ± s.d. n = 3. Statistical significance was measured by two-tailed Student’s t-test, see accompanying Source data. c RAD51 disassembly from ssDNA by FIGNL1 mutants. 0.2 μM of FIGNL1ΔN, FIGNL1ΔN-EE (F295E, A298E) or FIGNL1ΔN-KR (K447R) were added to ssDNA-pre-bound beads with RAD51. The binding of RAD51 was analyzed as described in a. Representative gel (top) and quantification (b) are shown. Data are mean ± s.d. n = 3. Statistical significance was measured by two-tailed student’s t-test, see accompanying Source Data. d RAD51 disassembly from ssDNA in the presence of FIGNL1ΔN and or SWSAP1. 0.5 μM of SWSAP1 and 0.5 μM of FIGNL1ΔN were pre-incubated for 30 min and added to the RAD51-bound ssDNA beads. The binding of RAD51 was analyzed as described in a. Representative gel (top) and quantification (b) are shown. Data are mean ± s.d., n = 6. Statistical significance was measured by two-tailed student’s t-test, see accompanying Source Data
