Fig. 6: Human RNF20 promotes hRad51-mediated strand exchange and counteracts the disrupting activities of FBH1.

a Immunoprecipitation showing the interaction between endogenous RNF20 and hRad51 in Hela cells. b GST pull-down showing the interaction between GST-RNF20 and 6xHis-hRad51. GST or GST-RNF20 was assessed by Coomassie blue staining. 6xHis-hRad51 was detected by Western blot. The asterisk denotes the full-length GST-RNF20. c, d An ssDNA pull-down assay and quantification showing the effect of RNF20N on hRad51 binding to ssDNA. ssDNA-bound proteins were detected by Western blot with the anti-GST or anti-His antibody. e, h Single-molecule MT monitoring the role of RNF20N on hRad51-ssDNA assembly (e) or on hRad51 replacement of ssDNA-bound hRPA (h). The average (dark-colored) and SD (lighter-colored) of the time courses at each condition were obtained from three independent molecules (n = 3). 150 nM of hRad51 and RNF20N were used for the experiment in (e). For testing hRPA replacement by hRad51, ssDNA was initially saturated with 400 nM of hRPA. Subsequently, 1 μM of hRad51 with or without RNF20N(400 nM) was used for the reaction in (h). f, g An ssDNA pull-down assay and quantification showing the effect of RNF20 on the replacement of ssDNA-bound hRPA by hRad51. i, j Analysis of the effect of RNF20 on the strand exchange reaction by EMSA. Quantification of the relative amount of strand exchange products is presented in (j). k, l EMSA and quantification showing the role of RNF20 in counteracting the effect of FBH1 on hRad51-mediated strand exchange. m Immunoprecipitation indicating the interaction between the ectopically expressed FLAG-RNF20 and HA-FBH1 (upper panel) or between the endogenous RNF20 and FBH1 (lower panel). n GST pull-down assay showing the interaction between GST-RNF20 and 6xHis-FBH1 in vitro. The asterisk indicates the full-length GST-RNF20 protein. Values in d, g, j, l are the mean ± SD of three independent experiments (n = 3). Statistical analysis was calculated with the Student t-test (two-tailed). n.s. no significance; *p < 0.05, **p < 0.01, ***p < 0.001. Source data are provided as a Source Data file.